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LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b-3p to up-regulate RCCD1
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b-3p to up-regulate RCCD1 Ziming Cheng, Shizhen Hou*, Yubing Wu, Xiangdong Wang, Yi Sun, Bing Liu, Maoxi Yuan Department of Thoracic Surgery, Linyi Central Hospital. No.17, Health Road, Linyi, Shandong, 276400, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 September 2019 Accepted 21 September 2019 Available online xxx
Lung adenocarcinoma (LUAD) is one of the most common type of lung cancer notorious for the high incidence and mortality around the world. Long non-coding RNAs (LncRNAs) are defined as a class of RNAs with length more than 200 nucleotides. Mounting studies have proved that lncRNAs are related closely to incidence of diseases and play crucial roles in cancer progression. Although LINC01419 has been studied in several cancers, the function and mechanism of LINC01419 in LUAD remains a mystery. Our findings showed that LINC01419 level was high in LUAD cells, and LINC01419 knockdown inhibited carcinogenesis via suppressing cell proliferation, migration as well as invasion. Moreover, bioinformatics prediction, luciferase reporter experiments and RIP assay were used to confirm miR-519-3p was sequestered and negatively regulated by LINC01419. Subsequently, RCCD1 was identified as a miR-519b3p target and had inverse relationship with miR-519b-3p. LINC01419 was positively related to RCCD1. Furthermore, rescue assays confirmed that miR-519b-3p inhibitor or RCCD1 overexpression could neutralize the effect of LINC01419 silenced in cell proliferation, migration and invasion. Taken together, all the results indicated that LINC01419 exhibited oncogenic behaviors LUAD via binding to miR-519b-3p to enhance the expression of RCCD1, manifesting the underlying therapy values of LINC01419 in LUAD. © 2019 Elsevier Inc. All rights reserved.
Keywords: LINC01419 miR-519b-3p Lung adenocarcinoma RCCD1
1. Introduction Lung cancer possesses high incidence and mortality rate among all the malignant tumors [1]. Lung cancer cases are classified into small cell and non-small cell lung cancer (NSCLC) [2]. Lung adenocarcinoma (LUAD) accounts for a large proportion of NSCLC [3], threatening human health around the world [4]. Despite that much therapeutic progress has been achieved, the prognosis of LUAD is still very poor [5]. Therefore, it is necessary to understand the pathogenetic factors to find out efficient therapeutic methods for LUAD. Emerging studies confirmed that lncRNAs served as an important role in the progression of diseases [6]. LOXL-AS1 was reported to mediate the doxorubicin resistance in prostate cancer by sponging miR-let-7a-5p to elevate EGFR [7]. NEF was considered to have inhibitory effect on cell migration and invasion via decreasing miR-21 in osteosarcoma [8]. Accumulating articles suggested the deep involvement of lncRNAs in LUAD. LncRNA TTN-AS1 promoted
* Corresponding author. E-mail address: [email protected] (S. Hou).
invasion and migration in LUAD cells via targeting miR-4677-3p and upregulating ZEB1 [9]. LncRNA HMMR-AS1 fostered proliferation and metastasis of LUAD by sponging miR-138 to enhance sirt6 [10]. The role of LINC01419 was analyzed in esophageal cell carcinoma [11]. However, never has the link between LINC01419 and LUAD been established. It was said that lncRNAs functioned as ceRNAs to sponge miRNAs in the process of cancers. RSU1P2 accelerated the tumor growth via playing as a ceRNA against let-7a in cervical cancer [12]. SMAD5-AS1 repressed cell growth in diffuse large B cell lymphoma through Wnt/b-catenin signaling by targeting miR-135b-5p/APC axis [13]. PTV1 worked as a ceRNA sequestering miR-199a-5p to regulate HIF1a in NSCLC upon hypoxia [14]. However, the interaction of LINC01419 with miR-159b-3p in LUAD has never been revealed. Our study investigated the role of LINC01419 in LUAD. It was evident that LINC01419 was highly expressed in LUAD cells. Knockdown of LINC01419 led to the decreased cell proliferation, migration and invasion in LUAD cells. LINC01419 could bind with miR-519b-3p and miR-519b-3p was negatively regulated by LINC01419. Moreover, LINC01419 promoted tumorigenesis via regulating the expression of RCCD1 by targeting miR-519b-3p in
https://doi.org/10.1016/j.bbrc.2019.09.090 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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LUAD cells, manifesting a new insight for the LUAD treatment. 2. Materials and methods 2.1. Cell lines and cell culture The Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) offered LUAD cell lines (SPC-A1, A549, H446 and H460) and normal pulmonary epithelial cell (BEAS-2B). Cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone Laboratories, Logan, UT, USA) enriched with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) filled with 100 U/mL penicillin and 100 mg/mL streptomycin in moist atmosphere at 37 C with 5% CO2. 2.2. Cell transfection Sh- LINC01419#1/2 was applied to knock down LINC01419. PcDNA3.1/RCCD1 was used to up-regulate RCCD1. MiR-519b-3p mimics were used to overexpress miR-519b-3p, and miR-519b-3p was down-regulated by miR-519b-3p inhibitor. All plasmids were purchased from Gene Pharmacy (Shanghai, China). Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, USA) was used for transfection. 2.3. RT-qPCR assay TRIpure Reagent (Labgene Biotech, Guangzhou, Guangdong, China) was utilized to extract total RNAs. Taq Man™ Advanced miRNA cDNA Synthesis Kit (Waltham, MA, USA) and the Omni script Reverse Transcription Kit (HaoranBio, Xuhui, Shanghai, China) were respectively used to carry out the reverse transcription. The SYBR Prime Script RT-PCR kit (TaKaRa, Dalian, China) was then exploited to conduct RT-qPCR assay on ABI 7500 System (Applied Biosystems, Carlsbad, California). Both GAPDH and U2 were internal controls. Relative expression levels of RNAs were calculated through the 2 DDCt method. 2.4. RNA pull-down assay LINC01419-WT, LINC01419-Mut and NC group were biotinylated to be Bio-LINC01419-WT, Bio-LINC01419-Mut and Bio-NC by Gene Pharma Company (Shanghai, China). Bio-LINC01419-WT, BioLINC01419-Mut and Bio-NC were transfected into SPC-A1and A549 cells. 48 h later, the lysates of cells were incubated with Dynabeads M 280 Streptavidin (Invitrogen, CA). Following purification, RNA complex was measured by RT-qPCR assay. 2.5. RIP assay RIP assay was carried out by using RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). LUAD cell lysates were cultured with RIP buffer that contained magnetic beads conjugated with Ago2 antibody (Millipore, Billerica, MA, USA) and IgG (Millipore, Billerica, MA, USA) was used as a negative control. Immunoprecipitate RNA was isolated by proteinase K and then the relative expression of RNAs was examined by RT-qPCR assay. 2.6. Nuclear-cytoplasmic fractionation The nuclear and cytoplasmic fractions of SPC-A1 and A549 cells were separated with a PARIS kit (Life Technologies).
2.7. CCK-8 assay SPC-A1 and A549 cells at a density of 1 104 cells/well were plated into 96-well plate. After the incubation at 24, 48 and 72, 96 h, the culture plate was taken out. The cell culture medium was removed and the plate was washed twice with PBS.10 mL CCK-8 solution (Keygen Biotech, Nanjing, China) and serum-free medium (1:10) mixture were added into each well. And the incubation lasted for 4 h. The cell growth curves were draw by measuring 450 nm absorbance using microplate readers (EL340; Bio-Tek Instruments, Hopkinton, MA, USA) at each indicated time point. 2.8. Luciferase reporter assay The LINC01419-WT or LINC01419-Mut reporters were cotransfected, respectively, with miR-519b-3p mimics into cells. LINC01419-WT or LINC01419-Mut underwent co-transfection with NC mimics or miR-519b-3p mimics into cells. Lipofectamine 2000 was applied to conduct transfection. Subsequent to 48 h, luciferase reporter assay system (Promega, Madison WI) was adopted to assess the relative luciferase activities. 2.9. Western blot assay Cells were lysed using RIPA lysis buffer (Beyotime biotech, Shanghai, China) for total protein extraction. Then total protein solution was quantified using BCA kit (Beyotime biotech, Shanghai, China). Protein extractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Sigma-Aldrich, USA). After being blocked with nonfat milk, the membranes were incubated with proper primary antibodies overnight at 4 C.Then they were incubated with secondary antibodies at 37 C for 1 h. GAPDH was served as the internal control in this research. 2.10. Wound healing assay Wound healing assay was employed to estimate cell migration capacity in LUAD cells. Cells were incubated onto 6-well plates and treated in DMEM containing 10% FBS at indoor temperature overnight. When reaching 80% confluence, a pipette tip was utilized to make a straight scratch. The cells were then washed by use of PBS for three times and then incubated in serum-free medium. Images of wounds at the same location were observed at indicated time points. 2.11. Transwell invasion assays As for cell migration and invasion capacities, transwell assays were implemented. The cells were starved with serum-free DMEM medium (HyClone Laboratories) in the upper chamber coated with Matrigel (Corning, NY, USA). 10% FBS DMEM medium (HyClone Laboratories) were filled in the lower chamber. After incubating for 48 h, cells stayed in upper chamber were gently abraded, while cells migrated or invaded into the lower chamber were fixed with methanol and stained by use of 0.1% crystal violet. Images were captured for counting the number of migrated or invaded cells under a microscope (Olympus, Japan). 2.12. Statistical analysis Each assay was performed in triplicate and data was presented as mean ± standard deviation. Student’s t-test was utilized to make comparison between two groups, and one-way analysis of variance (ANOVA) was used to make comparison between various groups.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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All analyses were conducted by using SPSS for Windows, Version 14.0. (SPSS Inc., Chicago). A value of p < 0.05 was regarded statistically significant. 3. Results 3.1. Knockdown of LINC01419 inhibited the development of LUAD To illustrate the function of LINC01419 on LUAD progression, RTqPCR data showed that the expression of LINC01419 was much higher in LUAD cell lines (SPC-A1, A549, H446 and H460) than that in normal pulmonary epithelial cells (BEAS-2B) (Fig. 1A). Moreover, the expression of LINC01419 in SPC-A1 and A549 cells was much higher than that in H446 and H460 cells. Thus, these two cells were chosen in the following experiments. To examine the subcellular distribution of LINC01419 in LUAD, nucleus and cytoplasm were isolated from SPC-A1 and A549 cells. Data from RT-qPCR showed that LINC01419 was mainly amassed in cytoplasm in SPC-A1 and A549 cells (Fig. 1B). In order to learn more about the biological role of LINC01419, we confirmed the knockdown of endogenous LINC01419 by sh-LINC01419#1 and sh-LINC01419#2 in SPC-A1 and A549 cells (Fig. 1C). Then, the cell proliferation ability was
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examined by CCK8 assay and colony formation assay. The results depicted that knockdown of LINC01419 inhibited the cell proliferation in two cells (Fig. 1D and E). Also, LINC01419 silence repressed cell migration in wound healing assay (Fig. 1F). Knockdown of LINC01419 suppressed the cell invasion in transwell assay (Fig. 1G). In conclusion, LINC01419 was highly expressed in LUAD and promoted the progression of LUAD by accelerating cell proliferation, migration and invasion. 3.2. MiR-519b-3p was a target of LINC01419 Emerging evidence has revealed that lncRNAs serve as sponges of miRNAs in tumor progression [15]. So we wondered whether LINC01419 functioned in LUAD in this manner. 4 miRNAs were predicted as potential targets for LINC01419 in Starbase (Fig. 2A). However, among the 4 candidates, only miR-519b-3p was dramatically enriched in the pulldown of bio-LINC01419-WT rather than bio-LINC01419 Mut or bio-NC (Fig. 2B), indicating the interaction between LINC01419 with miR-519b-3p. In addition, LINC01419 silence prominently increased miR-519b-3p level in comparison with normal control group (Fig. 2C). Later, RT-qPCR data verified that overexpression miR-519b-3p increased the
Fig. 1. Knockdown of LINC01419 inhibited the development of LUAD (A) The expression of LINC01419 in normal pulmonary epithelial cells (BEAS-2B) and LUAD cell lines (SPC-A1, A549, H446, H460) was measured by RT-qPCR assay. (B) Nuclear-cytoplasmic fractionation was conducted to examine the distribution of LINC01419 nucleolus and cytoplasm. (C) The knockdown efficiency of LINC01419 in SPC-A1 and A549 cells was examined by RT-qPCR assay. (D, E) Proliferation in LUAD cells was monitored by CCK8 experiments and colony formation. (F) Wound healing assay was performed to probe the cell migration. (G) Transwell assay was carried out to detect the cell invasion.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Fig. 2. MiR-519b-3p was a target of LINC01419 (A) StarBase database predicted possible miRNAs binding with LINC01419. (B) RNA pull down assay verified the binding of miR3121-3p, miR-519a/b/c-3p and LINC01419. (C) RT-qPCR data verified the overt knockdown of LINC01419 increased the expression of miR-519b-3p. (D) RT-qPCR assay showed the results of miR-519b-3p overexpression. (E) The predicted binding site between LINC01419 and miR-519b-3p and luciferase reporter assay confirmed that miR-519b-3p could bind with LINC01419. (F) RIP assays were used to confirm miR-519b-3p and LINC01419 coexisted in RNA-induced silencing complexes (RISCs).
expression of miR-519b-3p in SPC-A1 and A549 cells (Fig. 2D). The miR-159b-3p binding sites on LINC01419 were presented in Fig. 2E. The results of luciferase reporter assay revealed that miR-519b-3p mimics allowed the decline of luciferase activity of LINC01419WT reporter while no obvious change could be seen in LINC01419-Mut reporter (Fig. 2E). RIP assays demonstrated that LINC01419 and miR-519b-3p were both enriched in Ago2 group but
not in IgG group in two cells (Fig. 2F). Taken together, miR-519b-3p could bind with LINC01419 and negatively regulate LINC01419 expression. 3.3. RCCD1 was the target of miR-519b-3p Since, miRNAs are widely known to affect development by
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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targeting some specific genes, we further explored downstream targets for miR-519b-3p in LUAD. As presented in Venn diagram, intersection of prediction results RNA22, miRmap and microT databases indicated RCCD1, FAM102A and OCRL as potential targets for miR-519b-3p (Fig. 3A). Subsequently, we found that the level of RCCD1, rather than FAM102A and OCRL, decreased in SPC-A1 and A549 cells with miR-519b-3p upregulation (Fig. 3B), indicating that miR-519b-3p negatively modulated RCCD1 in LUAD. RCCD1 is reported to elicit promoting function in NSCLC. So, it was reasonable to deduce that RCCD1 was a target for miR-159b-3p in LUAD. According to the outcomes of RT-qPCR, RCCD1 expression was much higher in LUAD cell lines than that in normal pulmonary epithelial cells (Fig. 3C). Furthermore, results of RT-qPCR assays and Western blot assays exhibited that knockdown of LINC01419 decreased the expression of RCCD1 in two cells (Fig. 3D). Luciferase reporter assays indicated that luciferase activity of RCCD1-WT, instead of
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RCCD1-Mut, declined responding to miR-519b-3p mimics in both cells (Fig. 3E). RIP assays demonstrated that miR-519b-3p and RCCD1 were both enriched in Ago2 group but not in IgG group in two cells (Fig. 3F). All the results showed that RCCD1 was the downstream target of miR-519b-3p and had inverse relationship with miR-519b-3p. 3.4. LINC01419 accelerated LUAD development by targeting miR519b-3p/RCCD1 axis Finally, we tested whether miR-519b-3p/RCCD1 mediated the regulation of LINC01419 on LUAD proliferation, migration, and invasion via rescue assays. In SPC-A1 cell, we observed through RTqPCR assays that miR-519b-3p inhibitor reduced the expression of miR-519b-3p (Fig. 4A). And pcDNA3.1/RCCD1 could enhance the expression of RCCD1 in SPC-A1 cell (Fig. 4B). Subsequently, in CCK8
Fig. 3. RCCD1 was the target of miR-519b-3p (A) Venn diagram showed that RCCD1, FAM102A and OCRL were downstream targets of miR-519b-3p. (B) RT-qPCR data of RCCD1 downregulation, rather than FAM102A and OCRL, responding to overexpression of miR-519b-3p. (C) The expression of RCCD1 in normal pulmonary epithelial cells and LUAD cell lines was measured by RT-qPCR assay. (D) The mRNA and protein levels of RCCD1 in cells after sh-LINC01419#1 transfection were estimated by RT-qPCR assay and Western blot. (E) The binding site between RCCD1 and miR-519b-3p was predicted and luciferase reporter assay confirmed that miR-519b-3p could bind with RCCD1. (F) RIP assays were used to confirm miR-519b-3p coexisted in RISCs with LINC01419 or RCCD1.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Fig. 4. LINC01419 accelerated LUAD development by targeting miR-519b-3p/RCCD1 axis (A) Efficiency of miR-519b-3p inhibitor was determined by RT-qPCR. (B) The efficiency of RCCD1 overexpression was determined by RT-qPCR. (CeD) CCK-8 assay and colony formation assay were conducted to detect cell proliferation in rescue assay. (E) Migratory capacity of cells was monitored by scratch wound experiment in rescue assay. (F) Transwell assay was applied to evaluate cell invasion in rescue assay.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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assay and colony formation assay, the repressing effect of shLINC01419#1 on cell proliferation was counteracted by miR-519b3p inhibitor or RCCD1 overexpression in SPC-A1 cell (Fig. 4C and D). Moreover, in wound healing assay, RCCD1 overexpression or miR-519b-3p inhibitor recovered cell migration that was hindered by LINC01419 knockdown in SPC-A1 cell (Fig. 4E). Finally, in transwell assay, the cell invasion impeded by LINC01419 knockdown was renewed by RCCD1 overexpression or miR-519b-3p inhibitor in SPC-A1 cell (Fig. 4F). Overall, LINC01419 facilitated LUAD via targeting miR-519b-3p/RCCD1 axis.
Acknowledgement
4. Discussion
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Large quantities of studies have documented that lncRNAs took part in the development of cancers, such as cervical cancer, breast cancer, and gastric cancer [16e18]. LINC01419 has been demonstrated to serve as a carcinogen in multiple cancers, such as esophageal squamous cell carcinoma, hepatocellular carcinoma [11,19]. However, we firstly explored the function and mechanism of LINC01419 in LUAD. We confirmed that LINC01419 expression was high in LUAD cells and demonstrated through loss-of-function assays that LINC01419 silence restrained the proliferative, migratory, and invasive capabilities of LUAD cells. The results indicated that LINC01419 functioned as an oncogene by positively regulating cell proliferation, migration and invasion in LUAD cells. miRNAs are defined as short non-coding RNAs with 20e24 nucleotides, and they are tightly linked to the incidence and development of various diseases [20,21]. Till now, it has been validated that lncRNAs could modulate many diseases via sponging some specific miRNAs [13,22]. Nevertheless, the interaction between LINC01419 and target miRNAs in LUAD was firstly revealed in our study. We identified 4 miRNAs potentially targeted by LINC01419 through bioinformatics tools and confirmed the interaction of miR-519b-3p with LINC01419 by pulldown assay, indicating that miR-519b-3p was targeted by LINC01419 in LUAD. This is also the first time for miR-519b-3p to be explored in LUAD. Current study confirmed that LINC01419 could bind with miR-519b-3p. It has been reported that miRNAs took part in the development of cancers by regulating the expression of their downstream mRNAs [23,24]. Herein, we showed that LINC01419 was negatively regulated by miR-519b-3p in LUAD, firstly indicating that miR-519b-3p was a negative regulator in LUAD. RCCD1 has been validated as a tumor promoter in several cancers [25,26]. We identified RCCD1, FAM102A and OCRL as the common target of miR-519b-3p from RNA22, miRmap and microT, and RCCD was sorted out because only RCCD1 level was reduced by miR-519b-3p among 2 targets in LUAD cells. Besides, knockdown of LINC01419 could reduce the expression of RCCD1. Moreover, we confirmed that LINC01419 regulated the expression of RCCD1 by functioning as a ceRNA via sponging miR-519b-3p. Finally, we found that overexpression of RCCD1 or knockdown of miR-519b-3p could partially counteract the suppressive effect of LINC01419 silence on malignant phenotypes of LUAD cells. That meant LINC01419 promoted the progression of LUAD by targeting at miR519b-3p/RCCD1 axis. In a word, we elucidated that miR-519b-3p could bind with LINC01419 and RCCD1 was the downstream target of miR-519b-3p. Our study proved that LINC01419 silence repressed the development of LUAD via sponging miR-519b-3p to regulate RCCD1, further deepening the understanding of modulating mechanism in LUAD. Conflicts of interest Authors declare no conflicts of interest in this study.
We appreciate all members. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.090. References
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b-3p to up-regulate RCCD1 Ziming Cheng, Shizhen Hou*, Yubing Wu, Xiangdong Wang, Yi Sun, Bing Liu, Maoxi Yuan Department of Thoracic Surgery, Linyi Central Hospital. No.17, Health Road, Linyi, Shandong, 276400, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 September 2019 Accepted 21 September 2019 Available online xxx
Lung adenocarcinoma (LUAD) is one of the most common type of lung cancer notorious for the high incidence and mortality around the world. Long non-coding RNAs (LncRNAs) are defined as a class of RNAs with length more than 200 nucleotides. Mounting studies have proved that lncRNAs are related closely to incidence of diseases and play crucial roles in cancer progression. Although LINC01419 has been studied in several cancers, the function and mechanism of LINC01419 in LUAD remains a mystery. Our findings showed that LINC01419 level was high in LUAD cells, and LINC01419 knockdown inhibited carcinogenesis via suppressing cell proliferation, migration as well as invasion. Moreover, bioinformatics prediction, luciferase reporter experiments and RIP assay were used to confirm miR-519-3p was sequestered and negatively regulated by LINC01419. Subsequently, RCCD1 was identified as a miR-519b3p target and had inverse relationship with miR-519b-3p. LINC01419 was positively related to RCCD1. Furthermore, rescue assays confirmed that miR-519b-3p inhibitor or RCCD1 overexpression could neutralize the effect of LINC01419 silenced in cell proliferation, migration and invasion. Taken together, all the results indicated that LINC01419 exhibited oncogenic behaviors LUAD via binding to miR-519b-3p to enhance the expression of RCCD1, manifesting the underlying therapy values of LINC01419 in LUAD. © 2019 Elsevier Inc. All rights reserved.
Keywords: LINC01419 miR-519b-3p Lung adenocarcinoma RCCD1
1. Introduction Lung cancer possesses high incidence and mortality rate among all the malignant tumors [1]. Lung cancer cases are classified into small cell and non-small cell lung cancer (NSCLC) [2]. Lung adenocarcinoma (LUAD) accounts for a large proportion of NSCLC [3], threatening human health around the world [4]. Despite that much therapeutic progress has been achieved, the prognosis of LUAD is still very poor [5]. Therefore, it is necessary to understand the pathogenetic factors to find out efficient therapeutic methods for LUAD. Emerging studies confirmed that lncRNAs served as an important role in the progression of diseases [6]. LOXL-AS1 was reported to mediate the doxorubicin resistance in prostate cancer by sponging miR-let-7a-5p to elevate EGFR [7]. NEF was considered to have inhibitory effect on cell migration and invasion via decreasing miR-21 in osteosarcoma [8]. Accumulating articles suggested the deep involvement of lncRNAs in LUAD. LncRNA TTN-AS1 promoted
* Corresponding author. E-mail address: [email protected] (S. Hou).
invasion and migration in LUAD cells via targeting miR-4677-3p and upregulating ZEB1 [9]. LncRNA HMMR-AS1 fostered proliferation and metastasis of LUAD by sponging miR-138 to enhance sirt6 [10]. The role of LINC01419 was analyzed in esophageal cell carcinoma [11]. However, never has the link between LINC01419 and LUAD been established. It was said that lncRNAs functioned as ceRNAs to sponge miRNAs in the process of cancers. RSU1P2 accelerated the tumor growth via playing as a ceRNA against let-7a in cervical cancer [12]. SMAD5-AS1 repressed cell growth in diffuse large B cell lymphoma through Wnt/b-catenin signaling by targeting miR-135b-5p/APC axis [13]. PTV1 worked as a ceRNA sequestering miR-199a-5p to regulate HIF1a in NSCLC upon hypoxia [14]. However, the interaction of LINC01419 with miR-159b-3p in LUAD has never been revealed. Our study investigated the role of LINC01419 in LUAD. It was evident that LINC01419 was highly expressed in LUAD cells. Knockdown of LINC01419 led to the decreased cell proliferation, migration and invasion in LUAD cells. LINC01419 could bind with miR-519b-3p and miR-519b-3p was negatively regulated by LINC01419. Moreover, LINC01419 promoted tumorigenesis via regulating the expression of RCCD1 by targeting miR-519b-3p in
https://doi.org/10.1016/j.bbrc.2019.09.090 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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LUAD cells, manifesting a new insight for the LUAD treatment. 2. Materials and methods 2.1. Cell lines and cell culture The Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) offered LUAD cell lines (SPC-A1, A549, H446 and H460) and normal pulmonary epithelial cell (BEAS-2B). Cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone Laboratories, Logan, UT, USA) enriched with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) filled with 100 U/mL penicillin and 100 mg/mL streptomycin in moist atmosphere at 37 C with 5% CO2. 2.2. Cell transfection Sh- LINC01419#1/2 was applied to knock down LINC01419. PcDNA3.1/RCCD1 was used to up-regulate RCCD1. MiR-519b-3p mimics were used to overexpress miR-519b-3p, and miR-519b-3p was down-regulated by miR-519b-3p inhibitor. All plasmids were purchased from Gene Pharmacy (Shanghai, China). Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, USA) was used for transfection. 2.3. RT-qPCR assay TRIpure Reagent (Labgene Biotech, Guangzhou, Guangdong, China) was utilized to extract total RNAs. Taq Man™ Advanced miRNA cDNA Synthesis Kit (Waltham, MA, USA) and the Omni script Reverse Transcription Kit (HaoranBio, Xuhui, Shanghai, China) were respectively used to carry out the reverse transcription. The SYBR Prime Script RT-PCR kit (TaKaRa, Dalian, China) was then exploited to conduct RT-qPCR assay on ABI 7500 System (Applied Biosystems, Carlsbad, California). Both GAPDH and U2 were internal controls. Relative expression levels of RNAs were calculated through the 2 DDCt method. 2.4. RNA pull-down assay LINC01419-WT, LINC01419-Mut and NC group were biotinylated to be Bio-LINC01419-WT, Bio-LINC01419-Mut and Bio-NC by Gene Pharma Company (Shanghai, China). Bio-LINC01419-WT, BioLINC01419-Mut and Bio-NC were transfected into SPC-A1and A549 cells. 48 h later, the lysates of cells were incubated with Dynabeads M 280 Streptavidin (Invitrogen, CA). Following purification, RNA complex was measured by RT-qPCR assay. 2.5. RIP assay RIP assay was carried out by using RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). LUAD cell lysates were cultured with RIP buffer that contained magnetic beads conjugated with Ago2 antibody (Millipore, Billerica, MA, USA) and IgG (Millipore, Billerica, MA, USA) was used as a negative control. Immunoprecipitate RNA was isolated by proteinase K and then the relative expression of RNAs was examined by RT-qPCR assay. 2.6. Nuclear-cytoplasmic fractionation The nuclear and cytoplasmic fractions of SPC-A1 and A549 cells were separated with a PARIS kit (Life Technologies).
2.7. CCK-8 assay SPC-A1 and A549 cells at a density of 1 104 cells/well were plated into 96-well plate. After the incubation at 24, 48 and 72, 96 h, the culture plate was taken out. The cell culture medium was removed and the plate was washed twice with PBS.10 mL CCK-8 solution (Keygen Biotech, Nanjing, China) and serum-free medium (1:10) mixture were added into each well. And the incubation lasted for 4 h. The cell growth curves were draw by measuring 450 nm absorbance using microplate readers (EL340; Bio-Tek Instruments, Hopkinton, MA, USA) at each indicated time point. 2.8. Luciferase reporter assay The LINC01419-WT or LINC01419-Mut reporters were cotransfected, respectively, with miR-519b-3p mimics into cells. LINC01419-WT or LINC01419-Mut underwent co-transfection with NC mimics or miR-519b-3p mimics into cells. Lipofectamine 2000 was applied to conduct transfection. Subsequent to 48 h, luciferase reporter assay system (Promega, Madison WI) was adopted to assess the relative luciferase activities. 2.9. Western blot assay Cells were lysed using RIPA lysis buffer (Beyotime biotech, Shanghai, China) for total protein extraction. Then total protein solution was quantified using BCA kit (Beyotime biotech, Shanghai, China). Protein extractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Sigma-Aldrich, USA). After being blocked with nonfat milk, the membranes were incubated with proper primary antibodies overnight at 4 C.Then they were incubated with secondary antibodies at 37 C for 1 h. GAPDH was served as the internal control in this research. 2.10. Wound healing assay Wound healing assay was employed to estimate cell migration capacity in LUAD cells. Cells were incubated onto 6-well plates and treated in DMEM containing 10% FBS at indoor temperature overnight. When reaching 80% confluence, a pipette tip was utilized to make a straight scratch. The cells were then washed by use of PBS for three times and then incubated in serum-free medium. Images of wounds at the same location were observed at indicated time points. 2.11. Transwell invasion assays As for cell migration and invasion capacities, transwell assays were implemented. The cells were starved with serum-free DMEM medium (HyClone Laboratories) in the upper chamber coated with Matrigel (Corning, NY, USA). 10% FBS DMEM medium (HyClone Laboratories) were filled in the lower chamber. After incubating for 48 h, cells stayed in upper chamber were gently abraded, while cells migrated or invaded into the lower chamber were fixed with methanol and stained by use of 0.1% crystal violet. Images were captured for counting the number of migrated or invaded cells under a microscope (Olympus, Japan). 2.12. Statistical analysis Each assay was performed in triplicate and data was presented as mean ± standard deviation. Student’s t-test was utilized to make comparison between two groups, and one-way analysis of variance (ANOVA) was used to make comparison between various groups.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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All analyses were conducted by using SPSS for Windows, Version 14.0. (SPSS Inc., Chicago). A value of p < 0.05 was regarded statistically significant. 3. Results 3.1. Knockdown of LINC01419 inhibited the development of LUAD To illustrate the function of LINC01419 on LUAD progression, RTqPCR data showed that the expression of LINC01419 was much higher in LUAD cell lines (SPC-A1, A549, H446 and H460) than that in normal pulmonary epithelial cells (BEAS-2B) (Fig. 1A). Moreover, the expression of LINC01419 in SPC-A1 and A549 cells was much higher than that in H446 and H460 cells. Thus, these two cells were chosen in the following experiments. To examine the subcellular distribution of LINC01419 in LUAD, nucleus and cytoplasm were isolated from SPC-A1 and A549 cells. Data from RT-qPCR showed that LINC01419 was mainly amassed in cytoplasm in SPC-A1 and A549 cells (Fig. 1B). In order to learn more about the biological role of LINC01419, we confirmed the knockdown of endogenous LINC01419 by sh-LINC01419#1 and sh-LINC01419#2 in SPC-A1 and A549 cells (Fig. 1C). Then, the cell proliferation ability was
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examined by CCK8 assay and colony formation assay. The results depicted that knockdown of LINC01419 inhibited the cell proliferation in two cells (Fig. 1D and E). Also, LINC01419 silence repressed cell migration in wound healing assay (Fig. 1F). Knockdown of LINC01419 suppressed the cell invasion in transwell assay (Fig. 1G). In conclusion, LINC01419 was highly expressed in LUAD and promoted the progression of LUAD by accelerating cell proliferation, migration and invasion. 3.2. MiR-519b-3p was a target of LINC01419 Emerging evidence has revealed that lncRNAs serve as sponges of miRNAs in tumor progression [15]. So we wondered whether LINC01419 functioned in LUAD in this manner. 4 miRNAs were predicted as potential targets for LINC01419 in Starbase (Fig. 2A). However, among the 4 candidates, only miR-519b-3p was dramatically enriched in the pulldown of bio-LINC01419-WT rather than bio-LINC01419 Mut or bio-NC (Fig. 2B), indicating the interaction between LINC01419 with miR-519b-3p. In addition, LINC01419 silence prominently increased miR-519b-3p level in comparison with normal control group (Fig. 2C). Later, RT-qPCR data verified that overexpression miR-519b-3p increased the
Fig. 1. Knockdown of LINC01419 inhibited the development of LUAD (A) The expression of LINC01419 in normal pulmonary epithelial cells (BEAS-2B) and LUAD cell lines (SPC-A1, A549, H446, H460) was measured by RT-qPCR assay. (B) Nuclear-cytoplasmic fractionation was conducted to examine the distribution of LINC01419 nucleolus and cytoplasm. (C) The knockdown efficiency of LINC01419 in SPC-A1 and A549 cells was examined by RT-qPCR assay. (D, E) Proliferation in LUAD cells was monitored by CCK8 experiments and colony formation. (F) Wound healing assay was performed to probe the cell migration. (G) Transwell assay was carried out to detect the cell invasion.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Fig. 2. MiR-519b-3p was a target of LINC01419 (A) StarBase database predicted possible miRNAs binding with LINC01419. (B) RNA pull down assay verified the binding of miR3121-3p, miR-519a/b/c-3p and LINC01419. (C) RT-qPCR data verified the overt knockdown of LINC01419 increased the expression of miR-519b-3p. (D) RT-qPCR assay showed the results of miR-519b-3p overexpression. (E) The predicted binding site between LINC01419 and miR-519b-3p and luciferase reporter assay confirmed that miR-519b-3p could bind with LINC01419. (F) RIP assays were used to confirm miR-519b-3p and LINC01419 coexisted in RNA-induced silencing complexes (RISCs).
expression of miR-519b-3p in SPC-A1 and A549 cells (Fig. 2D). The miR-159b-3p binding sites on LINC01419 were presented in Fig. 2E. The results of luciferase reporter assay revealed that miR-519b-3p mimics allowed the decline of luciferase activity of LINC01419WT reporter while no obvious change could be seen in LINC01419-Mut reporter (Fig. 2E). RIP assays demonstrated that LINC01419 and miR-519b-3p were both enriched in Ago2 group but
not in IgG group in two cells (Fig. 2F). Taken together, miR-519b-3p could bind with LINC01419 and negatively regulate LINC01419 expression. 3.3. RCCD1 was the target of miR-519b-3p Since, miRNAs are widely known to affect development by
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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targeting some specific genes, we further explored downstream targets for miR-519b-3p in LUAD. As presented in Venn diagram, intersection of prediction results RNA22, miRmap and microT databases indicated RCCD1, FAM102A and OCRL as potential targets for miR-519b-3p (Fig. 3A). Subsequently, we found that the level of RCCD1, rather than FAM102A and OCRL, decreased in SPC-A1 and A549 cells with miR-519b-3p upregulation (Fig. 3B), indicating that miR-519b-3p negatively modulated RCCD1 in LUAD. RCCD1 is reported to elicit promoting function in NSCLC. So, it was reasonable to deduce that RCCD1 was a target for miR-159b-3p in LUAD. According to the outcomes of RT-qPCR, RCCD1 expression was much higher in LUAD cell lines than that in normal pulmonary epithelial cells (Fig. 3C). Furthermore, results of RT-qPCR assays and Western blot assays exhibited that knockdown of LINC01419 decreased the expression of RCCD1 in two cells (Fig. 3D). Luciferase reporter assays indicated that luciferase activity of RCCD1-WT, instead of
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RCCD1-Mut, declined responding to miR-519b-3p mimics in both cells (Fig. 3E). RIP assays demonstrated that miR-519b-3p and RCCD1 were both enriched in Ago2 group but not in IgG group in two cells (Fig. 3F). All the results showed that RCCD1 was the downstream target of miR-519b-3p and had inverse relationship with miR-519b-3p. 3.4. LINC01419 accelerated LUAD development by targeting miR519b-3p/RCCD1 axis Finally, we tested whether miR-519b-3p/RCCD1 mediated the regulation of LINC01419 on LUAD proliferation, migration, and invasion via rescue assays. In SPC-A1 cell, we observed through RTqPCR assays that miR-519b-3p inhibitor reduced the expression of miR-519b-3p (Fig. 4A). And pcDNA3.1/RCCD1 could enhance the expression of RCCD1 in SPC-A1 cell (Fig. 4B). Subsequently, in CCK8
Fig. 3. RCCD1 was the target of miR-519b-3p (A) Venn diagram showed that RCCD1, FAM102A and OCRL were downstream targets of miR-519b-3p. (B) RT-qPCR data of RCCD1 downregulation, rather than FAM102A and OCRL, responding to overexpression of miR-519b-3p. (C) The expression of RCCD1 in normal pulmonary epithelial cells and LUAD cell lines was measured by RT-qPCR assay. (D) The mRNA and protein levels of RCCD1 in cells after sh-LINC01419#1 transfection were estimated by RT-qPCR assay and Western blot. (E) The binding site between RCCD1 and miR-519b-3p was predicted and luciferase reporter assay confirmed that miR-519b-3p could bind with RCCD1. (F) RIP assays were used to confirm miR-519b-3p coexisted in RISCs with LINC01419 or RCCD1.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Fig. 4. LINC01419 accelerated LUAD development by targeting miR-519b-3p/RCCD1 axis (A) Efficiency of miR-519b-3p inhibitor was determined by RT-qPCR. (B) The efficiency of RCCD1 overexpression was determined by RT-qPCR. (CeD) CCK-8 assay and colony formation assay were conducted to detect cell proliferation in rescue assay. (E) Migratory capacity of cells was monitored by scratch wound experiment in rescue assay. (F) Transwell assay was applied to evaluate cell invasion in rescue assay.
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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assay and colony formation assay, the repressing effect of shLINC01419#1 on cell proliferation was counteracted by miR-519b3p inhibitor or RCCD1 overexpression in SPC-A1 cell (Fig. 4C and D). Moreover, in wound healing assay, RCCD1 overexpression or miR-519b-3p inhibitor recovered cell migration that was hindered by LINC01419 knockdown in SPC-A1 cell (Fig. 4E). Finally, in transwell assay, the cell invasion impeded by LINC01419 knockdown was renewed by RCCD1 overexpression or miR-519b-3p inhibitor in SPC-A1 cell (Fig. 4F). Overall, LINC01419 facilitated LUAD via targeting miR-519b-3p/RCCD1 axis.
Acknowledgement
4. Discussion
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Large quantities of studies have documented that lncRNAs took part in the development of cancers, such as cervical cancer, breast cancer, and gastric cancer [16e18]. LINC01419 has been demonstrated to serve as a carcinogen in multiple cancers, such as esophageal squamous cell carcinoma, hepatocellular carcinoma [11,19]. However, we firstly explored the function and mechanism of LINC01419 in LUAD. We confirmed that LINC01419 expression was high in LUAD cells and demonstrated through loss-of-function assays that LINC01419 silence restrained the proliferative, migratory, and invasive capabilities of LUAD cells. The results indicated that LINC01419 functioned as an oncogene by positively regulating cell proliferation, migration and invasion in LUAD cells. miRNAs are defined as short non-coding RNAs with 20e24 nucleotides, and they are tightly linked to the incidence and development of various diseases [20,21]. Till now, it has been validated that lncRNAs could modulate many diseases via sponging some specific miRNAs [13,22]. Nevertheless, the interaction between LINC01419 and target miRNAs in LUAD was firstly revealed in our study. We identified 4 miRNAs potentially targeted by LINC01419 through bioinformatics tools and confirmed the interaction of miR-519b-3p with LINC01419 by pulldown assay, indicating that miR-519b-3p was targeted by LINC01419 in LUAD. This is also the first time for miR-519b-3p to be explored in LUAD. Current study confirmed that LINC01419 could bind with miR-519b-3p. It has been reported that miRNAs took part in the development of cancers by regulating the expression of their downstream mRNAs [23,24]. Herein, we showed that LINC01419 was negatively regulated by miR-519b-3p in LUAD, firstly indicating that miR-519b-3p was a negative regulator in LUAD. RCCD1 has been validated as a tumor promoter in several cancers [25,26]. We identified RCCD1, FAM102A and OCRL as the common target of miR-519b-3p from RNA22, miRmap and microT, and RCCD was sorted out because only RCCD1 level was reduced by miR-519b-3p among 2 targets in LUAD cells. Besides, knockdown of LINC01419 could reduce the expression of RCCD1. Moreover, we confirmed that LINC01419 regulated the expression of RCCD1 by functioning as a ceRNA via sponging miR-519b-3p. Finally, we found that overexpression of RCCD1 or knockdown of miR-519b-3p could partially counteract the suppressive effect of LINC01419 silence on malignant phenotypes of LUAD cells. That meant LINC01419 promoted the progression of LUAD by targeting at miR519b-3p/RCCD1 axis. In a word, we elucidated that miR-519b-3p could bind with LINC01419 and RCCD1 was the downstream target of miR-519b-3p. Our study proved that LINC01419 silence repressed the development of LUAD via sponging miR-519b-3p to regulate RCCD1, further deepening the understanding of modulating mechanism in LUAD. Conflicts of interest Authors declare no conflicts of interest in this study.
We appreciate all members. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.090. References
Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090
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Please cite this article as: Z. Cheng et al., LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b3p to up-regulate RCCD1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.090