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miR-383 inhibits cell growth and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway
Biomedicine & Pharmacotherapy 120 (2019) 109551
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miR-383 inhibits cell growth and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway Jianchu Wang, Libai Lu, Zongjiang Luo, Wenchuan Li, Yuan Lu, Qianli Tang, Jian Pu
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Department of Hepatobiliary Surgery, Affiliated Hospital of Youjiang Medical University for Nationalities, No. 18 Zhongshan Road, Baise 533000, Guangxi Zhuang Autonomous Region, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-383 IL-17 p-Stat3 STAT3 signaling pathway
Objectives: Emerging microRNAs (miRNAs) are validated to take part in pathological processes, including numerous carcinomas. Currently, we focused on the functional role of miR-383 and interleukin-17 (IL-17) in hepatocellular carcinoma (HCC), and the underlying molecular mechanisms were also the emphases in our research. Methods: We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of miR-383 in 45 paired tumor tissues and adjacent non-tumor tissues extracted from patients with hepatocellular carcinoma. These tissues were also stained for IL-17 using immunohistochemical staining. Western blot was performed to detect the protein expressions of following protein-coding genes, including pStat3, Stat3 and GAPDH. A dual-luciferase activity was carried out to determine whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma development. The colony assay, CCK8 assay, and apoptosis assay were used to explore the detailed regulatory effects of miR-383/IL-17 axis in the cellular processes of hepatocellular carcinoma separately. Results: miR-383 was down-regulated significantly in tumor tissues, while IL-17 was up-regulated. IL-17 was certificated to act as the downstream gene of miR-383. Furthermore, overexpression of miR-383 suppressed cell proliferation and promoted apoptosis in hepatocellular carcinoma. However, the raised IL-17 attenuated the inhibition effect of miR-383 in hepatocellular carcinoma. In addition, we found that p-Stat3 was repressed by miR-383, and the up-regulation of IL-17 reversed the suppression effect in hepatocellular carcinoma. Conclusions: miR-383 may play a anti-tumor role in the pathogenesis of hepatocellular carcinoma by targeting IL-17 through STAT3 signaling pathway. miR-383/IL-17 axis maybe a potent target for the clinical diagnosis and treatment of hepatocellular carcinoma.
1. Introduction Hepatocellular carcinoma consists of 85%˜ 90% of liver cancer [1], which is a common type of malignant cancers with the sixth incidence (4.7%) and the fourth mortality (8.7%) in cancer-related diseases worldwide, according to GLOBOCAN 2018. There are many risk factors leading to HCC, such as, mainly hepatitis virus B or C, diabetes, alcoholic or nonalcoholic steatohepatitis and genetic metabolic diseases [1,2]. Recent years, despite advancements in clinical diagnosis and treatments of liver cancer, due to the long incubation period that HCC is still hard to be diagnosed in the early stage, the finite therapeutic targets, and also the poor prognosis, as well as the high occurrence rate, the mortality of liver cancer still stay in a high level without going down much more. For instance, the mortality of liver cancer in 2012
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was 9.1% [3], only 4.4% decreased, compared with the mortality of 2018. And the five-year survival rate for HCC patients is still quite low (−30%) [3], almost 70,000 patients suffering liver cancer died every year across the world [4]. So the research for explaining molecular heterogeneity and detecting more biomarkers in HCC is urgent. Since 2006, Murakami and his colleagues proposed that alteration of microRNAs expression was found commonly in liver cancer firstly [5]. The functional effects and underlying molecular mechanisms of miRNAs in HCC draw our attentions in numerous studies in decades. MicroRNAs (miRNAs) are a class of short (approximately 22nts), single-strand and non-coding RNAs transcribed from particular regions of protein-coding genes [6]. miRNAs have been proven to be involved in post-transcription processes by interacting with target mRNA 3′UTR and interrupting protein generating [7]. Based on previous studies,
Corresponding author. E-mail address: [email protected] (J. Pu).
https://doi.org/10.1016/j.biopha.2019.109551 Received 30 July 2019; Received in revised form 9 October 2019; Accepted 9 October 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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CCAGCCCATGATGGTTCTGAT-3′ (reverse). TP53 primer was 5′-ACCT ATGGAAACTACTTCCTGAAA-3′ (forward) and 5′-CTGGCATTCTGGGA GCTTCA-3′ (reverse). MMP-2 primer was 5′-CAGGACATTGTCTTTGAT GGCATCGC-3′ (forward) and 5′-TGAAGAAGTAGCTATGACCACC GCC-3′ (reverse). MMP-9 primer was 5′-ATCCCCCAACCTTTACCA-3′ (forward) and 5′-TCAGAACCGACCCTACAA-3′ (reverse). GAPDH primer was 5′-GACTCATGACCACAGTCCATGC-3′ (forward) and 5′-AGAGGCAGGGATGATGTTCTG-3′ (reverse). The Applied Biosystems 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) was used to perform above quantitative experiments.
miRNAs were found expressed abnormally and conducted as a oncegene or inhibitor in multitudinous kinds of human diseases, including lung cancer, ovarian cancer, and liver cancer [8]. miRNAs act as either promotor or inhibitor in pathogenesis of diseases through participating in diverse cellular processes, such as, cell growth, differentiation, apoptosis and metastasis [9]. In the case of HCC, Klingenberg et al summaried 16 miRNA expressions were dysregulated, mainly upregulated [10]. Integrating with many studies concerning the functional role of miRNA in HCC, oncogenetic miRNAs were revealed, including miR-184 [11], miR-10b-3p [12]. Meanwhile, suppressive miRNAs were following, miR-122 [13], miR-146a-5p [14], miR-143 [15], miR-1 [16], miR-144 [17], miR-940 [18]. As shown above, miRNAs were closely linked with HCC. Cytokines are a group of bioactive small molecule protein produced by immune cells mainly in response to stimuli and are vital immune regulators in biological activities, including innate immunity, autoimmune pathology and cancer immunity process [19]. Numerous cytokines have certificated to be involved in cancer pathogenesis. Inflammatory cytokines IL-4 and IL-13 were related with cancer stem cells [20]. IL-8 and IL-12 could act as biomarkers for justifying metastatic breast cancer [21]. IL-1b, IL-6, IL-10, IL-23 and TNF-αare commonly correlated with HCC [22]. The effect of cytokines in HCC also could not be ignored. In our current study, we focused on the relationship between miR383, IL-17 and HCC, and aimed to investigate the functional effect, regulatory role and potential signaling pathway of miR-383/IL-17 axis on the process of HCC.
2.4. Immunohistochemistry staining (IHC) Paraffin-embedded, formalin fixed liver tissue Sections (4) um were incubated at 65℃overnight. Then slides were blocked with 3% hydrogen peroxide (H2O2) for 35 min. After incubating in 5% BSA for 40 min. The slides were incubated with a 1:300 dilution of primary antibody against human IL-17 (R&D Systems, Minneapolis, MN) at 4 ℃ overnight and were then subjected to secondary antibody incubation. Diaminobenzidine tetrahydrochloride was used for the chromogenic reactionas. Two independent investigators counted the positively stained cells by using microscope. 2.5. Transfection miR-383 mimics (mimic-miR-383) and its negative control (mimicNC) obtained from Genepharma (Shanghai, China) were transfected into 293 T cells to perform luciferase activity. LV-miR-383 or the negative control (LV-NC) were transfected into HepG2 and Huh7 cells to observe the colony numbers and apoptotic rate. Above lentivirses were bought from Genepharma (Shanghai, China).
2. Materials and methods 2.1. Samples and tissues From August 2017 to February 2019, 45 patients suffering hepatocellular carcinoma were admitted in our hospital. Based on the TumorNode Metastasis (TNM) Classification of malignant cancers following International Union Against Cancer (UICC). In all of the 45 hepatocellular carcinoma cases, 11 cases were in stage I-II, 18 cases were in stage III, 16 cases were in stage IV. The tumor tissues and paired adjacent non-tumor tissues were extracted from the 45 HCC patients. Total tissues were rapidly frozen in liquid nitrogen and then stored at -80 ℃ medical refrigerator for the following experiments. All the usage of tissues were conformed to ethical standards of our hospital.
2.6. Luciferase activity assay The pGL3 vector encompassing luciferase reporter gene was inserted with wild type of IL-17 3′UTR (WT-IL-17 3′UTR) or mutant type of IL-17 3′UTR (MUT-IL-17 3′UTR) separately for a dual-luciferase activity assay. Above vector and sequences were all achieved from Genepharam (Shanghai, China). Then, pGL3-WT-IL-17 3′UTR and pGL3-MUT-IL-17 3′UTR were transfected into 293 T cells placed in 6well plate in advance separately. Next, miR-383 mimics (mimic-miR383) and its negative control (mimic-NC) with Lipofectamine 2000 reagent were transfected into above 293 T cells. 2 days later, We used a dual-luciferase reporter system (Promega Corporation, Madison, WI, USA) to detect the luciferase activity according to manufacturer’s instructions.
2.2. Cell culture Human HCC cell lines, HepG2 and Huh7, as well as 293 T cells were all obtained from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), and cultured in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubators with 5% CO2 at 37℃.
2.7. ELISA The protein expression of IL-17 were evaluated using ELISA kits (R& D Systems, Minneapolis, MN, USA).
2.3. RT-qPCR
2.8. Western blot
Firstly, total RNA were extracted from abraded tissues including tumor tissues and matched adjacent non-tumor tissues using TRIZOL (Sigma, USA). Secondly, RNA were reverse transcribed to cDNA with the support of PrimeScript™RT reagent Kit (TaKaRaBio Technology). Taqman MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) was used to measure the expression levels of miR-383. Caspase-3 primer was 5′-CATGGAAGCGAATCAATGGACT-3′ (forward) and 5′-CTGTACC AGACCGAGATGTCA-3′ (reverse). Caspase-9 primer was 5′-ATGTCGG ACTACGAGAACGAT-3′ (forward) and 5′-TGATGCGTGAGGGGTC GAT-3′ (reverse). Bcl-2 primer was 5′-TTCTTTGAGTTCGGTGGGGTC-3′ (forward) and 5′-TGCATATTTGTTTGGGGCAGG-3′ (reverse). Bax primer was 5′−CCCGAGAGGTCTTTTTCCGAG-3′ (forward) and 5′−
HepG2, Huh7 cells transfected with lentiviruses were lysed using radio immunoprecipitation assay buffer (RIPA, Beyotime Biotechnology, China). Proteins were separately by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and then transferred to Poly Vinylidene Difluoride (PVDF) membrane. After above membranes were blocked in 5% defatted milk at ordinary temperature, the membranes were incubated with specific primary antibodies (Stat3, p-Stat3 and GAPDH primary antibody) at 4 °C overnight. Membranes were then washed by TBST three times and incubated with secondary antibodies for 2 h at room temperature to visualize the proteins. All antibodies used were purchased from Abcam (ambridge, UK). Protein concentrations were quantified by a bicinchoninic acid protein assay kit 2
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(Beyotime Institute of Biotechnology).
3.2. IL-17 was up-regulated in hepatocellular carcinoma tumor tissues and was correlated with miR-383 negatively
2.9. Colony formation assay
IL-17 was chosen to be a potential target gene of miR-383 via TargetScan website (http://www.targetscan.org/). To explore whether IL-17 was in relation to hepatocellular carcinoma, IL-17 positive cells were counted in 45 paired tumor tissues and adjacent normal tissues using immunohistochemical staining. The results showed that IL-17 positive cells were much more in hepatocellular carcinoma tumor tissues, compared with normal tissues (Fig. 1C). Univariate regression analysis was established to observe the relationship between miR-383 expression and IL-17 in 45 cases. The results showed the slope of regression line was negative (Fig. 1D). These results illustrated that IL-17 was up-regulated in hepatocellular carcinoma and had a negative correlation with miR-383.
HepG2 and Huh7 cells transfected with LV-NC, LV-miR-383 or LVmiR-383 with IL-17 (20 ng/ml, Cat# 7955-IL-025/CF, Fisher Scientific, Pittsburgh, PA) were seeded into 6-well plate rounded with agar medium. Then, fresh medium was added into HepG2 and Huh7 cells every 3 days, which was cultured in a humidified incubator with 5% CO2 at 37℃. 12 days later, we obtained the image of colonies in each well. Then the numbers of colony were counted with the help of Image J 1.49v software (National Institutes of Health, Bethesda, MD, USA).
2.10. Cell proliferation assay
3.3. IL-17 was targeted directly by miR-383 and was regulated by miR-383 negatively
To evaluate the cell proliferation, CCK-8 assay was employed to detect the cell growth. A 96-well plates was prepared for HepG2 and Huh7 cells transfected with mimic-NC, mimic-miR-383, LV-NC, LVmiR-383, LV-miR-383+IL-17 or LV-miR-383+IL-17 with AG490 (15 ug/ml, Sigma, USA). The cells were kept at 37 °C with a 5% CO2. For every 24 h, the cells were counted and OD450 value was collected. We drew a cell growth curve after incubtion of 5 days.
To investigate whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma, luciferase activity assay was carried out. The sequence of WT-IL-17 3′UTR could bind to miR-383, while MUT-IL17 3′UTR could not (Fig. 2A). In the case of pGL3 vector constructed with IL-17 3′UTRwt, the luciferase activity of 293 T cell transfected with mimic-miR-383 was lower than that transfected with mimic-NC (Fig. 2B). However, in the case of pGL3 vector constructed with IL-17 3′UTRmut, the luciferase activity of 293 T cell transfected with mimicmiR-383 was the same with that transfected with mimic-NC (Fig. 2C). Then, ELISA was performed to explore the regulatory effect of miR-383 on IL-17 expression. The results exhibited that IL-17 protein expression levels in HepG2 and Huh7 cells transfected with mimic-miR-383 were both less than the expression levels in HepG2 and Huh7 cells transfected with mimic-NC significantly (Fig. 2D and 2E). These results explained that IL-17 was targeted directly by miR-383 and the negative correlation between miR-383 and IL-17 was certificated a step furtherly.
2.11. Apoptosis assay After HepG2 and Huh7 cells transfected with LV-NC, LV-miR-383, LV-miR-383+IL-17 or LV-miR-383+IL-17+AG490 were seeded into 6well plate and cultured 48 h later, above cells were washed three times by PBS and then resuspended in 500 u L binding buffer (KeyGen). 5 u L Annexin V-FITC (KeyGen) and 5 u L propidium iodide (PI) were supplemented into above buffer and mixed at room temperature without light for 12 min. We used the flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) to analyze the apoptosis rate of cells.
3.4. miR-383 represses tumor cell growth and IL-17 attenuates the inhibition effect of miR-383 on hepatocellular carcinoma cell growth in vitro
2.12. Statistical analysis The values were represented as the mean ± standard deviation (SD) and were the average of three experiments. We carried out Student t-test and One-Way ANOVA analysis to measure the differences between quantitative variables. Univariate regression was used to describe the correlation relationship between miR-383 and IL-17. Above statistical methods were performed with the help of Prism (7.0) and R studio (3.6.1). Two-tailed P-value < 0.05 was considered to be significant statistically.
To identify the functional effect of IL-17 on the development of hepatocellular carcinoma, LV-NC, LV-miR-383 or LV-miR-383 with IL17 was transfected into HepG2 and Huh7 cells. Colony formation assay was performed in the aim of confirming the effect of miR-383 on hepatocellular carcinoma cell growth. The results pointed out that the colony numbers of HepG2 and Huh7 cells transfected with LV-miR-383 was both fewer than the colony numbers of HepG2 and Huh7 cells transfected with LV-NC. In addition, the colony numbers of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was more than the colony numbers of HepG2 and Huh7 cells transfected with LVmiR-383 only (Fig. 3A and B). CCK-8 assay results identified that HepG2 and Huh7 cells transfected with LV-miR-383 had lower cell viability, compared with HepG2 and Huh7 cells transfected with LVNC. In addition, the cell proliferation rate of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was higher than that transfected with LV-miR-383 (Fig. 3C and D). The results demonstrated that miR-383 repressed cells growth while IL-17 reversed the inhibition effect of miR-383 on cells growth in HepG2 and Huh7 cells.
3. Results 3.1. miR-383 was down-regulated in vivo To identify whether miR-383 was abnormally expressed in hepatocellular carcinoma, we measured the expression levels of 45 paired tumor tissues and adjacent normal tissues using RT-qPCR. The results demonstrated that miR-383 was down-regulated obviously in tumor tissues, compared with normal tissues (Fig. 1A). Furthermore, according to clinicalpathological stage, we grouped 45 tumor tissues into three groups, including stage I-II, stage III, stage IV. Correspondingly, miR-383 expression levels were separately into three groups. The boxplot of each group showed that stage IV had the lowest miR-383 expression level, and stage I-II had the highest miR-383 expression level (Fig. 1B). Above findings indicated miR-383 was associated with hepatocellular carcinoma and had a negative correlation with clinicalpathological stage.
3.5. miR-383 promotes tumor cell apoptosis and IL-17 reverses the promote effect of miR-383 on tumor cell apoptosis in vitro To verify whether miR-383 was involved in the process of tumor cell apoptosis in hepatocellular carcinoma, apoptosis assay was carried out in HepG2 and Huh7 separately. The results showed that the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected 3
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Fig. 1. miR-383 and IL-17 protein expression levels in HCC tissues. (A) miR-383 expression levels were measured in 45 HCC tumor tissues and matched normal tissues using RT-qPCR. (B) miR-383 expression levels of 45 HCC tumor tissues were grouped into Stage I–II, Stage II and Stage III. (C) Representative images of immunohistochemical staining of IL-17 expression in 45 HCC tumor tissues and matched normal tissues (50um). The frequency of IL-17 positive cells in tumor tissues was significantly higher than that in non-tumor tissues. (D) Univariate regression analysis between miR-383 expression and corresponding IL-17 positive cells in 45 HCC tumor tissues. *p < 0.05.
HepG2 and Huh7 cells transfected with LV-miR-383 were both lower than that in HepG2 and Huh7 cells transfected with LV-NC, and both of them were lower than the expression levels in HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17. However, Stat3 protein expression levels in HepG2 and Huh7 cells transfected with LV-miR383, LV-miR-383 with LV-IL-17 or LV-NV were all identical (Fig. 5A–C). We employed RT-qPCR to find the function of miR-383 on p-Stat3 downstream factors TP53, MMP2 and MMP9. Overexpression of miR383 increased TP53 expression (Fig. 5D) in HepG2 and Huh7 cells. And upgulation of miR-383 inhibited MMP2 and MMP9 expression (Fig. 5E and F). What’s more, loss of STAT3 had the similar effect on tumor growth and apoptosis as miR-383 (Fig. 5G–I). Above findings certificated that STAT3 phosphorylation was down-regulated by miR-383 while IL-17 reversed the down-regulation effect of miR-383 on p-Stat3. Furthermore, STAT3 was associated with miR-383 and IL-17 and might play vital role in hepatocellular carcinoma development.
with LV-miR-383 were both more than the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected with LV-NC. And, the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was fewer than the apoptosis numbers and apoptotic cell rate of HepG2 and Huh7 cells transfected with LV-miR-383 only (Fig. 4A–D). To elucidate the molecular basis of apoptosis we examined the apoptosis mediators and molecular markers. As shown in Fig. 4E and F, decreased expression level of Bcl-2 and increased expression level of caspase-3, caspase-9 and BAX was detected in HepG2 and Huh7 cells transfected with LV-miR383 compared with HepG2 and Huh7 cells transfected with LV-NC. In addition, increased expression level of Bcl-2 and decreased expression level of caspase-3, caspase-9 and BAX was detected in HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 than that transfected with LV-miR-383. Above results revealed that miR-383 promoted tumor cell apoptosis while IL-17 reversed the positive effect of miR-383 on tumor cell apoptosis in the process of hepatocellular carcinoma in vitro.
4. Discussion Currently, our data and images showed that miR-383 was downregulated and had a negative correlation with TNM stage and IL-17 expression separately in 45 HCC tumor tissues. So miR-383 expression provided a potent target for the diagnosis of HCC patients and might be a meaningful reference for determining clinicalpathological stage in order to accept pertinent treatment timely. In addition, IL-17 was validated to be the downstream gene of miR-383 in HCC. Furthermore,
3.6. p-Stat3 is down-regulated by the overexpression of miR-383 and IL-17 reverses the down-regulation effect of miR-383 on p-Stat3 in vitro To identify whether STAT3 phosphorylation was associated with miR-383 and IL-17 in hepatocellular carcinoma development, p-Stat3 and Stat3 protein expression levels were measured through western blot. The results indicated that p-Stat3 protein expression levels in 4
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Fig. 2. IL-17 was targeted by miR-383 directly. (A) The sequences of miR-383, wild type IL-17 3′UTR and mutant type IL-17 3′UTR. (B) The luciferase activities were detected in 293 T cells co-transfected with mimic-miR-383 or mimic-NC and pGL3 vector containing IL-17 3′UTRwt via luciferase activity assay. (C) The luciferase activities were detected in 293 T cells co-transfected with mimic-miR-383 or mimic-NC and pGL3 vector containing IL-17 3′UTRmut via luciferase activity assay. All quantitative values were the average of triple experiments. (D) The expression of IL-17 was determined by ELISA in HepG2 cells transfected with mimic-miR-383 or mimic-NC. (E) The expression of IL-17 was determined by ELISA in Huh7 cells transfected with mimic-miR-383 or mimic-NC. *p < 0.05. ns, not significant.
383 and LDHA axis repress cell proliferation, invasion and aerobic glycolysis [24]. miR-383-5p-TRIM27 combination inhibit cell proliferation and promote chemosensitivity [25]. Considering glioma, miR383 inhibits cell invasion by targeting IGF1R [26]. With regard to cholangiocarcinoma, which was the another type of liver cancer, the tumor-suppressive effect of miR-383 was also illustrated [27]. In our study, miR-383 was investigated as a tumor inhibitor in HCC development, this finding was compatible with above experimental results in other carcinomas. The anti-tumor role of miR-383 in a wide range of carcinomas or other pathological diseases is valuable and need more research. Interleukin-17 (IL-17) first found in rodents T cells at 1993 belongs to IL family. It is a kind of cytokines, through binding to IL-17 receptor (IL-17R) thus activating signal transduction pathway, including but not
miR-383 repressed tumor cell growth and promoted cell apoptosis in HepG2 and Huh7 cell lines, while IL-17 attenuated the inhibition effects, showing that miR-383 was an inhibitor in the progression of HCC, while IL-17 was a promotor. What’s more, p-Stat3 was found to be down-regulated by the overexpression of miR-383. IL-17 reversed the down-regulation effect of miR-383 on p-Stat3 in HepG2 and Huh7 cell lines. Taken together, we hypothesis that miR-383 inhibits cell proliferation and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway. These findings may provide a novel therapeutic approach for HCC. miR-383 have been reported to play a anti-tumor role in several types of malignant tumors. In cervical cancer, Teng et al revealed that miR-383 acted as a tumor suppressor by inhibiting PARP2+ and PI3KAKT-MTOR signaling pathway [23]. In the case of ovarian cancer, miR5
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Fig. 3. Overexpression of miR-383 inhibited tumor cell growth and up-regulation of IL-17 attenuated the inhibition effect in HepG2 and Huh7 cells. (A) The images of colonies were captured for HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC with the help of colony formation assay separately. (B) The number of colonies were counted for HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC separately. (C) Cell viability in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (D) Cell viability in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. All quantitative values were the average of triple experiments. *p < 0.05.
limited, NF-B, MAPK, TRAF6, JAK/STAT, TAK1, finally, altering target genes transcription, mainly pro-inflammatory chemokines, hematopoietic cytokines, acute phase response genes and anti-microbial substances [28,29]. For liver cancer, cytokines like IL-1α, IL-1β, IL-6, IL-8 and TNF-α have been certifated to associated with chronic hepatic inflammation, and IL-6 is highlighted in human hepatocarcinogenesis [22,30]. Whether IL-17 is related with HCC also have been paid much attention by researchers. Liu et al reported that plasma IL-17 was highly expressed in 39 HCC group compared with control group [31]. Yang et al found IL-17 serve as a protumor role in mice hepatocarcinoma model [32]. Li et al demonstrated that IL-17A act as a promotor in hepatocellular carcinoma invasion and migration [33]. Take our results into consideration, up-regulation of IL-17 promoted cell growth and inhibited cell apoptosis in HCC, the involvement of IL-17 in HCC got supports by previous studies. Emerging studies have elaborated that miRNAs functions in pathologiacal and progression of diseases by modulating the expression levels of cytokines directly or indirectly [19]. For IL-17, miR-340 targets IL-17 directly thus relieve psoriasis in mice [34]. miR-20 repress IL-17 expression by inhibiting T17 differentiation through targeting STAT3 in autoimmune encephalomyelitis [35]. Similarly, our study illustrated that miR-383 inhibits cell proliferation and promotes cell apoptosis in HCC by targeting IL-17. Therefore, miR-383/IL-17 axis in tumorgenesis of HCC is consistent with the above findings. However, the up-stream or down-stream mechnisms of miR-383/IL-17 is not clear enough. Due to the special ability of IL-17 that could activate signaling transduction pathway, we focused on whether STAT3 signaling pathway was enlightened by miR-383/IL-17 axis in HCC for our study. STAT3 pertains to STAT protein family, is a transcription factor, which
could be phosphorylated by JAKs when stimulated by cytokines and growth factors [36]. STAT3 have been reported to take a place in pathogenesis of various diseases, including hematological disease [36], lung cancer [37], liver fibrosis [38], psoriatic arthritis [39], hepatocellular carcinoma [40]. In addition, miRNAs have been validated to be involved in the STAT3 signaling pathway. Firstly, JAK2 is activated through the combination of IL-6 receptors (IL-6R) and IL-6. Secondly, STAT3 is transformed to p-Stat3 with the activation of JAK2. Finally, Stat3 act as a transcription factor to activate target genes, including miRNAs. For instance, STAT3 regulates miR-21 expression directly with the mediation of IL-6, which promotes miR-21 through binding STAT3 to an upstream enhancer of miR-21 [41] [10]. On the other hand, STAT3 could binds to the IL-17 in T cells and enhance the production of IL-17 [30]. IL-17-STAT3 mechanisms were also observed in liver fibrosis [38], psoriatic arthritis [39], hepatocarcinoma [42]. Above elucidates about miR-STAT3 and IL-17-STAT3 provide potent evidences that STAT3 signaling pathway has a great chance in theory to correlate with miR-383/IL-17 axis in HCC. Our results showed p-STAT3 was down-regulated by overexpression of miR-383 and was overexpressed by the up-regulation of IL-17 in HCC, which indicated that STAT3 pathway might be the potent signaling pathway of miR-383/IL-17 axis in regulating HCC tumor cell growth and anti-apoptosis. Indeed, there have been several studies about the functional effect of miR-383 in HCC earlier time. Chen et al found miR-383 targets APRIL to exert its inhibition effect in HCC [43]. Fang et al revealed that miR-383 serves as a anti-tumor role via targeting LDHA in HCC [44]. AKR1B10 is elucidated to promote the progression of HCC targeted by miR-383 [45]. miR-383 suppressed doxorubicin resistance in HCC by targeting EIF5A2 [46]. Above previous studies supported that miR-383 6
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Fig. 4. Overexpression of miR-383 promoted tumor cell apoptosis and up-regulation of IL-17 relieved the enhance effect in HepG2 and Huh7 cells. (A) The apoptosis images were captured for HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (B) The apoptotic rates in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (C) The apoptosis images were captured for Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (D) The apoptotic rates in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (E) The expression of apoptosis marker caspase-3, caspase-9, Bcl-2 and Bax was tested by RT-qPCR in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (F) The expression of apoptosis marker caspase-3, caspase-9, Bcl-2 and Bax was tested by RT-qPCR in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. All quantitative values were the average of triple experiments. *p < 0.05.
detected for exploring more effective biomarkers for HCC. Overall, the findings of miR-383/IL-17 functional role in HCC provide a novel insight into HCC diagnosis and therapeutic target.
has various target genes in HCC. Our study demonstrated IL-17 was directly targeted by miR-383 in HCC tumorigenesis. Additional target genes of miR-383 still needed to be explored to clarify the molecular mechanisms network of miR-383 in HCC. Generally, our study illustrated miR-383 act as a suppressor in HCC tumor cell growth and anti-apoptosis by targeting IL-17. Functional experiments certificated that STAT3 signaling pathway was also involved in miR-383 and IL-17 axis in regulating HCC development. On the whole, we proposed the miR-383 inhibits cell growth and promotes cell apoptosis in HCC by targeting IL-17 via STAT3 signaling pathway. However, more underlying molecular mechnisms as well as in vivo experiments between miR-383/IL-17 and STAT3 signaling pathway in HCC still need further research to confirm our data. What's more, alternative downstream genes of miR-383 in HCC are also worth to be
Funding This work was supported by the Guangxi clinic medicine research center of hepatobiliary diseases (AD17129025). Declaration of Competing Interest All authors declare that they have no financial and non-financial conflicts of interest. 7
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Fig. 5. Overexpression of miR-383 suppressed STAT3 phosphorylation and up-regulation of IL-17 attenuated the inhibition effect in HepG2 and Huh7 cells. (A) pStat3 and Stat3 protein expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC by western blot separately. (B) Quantitative protein expression levels of p-Stat3 and Stat3 in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (C) Quantitative protein expression levels of p-Stat3 and Stat3 in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (D) TP53 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17 + AG490 using RT-qPCR. (E) MMP-2 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17+AG490 using RT-qPCR. (F) MMP-9 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL-17 + AG490 using RT-qPCR. (G) Cell viability in HepG2 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17 + AG490 via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (H) Cell viability in Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL-17 + AG490 via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (I) The apoptotic rates in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL17 + AG490 using apoptosis assay. All quantitative values were the average of triple experiments. *p < 0.05. ns, not significant.
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miR-383 inhibits cell growth and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway Jianchu Wang, Libai Lu, Zongjiang Luo, Wenchuan Li, Yuan Lu, Qianli Tang, Jian Pu
T
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Department of Hepatobiliary Surgery, Affiliated Hospital of Youjiang Medical University for Nationalities, No. 18 Zhongshan Road, Baise 533000, Guangxi Zhuang Autonomous Region, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hepatocellular carcinoma miR-383 IL-17 p-Stat3 STAT3 signaling pathway
Objectives: Emerging microRNAs (miRNAs) are validated to take part in pathological processes, including numerous carcinomas. Currently, we focused on the functional role of miR-383 and interleukin-17 (IL-17) in hepatocellular carcinoma (HCC), and the underlying molecular mechanisms were also the emphases in our research. Methods: We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of miR-383 in 45 paired tumor tissues and adjacent non-tumor tissues extracted from patients with hepatocellular carcinoma. These tissues were also stained for IL-17 using immunohistochemical staining. Western blot was performed to detect the protein expressions of following protein-coding genes, including pStat3, Stat3 and GAPDH. A dual-luciferase activity was carried out to determine whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma development. The colony assay, CCK8 assay, and apoptosis assay were used to explore the detailed regulatory effects of miR-383/IL-17 axis in the cellular processes of hepatocellular carcinoma separately. Results: miR-383 was down-regulated significantly in tumor tissues, while IL-17 was up-regulated. IL-17 was certificated to act as the downstream gene of miR-383. Furthermore, overexpression of miR-383 suppressed cell proliferation and promoted apoptosis in hepatocellular carcinoma. However, the raised IL-17 attenuated the inhibition effect of miR-383 in hepatocellular carcinoma. In addition, we found that p-Stat3 was repressed by miR-383, and the up-regulation of IL-17 reversed the suppression effect in hepatocellular carcinoma. Conclusions: miR-383 may play a anti-tumor role in the pathogenesis of hepatocellular carcinoma by targeting IL-17 through STAT3 signaling pathway. miR-383/IL-17 axis maybe a potent target for the clinical diagnosis and treatment of hepatocellular carcinoma.
1. Introduction Hepatocellular carcinoma consists of 85%˜ 90% of liver cancer [1], which is a common type of malignant cancers with the sixth incidence (4.7%) and the fourth mortality (8.7%) in cancer-related diseases worldwide, according to GLOBOCAN 2018. There are many risk factors leading to HCC, such as, mainly hepatitis virus B or C, diabetes, alcoholic or nonalcoholic steatohepatitis and genetic metabolic diseases [1,2]. Recent years, despite advancements in clinical diagnosis and treatments of liver cancer, due to the long incubation period that HCC is still hard to be diagnosed in the early stage, the finite therapeutic targets, and also the poor prognosis, as well as the high occurrence rate, the mortality of liver cancer still stay in a high level without going down much more. For instance, the mortality of liver cancer in 2012
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was 9.1% [3], only 4.4% decreased, compared with the mortality of 2018. And the five-year survival rate for HCC patients is still quite low (−30%) [3], almost 70,000 patients suffering liver cancer died every year across the world [4]. So the research for explaining molecular heterogeneity and detecting more biomarkers in HCC is urgent. Since 2006, Murakami and his colleagues proposed that alteration of microRNAs expression was found commonly in liver cancer firstly [5]. The functional effects and underlying molecular mechanisms of miRNAs in HCC draw our attentions in numerous studies in decades. MicroRNAs (miRNAs) are a class of short (approximately 22nts), single-strand and non-coding RNAs transcribed from particular regions of protein-coding genes [6]. miRNAs have been proven to be involved in post-transcription processes by interacting with target mRNA 3′UTR and interrupting protein generating [7]. Based on previous studies,
Corresponding author. E-mail address: [email protected] (J. Pu).
https://doi.org/10.1016/j.biopha.2019.109551 Received 30 July 2019; Received in revised form 9 October 2019; Accepted 9 October 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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CCAGCCCATGATGGTTCTGAT-3′ (reverse). TP53 primer was 5′-ACCT ATGGAAACTACTTCCTGAAA-3′ (forward) and 5′-CTGGCATTCTGGGA GCTTCA-3′ (reverse). MMP-2 primer was 5′-CAGGACATTGTCTTTGAT GGCATCGC-3′ (forward) and 5′-TGAAGAAGTAGCTATGACCACC GCC-3′ (reverse). MMP-9 primer was 5′-ATCCCCCAACCTTTACCA-3′ (forward) and 5′-TCAGAACCGACCCTACAA-3′ (reverse). GAPDH primer was 5′-GACTCATGACCACAGTCCATGC-3′ (forward) and 5′-AGAGGCAGGGATGATGTTCTG-3′ (reverse). The Applied Biosystems 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) was used to perform above quantitative experiments.
miRNAs were found expressed abnormally and conducted as a oncegene or inhibitor in multitudinous kinds of human diseases, including lung cancer, ovarian cancer, and liver cancer [8]. miRNAs act as either promotor or inhibitor in pathogenesis of diseases through participating in diverse cellular processes, such as, cell growth, differentiation, apoptosis and metastasis [9]. In the case of HCC, Klingenberg et al summaried 16 miRNA expressions were dysregulated, mainly upregulated [10]. Integrating with many studies concerning the functional role of miRNA in HCC, oncogenetic miRNAs were revealed, including miR-184 [11], miR-10b-3p [12]. Meanwhile, suppressive miRNAs were following, miR-122 [13], miR-146a-5p [14], miR-143 [15], miR-1 [16], miR-144 [17], miR-940 [18]. As shown above, miRNAs were closely linked with HCC. Cytokines are a group of bioactive small molecule protein produced by immune cells mainly in response to stimuli and are vital immune regulators in biological activities, including innate immunity, autoimmune pathology and cancer immunity process [19]. Numerous cytokines have certificated to be involved in cancer pathogenesis. Inflammatory cytokines IL-4 and IL-13 were related with cancer stem cells [20]. IL-8 and IL-12 could act as biomarkers for justifying metastatic breast cancer [21]. IL-1b, IL-6, IL-10, IL-23 and TNF-αare commonly correlated with HCC [22]. The effect of cytokines in HCC also could not be ignored. In our current study, we focused on the relationship between miR383, IL-17 and HCC, and aimed to investigate the functional effect, regulatory role and potential signaling pathway of miR-383/IL-17 axis on the process of HCC.
2.4. Immunohistochemistry staining (IHC) Paraffin-embedded, formalin fixed liver tissue Sections (4) um were incubated at 65℃overnight. Then slides were blocked with 3% hydrogen peroxide (H2O2) for 35 min. After incubating in 5% BSA for 40 min. The slides were incubated with a 1:300 dilution of primary antibody against human IL-17 (R&D Systems, Minneapolis, MN) at 4 ℃ overnight and were then subjected to secondary antibody incubation. Diaminobenzidine tetrahydrochloride was used for the chromogenic reactionas. Two independent investigators counted the positively stained cells by using microscope. 2.5. Transfection miR-383 mimics (mimic-miR-383) and its negative control (mimicNC) obtained from Genepharma (Shanghai, China) were transfected into 293 T cells to perform luciferase activity. LV-miR-383 or the negative control (LV-NC) were transfected into HepG2 and Huh7 cells to observe the colony numbers and apoptotic rate. Above lentivirses were bought from Genepharma (Shanghai, China).
2. Materials and methods 2.1. Samples and tissues From August 2017 to February 2019, 45 patients suffering hepatocellular carcinoma were admitted in our hospital. Based on the TumorNode Metastasis (TNM) Classification of malignant cancers following International Union Against Cancer (UICC). In all of the 45 hepatocellular carcinoma cases, 11 cases were in stage I-II, 18 cases were in stage III, 16 cases were in stage IV. The tumor tissues and paired adjacent non-tumor tissues were extracted from the 45 HCC patients. Total tissues were rapidly frozen in liquid nitrogen and then stored at -80 ℃ medical refrigerator for the following experiments. All the usage of tissues were conformed to ethical standards of our hospital.
2.6. Luciferase activity assay The pGL3 vector encompassing luciferase reporter gene was inserted with wild type of IL-17 3′UTR (WT-IL-17 3′UTR) or mutant type of IL-17 3′UTR (MUT-IL-17 3′UTR) separately for a dual-luciferase activity assay. Above vector and sequences were all achieved from Genepharam (Shanghai, China). Then, pGL3-WT-IL-17 3′UTR and pGL3-MUT-IL-17 3′UTR were transfected into 293 T cells placed in 6well plate in advance separately. Next, miR-383 mimics (mimic-miR383) and its negative control (mimic-NC) with Lipofectamine 2000 reagent were transfected into above 293 T cells. 2 days later, We used a dual-luciferase reporter system (Promega Corporation, Madison, WI, USA) to detect the luciferase activity according to manufacturer’s instructions.
2.2. Cell culture Human HCC cell lines, HepG2 and Huh7, as well as 293 T cells were all obtained from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), and cultured in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubators with 5% CO2 at 37℃.
2.7. ELISA The protein expression of IL-17 were evaluated using ELISA kits (R& D Systems, Minneapolis, MN, USA).
2.3. RT-qPCR
2.8. Western blot
Firstly, total RNA were extracted from abraded tissues including tumor tissues and matched adjacent non-tumor tissues using TRIZOL (Sigma, USA). Secondly, RNA were reverse transcribed to cDNA with the support of PrimeScript™RT reagent Kit (TaKaRaBio Technology). Taqman MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) was used to measure the expression levels of miR-383. Caspase-3 primer was 5′-CATGGAAGCGAATCAATGGACT-3′ (forward) and 5′-CTGTACC AGACCGAGATGTCA-3′ (reverse). Caspase-9 primer was 5′-ATGTCGG ACTACGAGAACGAT-3′ (forward) and 5′-TGATGCGTGAGGGGTC GAT-3′ (reverse). Bcl-2 primer was 5′-TTCTTTGAGTTCGGTGGGGTC-3′ (forward) and 5′-TGCATATTTGTTTGGGGCAGG-3′ (reverse). Bax primer was 5′−CCCGAGAGGTCTTTTTCCGAG-3′ (forward) and 5′−
HepG2, Huh7 cells transfected with lentiviruses were lysed using radio immunoprecipitation assay buffer (RIPA, Beyotime Biotechnology, China). Proteins were separately by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and then transferred to Poly Vinylidene Difluoride (PVDF) membrane. After above membranes were blocked in 5% defatted milk at ordinary temperature, the membranes were incubated with specific primary antibodies (Stat3, p-Stat3 and GAPDH primary antibody) at 4 °C overnight. Membranes were then washed by TBST three times and incubated with secondary antibodies for 2 h at room temperature to visualize the proteins. All antibodies used were purchased from Abcam (ambridge, UK). Protein concentrations were quantified by a bicinchoninic acid protein assay kit 2
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(Beyotime Institute of Biotechnology).
3.2. IL-17 was up-regulated in hepatocellular carcinoma tumor tissues and was correlated with miR-383 negatively
2.9. Colony formation assay
IL-17 was chosen to be a potential target gene of miR-383 via TargetScan website (http://www.targetscan.org/). To explore whether IL-17 was in relation to hepatocellular carcinoma, IL-17 positive cells were counted in 45 paired tumor tissues and adjacent normal tissues using immunohistochemical staining. The results showed that IL-17 positive cells were much more in hepatocellular carcinoma tumor tissues, compared with normal tissues (Fig. 1C). Univariate regression analysis was established to observe the relationship between miR-383 expression and IL-17 in 45 cases. The results showed the slope of regression line was negative (Fig. 1D). These results illustrated that IL-17 was up-regulated in hepatocellular carcinoma and had a negative correlation with miR-383.
HepG2 and Huh7 cells transfected with LV-NC, LV-miR-383 or LVmiR-383 with IL-17 (20 ng/ml, Cat# 7955-IL-025/CF, Fisher Scientific, Pittsburgh, PA) were seeded into 6-well plate rounded with agar medium. Then, fresh medium was added into HepG2 and Huh7 cells every 3 days, which was cultured in a humidified incubator with 5% CO2 at 37℃. 12 days later, we obtained the image of colonies in each well. Then the numbers of colony were counted with the help of Image J 1.49v software (National Institutes of Health, Bethesda, MD, USA).
2.10. Cell proliferation assay
3.3. IL-17 was targeted directly by miR-383 and was regulated by miR-383 negatively
To evaluate the cell proliferation, CCK-8 assay was employed to detect the cell growth. A 96-well plates was prepared for HepG2 and Huh7 cells transfected with mimic-NC, mimic-miR-383, LV-NC, LVmiR-383, LV-miR-383+IL-17 or LV-miR-383+IL-17 with AG490 (15 ug/ml, Sigma, USA). The cells were kept at 37 °C with a 5% CO2. For every 24 h, the cells were counted and OD450 value was collected. We drew a cell growth curve after incubtion of 5 days.
To investigate whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma, luciferase activity assay was carried out. The sequence of WT-IL-17 3′UTR could bind to miR-383, while MUT-IL17 3′UTR could not (Fig. 2A). In the case of pGL3 vector constructed with IL-17 3′UTRwt, the luciferase activity of 293 T cell transfected with mimic-miR-383 was lower than that transfected with mimic-NC (Fig. 2B). However, in the case of pGL3 vector constructed with IL-17 3′UTRmut, the luciferase activity of 293 T cell transfected with mimicmiR-383 was the same with that transfected with mimic-NC (Fig. 2C). Then, ELISA was performed to explore the regulatory effect of miR-383 on IL-17 expression. The results exhibited that IL-17 protein expression levels in HepG2 and Huh7 cells transfected with mimic-miR-383 were both less than the expression levels in HepG2 and Huh7 cells transfected with mimic-NC significantly (Fig. 2D and 2E). These results explained that IL-17 was targeted directly by miR-383 and the negative correlation between miR-383 and IL-17 was certificated a step furtherly.
2.11. Apoptosis assay After HepG2 and Huh7 cells transfected with LV-NC, LV-miR-383, LV-miR-383+IL-17 or LV-miR-383+IL-17+AG490 were seeded into 6well plate and cultured 48 h later, above cells were washed three times by PBS and then resuspended in 500 u L binding buffer (KeyGen). 5 u L Annexin V-FITC (KeyGen) and 5 u L propidium iodide (PI) were supplemented into above buffer and mixed at room temperature without light for 12 min. We used the flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) to analyze the apoptosis rate of cells.
3.4. miR-383 represses tumor cell growth and IL-17 attenuates the inhibition effect of miR-383 on hepatocellular carcinoma cell growth in vitro
2.12. Statistical analysis The values were represented as the mean ± standard deviation (SD) and were the average of three experiments. We carried out Student t-test and One-Way ANOVA analysis to measure the differences between quantitative variables. Univariate regression was used to describe the correlation relationship between miR-383 and IL-17. Above statistical methods were performed with the help of Prism (7.0) and R studio (3.6.1). Two-tailed P-value < 0.05 was considered to be significant statistically.
To identify the functional effect of IL-17 on the development of hepatocellular carcinoma, LV-NC, LV-miR-383 or LV-miR-383 with IL17 was transfected into HepG2 and Huh7 cells. Colony formation assay was performed in the aim of confirming the effect of miR-383 on hepatocellular carcinoma cell growth. The results pointed out that the colony numbers of HepG2 and Huh7 cells transfected with LV-miR-383 was both fewer than the colony numbers of HepG2 and Huh7 cells transfected with LV-NC. In addition, the colony numbers of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was more than the colony numbers of HepG2 and Huh7 cells transfected with LVmiR-383 only (Fig. 3A and B). CCK-8 assay results identified that HepG2 and Huh7 cells transfected with LV-miR-383 had lower cell viability, compared with HepG2 and Huh7 cells transfected with LVNC. In addition, the cell proliferation rate of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was higher than that transfected with LV-miR-383 (Fig. 3C and D). The results demonstrated that miR-383 repressed cells growth while IL-17 reversed the inhibition effect of miR-383 on cells growth in HepG2 and Huh7 cells.
3. Results 3.1. miR-383 was down-regulated in vivo To identify whether miR-383 was abnormally expressed in hepatocellular carcinoma, we measured the expression levels of 45 paired tumor tissues and adjacent normal tissues using RT-qPCR. The results demonstrated that miR-383 was down-regulated obviously in tumor tissues, compared with normal tissues (Fig. 1A). Furthermore, according to clinicalpathological stage, we grouped 45 tumor tissues into three groups, including stage I-II, stage III, stage IV. Correspondingly, miR-383 expression levels were separately into three groups. The boxplot of each group showed that stage IV had the lowest miR-383 expression level, and stage I-II had the highest miR-383 expression level (Fig. 1B). Above findings indicated miR-383 was associated with hepatocellular carcinoma and had a negative correlation with clinicalpathological stage.
3.5. miR-383 promotes tumor cell apoptosis and IL-17 reverses the promote effect of miR-383 on tumor cell apoptosis in vitro To verify whether miR-383 was involved in the process of tumor cell apoptosis in hepatocellular carcinoma, apoptosis assay was carried out in HepG2 and Huh7 separately. The results showed that the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected 3
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Fig. 1. miR-383 and IL-17 protein expression levels in HCC tissues. (A) miR-383 expression levels were measured in 45 HCC tumor tissues and matched normal tissues using RT-qPCR. (B) miR-383 expression levels of 45 HCC tumor tissues were grouped into Stage I–II, Stage II and Stage III. (C) Representative images of immunohistochemical staining of IL-17 expression in 45 HCC tumor tissues and matched normal tissues (50um). The frequency of IL-17 positive cells in tumor tissues was significantly higher than that in non-tumor tissues. (D) Univariate regression analysis between miR-383 expression and corresponding IL-17 positive cells in 45 HCC tumor tissues. *p < 0.05.
HepG2 and Huh7 cells transfected with LV-miR-383 were both lower than that in HepG2 and Huh7 cells transfected with LV-NC, and both of them were lower than the expression levels in HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17. However, Stat3 protein expression levels in HepG2 and Huh7 cells transfected with LV-miR383, LV-miR-383 with LV-IL-17 or LV-NV were all identical (Fig. 5A–C). We employed RT-qPCR to find the function of miR-383 on p-Stat3 downstream factors TP53, MMP2 and MMP9. Overexpression of miR383 increased TP53 expression (Fig. 5D) in HepG2 and Huh7 cells. And upgulation of miR-383 inhibited MMP2 and MMP9 expression (Fig. 5E and F). What’s more, loss of STAT3 had the similar effect on tumor growth and apoptosis as miR-383 (Fig. 5G–I). Above findings certificated that STAT3 phosphorylation was down-regulated by miR-383 while IL-17 reversed the down-regulation effect of miR-383 on p-Stat3. Furthermore, STAT3 was associated with miR-383 and IL-17 and might play vital role in hepatocellular carcinoma development.
with LV-miR-383 were both more than the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected with LV-NC. And, the apoptosis cell numbers and apoptotic rate of HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 was fewer than the apoptosis numbers and apoptotic cell rate of HepG2 and Huh7 cells transfected with LV-miR-383 only (Fig. 4A–D). To elucidate the molecular basis of apoptosis we examined the apoptosis mediators and molecular markers. As shown in Fig. 4E and F, decreased expression level of Bcl-2 and increased expression level of caspase-3, caspase-9 and BAX was detected in HepG2 and Huh7 cells transfected with LV-miR383 compared with HepG2 and Huh7 cells transfected with LV-NC. In addition, increased expression level of Bcl-2 and decreased expression level of caspase-3, caspase-9 and BAX was detected in HepG2 and Huh7 cells transfected with LV-miR-383 added with IL-17 than that transfected with LV-miR-383. Above results revealed that miR-383 promoted tumor cell apoptosis while IL-17 reversed the positive effect of miR-383 on tumor cell apoptosis in the process of hepatocellular carcinoma in vitro.
4. Discussion Currently, our data and images showed that miR-383 was downregulated and had a negative correlation with TNM stage and IL-17 expression separately in 45 HCC tumor tissues. So miR-383 expression provided a potent target for the diagnosis of HCC patients and might be a meaningful reference for determining clinicalpathological stage in order to accept pertinent treatment timely. In addition, IL-17 was validated to be the downstream gene of miR-383 in HCC. Furthermore,
3.6. p-Stat3 is down-regulated by the overexpression of miR-383 and IL-17 reverses the down-regulation effect of miR-383 on p-Stat3 in vitro To identify whether STAT3 phosphorylation was associated with miR-383 and IL-17 in hepatocellular carcinoma development, p-Stat3 and Stat3 protein expression levels were measured through western blot. The results indicated that p-Stat3 protein expression levels in 4
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Fig. 2. IL-17 was targeted by miR-383 directly. (A) The sequences of miR-383, wild type IL-17 3′UTR and mutant type IL-17 3′UTR. (B) The luciferase activities were detected in 293 T cells co-transfected with mimic-miR-383 or mimic-NC and pGL3 vector containing IL-17 3′UTRwt via luciferase activity assay. (C) The luciferase activities were detected in 293 T cells co-transfected with mimic-miR-383 or mimic-NC and pGL3 vector containing IL-17 3′UTRmut via luciferase activity assay. All quantitative values were the average of triple experiments. (D) The expression of IL-17 was determined by ELISA in HepG2 cells transfected with mimic-miR-383 or mimic-NC. (E) The expression of IL-17 was determined by ELISA in Huh7 cells transfected with mimic-miR-383 or mimic-NC. *p < 0.05. ns, not significant.
383 and LDHA axis repress cell proliferation, invasion and aerobic glycolysis [24]. miR-383-5p-TRIM27 combination inhibit cell proliferation and promote chemosensitivity [25]. Considering glioma, miR383 inhibits cell invasion by targeting IGF1R [26]. With regard to cholangiocarcinoma, which was the another type of liver cancer, the tumor-suppressive effect of miR-383 was also illustrated [27]. In our study, miR-383 was investigated as a tumor inhibitor in HCC development, this finding was compatible with above experimental results in other carcinomas. The anti-tumor role of miR-383 in a wide range of carcinomas or other pathological diseases is valuable and need more research. Interleukin-17 (IL-17) first found in rodents T cells at 1993 belongs to IL family. It is a kind of cytokines, through binding to IL-17 receptor (IL-17R) thus activating signal transduction pathway, including but not
miR-383 repressed tumor cell growth and promoted cell apoptosis in HepG2 and Huh7 cell lines, while IL-17 attenuated the inhibition effects, showing that miR-383 was an inhibitor in the progression of HCC, while IL-17 was a promotor. What’s more, p-Stat3 was found to be down-regulated by the overexpression of miR-383. IL-17 reversed the down-regulation effect of miR-383 on p-Stat3 in HepG2 and Huh7 cell lines. Taken together, we hypothesis that miR-383 inhibits cell proliferation and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway. These findings may provide a novel therapeutic approach for HCC. miR-383 have been reported to play a anti-tumor role in several types of malignant tumors. In cervical cancer, Teng et al revealed that miR-383 acted as a tumor suppressor by inhibiting PARP2+ and PI3KAKT-MTOR signaling pathway [23]. In the case of ovarian cancer, miR5
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Fig. 3. Overexpression of miR-383 inhibited tumor cell growth and up-regulation of IL-17 attenuated the inhibition effect in HepG2 and Huh7 cells. (A) The images of colonies were captured for HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC with the help of colony formation assay separately. (B) The number of colonies were counted for HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC separately. (C) Cell viability in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (D) Cell viability in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. All quantitative values were the average of triple experiments. *p < 0.05.
limited, NF-B, MAPK, TRAF6, JAK/STAT, TAK1, finally, altering target genes transcription, mainly pro-inflammatory chemokines, hematopoietic cytokines, acute phase response genes and anti-microbial substances [28,29]. For liver cancer, cytokines like IL-1α, IL-1β, IL-6, IL-8 and TNF-α have been certifated to associated with chronic hepatic inflammation, and IL-6 is highlighted in human hepatocarcinogenesis [22,30]. Whether IL-17 is related with HCC also have been paid much attention by researchers. Liu et al reported that plasma IL-17 was highly expressed in 39 HCC group compared with control group [31]. Yang et al found IL-17 serve as a protumor role in mice hepatocarcinoma model [32]. Li et al demonstrated that IL-17A act as a promotor in hepatocellular carcinoma invasion and migration [33]. Take our results into consideration, up-regulation of IL-17 promoted cell growth and inhibited cell apoptosis in HCC, the involvement of IL-17 in HCC got supports by previous studies. Emerging studies have elaborated that miRNAs functions in pathologiacal and progression of diseases by modulating the expression levels of cytokines directly or indirectly [19]. For IL-17, miR-340 targets IL-17 directly thus relieve psoriasis in mice [34]. miR-20 repress IL-17 expression by inhibiting T17 differentiation through targeting STAT3 in autoimmune encephalomyelitis [35]. Similarly, our study illustrated that miR-383 inhibits cell proliferation and promotes cell apoptosis in HCC by targeting IL-17. Therefore, miR-383/IL-17 axis in tumorgenesis of HCC is consistent with the above findings. However, the up-stream or down-stream mechnisms of miR-383/IL-17 is not clear enough. Due to the special ability of IL-17 that could activate signaling transduction pathway, we focused on whether STAT3 signaling pathway was enlightened by miR-383/IL-17 axis in HCC for our study. STAT3 pertains to STAT protein family, is a transcription factor, which
could be phosphorylated by JAKs when stimulated by cytokines and growth factors [36]. STAT3 have been reported to take a place in pathogenesis of various diseases, including hematological disease [36], lung cancer [37], liver fibrosis [38], psoriatic arthritis [39], hepatocellular carcinoma [40]. In addition, miRNAs have been validated to be involved in the STAT3 signaling pathway. Firstly, JAK2 is activated through the combination of IL-6 receptors (IL-6R) and IL-6. Secondly, STAT3 is transformed to p-Stat3 with the activation of JAK2. Finally, Stat3 act as a transcription factor to activate target genes, including miRNAs. For instance, STAT3 regulates miR-21 expression directly with the mediation of IL-6, which promotes miR-21 through binding STAT3 to an upstream enhancer of miR-21 [41] [10]. On the other hand, STAT3 could binds to the IL-17 in T cells and enhance the production of IL-17 [30]. IL-17-STAT3 mechanisms were also observed in liver fibrosis [38], psoriatic arthritis [39], hepatocarcinoma [42]. Above elucidates about miR-STAT3 and IL-17-STAT3 provide potent evidences that STAT3 signaling pathway has a great chance in theory to correlate with miR-383/IL-17 axis in HCC. Our results showed p-STAT3 was down-regulated by overexpression of miR-383 and was overexpressed by the up-regulation of IL-17 in HCC, which indicated that STAT3 pathway might be the potent signaling pathway of miR-383/IL-17 axis in regulating HCC tumor cell growth and anti-apoptosis. Indeed, there have been several studies about the functional effect of miR-383 in HCC earlier time. Chen et al found miR-383 targets APRIL to exert its inhibition effect in HCC [43]. Fang et al revealed that miR-383 serves as a anti-tumor role via targeting LDHA in HCC [44]. AKR1B10 is elucidated to promote the progression of HCC targeted by miR-383 [45]. miR-383 suppressed doxorubicin resistance in HCC by targeting EIF5A2 [46]. Above previous studies supported that miR-383 6
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Fig. 4. Overexpression of miR-383 promoted tumor cell apoptosis and up-regulation of IL-17 relieved the enhance effect in HepG2 and Huh7 cells. (A) The apoptosis images were captured for HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (B) The apoptotic rates in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (C) The apoptosis images were captured for Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (D) The apoptotic rates in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC using apoptosis assay separately. (E) The expression of apoptosis marker caspase-3, caspase-9, Bcl-2 and Bax was tested by RT-qPCR in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (F) The expression of apoptosis marker caspase-3, caspase-9, Bcl-2 and Bax was tested by RT-qPCR in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. All quantitative values were the average of triple experiments. *p < 0.05.
detected for exploring more effective biomarkers for HCC. Overall, the findings of miR-383/IL-17 functional role in HCC provide a novel insight into HCC diagnosis and therapeutic target.
has various target genes in HCC. Our study demonstrated IL-17 was directly targeted by miR-383 in HCC tumorigenesis. Additional target genes of miR-383 still needed to be explored to clarify the molecular mechanisms network of miR-383 in HCC. Generally, our study illustrated miR-383 act as a suppressor in HCC tumor cell growth and anti-apoptosis by targeting IL-17. Functional experiments certificated that STAT3 signaling pathway was also involved in miR-383 and IL-17 axis in regulating HCC development. On the whole, we proposed the miR-383 inhibits cell growth and promotes cell apoptosis in HCC by targeting IL-17 via STAT3 signaling pathway. However, more underlying molecular mechnisms as well as in vivo experiments between miR-383/IL-17 and STAT3 signaling pathway in HCC still need further research to confirm our data. What's more, alternative downstream genes of miR-383 in HCC are also worth to be
Funding This work was supported by the Guangxi clinic medicine research center of hepatobiliary diseases (AD17129025). Declaration of Competing Interest All authors declare that they have no financial and non-financial conflicts of interest. 7
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Fig. 5. Overexpression of miR-383 suppressed STAT3 phosphorylation and up-regulation of IL-17 attenuated the inhibition effect in HepG2 and Huh7 cells. (A) pStat3 and Stat3 protein expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC by western blot separately. (B) Quantitative protein expression levels of p-Stat3 and Stat3 in HepG2 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (C) Quantitative protein expression levels of p-Stat3 and Stat3 in Huh7 cells transfected with LV-miR-383, LV-miR-383 with IL-17, or LV-NC. (D) TP53 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17 + AG490 using RT-qPCR. (E) MMP-2 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17+AG490 using RT-qPCR. (F) MMP-9 expression levels were measured in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL-17 + AG490 using RT-qPCR. (G) Cell viability in HepG2 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383+IL-17 + AG490 via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (H) Cell viability in Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL-17 + AG490 via CCK-8 assay from Day 1 to Day 5 with the time difference of 24 h. (I) The apoptotic rates in HepG2 and Huh7 cells transfected with LV-miR-383, LV-miR-383 + IL-17, or LV-miR-383 + IL17 + AG490 using apoptosis assay. All quantitative values were the average of triple experiments. *p < 0.05. ns, not significant.
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