- Email: [email protected]
IGF2 axis
International Immunopharmacology 78 (2020) 106065
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Long non-coding RNA SNHG16 promotes lipopolysaccharides-induced acute pneumonia in A549 cells via targeting miR-370-3p/IGF2 axis Ju Zhang, Fengxia Mao, Gai Zhao, Haixia Wang, Xiaomin Yan, Qian Zhang
T
⁎
Department of Pediatrics, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: SNHG16 miR-370-3p IGF2 Acute pneumonia LPS A549 cells
Background: Pneumonia is an infectious lung inflammation in children with high mortality and morbidity rates. Small nucleolar RNA host gene 16 (SNHG16) has been verified to accelerate the progression of acute pneumonia. However, the role of SNHG16 in acute pneumonia has not yet been fully elucidated. The study was aimed to explore the regulatory mechanism of SNHG16 in LPS-induced acute pneumonia in A549 cells. Methods: The levels of SNHG16, miR-370-3p and IGF2 in serum samples and LPS-induced A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptosis of A549 cells were examined by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The levels of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by enzymelinked immunosorbent assay (ELISA). The binding relationships among SNHG16, miR-370-3p and IGF2 were predicted by online database and verified by Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The protein levels of IGF2 were tested by Western blot. Results: SNHG16 and IGF2 were upregulated while miR-370-3p was downregulated in serum of acute pneumonia patients and LPS-induced A549 cells. SNHG16 regulated proliferation, apoptosis and inflammatory cytokines by inhibiting miR-370-3p in LPS-induced A549 cells. MiR-370-3p targeted IGF2 and inhibited LPS-induced inflammatory injury via IGF2 in A549 cells. Furthermore, SNHG16 was verified to promote IGF2 expression by sponging miR-370-3p in A549 cells. Conclusion: SNHG16 impeded cell viability and promoted apoptosis, inflammatory injury by targeting IGF2 mediated by miR-370-3p in LPS-induced A549 cells.
1. Introduction Pneumonia is an inflammatory disease of the lung, which is triggered by different pathogens infection [1,2]. The typical clinical features of pneumonia are fever, cough, dyspnea, somnolence and chest pain [3,4]. As a main infectious disease, it has a high mortality rate in the elderly and children [5,6]. Lipopolysaccharide (LPS) is the major biologically active component of the cell wall, which is produced by Gram-negative bacteria and belongs to the endotoxins, and it is pivotal for the inflammatory response related to pneumonia [7–9]. Despite substantial progress has been made in anti-inflammatory treatment for pneumonia, the therapy method still need to be further improved. Therefore, it is of great importance to explore the underlying mechanism and search for the novel effective anti-inflammatory medicine to cure pneumonia. Long non-coding RNAs (lncRNAs) are conserved non-coding RNAs
which have no ability to encode protein [10,11]. A growing bed of evidence have suggested that plenty of lncRNAs perform a vital role in multifarious human diseases, including neurodegenerative diseases [12], cancers [13] and cardiovascular diseases [14]. LncRNA small nucleolar RNA host gene 16 (SNHG16) has been reported to be upregulated and can promote the progression of numerous cancers, for instance, pancreatic cancer [15], hepatocellular carcinoma [16], osteosarcoma [17], and so on. Meanwhile, previous study also reported that SNHG16 was overexpressed in the serum of acute pneumonia patients and it regulated LPS-induced apoptosis and inflammation in LPS-induced WI-38 cells [18]. However, the regulatory mechanism of SNHG16 in acute pneumonia is very complicated and has not yet been well elucidated. MicroRNAs (miRNAs) are short non-coding RNAs that contribute to degrading mRNA or repressing translation by binding to 3′-untranslated region (3′-UTR) of mRNAs [19,20]. Various evidences supported that
⁎ Corresponding author at: Department of Pediatrics, First Affiliated Hospital of Zhengzhou University, Henan Province, No. 1 Jianshe East Road, Erqi District, Zhengzhou City, Zhengzhou 450000, China. E-mail address: [email protected] (Q. Zhang).
https://doi.org/10.1016/j.intimp.2019.106065 Received 20 August 2019; Received in revised form 23 October 2019; Accepted 17 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
manufacturer. SNHG16 and IGF2 were normalized to β-actin, while miR-370-3p was normalized to U6. The relative transcriptional folds were quantified and calculated using the comparative threshold (Ct) cycle (2-ΔΔCt) method. The primers were obtained from Sangon Biotech and listed as follows: SNHG16: 5′-CCCAAGCTTGCGTTCTTTTCGAGGT CGGC-3′ (forward) and 5′-CCGGAATTCTGACGGTAGTTTCCCAAGTT-3′ (reverse); miR-370-3p: 5′-TGTAACCAGAGAGCGGGATGT-3′ (forward) and 5′-TTTTGGCATAACTAAGGCCGAA-3′ (reverse); IGF2: 5′-CTTGGA CTTTGAGTCAAATTGG-3′ (forward) and 5′-GGTCGTGCCAATTACATT TCA-3′ (reverse); U6: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ (forward) and 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); β-actin: 5′-TGGAATC CTGTGGCATCCATGAAAC-3′ (forward) and 5′-ACGCAGCTCAGTAACA GTCCG-3′ (reverse).
deregulated miRNAs were closely linked to many diseases, including cardiovascular disease [21], diabetes and autoimmune diseases [22]. Furthermore, miRNAs also played a vital part in lung inflammation and lung cancer [23]. MiR-370-3p acted as an anti-oncogene in various cancers, including glioma [24], cerebral aneurysm [25], and so on. Moreover, miR-370-3p had a regulatory influence on LPS-induced inflammatory injury in acute pneumonia [26]. IGF2 (Insulin-like growth factor 2) is a necessary ingredient of the stem cell niche [27,28], and it involved in drug resistance and cancers [28]. Moreover, IGF2 knockdown obviously mitigated LPS-induced cell injury of A549 cells in acute pneumonia [29]. However, the interaction between miR-370-3p and IGF2 as well as their roles in pneumonia have not been completely explained. In this study, the expression of SNHG16 in the serum of acute pneumonia patients and LPS-induced A549 cell model was firstly explored. Furtherly, the function of SNHG16 in LPS-induced inflammatory injury of A549 cells and the latent molecular mechanism were verified by gain- and loss-of-function experiments.
2.5. Cell counting Kit-8 (CCK-8) assay The cell viability of A549 cells was explored using CCK-8 (Sangon Biotech) after LPS treatment. Briefly, A549 cells were seeded into 96well plate with approximately 5000 cells per well and then subjected to LPS treatment or transfection. Later, CCK-8 solution was added to each well and incubated for 1 h at 37 °C in an incubator with 5% CO2. The absorbance of each well was measured at 450 nm with a microplate reader, and the cell viability (%) was estimated by average absorbance ratio of treatment (or transfection) group with control group.
2. Materials and methods 2.1. Patients and blood collection In this study, 32 patients with acute pneumonia and 10 healthy individuals from First Affiliated Hospital of Zhengzhou University were recruited. The patients with acute pneumonia in observation group were enrolled according to the diagnostic criteria of acute pediatric pneumonia, and the healthy individuals in the control group had normal physical examination results. General data of the enrolled subjects in the two groups were comparable. The patients involved in this research had not received anti-inflammatory therapy and the patients with other complications were ruled out. Serum was obtained from blood samples of acute pneumonia patients and healthy control individuals, and later kept at −80 °C. This research had gained an approval from the Ethics Committee of First Affiliated Hospital of Zhengzhou University and all participators signed informed consent.
2.6. Apoptosis assay The apoptosis rate of A549 cells was determined using Annexin Vfluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis assay kit (Invitrogen). Briefly, A549 cells were subjected to LPS treatment and transfection and later dyed with Annexin V-FITC and PI for 25 min in the dark. Afterwards, apoptotic cells were tested by flow cytometer. 2.7. Enzyme-linked immunosorbent assay (ELISA)
2.2. Cell culture and LPS treatment To measure the levels of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in culture supernatant of A549 cells, ELISA was conducted by the ELISA kits for IL-1β, IL-6 and TNF-α (Abcam Biotechnology, Cambridge, MA, USA) following the instructions of manufacturer. Absorbance was tested using a microplate reader at 450 nm.
The human pulmonary epithelianl cell line A549 was obtained from the (Sangon Biotech, Shanghai, China) and then incubated in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) replenished with 10% fetal bovine serum (FBS; Sangon Biotech) in a humidified incubator with 5% CO2 at 37 °C. After cells were cultivated in 96-well plates for 24 h, LPS (Solarbio, Beijing, China) at 5, 10, 20 µg/ mL or control dimethyl sulfoxide (DMSO) (Solarbio) was added in DMEM medium for 12 h to produce cell injury model.
2.8. Dual-luciferase reporter assay The Dual-luciferase reporter vectors of wild type SNHG16 (SNHG16-WT) or wild type (IGF2-WT) containing the potential targeted sites of miR-370-3p and mutant-type SNHG16 (SNHG16-MUT) or mutant type IGF2-MUT were established. A549 cells were transfected with above reporter vectors together with miR-370-3p or miR-NC using Lipofectamine 3000 reagent (Invitrogen). The luciferase activity was examined by the Dual-luciferase reporter assay system (Promega, Madison, WI, USA).
2.3. Cell transfection Small interfering RNA against SNHG16 (si-SNHG16) and matched negative control (si-NC), pcDNA-SNHG16 (SNHG16), pcDNA-IGF2 (IGF2) and control (pcDNA), miR-370-3p mimic (miR-370-3p), miR370-3p inhibitor (anti-miR-370-3p) and matched controls (miR-NC or anti-miR-NC) were acquired from RiboBio Co., Ltd (Guangzhou, China). A549 cells were transfected using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) referring to user guide.
2.9. RNA immunoprecipitation (RIP) assay 2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) RIP assay was performed using RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) following the protocols of manufacturer. A549 cells were lysed and co-incubated with immunoglobulin G (IgG) antibody conjugated magnetic beads (ab133470) (Abcam Biotechnology) or Argonaute2 (Ago2) antibody (ab32381) (Abcam Biotechnology). The expression of SNHG16, miR370-3p and IGF2 was analyzed by qRT-PCR.
TRIzol reagent (Invitrogen) was employed to isolate total RNA from serum samples and A549 cells. The first strand of cDNA was reversely transcribed from extractive RNA using PrimeScript RT reagent Kit (Takara, Dalian, China). QRT-PCR was adopted using 2X SYBR Green Abstart PCR Mix (Sangon Biotech) on an ABI 7300 PCR System (Applied Biosystems, Carlsbad, CA, USA) based on the instructions of 2
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
3.2. Knockdown of SNHG16 alleviated inflammatory injury in LPS-induced A549 cells
2.10. Western blot assay Total protein was isolated from LPS-stimulated or transfected A549 cells using RIPA buffer (Sangon Biotech) and then quantified by Bradford Protein Assay Kit (Sangon Biotech). Then, proteins were isolated by 10% SDS-PAGE, and later transferred to PVDF membrane (Sangon Biotech). After blocked for 1 h in 5% dried skim milk, the membrane was added with primary antibodies against IGF2 and β-actin (1:1000, Abcam Biotechnology) at 4 °C for 24 h. Subsequently, the PVDF membrane was co-cultured with horseradish peroxidase (HRP)conjugated secondary antibody (1:5000; Abcam Biotechnology) for 2 h following rinsing twice with Tris-buffered saline and Tween 20 (TBST; Beyotime Biotechnology, Shanghai, China). Finally, the protein bands were visualized by ECL detection kit (Beyotime Biotechnology), and the signal intensity of bands was quantified by ImageJ software normalized to β-actin.
To explore the role of SNHG16 in LPS-induced inflammatory injury, A549 cells were transfected with si-SNHG16 or negative control si-NC. The expression of SNHG16 in LPS-induced A549 cells transfected with si-SNHG16 was strikingly inhibited compared with si-NC group detected by qRT-PCR assay, which evidenced high transfection efficiency (Fig. 2A). CCK-8 assay revealed that the cell viability of A549 cells was significantly reduced by LPS treatment, while si-SNHG16-mediated SNHG16 silencing triggered a remarkable increase of cell viability in LPS-induced A549 cells compared with si-NC-transfected cells (Fig. 2B). And knockdown of SNHG16 visibly decreased apoptotic cell rates in A549 cells subjected to LPS treatment compared with si-NC-transfected cells tested by flow cytometry analysis using Annexin V-FITC/PI Apoptosis Detection Kit (Fig. 2C and D). Meanwhile, SNHG16 silencing also strongly decreased the production levels of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells tested by ELISA assay (Fig. 2E–G). These data suggested that SNHG16 silencing could relieve inflammatory injury of LPS-induced A549 cells.
2.11. Statistical analysis All experiments were performed more than three replicates and the data in this study was exhibited as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 19.0. The comparison significances between different groups were evaluated by Student’s t-test and one-way analysis of variance. P < 0.05 was designated as statistically significant.
3.3. SNHG16 targeted miR-370-3p and suppressed miR-370-3p expression in LPS-induced A549 cells Aiming to explain the function of SNHG16 in LPS-induced A549 cells and the underlying mechanisms, LncBase v.2 database was used to predict the putative association between SNHG16 and miR-370-3p and the binding sites between them were exhibited (Fig. 3A). Then the luciferase reporter vectors wild-type SNHG16 (SNHG16-WT) containing the wild-type miR-370-3p-binding sequence in SNHG16 and mutanttype SNHG16 (SNHG16-MUT) were constructed and co-transfected with miR-370-3p or miR-NC into A549 cells. Dual-luciferase reporter assay indicated that the luciferase activity of SNHG16-WT was significantly reduced in A549 cells co-transfected with miR-370-3p, whereas the luciferase activity of SNHG16-MUT was insusceptible (Fig. 3B). The RIP assay was carried out to further validate the direct interaction between SNHG16 and miR-370-3p using A549 cells extracts and Ago2 antibody. The RIP results showed that SNHG16 and miR-3703p exerted more remarkable enrichment in the Ago2-containing miRNA ribonucleoprotein complexes (miRNPs) than negative control IgG group (Fig. 3C). Meanwhile, miR-370-3p was significantly down-regulated in the serum of acute pneumonia patients (n = 32) in comparison with that of healthy controls (n = 10) (Fig. 3D). Consistently, the level of miR-370-3p was overtly reduced with LPS induction in A549 cells in a concentration-dependent manner tested by qRT-PCR (Fig. 3E). Furthermore, Gain- and loss-of-function assays disclosed that the expression of miR-370-3p in A549 cells transfected with SNHG16 was evidently downregulated compared with that of pcDNA-transfected cells,
3. Results 3.1. SNHG16 expression was increased in serum of acute pneumonia patients and LPS-induced A549 cells To examine the level of SNHG16 in pneumonia, qRT-PCR assay was performed. The results suggested that the level of SNHG16 was notably upregulated in the serum of acute pneumonia patients (n = 32) relative to that of healthy controls (n = 10) (Fig. 1A). Meanwhile, the expression of SNHG16 in human pulmonary epithelial cell line A549 treated with different concentrations of LPS (0, 5, 10, and 20 µg/mL) was further measured. As shown in Fig. 1B, the expression of SNHG16 in LPS-induced A549 cells was strikingly increased in dose-dependent manner, with statistically significant effects at 5 µg/mL (*P < 0.05), and 10 µg/mL (***P < 0.001) concentrations. Therefore, 10 µg/mL was chosen as LPS-stimulating condition for application in the following experiments. Hence, we speculated that SNHG16 might perform a latent role in regulating acute pneumonia development.
Fig. 1. The expression of SNHG16 was upregulated in serum of acute pneumonia patients and LPS-induced A549 cells. (A) The expression of SNHG16 in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was determined by qRTPCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (B) The expression of SNHG16 in human pulmonary epithelial cell line A549 treated with different concentrations of LPS (0, 5, 10, and 20 µg/mL) was tested by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA.
3
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 2. SNHG16 knockdown alleviated LPS-induced inflammatory injury in A549 cells. (A) The expression of SNHG16 in LPS-induced A549 cells transfected with siSNHG16 or negative controls was detected by qRT-PCR assay. (B) The cell viability of LPS-induced A549 cells transfected with si-SNHG16 or negative controls was tested by CCK-8 assay. (C-D) The apoptotic rate of LPS-induced A549 cells transfected with si-SNHG16 or negative controls was examined by Annexin V-FITC/PI Apoptosis Detection Kit using flow cytometry. (E-G) The levels of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with siSNHG16 or negative controls were estimated by ELISA assay. * indicated P < 0.05, ** indicated P < 0.01 and *** indicated P < 0.001 vs. control or LPS-induced A549 cells transfected with si-NC for A–G, analyzed by Student’s t-test.
(Fig. 4D–F). Above data suggested that SNHG16 could increase LPSinduced injury by targeting miR-370-3p in A549 cells.
while the expression of miR-370-3p in A549 cells transfected with siSNHG16 was obviously upregulated relative to that of si-NC group (Fig. 3F). Thus, it could be concluded that miR-370-3p was a target of SNHG16 and SNHG16 negatively regulated miR-370-3p expression in A549 cells.
3.5. IGF2 was a target of miR-370-3p in A549 cells For the purpose of exploring the role of miR-370-3p in A549 cells, microT-CDS database was employed to predict the targeted binding between miR-370-3p and IGF2. The results indicated that there was a binding sequence between miR-370-3p and IGF2 (Fig. 5A). The reporter plasmids IGF2-WT containing the predicted miR-370-3p binding sites and mutant IGF2-MUT were constructed. As shown in Fig. 5B, the luciferase activity of IGF2-WT co-transfected with miR-370-3p in A549 cells was strongly limited, while the luciferase activity of IGF2-MUT cotransfected with miR-370-3p was unchanged. The direct interaction between miR-370-3p and IGF2 was further validated in RIP assay and the results evinced that IGF2 and miR-370-3p were preferentially enriched in Ago2-containing miRNPs compared with control IgG immunoprecipitates (Fig. 5C). Simultaneously, the mRNA level of IGF2 was increased in the serum of acute pneumonia patients (n = 32) compared with that in healthy controls (n = 10) (Fig. 5D) and was gradually raised with the concentration increasing of LPS examined by qRT-PCR (Fig. 5E). Moreover, function assays verified that the protein
3.4. SNHG16 regulated proliferation, apoptosis and inflammatory damage via miR-370-3p in LPS-induced A549 cells To verify the regulatory relation between SNHG16 and miR-370-3p, SNHG16 was down-regulated by its siRNA, and miR-370-3p was further knocked down by its inhibitor in LPS-induced A549 cells. As expected, the expression of miR-370-3p in LPS-induced A549 cells transfected with si-SNHG16 was obviously upregulated compared with that of siNC group detected by qRT-PCR, while the stimulative effect was broadly inhibited by anti-miR-370-3p (Fig. 4A). Moreover, si-SNHG16 improved cell viability and reduced apoptosis in LPS-induced A549 cells, whereas the effect of SNHG16 silencing was largely abolished by inhibition of miR-370-3p (Fig. 4B and C). In addition, the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) were decreased in LPS-induced A549 cells transfected with si-SNHG16, while the effects were partly reversed by co-transfection with miR-370-3p inhibitor 4
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 3. SNHG16 targeted miR-370-3p and inhibited miR-370-3p expression in A549 cells. (A) The complementary sequence between SNHG16 and miR-370-3p was predicted by LncBase v.2 database. (B) The luciferase activity of SNHG16-WT or SNHG16-MUT in A549 cells co-transfected with miR-370-3p or miR-NC was detected by Dual-luciferase reporter assay. *** indicated P < 0.001 vs. cells transfected with miR-NC, analyzed by Student’s t-test. (C) Association of SNHG16 and miR-3703p with Ago2 was performed by RIP assay. The RNA levels of SNHG16 and miR-370-3p were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). *** indicated P < 0.001 vs. IgG group, analyzed by ANOVA. (D) The expression of miR-370-3p in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was tested by qRT-PCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (E) The expression of miR-370-3p in A549 cells treated with different doses of LPS was tested by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA. (F) The expression of miR-370-3p in A549 cells transfected with SNHG16, si-SNHG16 or corresponding negative controls was determined by qRT-PCR. *** indicated P < 0.001 vs. cells transfected with pcDNA or si-NC, analyzed by Student’s t-test.
3p targeted IGF2 and suppressed IGF2 expression in A549 cells.
level of IGF2 in A549 cells was clearly reduced when overexpression of miR-370-3p compared with miR-NC-transfected cells, while the level of IGF2 was strikingly elevated by anti-miR-370-3p relative to that of antimiR-NC group (Fig. 5F). All these findings demonstrated that miR-370-
Fig. 4. SNHG16 modulated proliferation, apoptosis and inflammatory injury via miR-370-3p in LPS-induced A549 cells. (A) The expression of miR-370-3p in LPSinduced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p or corresponding negative controls was tested by qRT-PCR assay. (B) The cell viability of LPS-induced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p, or corresponding negative controls was detected by CCK-8 assay. (C) The apoptotic rate of LPS-induced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p, or corresponding negative controls was determined by Annexin V-FITC/PI Apoptosis Detection Kit using flow cytometry. (D–F) The levels of IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with si-SNHG16, siSNHG16+ anti-miR-370-3p, or corresponding negative controls were examined by ELISA assay. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for Fig. 4A-2F, analyzed by Student’s t-test. 5
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 5. IGF2 was a target of miR-370-3p in A549 cells. (A) The binding sites between miR-370-3p and IGF2 were predicted by microT-CDS database. (B) The luciferase activity of IGF2-WT or IGF2-MUT in A549 cells co-transfected with miR-370-3p or miR-NC was examined by Dual-luciferase reporter assay. *** indicated P < 0.001 vs. cells transfected with miR-NC, analyzed by Student’s t-test. (C) The direct interaction between miR-370-3p and IGF2 was validated by RIP assay. *** indicated P < 0.001 vs. IgG group, analyzed by ANOVA. (D) The relative IGF2 mRNA level in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was tested by qRT-PCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (E) The mRNA level of IGF2 in A549 cells subjected to different concentration of LPS was determined by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA. (F) The protein level of IGF2 in A549 cells transfected with miR-370-3p, anti-miR-370-3p or corresponding negative controls was tested by Western blot. *** indicated P < 0.001 vs. cells transfected with miR-NC or anti-miR-NC, analyzed by Student’s t-test.
Fig. 6. MiR-370-3p suppressed inflammatory injury by targeting IGF2 in LPS-induced A549 cells. (A) The protein level of IGF2 in LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls was explored by Western blot. (B–C) The cell viability and apoptosis of LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls were evaluated by CCK-8 assay or flow cytometry, respectively. (D–F) The levels of IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls were tested by ELISA assay. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for A–F, analyzed by Student’s t-test.
3p + pcDNA or miR-370-3p + IGF2. The level of IGF2 in LPS-induced A549 cells transfected with miR-370-3p was significantly declined compared with miR-NC-transfected cells, while the effect was distinctly weakened by IGF2 overexpression (Fig. 6A). Meanwhile, the cell viability was elevated and cell apoptosis was reduced in LPS-induced A549
3.6. MiR-370-3p inhibited inflammatory injury by targeting IGF2 in LPSinduced A549 cells In order to study the function of miR-370-3p in depth, LPS-induced A549 cells were transfected with miR-NC, miR-370-3p, miR-3706
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 7. SNHG16 regulated the expression of IGF2 through miR-370-3p in A549 cells. (A) The expression of miR-370-3p in A549 cells transfected with SNHG16, SNHG16+ miR-370-3p or corresponding negative controls was assessed by qRT-PCR. (B) The expression of miR-370-3p in A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p or corresponding negative controls was tested by qRTPCR. (C) The protein level of IGF2 in A549 cells transfected with SNHG16, SNHG16+ miR-370-3p or corresponding negative controls was detected by Western blot. (D) The protein level of IGF2 in A549 cells transfected with si-SNHG16, si-SNHG16+ antimiR-370-3p or corresponding negative controls was determined by Western blot. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for A–D, analyzed by Student’s ttest.
cells transfected with miR-370-3p relative to that in miR-NC group, while the effects were significantly reversed by co-transfection with IGF2 (Fig. 6B and C). Correspondingly, the levels of IL-1β, IL-6 and TNF-α were declined in LPS-induced A549 cells transfected with miR370-3p, whereas the effects were largely overturned by IGF2 overexpression (Fig. 6D–F). All these evidences confirmed that miR-370-3p suppressed LPS-induced inflammatory injury through IGF2 in A549 cells. 3.7. SNHG16 regulated the expression of IGF2 by sponging miR-370-3p in A549 cells To further explore the relationship among SNHG16, miR-370-3p and IGF2, gain- and loss-function experiments were proceeded. The results demonstrated that the expression of miR-370-3p was notably downregulated and the protein level of IGF2 was significantly increased in A549 cells transfected with SNHG16, whereas the influences were effectively overturned by co-transfection with miR-370-3p (Fig. 7A and C). Expectedly, the level of miR-370-3p was overtly enhanced and the protein level of IGF2 was strikingly weakened in A549 cells transfected with si-SNHG16, while the effects were vastly eliminated when cotransfection with si-SNHG16 and anti-miR-370-3p (Fig. 7B and D). From the above, the findings indicated that SNHG16 regulated IGF2 expression via miR-370-3p in A549 cells (see Fig. 8).
Fig. 8. The mechanism schematic model by SNHG16/miR-370-3p/IGF2 axis in pneumonia (LPS-induced A549 cells). The expression of SNHG16 and IGF2 was upregulated, while miR-370-3p was downregulated in pneumonia. Upregulated SNHG16 impeded cell viability while promoted apoptosis and inflammation through increasing the expression of IGF2 via inhibiting miR-370-3p in pneumonia (LPS-induced A549 cells).
4. Discussion Pneumonia is one of lower respiratory illnesses and a common 7
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
LPS-induced A549 cells. Therefore, this study was helpful for better understanding of the role of SNHG16 and the underlying mechanism in LPS-induced A549 cells, in order to provide novel targets for pneumonia therapy.
hospital-acquired fatal infections [2,30]. It has been reported that the progression of pneumonia is related to pro-inflammatory cytokines, for instance, TNF-α, IL-1β and IL-6 which activate the immune system and take part in the acute inflammatory response [31,32]. Thereby, searching for novel therapeutic targets to block inflammatory response and cell injury in order to cure pneumonia is a very pressing task. As we all know, a large number of lncRNAs can serve as novel biomarkers for early diagnosis of diseases through acting as miRNAs sponges. Similarly, lncRNA SNHG16 could promote cancer progression via sponging different miRNAs. For example, SNHG16 could spong miR-140-5p to accelerate human retinoblastoma progression [33], and facilitated glioma tumorigenicity by targeting miR-373 [34]. Previous research also revealed that SNHG16 was upregulated and involved in tumor progression in non-small cell lung cancer by sponging miR-146a [35]. Moreover, the expression of SNHG16 was elevated in the serum of patients infected with acute pneumonia, and knockdown of SNHG16 alleviated the LPS-induced cell damage by accelerating cell viability, blocking apoptosis and production of inflammatory cytokines via miR146a-5p/CCL5 axis [18]. Consistent with above research, this study demonstrated that the level of SNHG16 was raised in serum of acute pneumonia patients and LPS-induced A549 cells. SNHG16 silencing notably improved cell viability, reduced apoptosis and the production of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells. Moreover, target prediction database and dual-luciferase reporter assay revealed that SNHG16 was a spong of miR-370-3p, which was further verified by RIP assay. All these data confirmed that SNHG16 participated in inflammatory response in LPS-induced A549 cells and might act as a negative regulator through sponging miR-370-3p in acute pneumonia. Growing evidence have indicated that miRNAs are especially important to maintain the homeostasis and development of lung, and they are involved in the pathogenesis of pulmonary diseases, for example, sarcoidosis, asthma and lung cancer [23,36]. MiR-370-3p could act as an anti-oncogene by regulating different targeted genes in various cancers. For instance, miR-370-3p was downregulated and inhibited the progression of nonfunctional pituitary adenomas by targeting HMGA2 [37], and also suppressed cell proliferation of glioma via targeting βcatenin [24]. However, the studies about the role of miR-370-3p in pneumonia was very lacking. Zhang et al. found that miR-370-3p weakened the LPS-induced WI-38 cells inflammation injury and cell apoptosis in acute pneumonia by targeting downstream gene TLR4 [26]. In this study, it was verified that miR-370-3p was a target of SNHG16 and inhibition of miR-370-3p reversed the effects of SNHG16 knockdown on proliferation, apoptosis and the production of inflammatory cytokines in LPS-induced A549 cells. Taken together, these findings indicated that miR-370-3p was a tumor suppressor gene in LPSinduced A549 cells. IGF2 was verified to be involved in LPS-induced cell injury of A549 cells targeted by miRNA-3941 in acute pneumonia [29]. What is more, IGF2 was evinced as a target gene of miR-370-3p by on line database and functional experiments in this study. Besides, overexpression of IGF2 strongly abolished the influence of miR-370-3p on cell injury, and the elevated protein level of IGF2 in A549 cells transfected with SNHG16 was vastly weakened by miR-370-3p, while the decreased protein level of IGF2 in A549 cells induced by si-SNHG16 was obviously enhanced by miR-370-3p knockdown. Hence, it could be speculated that SNHG16 could restore the expression of IGF2 by sponging miR370-3p. Thus, it was concluded that SNHG16 might take part in cell proliferation, apoptosis and inflammatory damage via miR-370-3p/ IGF2 axis in LPS-induced A549 cells.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement None. Fund None. References [1] C. Cilloniz, A. Torres, M. Niederman, M. van der Eerden, J. Chalmers, T. Welte, F. Blasi, Community-acquired pneumonia related to intracellular pathogens, Intensive Care Med. 42 (9) (2016) 1374–1386. [2] L.A. Mandell, Community-acquired pneumonia: An overview, Postgrad. Med. 127 (6) (2015) 607–615. [3] J.T. Mattila, M.J. Fine, A.H. Limper, P.R. Murray, B.B. Chen, P.L. Lin, Pneumonia. Treatment and diagnosis, Ann. Am. Thorac. Soc. 11 (Suppl 4) (2014) S189–S192. [4] R.R. Watkins, T.L. Lemonovich, Diagnosis and management of community-acquired pneumonia in adults, Am. Fam. Physician 83 (11) (2011) 1299–1306. [5] P. Faverio, S. Aliberti, G. Bellelli, G. Suigo, S. Lonni, A. Pesci, M.I. Restrepo, The management of community-acquired pneumonia in the elderly, Eur. J. Int. Med. 25 (4) (2014) 312–319. [6] T. Wardlaw, P. Salama, E.W. Johansson, E. Mason, Pneumonia: the leading killer of children, Lancet 368 (9541) (2006) 1048–1050. [7] D. Dreyfuss, J.D. Ricard, Acute lung injury and bacterial infection, Clin. Chest Med. 26 (1) (2005) 105–112. [8] N. Meng, J. Zhao, L. Su, B. Zhao, Y. Zhang, S. Zhang, J. Miao, A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells, Int. J. Biochem. Cell Biol. 44 (2) (2012) 311–319. [9] M. Rojas, C.R. Woods, A.L. Mora, J. Xu, K.L. Brigham, Endotoxin-induced lung injury in mice: structural, functional, and biochemical responses, Am. J. Physiol. Lung Cell. Mol. Physiol. 288 (2) (2005) L333–L341. [10] C. Ernst, C.C. Morton, Identification and function of long non-coding RNA, Front. Cell. Neurosci. 7 (2013) 168. [11] C.P. Ponting, P.L. Oliver, W. Reik, Evolution and functions of long noncoding RNAs, Cell 136 (4) (2009) 629–641. [12] C.W. Wei, T. Luo, S.S. Zou, A.S. Wu, The role of long noncoding RNAs in central nervous system and neurodegenerative diseases, Front. Behav. Neurosci. 12 (2018) 175. [13] R. Spizzo, M.I. Almeida, A. Colombatti, G.A. Calin, Long non-coding RNAs and cancer: a new frontier of translational research? Oncogene 31 (43) (2012) 4577–4587. [14] S. Uchida, S. Dimmeler, Long noncoding RNAs in cardiovascular diseases, Circ. Res. 116 (4) (2015) 737–750. [15] S. Liu, W. Zhang, K. Liu, Y. Liu, LncRNA SNHG16 promotes tumor growth of pancreatic cancer by targeting miR-218-5p, Biomed. Pharmacother. 114 (2019) 108862. [16] J.H. Zhong, X. Xiang, Y.Y. Wang, X. Liu, L.N. Qi, C.P. Luo, W.E. Wei, X.M. You, L. Ma, B.D. Xiang, L.Q. Li, The lncRNA SNHG16 affects prognosis in hepatocellular carcinoma by regulating p62 expression, J. Cell. Physiol. (2019). [17] P. Su, S. Mu, Z. Wang, Long noncoding RNA SNHG16 promotes osteosarcoma cells migration and invasion via sponging miRNA-340, DNA Cell Biol. 38 (2) (2019) 170–175. [18] Z. Zhou, Y. Zhu, G. Gao, Y. Zhang, Long noncoding RNA SNHG16 targets miR-146a5p/CCL5 to regulate LPS-induced WI-38 cell apoptosis and inflammation in acute pneumonia, Life Sci. 228 (2019) 189–197. [19] M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, Identification of novel genes coding for small expressed RNAs, Science 294 (5543) (2001) 853–858. [20] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2) (2004) 281–297. [21] T. Barwari, A. Joshi, M. Mayr, MicroRNAs in cardiovascular disease, J. Am. Coll. Cardiol. 68 (23) (2016) 2577–2584. [22] E.N. Kontomanolis, A. Koukouli, G. Liberis, H. Stanulov, A. Achouhan, A. Pagkalos, MiRNAs: regulators of human disease, Eur. J. Gynaecol. Oncol. 37 (6) (2016) 759–765. [23] A. Sittka, B. Schmeck, MicroRNAs in the lung, Adv. Exp. Med. Biol. 774 (2013) 121–134.
5. Conclusion This study verified the involvement of SNHG16 in LPS-induced A549 cells. SNHG16 may be involved in proliferation, apoptosis and inflammatory response by modulating IGF2 mediated by miR-370-3p in 8
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
[32] G. Ilonidis, E. Parapanisiou, A. Anogeianaki, I. Giavazis, E.K. Theofilogiannakos, P. Tsekoura, K. Kidonopoulou, C. Trakatelli, Z. Polimenidis, P. Conti, G. Anogianakis, Interleukin -1beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor (TNF) in plasma and pleural fluid of pneumonia, lung cancer and tuberculous pleuritis, J. Biol. Regul. Homeost. Agents 20 (1–2) (2006) 41–46. [33] C. Xu, C. Hu, Y. Wang, S. Liu, Long noncoding RNA SNHG16 promotes human retinoblastoma progression via sponging miR-140-5p, Biomed. Pharmacother. 117 (2019) 109153. [34] X.Y. Zhou, H. Liu, Z.B. Ding, H.P. Xi, G.W. Wang, lncRNA SNHG16 promotes glioma tumorigenicity through miR-373/EGFR axis by activating PI3K/AKT pathway, Genomics (2019). [35] W. Han, X. Du, M. Liu, J. Wang, L. Sun, Y. Li, Increased expression of long noncoding RNA SNHG16 correlates with tumor progression and poor prognosis in nonsmall cell lung cancer, Int. J. Biol. Macromol. 121 (2019) 270–278. [36] A. Ebrahimi, E. Sadroddiny, MicroRNAs in lung diseases: Recent findings and their pathophysiological implications, Pulm. Pharmacol. Ther. 34 (2015) 55–63. [37] F. Cai, C. Dai, S. Chen, Q. Wu, X. Liu, Y. Hong, Z. Wang, L. Li, W. Yan, R. Wang, J. Zhang, CXCL12-regulated miR-370-3p functions as a tumor suppressor gene by targeting HMGA2 in nonfunctional pituitary adenomas, Mol. Cell. Endocrinol. 488 (2019) 25–35.
[24] Z. Peng, T. Wu, Y. Li, Z. Xu, S. Zhang, B. Liu, Q. Chen, D. Tian, MicroRNA-370-3p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting beta-catenin, Brain Res. 1644 (2016) 53–61. [25] W.Z. Hou, X.L. Chen, W. Wu, C.H. Hang, MicroRNA-370-3p inhibits human vascular smooth muscle cell proliferation via targeting KDR/AKT signaling pathway in cerebral aneurysm, Eur. Rev. Med. Pharmacol. Sci. 21 (5) (2017) 1080–1087. [26] Y. Zhang, Y. Zhu, G. Gao, Z. Zhou, Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia, Cell Biochem. Funct. 37 (5) (2019) 348–358. [27] C. Wang, X. Li, H. Dang, P. Liu, B.O. Zhang, F. Xu, Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR, Cytotherapy 21 (6) (2019) 619–630. [28] J. Brouwer-Visser, G.S. Huang, IGF2 signaling and regulation in cancer, Cytokine Growth Factor Rev. 26 (3) (2015) 371–377. [29] S. Fei, L. Cao, L. Pan, microRNA3941 targets IGF2 to control LPSinduced acute pneumonia in A549 cells, Mol. Med. Rep. 17 (3) (2018) 4019–4026. [30] M.C. Tracy, R. Mathew, Complicated pneumonia: current concepts and state of the art, Curr. Opin. Pediatr. 30 (3) (2018) 384–392. [31] B. Moldoveanu, P. Otmishi, P. Jani, J. Walker, X. Sarmiento, J. Guardiola, M. Saad, J. Yu, Inflammatory mechanisms in the lung, J Inflamm Res 2 (2009) 1–11.
9
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Long non-coding RNA SNHG16 promotes lipopolysaccharides-induced acute pneumonia in A549 cells via targeting miR-370-3p/IGF2 axis Ju Zhang, Fengxia Mao, Gai Zhao, Haixia Wang, Xiaomin Yan, Qian Zhang
T
⁎
Department of Pediatrics, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: SNHG16 miR-370-3p IGF2 Acute pneumonia LPS A549 cells
Background: Pneumonia is an infectious lung inflammation in children with high mortality and morbidity rates. Small nucleolar RNA host gene 16 (SNHG16) has been verified to accelerate the progression of acute pneumonia. However, the role of SNHG16 in acute pneumonia has not yet been fully elucidated. The study was aimed to explore the regulatory mechanism of SNHG16 in LPS-induced acute pneumonia in A549 cells. Methods: The levels of SNHG16, miR-370-3p and IGF2 in serum samples and LPS-induced A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptosis of A549 cells were examined by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The levels of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by enzymelinked immunosorbent assay (ELISA). The binding relationships among SNHG16, miR-370-3p and IGF2 were predicted by online database and verified by Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The protein levels of IGF2 were tested by Western blot. Results: SNHG16 and IGF2 were upregulated while miR-370-3p was downregulated in serum of acute pneumonia patients and LPS-induced A549 cells. SNHG16 regulated proliferation, apoptosis and inflammatory cytokines by inhibiting miR-370-3p in LPS-induced A549 cells. MiR-370-3p targeted IGF2 and inhibited LPS-induced inflammatory injury via IGF2 in A549 cells. Furthermore, SNHG16 was verified to promote IGF2 expression by sponging miR-370-3p in A549 cells. Conclusion: SNHG16 impeded cell viability and promoted apoptosis, inflammatory injury by targeting IGF2 mediated by miR-370-3p in LPS-induced A549 cells.
1. Introduction Pneumonia is an inflammatory disease of the lung, which is triggered by different pathogens infection [1,2]. The typical clinical features of pneumonia are fever, cough, dyspnea, somnolence and chest pain [3,4]. As a main infectious disease, it has a high mortality rate in the elderly and children [5,6]. Lipopolysaccharide (LPS) is the major biologically active component of the cell wall, which is produced by Gram-negative bacteria and belongs to the endotoxins, and it is pivotal for the inflammatory response related to pneumonia [7–9]. Despite substantial progress has been made in anti-inflammatory treatment for pneumonia, the therapy method still need to be further improved. Therefore, it is of great importance to explore the underlying mechanism and search for the novel effective anti-inflammatory medicine to cure pneumonia. Long non-coding RNAs (lncRNAs) are conserved non-coding RNAs
which have no ability to encode protein [10,11]. A growing bed of evidence have suggested that plenty of lncRNAs perform a vital role in multifarious human diseases, including neurodegenerative diseases [12], cancers [13] and cardiovascular diseases [14]. LncRNA small nucleolar RNA host gene 16 (SNHG16) has been reported to be upregulated and can promote the progression of numerous cancers, for instance, pancreatic cancer [15], hepatocellular carcinoma [16], osteosarcoma [17], and so on. Meanwhile, previous study also reported that SNHG16 was overexpressed in the serum of acute pneumonia patients and it regulated LPS-induced apoptosis and inflammation in LPS-induced WI-38 cells [18]. However, the regulatory mechanism of SNHG16 in acute pneumonia is very complicated and has not yet been well elucidated. MicroRNAs (miRNAs) are short non-coding RNAs that contribute to degrading mRNA or repressing translation by binding to 3′-untranslated region (3′-UTR) of mRNAs [19,20]. Various evidences supported that
⁎ Corresponding author at: Department of Pediatrics, First Affiliated Hospital of Zhengzhou University, Henan Province, No. 1 Jianshe East Road, Erqi District, Zhengzhou City, Zhengzhou 450000, China. E-mail address: [email protected] (Q. Zhang).
https://doi.org/10.1016/j.intimp.2019.106065 Received 20 August 2019; Received in revised form 23 October 2019; Accepted 17 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
manufacturer. SNHG16 and IGF2 were normalized to β-actin, while miR-370-3p was normalized to U6. The relative transcriptional folds were quantified and calculated using the comparative threshold (Ct) cycle (2-ΔΔCt) method. The primers were obtained from Sangon Biotech and listed as follows: SNHG16: 5′-CCCAAGCTTGCGTTCTTTTCGAGGT CGGC-3′ (forward) and 5′-CCGGAATTCTGACGGTAGTTTCCCAAGTT-3′ (reverse); miR-370-3p: 5′-TGTAACCAGAGAGCGGGATGT-3′ (forward) and 5′-TTTTGGCATAACTAAGGCCGAA-3′ (reverse); IGF2: 5′-CTTGGA CTTTGAGTCAAATTGG-3′ (forward) and 5′-GGTCGTGCCAATTACATT TCA-3′ (reverse); U6: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ (forward) and 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); β-actin: 5′-TGGAATC CTGTGGCATCCATGAAAC-3′ (forward) and 5′-ACGCAGCTCAGTAACA GTCCG-3′ (reverse).
deregulated miRNAs were closely linked to many diseases, including cardiovascular disease [21], diabetes and autoimmune diseases [22]. Furthermore, miRNAs also played a vital part in lung inflammation and lung cancer [23]. MiR-370-3p acted as an anti-oncogene in various cancers, including glioma [24], cerebral aneurysm [25], and so on. Moreover, miR-370-3p had a regulatory influence on LPS-induced inflammatory injury in acute pneumonia [26]. IGF2 (Insulin-like growth factor 2) is a necessary ingredient of the stem cell niche [27,28], and it involved in drug resistance and cancers [28]. Moreover, IGF2 knockdown obviously mitigated LPS-induced cell injury of A549 cells in acute pneumonia [29]. However, the interaction between miR-370-3p and IGF2 as well as their roles in pneumonia have not been completely explained. In this study, the expression of SNHG16 in the serum of acute pneumonia patients and LPS-induced A549 cell model was firstly explored. Furtherly, the function of SNHG16 in LPS-induced inflammatory injury of A549 cells and the latent molecular mechanism were verified by gain- and loss-of-function experiments.
2.5. Cell counting Kit-8 (CCK-8) assay The cell viability of A549 cells was explored using CCK-8 (Sangon Biotech) after LPS treatment. Briefly, A549 cells were seeded into 96well plate with approximately 5000 cells per well and then subjected to LPS treatment or transfection. Later, CCK-8 solution was added to each well and incubated for 1 h at 37 °C in an incubator with 5% CO2. The absorbance of each well was measured at 450 nm with a microplate reader, and the cell viability (%) was estimated by average absorbance ratio of treatment (or transfection) group with control group.
2. Materials and methods 2.1. Patients and blood collection In this study, 32 patients with acute pneumonia and 10 healthy individuals from First Affiliated Hospital of Zhengzhou University were recruited. The patients with acute pneumonia in observation group were enrolled according to the diagnostic criteria of acute pediatric pneumonia, and the healthy individuals in the control group had normal physical examination results. General data of the enrolled subjects in the two groups were comparable. The patients involved in this research had not received anti-inflammatory therapy and the patients with other complications were ruled out. Serum was obtained from blood samples of acute pneumonia patients and healthy control individuals, and later kept at −80 °C. This research had gained an approval from the Ethics Committee of First Affiliated Hospital of Zhengzhou University and all participators signed informed consent.
2.6. Apoptosis assay The apoptosis rate of A549 cells was determined using Annexin Vfluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis assay kit (Invitrogen). Briefly, A549 cells were subjected to LPS treatment and transfection and later dyed with Annexin V-FITC and PI for 25 min in the dark. Afterwards, apoptotic cells were tested by flow cytometer. 2.7. Enzyme-linked immunosorbent assay (ELISA)
2.2. Cell culture and LPS treatment To measure the levels of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in culture supernatant of A549 cells, ELISA was conducted by the ELISA kits for IL-1β, IL-6 and TNF-α (Abcam Biotechnology, Cambridge, MA, USA) following the instructions of manufacturer. Absorbance was tested using a microplate reader at 450 nm.
The human pulmonary epithelianl cell line A549 was obtained from the (Sangon Biotech, Shanghai, China) and then incubated in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) replenished with 10% fetal bovine serum (FBS; Sangon Biotech) in a humidified incubator with 5% CO2 at 37 °C. After cells were cultivated in 96-well plates for 24 h, LPS (Solarbio, Beijing, China) at 5, 10, 20 µg/ mL or control dimethyl sulfoxide (DMSO) (Solarbio) was added in DMEM medium for 12 h to produce cell injury model.
2.8. Dual-luciferase reporter assay The Dual-luciferase reporter vectors of wild type SNHG16 (SNHG16-WT) or wild type (IGF2-WT) containing the potential targeted sites of miR-370-3p and mutant-type SNHG16 (SNHG16-MUT) or mutant type IGF2-MUT were established. A549 cells were transfected with above reporter vectors together with miR-370-3p or miR-NC using Lipofectamine 3000 reagent (Invitrogen). The luciferase activity was examined by the Dual-luciferase reporter assay system (Promega, Madison, WI, USA).
2.3. Cell transfection Small interfering RNA against SNHG16 (si-SNHG16) and matched negative control (si-NC), pcDNA-SNHG16 (SNHG16), pcDNA-IGF2 (IGF2) and control (pcDNA), miR-370-3p mimic (miR-370-3p), miR370-3p inhibitor (anti-miR-370-3p) and matched controls (miR-NC or anti-miR-NC) were acquired from RiboBio Co., Ltd (Guangzhou, China). A549 cells were transfected using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) referring to user guide.
2.9. RNA immunoprecipitation (RIP) assay 2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) RIP assay was performed using RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) following the protocols of manufacturer. A549 cells were lysed and co-incubated with immunoglobulin G (IgG) antibody conjugated magnetic beads (ab133470) (Abcam Biotechnology) or Argonaute2 (Ago2) antibody (ab32381) (Abcam Biotechnology). The expression of SNHG16, miR370-3p and IGF2 was analyzed by qRT-PCR.
TRIzol reagent (Invitrogen) was employed to isolate total RNA from serum samples and A549 cells. The first strand of cDNA was reversely transcribed from extractive RNA using PrimeScript RT reagent Kit (Takara, Dalian, China). QRT-PCR was adopted using 2X SYBR Green Abstart PCR Mix (Sangon Biotech) on an ABI 7300 PCR System (Applied Biosystems, Carlsbad, CA, USA) based on the instructions of 2
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
3.2. Knockdown of SNHG16 alleviated inflammatory injury in LPS-induced A549 cells
2.10. Western blot assay Total protein was isolated from LPS-stimulated or transfected A549 cells using RIPA buffer (Sangon Biotech) and then quantified by Bradford Protein Assay Kit (Sangon Biotech). Then, proteins were isolated by 10% SDS-PAGE, and later transferred to PVDF membrane (Sangon Biotech). After blocked for 1 h in 5% dried skim milk, the membrane was added with primary antibodies against IGF2 and β-actin (1:1000, Abcam Biotechnology) at 4 °C for 24 h. Subsequently, the PVDF membrane was co-cultured with horseradish peroxidase (HRP)conjugated secondary antibody (1:5000; Abcam Biotechnology) for 2 h following rinsing twice with Tris-buffered saline and Tween 20 (TBST; Beyotime Biotechnology, Shanghai, China). Finally, the protein bands were visualized by ECL detection kit (Beyotime Biotechnology), and the signal intensity of bands was quantified by ImageJ software normalized to β-actin.
To explore the role of SNHG16 in LPS-induced inflammatory injury, A549 cells were transfected with si-SNHG16 or negative control si-NC. The expression of SNHG16 in LPS-induced A549 cells transfected with si-SNHG16 was strikingly inhibited compared with si-NC group detected by qRT-PCR assay, which evidenced high transfection efficiency (Fig. 2A). CCK-8 assay revealed that the cell viability of A549 cells was significantly reduced by LPS treatment, while si-SNHG16-mediated SNHG16 silencing triggered a remarkable increase of cell viability in LPS-induced A549 cells compared with si-NC-transfected cells (Fig. 2B). And knockdown of SNHG16 visibly decreased apoptotic cell rates in A549 cells subjected to LPS treatment compared with si-NC-transfected cells tested by flow cytometry analysis using Annexin V-FITC/PI Apoptosis Detection Kit (Fig. 2C and D). Meanwhile, SNHG16 silencing also strongly decreased the production levels of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells tested by ELISA assay (Fig. 2E–G). These data suggested that SNHG16 silencing could relieve inflammatory injury of LPS-induced A549 cells.
2.11. Statistical analysis All experiments were performed more than three replicates and the data in this study was exhibited as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 19.0. The comparison significances between different groups were evaluated by Student’s t-test and one-way analysis of variance. P < 0.05 was designated as statistically significant.
3.3. SNHG16 targeted miR-370-3p and suppressed miR-370-3p expression in LPS-induced A549 cells Aiming to explain the function of SNHG16 in LPS-induced A549 cells and the underlying mechanisms, LncBase v.2 database was used to predict the putative association between SNHG16 and miR-370-3p and the binding sites between them were exhibited (Fig. 3A). Then the luciferase reporter vectors wild-type SNHG16 (SNHG16-WT) containing the wild-type miR-370-3p-binding sequence in SNHG16 and mutanttype SNHG16 (SNHG16-MUT) were constructed and co-transfected with miR-370-3p or miR-NC into A549 cells. Dual-luciferase reporter assay indicated that the luciferase activity of SNHG16-WT was significantly reduced in A549 cells co-transfected with miR-370-3p, whereas the luciferase activity of SNHG16-MUT was insusceptible (Fig. 3B). The RIP assay was carried out to further validate the direct interaction between SNHG16 and miR-370-3p using A549 cells extracts and Ago2 antibody. The RIP results showed that SNHG16 and miR-3703p exerted more remarkable enrichment in the Ago2-containing miRNA ribonucleoprotein complexes (miRNPs) than negative control IgG group (Fig. 3C). Meanwhile, miR-370-3p was significantly down-regulated in the serum of acute pneumonia patients (n = 32) in comparison with that of healthy controls (n = 10) (Fig. 3D). Consistently, the level of miR-370-3p was overtly reduced with LPS induction in A549 cells in a concentration-dependent manner tested by qRT-PCR (Fig. 3E). Furthermore, Gain- and loss-of-function assays disclosed that the expression of miR-370-3p in A549 cells transfected with SNHG16 was evidently downregulated compared with that of pcDNA-transfected cells,
3. Results 3.1. SNHG16 expression was increased in serum of acute pneumonia patients and LPS-induced A549 cells To examine the level of SNHG16 in pneumonia, qRT-PCR assay was performed. The results suggested that the level of SNHG16 was notably upregulated in the serum of acute pneumonia patients (n = 32) relative to that of healthy controls (n = 10) (Fig. 1A). Meanwhile, the expression of SNHG16 in human pulmonary epithelial cell line A549 treated with different concentrations of LPS (0, 5, 10, and 20 µg/mL) was further measured. As shown in Fig. 1B, the expression of SNHG16 in LPS-induced A549 cells was strikingly increased in dose-dependent manner, with statistically significant effects at 5 µg/mL (*P < 0.05), and 10 µg/mL (***P < 0.001) concentrations. Therefore, 10 µg/mL was chosen as LPS-stimulating condition for application in the following experiments. Hence, we speculated that SNHG16 might perform a latent role in regulating acute pneumonia development.
Fig. 1. The expression of SNHG16 was upregulated in serum of acute pneumonia patients and LPS-induced A549 cells. (A) The expression of SNHG16 in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was determined by qRTPCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (B) The expression of SNHG16 in human pulmonary epithelial cell line A549 treated with different concentrations of LPS (0, 5, 10, and 20 µg/mL) was tested by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA.
3
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 2. SNHG16 knockdown alleviated LPS-induced inflammatory injury in A549 cells. (A) The expression of SNHG16 in LPS-induced A549 cells transfected with siSNHG16 or negative controls was detected by qRT-PCR assay. (B) The cell viability of LPS-induced A549 cells transfected with si-SNHG16 or negative controls was tested by CCK-8 assay. (C-D) The apoptotic rate of LPS-induced A549 cells transfected with si-SNHG16 or negative controls was examined by Annexin V-FITC/PI Apoptosis Detection Kit using flow cytometry. (E-G) The levels of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with siSNHG16 or negative controls were estimated by ELISA assay. * indicated P < 0.05, ** indicated P < 0.01 and *** indicated P < 0.001 vs. control or LPS-induced A549 cells transfected with si-NC for A–G, analyzed by Student’s t-test.
(Fig. 4D–F). Above data suggested that SNHG16 could increase LPSinduced injury by targeting miR-370-3p in A549 cells.
while the expression of miR-370-3p in A549 cells transfected with siSNHG16 was obviously upregulated relative to that of si-NC group (Fig. 3F). Thus, it could be concluded that miR-370-3p was a target of SNHG16 and SNHG16 negatively regulated miR-370-3p expression in A549 cells.
3.5. IGF2 was a target of miR-370-3p in A549 cells For the purpose of exploring the role of miR-370-3p in A549 cells, microT-CDS database was employed to predict the targeted binding between miR-370-3p and IGF2. The results indicated that there was a binding sequence between miR-370-3p and IGF2 (Fig. 5A). The reporter plasmids IGF2-WT containing the predicted miR-370-3p binding sites and mutant IGF2-MUT were constructed. As shown in Fig. 5B, the luciferase activity of IGF2-WT co-transfected with miR-370-3p in A549 cells was strongly limited, while the luciferase activity of IGF2-MUT cotransfected with miR-370-3p was unchanged. The direct interaction between miR-370-3p and IGF2 was further validated in RIP assay and the results evinced that IGF2 and miR-370-3p were preferentially enriched in Ago2-containing miRNPs compared with control IgG immunoprecipitates (Fig. 5C). Simultaneously, the mRNA level of IGF2 was increased in the serum of acute pneumonia patients (n = 32) compared with that in healthy controls (n = 10) (Fig. 5D) and was gradually raised with the concentration increasing of LPS examined by qRT-PCR (Fig. 5E). Moreover, function assays verified that the protein
3.4. SNHG16 regulated proliferation, apoptosis and inflammatory damage via miR-370-3p in LPS-induced A549 cells To verify the regulatory relation between SNHG16 and miR-370-3p, SNHG16 was down-regulated by its siRNA, and miR-370-3p was further knocked down by its inhibitor in LPS-induced A549 cells. As expected, the expression of miR-370-3p in LPS-induced A549 cells transfected with si-SNHG16 was obviously upregulated compared with that of siNC group detected by qRT-PCR, while the stimulative effect was broadly inhibited by anti-miR-370-3p (Fig. 4A). Moreover, si-SNHG16 improved cell viability and reduced apoptosis in LPS-induced A549 cells, whereas the effect of SNHG16 silencing was largely abolished by inhibition of miR-370-3p (Fig. 4B and C). In addition, the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) were decreased in LPS-induced A549 cells transfected with si-SNHG16, while the effects were partly reversed by co-transfection with miR-370-3p inhibitor 4
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 3. SNHG16 targeted miR-370-3p and inhibited miR-370-3p expression in A549 cells. (A) The complementary sequence between SNHG16 and miR-370-3p was predicted by LncBase v.2 database. (B) The luciferase activity of SNHG16-WT or SNHG16-MUT in A549 cells co-transfected with miR-370-3p or miR-NC was detected by Dual-luciferase reporter assay. *** indicated P < 0.001 vs. cells transfected with miR-NC, analyzed by Student’s t-test. (C) Association of SNHG16 and miR-3703p with Ago2 was performed by RIP assay. The RNA levels of SNHG16 and miR-370-3p were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates (lower panel). *** indicated P < 0.001 vs. IgG group, analyzed by ANOVA. (D) The expression of miR-370-3p in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was tested by qRT-PCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (E) The expression of miR-370-3p in A549 cells treated with different doses of LPS was tested by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA. (F) The expression of miR-370-3p in A549 cells transfected with SNHG16, si-SNHG16 or corresponding negative controls was determined by qRT-PCR. *** indicated P < 0.001 vs. cells transfected with pcDNA or si-NC, analyzed by Student’s t-test.
3p targeted IGF2 and suppressed IGF2 expression in A549 cells.
level of IGF2 in A549 cells was clearly reduced when overexpression of miR-370-3p compared with miR-NC-transfected cells, while the level of IGF2 was strikingly elevated by anti-miR-370-3p relative to that of antimiR-NC group (Fig. 5F). All these findings demonstrated that miR-370-
Fig. 4. SNHG16 modulated proliferation, apoptosis and inflammatory injury via miR-370-3p in LPS-induced A549 cells. (A) The expression of miR-370-3p in LPSinduced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p or corresponding negative controls was tested by qRT-PCR assay. (B) The cell viability of LPS-induced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p, or corresponding negative controls was detected by CCK-8 assay. (C) The apoptotic rate of LPS-induced A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p, or corresponding negative controls was determined by Annexin V-FITC/PI Apoptosis Detection Kit using flow cytometry. (D–F) The levels of IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with si-SNHG16, siSNHG16+ anti-miR-370-3p, or corresponding negative controls were examined by ELISA assay. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for Fig. 4A-2F, analyzed by Student’s t-test. 5
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 5. IGF2 was a target of miR-370-3p in A549 cells. (A) The binding sites between miR-370-3p and IGF2 were predicted by microT-CDS database. (B) The luciferase activity of IGF2-WT or IGF2-MUT in A549 cells co-transfected with miR-370-3p or miR-NC was examined by Dual-luciferase reporter assay. *** indicated P < 0.001 vs. cells transfected with miR-NC, analyzed by Student’s t-test. (C) The direct interaction between miR-370-3p and IGF2 was validated by RIP assay. *** indicated P < 0.001 vs. IgG group, analyzed by ANOVA. (D) The relative IGF2 mRNA level in serum of acute pneumonia patients (n = 32) and healthy controls (n = 10) was tested by qRT-PCR. *** indicated P < 0.001 vs. healthy controls, analyzed by Student’s t-test. (E) The mRNA level of IGF2 in A549 cells subjected to different concentration of LPS was determined by qRT-PCR. * indicated P < 0.05 and *** indicated P < 0.001 vs. blank group (0 µg/mL LPS treatment), analyzed by ANOVA. (F) The protein level of IGF2 in A549 cells transfected with miR-370-3p, anti-miR-370-3p or corresponding negative controls was tested by Western blot. *** indicated P < 0.001 vs. cells transfected with miR-NC or anti-miR-NC, analyzed by Student’s t-test.
Fig. 6. MiR-370-3p suppressed inflammatory injury by targeting IGF2 in LPS-induced A549 cells. (A) The protein level of IGF2 in LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls was explored by Western blot. (B–C) The cell viability and apoptosis of LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls were evaluated by CCK-8 assay or flow cytometry, respectively. (D–F) The levels of IL-1β, IL-6 and TNF-α in LPS-induced A549 cells transfected with miR-370-3p, miR-370-3p + IGF2 or corresponding negative controls were tested by ELISA assay. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for A–F, analyzed by Student’s t-test.
3p + pcDNA or miR-370-3p + IGF2. The level of IGF2 in LPS-induced A549 cells transfected with miR-370-3p was significantly declined compared with miR-NC-transfected cells, while the effect was distinctly weakened by IGF2 overexpression (Fig. 6A). Meanwhile, the cell viability was elevated and cell apoptosis was reduced in LPS-induced A549
3.6. MiR-370-3p inhibited inflammatory injury by targeting IGF2 in LPSinduced A549 cells In order to study the function of miR-370-3p in depth, LPS-induced A549 cells were transfected with miR-NC, miR-370-3p, miR-3706
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
Fig. 7. SNHG16 regulated the expression of IGF2 through miR-370-3p in A549 cells. (A) The expression of miR-370-3p in A549 cells transfected with SNHG16, SNHG16+ miR-370-3p or corresponding negative controls was assessed by qRT-PCR. (B) The expression of miR-370-3p in A549 cells transfected with si-SNHG16, si-SNHG16+ anti-miR-370-3p or corresponding negative controls was tested by qRTPCR. (C) The protein level of IGF2 in A549 cells transfected with SNHG16, SNHG16+ miR-370-3p or corresponding negative controls was detected by Western blot. (D) The protein level of IGF2 in A549 cells transfected with si-SNHG16, si-SNHG16+ antimiR-370-3p or corresponding negative controls was determined by Western blot. ** indicated P < 0.01 and *** indicated P < 0.001 vs. corresponding negative controls for A–D, analyzed by Student’s ttest.
cells transfected with miR-370-3p relative to that in miR-NC group, while the effects were significantly reversed by co-transfection with IGF2 (Fig. 6B and C). Correspondingly, the levels of IL-1β, IL-6 and TNF-α were declined in LPS-induced A549 cells transfected with miR370-3p, whereas the effects were largely overturned by IGF2 overexpression (Fig. 6D–F). All these evidences confirmed that miR-370-3p suppressed LPS-induced inflammatory injury through IGF2 in A549 cells. 3.7. SNHG16 regulated the expression of IGF2 by sponging miR-370-3p in A549 cells To further explore the relationship among SNHG16, miR-370-3p and IGF2, gain- and loss-function experiments were proceeded. The results demonstrated that the expression of miR-370-3p was notably downregulated and the protein level of IGF2 was significantly increased in A549 cells transfected with SNHG16, whereas the influences were effectively overturned by co-transfection with miR-370-3p (Fig. 7A and C). Expectedly, the level of miR-370-3p was overtly enhanced and the protein level of IGF2 was strikingly weakened in A549 cells transfected with si-SNHG16, while the effects were vastly eliminated when cotransfection with si-SNHG16 and anti-miR-370-3p (Fig. 7B and D). From the above, the findings indicated that SNHG16 regulated IGF2 expression via miR-370-3p in A549 cells (see Fig. 8).
Fig. 8. The mechanism schematic model by SNHG16/miR-370-3p/IGF2 axis in pneumonia (LPS-induced A549 cells). The expression of SNHG16 and IGF2 was upregulated, while miR-370-3p was downregulated in pneumonia. Upregulated SNHG16 impeded cell viability while promoted apoptosis and inflammation through increasing the expression of IGF2 via inhibiting miR-370-3p in pneumonia (LPS-induced A549 cells).
4. Discussion Pneumonia is one of lower respiratory illnesses and a common 7
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
LPS-induced A549 cells. Therefore, this study was helpful for better understanding of the role of SNHG16 and the underlying mechanism in LPS-induced A549 cells, in order to provide novel targets for pneumonia therapy.
hospital-acquired fatal infections [2,30]. It has been reported that the progression of pneumonia is related to pro-inflammatory cytokines, for instance, TNF-α, IL-1β and IL-6 which activate the immune system and take part in the acute inflammatory response [31,32]. Thereby, searching for novel therapeutic targets to block inflammatory response and cell injury in order to cure pneumonia is a very pressing task. As we all know, a large number of lncRNAs can serve as novel biomarkers for early diagnosis of diseases through acting as miRNAs sponges. Similarly, lncRNA SNHG16 could promote cancer progression via sponging different miRNAs. For example, SNHG16 could spong miR-140-5p to accelerate human retinoblastoma progression [33], and facilitated glioma tumorigenicity by targeting miR-373 [34]. Previous research also revealed that SNHG16 was upregulated and involved in tumor progression in non-small cell lung cancer by sponging miR-146a [35]. Moreover, the expression of SNHG16 was elevated in the serum of patients infected with acute pneumonia, and knockdown of SNHG16 alleviated the LPS-induced cell damage by accelerating cell viability, blocking apoptosis and production of inflammatory cytokines via miR146a-5p/CCL5 axis [18]. Consistent with above research, this study demonstrated that the level of SNHG16 was raised in serum of acute pneumonia patients and LPS-induced A549 cells. SNHG16 silencing notably improved cell viability, reduced apoptosis and the production of inflammatory cytokines IL-1β, IL-6 and TNF-α in LPS-induced A549 cells. Moreover, target prediction database and dual-luciferase reporter assay revealed that SNHG16 was a spong of miR-370-3p, which was further verified by RIP assay. All these data confirmed that SNHG16 participated in inflammatory response in LPS-induced A549 cells and might act as a negative regulator through sponging miR-370-3p in acute pneumonia. Growing evidence have indicated that miRNAs are especially important to maintain the homeostasis and development of lung, and they are involved in the pathogenesis of pulmonary diseases, for example, sarcoidosis, asthma and lung cancer [23,36]. MiR-370-3p could act as an anti-oncogene by regulating different targeted genes in various cancers. For instance, miR-370-3p was downregulated and inhibited the progression of nonfunctional pituitary adenomas by targeting HMGA2 [37], and also suppressed cell proliferation of glioma via targeting βcatenin [24]. However, the studies about the role of miR-370-3p in pneumonia was very lacking. Zhang et al. found that miR-370-3p weakened the LPS-induced WI-38 cells inflammation injury and cell apoptosis in acute pneumonia by targeting downstream gene TLR4 [26]. In this study, it was verified that miR-370-3p was a target of SNHG16 and inhibition of miR-370-3p reversed the effects of SNHG16 knockdown on proliferation, apoptosis and the production of inflammatory cytokines in LPS-induced A549 cells. Taken together, these findings indicated that miR-370-3p was a tumor suppressor gene in LPSinduced A549 cells. IGF2 was verified to be involved in LPS-induced cell injury of A549 cells targeted by miRNA-3941 in acute pneumonia [29]. What is more, IGF2 was evinced as a target gene of miR-370-3p by on line database and functional experiments in this study. Besides, overexpression of IGF2 strongly abolished the influence of miR-370-3p on cell injury, and the elevated protein level of IGF2 in A549 cells transfected with SNHG16 was vastly weakened by miR-370-3p, while the decreased protein level of IGF2 in A549 cells induced by si-SNHG16 was obviously enhanced by miR-370-3p knockdown. Hence, it could be speculated that SNHG16 could restore the expression of IGF2 by sponging miR370-3p. Thus, it was concluded that SNHG16 might take part in cell proliferation, apoptosis and inflammatory damage via miR-370-3p/ IGF2 axis in LPS-induced A549 cells.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement None. Fund None. References [1] C. Cilloniz, A. Torres, M. Niederman, M. van der Eerden, J. Chalmers, T. Welte, F. Blasi, Community-acquired pneumonia related to intracellular pathogens, Intensive Care Med. 42 (9) (2016) 1374–1386. [2] L.A. Mandell, Community-acquired pneumonia: An overview, Postgrad. Med. 127 (6) (2015) 607–615. [3] J.T. Mattila, M.J. Fine, A.H. Limper, P.R. Murray, B.B. Chen, P.L. Lin, Pneumonia. Treatment and diagnosis, Ann. Am. Thorac. Soc. 11 (Suppl 4) (2014) S189–S192. [4] R.R. Watkins, T.L. Lemonovich, Diagnosis and management of community-acquired pneumonia in adults, Am. Fam. Physician 83 (11) (2011) 1299–1306. [5] P. Faverio, S. Aliberti, G. Bellelli, G. Suigo, S. Lonni, A. Pesci, M.I. Restrepo, The management of community-acquired pneumonia in the elderly, Eur. J. Int. Med. 25 (4) (2014) 312–319. [6] T. Wardlaw, P. Salama, E.W. Johansson, E. Mason, Pneumonia: the leading killer of children, Lancet 368 (9541) (2006) 1048–1050. [7] D. Dreyfuss, J.D. Ricard, Acute lung injury and bacterial infection, Clin. Chest Med. 26 (1) (2005) 105–112. [8] N. Meng, J. Zhao, L. Su, B. Zhao, Y. Zhang, S. Zhang, J. Miao, A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells, Int. J. Biochem. Cell Biol. 44 (2) (2012) 311–319. [9] M. Rojas, C.R. Woods, A.L. Mora, J. Xu, K.L. Brigham, Endotoxin-induced lung injury in mice: structural, functional, and biochemical responses, Am. J. Physiol. Lung Cell. Mol. Physiol. 288 (2) (2005) L333–L341. [10] C. Ernst, C.C. Morton, Identification and function of long non-coding RNA, Front. Cell. Neurosci. 7 (2013) 168. [11] C.P. Ponting, P.L. Oliver, W. Reik, Evolution and functions of long noncoding RNAs, Cell 136 (4) (2009) 629–641. [12] C.W. Wei, T. Luo, S.S. Zou, A.S. Wu, The role of long noncoding RNAs in central nervous system and neurodegenerative diseases, Front. Behav. Neurosci. 12 (2018) 175. [13] R. Spizzo, M.I. Almeida, A. Colombatti, G.A. Calin, Long non-coding RNAs and cancer: a new frontier of translational research? Oncogene 31 (43) (2012) 4577–4587. [14] S. Uchida, S. Dimmeler, Long noncoding RNAs in cardiovascular diseases, Circ. Res. 116 (4) (2015) 737–750. [15] S. Liu, W. Zhang, K. Liu, Y. Liu, LncRNA SNHG16 promotes tumor growth of pancreatic cancer by targeting miR-218-5p, Biomed. Pharmacother. 114 (2019) 108862. [16] J.H. Zhong, X. Xiang, Y.Y. Wang, X. Liu, L.N. Qi, C.P. Luo, W.E. Wei, X.M. You, L. Ma, B.D. Xiang, L.Q. Li, The lncRNA SNHG16 affects prognosis in hepatocellular carcinoma by regulating p62 expression, J. Cell. Physiol. (2019). [17] P. Su, S. Mu, Z. Wang, Long noncoding RNA SNHG16 promotes osteosarcoma cells migration and invasion via sponging miRNA-340, DNA Cell Biol. 38 (2) (2019) 170–175. [18] Z. Zhou, Y. Zhu, G. Gao, Y. Zhang, Long noncoding RNA SNHG16 targets miR-146a5p/CCL5 to regulate LPS-induced WI-38 cell apoptosis and inflammation in acute pneumonia, Life Sci. 228 (2019) 189–197. [19] M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl, Identification of novel genes coding for small expressed RNAs, Science 294 (5543) (2001) 853–858. [20] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2) (2004) 281–297. [21] T. Barwari, A. Joshi, M. Mayr, MicroRNAs in cardiovascular disease, J. Am. Coll. Cardiol. 68 (23) (2016) 2577–2584. [22] E.N. Kontomanolis, A. Koukouli, G. Liberis, H. Stanulov, A. Achouhan, A. Pagkalos, MiRNAs: regulators of human disease, Eur. J. Gynaecol. Oncol. 37 (6) (2016) 759–765. [23] A. Sittka, B. Schmeck, MicroRNAs in the lung, Adv. Exp. Med. Biol. 774 (2013) 121–134.
5. Conclusion This study verified the involvement of SNHG16 in LPS-induced A549 cells. SNHG16 may be involved in proliferation, apoptosis and inflammatory response by modulating IGF2 mediated by miR-370-3p in 8
International Immunopharmacology 78 (2020) 106065
J. Zhang, et al.
[32] G. Ilonidis, E. Parapanisiou, A. Anogeianaki, I. Giavazis, E.K. Theofilogiannakos, P. Tsekoura, K. Kidonopoulou, C. Trakatelli, Z. Polimenidis, P. Conti, G. Anogianakis, Interleukin -1beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor (TNF) in plasma and pleural fluid of pneumonia, lung cancer and tuberculous pleuritis, J. Biol. Regul. Homeost. Agents 20 (1–2) (2006) 41–46. [33] C. Xu, C. Hu, Y. Wang, S. Liu, Long noncoding RNA SNHG16 promotes human retinoblastoma progression via sponging miR-140-5p, Biomed. Pharmacother. 117 (2019) 109153. [34] X.Y. Zhou, H. Liu, Z.B. Ding, H.P. Xi, G.W. Wang, lncRNA SNHG16 promotes glioma tumorigenicity through miR-373/EGFR axis by activating PI3K/AKT pathway, Genomics (2019). [35] W. Han, X. Du, M. Liu, J. Wang, L. Sun, Y. Li, Increased expression of long noncoding RNA SNHG16 correlates with tumor progression and poor prognosis in nonsmall cell lung cancer, Int. J. Biol. Macromol. 121 (2019) 270–278. [36] A. Ebrahimi, E. Sadroddiny, MicroRNAs in lung diseases: Recent findings and their pathophysiological implications, Pulm. Pharmacol. Ther. 34 (2015) 55–63. [37] F. Cai, C. Dai, S. Chen, Q. Wu, X. Liu, Y. Hong, Z. Wang, L. Li, W. Yan, R. Wang, J. Zhang, CXCL12-regulated miR-370-3p functions as a tumor suppressor gene by targeting HMGA2 in nonfunctional pituitary adenomas, Mol. Cell. Endocrinol. 488 (2019) 25–35.
[24] Z. Peng, T. Wu, Y. Li, Z. Xu, S. Zhang, B. Liu, Q. Chen, D. Tian, MicroRNA-370-3p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting beta-catenin, Brain Res. 1644 (2016) 53–61. [25] W.Z. Hou, X.L. Chen, W. Wu, C.H. Hang, MicroRNA-370-3p inhibits human vascular smooth muscle cell proliferation via targeting KDR/AKT signaling pathway in cerebral aneurysm, Eur. Rev. Med. Pharmacol. Sci. 21 (5) (2017) 1080–1087. [26] Y. Zhang, Y. Zhu, G. Gao, Z. Zhou, Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia, Cell Biochem. Funct. 37 (5) (2019) 348–358. [27] C. Wang, X. Li, H. Dang, P. Liu, B.O. Zhang, F. Xu, Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR, Cytotherapy 21 (6) (2019) 619–630. [28] J. Brouwer-Visser, G.S. Huang, IGF2 signaling and regulation in cancer, Cytokine Growth Factor Rev. 26 (3) (2015) 371–377. [29] S. Fei, L. Cao, L. Pan, microRNA3941 targets IGF2 to control LPSinduced acute pneumonia in A549 cells, Mol. Med. Rep. 17 (3) (2018) 4019–4026. [30] M.C. Tracy, R. Mathew, Complicated pneumonia: current concepts and state of the art, Curr. Opin. Pediatr. 30 (3) (2018) 384–392. [31] B. Moldoveanu, P. Otmishi, P. Jani, J. Walker, X. Sarmiento, J. Guardiola, M. Saad, J. Yu, Inflammatory mechanisms in the lung, J Inflamm Res 2 (2009) 1–11.
9