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Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma
Accepted Manuscript Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma
Dongli Huang, Yuying Wei, Juxia Zhu, Fengyong Wang PII: DOI: Article Number: Reference:
S0378-1119(19)30653-5 https://doi.org/10.1016/j.gene.2019.143994 143994 GENE 143994
To appear in:
Gene
Received date: Revised date: Accepted date:
5 February 2019 17 July 2019 18 July 2019
Please cite this article as: D. Huang, Y. Wei, J. Zhu, et al., Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma, Gene, https://doi.org/10.1016/ j.gene.2019.143994
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ACCEPTED MANUSCRIPT Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma Dongli Huang1, Yuying Wei2, Juxia Zhu2, Fengyong Wang 3* Department of Hepatobiliary Surgery, Changyi People's Hospital of Shandong Province
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Department of Infectious Disease, Changyi People's Hospital of Shandong Province
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Department of general surgery, Tongde Hospital of Zhejiang Province
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*Correspondence to: Fengyong Wang; email: [email protected]
ACCEPTED MANUSCRIPT Abstract Long non-coding RNA (lncRNA) potentially regulates tumorigenesis. LncRNA small nucleolar RNA host gene 1 (SNHG1) expression remains high in hepatocellular carcinoma cells; however, its biological mechanism in hepatocellular carcinoma remains unknown. In this study, SNHG1 expression in hepatocellular carcinoma cells
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was detected by qRT-PCR. Proliferative and migratory potentials of hepatocellular carcinoma cells were determined by CCK-8 and Transwell assay, respectively. Then,
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the nude mice model of xenograft was employed to verify the effect of SNHG1 on tumor formation in vivo. We identified the potential target of SNHG1 through
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bioinformatics and dual-luciferase reporter gene. Furthermore, Western blot and RIP assay was used for clarifying their interaction and functions in regulating the
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development of hepatocellular carcinoma. Our results indicated a high expression of SNHG1 in hepatocellular carcinoma cells. Downregulation of SNHG1 inhibited
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proliferative and migratory potentials of hepatocellular carcinoma cells in vitro and in vivo. Moreover, the expression of programmed cell death 4 (PDCD4) was positively
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regulated by SNHG1 through competing with miR-195-5p. These results indicated that
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SNHG1 participated in the development of hepatocellular carcinoma as a ceRNA to competitively bind to miR-195-5p and thus mediate PDCD4 expression.
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Keywords: SNHG1; cell proliferation; miRNA; hepatocellular carcinoma
ACCEPTED MANUSCRIPT Introduction Hepatocellular carcinoma is the fifth most common tumor in patients worldwide and the third most common cause of cancer-related death next to lung and stomach cancer(Coskun, 2017). The 5-year survival rate of hepatocellular carcinoma is only about 60%, with the incidence increasing year by year in recent years(Omura, 2014;
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Gharat et al., 2016). Hepatocellular carcinoma related mortality accounted for the highest proportion among all cancers. Moreover, 90% of all hepatocellular carcinomas
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developed from liver cirrhosis(Costentin, 2017). The hepatocellular carcinoma may be diagnosed frequently and exclusively by cross-sectional imaging based on
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characteristic multiphase contrast instead of strict need for tissue collection. In spite of the progress in medical, locoregional and surgical therapies, hepatocellular carcinoma
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remained one of the most common causes of cancer-related death in the world (Hartke et al., 2017). The current studies on hepatocellular carcinoma have been extensive and
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gradually deepened to the level of genes, molecules and proteins. For example, Xiao J et al found that lncRNA HANR promoted tumorigenesis and increased the
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chemoresistance in hepatocellular carcinoma(Xiao et al., 2017). Wang F et al
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discovered that upregulated lncRNA-UCA1 was associated with the progression of hepatocellular carcinoma through inhibiting miR-216b and activating FGFR1/ERK signaling pathway(Chen et al., 2015a). To further explore the pathogenesis of
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hepatocellular carcinoma, we aim to elucidate mechanisms of the occurrence, progression, invasion and metastasis of hepatocellular carcinoma at the epigenetic level.
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Our results are of great significance to improve the diagnostic and therapeutic approaches of hepatocellular carcinoma. Long non-coding RNAs (lncRNAs) are non-coding RNAs with over 200 nucleotides that are capable of regulating gene expressions(Lorenzen and Thum, 2016; Sun et al., 2017a). They have been widely considered in recent years due to their complex biological functions. It is reported that certain lncRNAs exert their crucial role in proliferation, apoptosis, invasion and infiltration of various tumor cells(Min et al., 2016; Chen et al., 2017; Mao et al., 2017; Wang et al., 2017). LncRNA SNHG1 participates in the pathogenesis of many diseases, such as nervous system diseases, cardiovascular
ACCEPTED MANUSCRIPT diseases and various tumors(Cui et al., 2017; Hu et al., 2017; Sun et al., 2017b; Xu et al., 2017; Wang et al., 2018). It has been identified that SNHG1 plays promotive role in colorectal cancer(Xiao et al., 2018) and laryngeal cancer(Zheng et al., 2018). The biological effect of SNHG1 in the pathological process of hepatocellular carcinoma has been pointed out(Zhang et al., 2016b). Zhang H et al reported that expression of
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lncRNA small nucleolar RNA host gene 1 (SNHG1) exacerbated hepatocellular carcinoma by virtue of miR-195 suppression. They concluded that SNHG1 may be a
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potential oncogene in hepatocellular carcinoma(Zhang et al., 2016a). However, the specific effect of SNHG1 in the development of hepatocellular carcinoma remains
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unclear. So, further studies are warranted to explore the specific mechanism of SNHG1 involvement in hepatocellular carcinoma development.
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Our results showed that SNHG1 was highly expressed in hepatocellular carcinoma cell lines. SNHG1 overexpression accelerated proliferative and migratory potentials of
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H-97 and HuH7 cells. To sum up, we verified that SNHG1 participated in the development of hepatocellular carcinoma by competitively binding to miR-195-5p to
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mediate PDCD4 expression, which creates a new perspective for studying the
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pathogenesis of hepatocellular carcinoma.
Materials and Methods
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Cell culture and transfection
Human normal liver cell line HL-7702 and hepatocellular carcinoma cell lines Li-7,
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HuH7, HHCC, H-97, Hep3b and SMMC-7721 were purchased from ATCC (Manassas VA, USA). Cells were cultured in DMEM containing 10% FBS (Beyotime, Nantong, China), 100 μg/ml streptomycin and 100 IU/ml penicillin (Invitrogen, USA), and maintained at 37 °C 5% CO2. SNHG1 overexpression plasmid, SNHG1 siRNA, miR195-5p mimics and miR-195-5p inhibitor were all constructed by GenePharma (Shanghai, China). The transfection experiments were performed using Lipofectamine 2000 Reagent (Invitrogen, CA, USA) following the manufacturers’ instruction. All cells, which were cultured to about 50%–60% view of cell-culture dish, were transfected with in serum-free medium for 24 hours.
ACCEPTED MANUSCRIPT RNA extraction and qRT-PCR LncRNA and mRNA were reversely transcribed to cDNA with the Reverse Transcription Kit (Takara, Tokyo, Japan). For miRNA, cDNA was synthesized using a miRNA First Strand cDNA Synthesis Kit (Sangon Biotech, China). Subsequently, the cDNA was subjected to real-time PCR on a Quantstudio™ DX system (Applied Biosystems, Singapore). LncRNA and mRNA were quantified through normalizing to
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GAPDH using 2−ΔΔCT method. The expression of miRNA was normalized to small
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nuclear U6. The 2-ΔΔCT method was used to calculate relative expression. Each experiment was independently conducted in triplicate. All the PCR primers were listed
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in Table 1. Cell proliferation assays
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Approximately 1.0 × 103 HuH7 and H-97 cells that were transfected were cultured in 96-well plates and incubated with CCK-8 reagent (Beyotime, Nantong, China) for one
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hour. The absorbance at 450 nm was recorded using a TECAN infinite M200 Multimode microplate reader (Tecan, Mechelen, Belgium).
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Cell migration determination
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Migratory potential was measured with Transwell chamber (Millipore Corporation, Billerica, MA). 100 μL suspension containing 5×105 transfected cells was added to the apical chamber, whereas 600 μL medium containing 10% FBS was added to the
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basolateral chamber. After 24 hours of incubation, 10-minute fixation in 4% polymethanol and 20-minute dyeing using 0.1% crystal violet (Beyotime, Nantong,
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China) were performed. Cells were captured with ten fields in each well (magnification 200×). Metastatic cells were calculated by Image-pro Plus 6.0 (Media Cybernetics, USA).
Cell apoptosis assays Two hole of full view HuH7 and H-97 cells in 6-well plates that were transfected were collected. For the cell apoptosis, annexin-V mixed with PI (KeyGEN, Nanjing, China) was used to stain the treated cells. All assays were conducted and analysed with a flow cytometer (FACScan; BD Biosciences, USA) equipped with Cell Quest software (BD Biosciences).
ACCEPTED MANUSCRIPT Subcellular distribution RNA extraction was performed in cytoplasm and nucleus using the PARIS Kit (Life Technologies, USA). Total RNA in each fraction was quantified by qRT-RCR, with GAPDH and U6 utilized as cytoplasm and nucleus internal references, respectively. Dual-luciferase reporter gene assay
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We constructed wild-type plasmids SNHG1-WT and PDCD4-WT, as well as mutanttype plasmids SNHG1-MUT and PDCD4-MUT. H-97 and HuH7 cells seeded into 24-
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well plates were co-transfected with 50 nM miR-195-5p mimics or a negative control and wild-type or mutant-type plasmid using Lipofectamine 2000. 5 ng of pRL-SV40
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was added per 80 ng of plasmid. Dual-luciferase reporter assay kit (Promega, Madison, WI, USA) was used for determining luciferase intensity on a microplate reader.
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RNA immunoprecipitation (RIP)
RIP assay was conducted with Magna Nuclear RIP™ (Native) Nuclear RNA-Binding
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Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Cell lysis was performed in complete RIPA buffer containing protease inhibitor cocktail and an RNase
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inhibitor. Cell extract was subject to incubation with RIP buffer containing magnetic
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beads conjugated to human anti-AGO2 antibody (Millipore) or IgG control. Immunoprecipitated RNA was obtained from protein digestion. Finally, the purified RNA was quantified by qRT-PCR. Anti-SNHG1 used for RIP assay was purchased from
Western blot
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Abcam (Cambridge, MA, USA).
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Cells were lysed in RIPA buffer (CWBIO, China) with protease and phosphatase inhibitors (CWBIO, China). Identical quantities of proteins were electrophoresed by SDS-PAGE, transferred onto PVDF membranes and incubated with primary antibodies specific for PDCD4 (1:1000 dilution, Proteintech, USA), GAPDH (1:1000 dilution, ABclonal, China) at 4 °C overnight, followed by incubation with appropriate HRPconjugated secondary antibodies at room temperature for 1 h. Signals were detected by Immobilon ECL substrate (Millipore, Germany), and the images were acquired using an Optimax X-ray Film Processor (Protec, Germany). Construction of xenograft models
ACCEPTED MANUSCRIPT BALB/c nude mice (male, 4–5-week-old, 18-20g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and randomly divided into two groups (n=3 per group). H97 cells (1×106 per injection) that were transfected with si-SNHG1 or si-NC, respectively, were implanted into the right flank of the mice via subcutaneous injection. Tumor volumes were measured every 7 days after being apparently observed
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and calculated with the following formula: Volume= (length × width2)/2. After 4 weeks, all mice were sacrificed under anesthesia. Tumor tissues were harvested for further
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analysis. The animal experiments were approved by the Animal Care and Use Committee of Changyi People's Hospital of Shandong Province.
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Statistical processing
SPSS 20.0 software and GraphPad Prism 6.0 were utilized for statistical analyses.
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Quantitative data were represented as mean ± SD. Two-tailed Student’s t-test, Wilcoxon rank-sum test, or Mann-Whitney U-test were used to determine statistically
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considered statistically significant.
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significant differences between two groups, as appropriate. P value<0.05 was
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Results
SNHG1 expression and function in hepatocellular carcinoma cell lines qRT-PCR was used for detecting the SNHG1 expression in hepatocellular carcinoma
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cells (Li-7, HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cells HL-7702. As with the results of Zhang H et al, SNHG1 was highly expressed in
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hepatocellular carcinoma cells. In particular, the SNHG1 expression was the highest in H-97 cells and the lowest in HuH7 cells among the selected hepatocellular carcinoma cell lines, which were chosen for the subsequent experiments (Figure 1A). Transfection efficiency of SNHG1 siRNA or overexpression plasmid were shown in Supplementary Figure 1A. CCK-8 assay indicated that SNHG1 downregulation markedly decreased the proliferative ability of hepatocellular carcinoma cells. SNHG1 overexpression accelerated the proliferative rate of hepatocellular carcinoma cells (Figure 1B). In addition, cell migration experiment showed that as opposed to SNHG1 overexpression, SNHG1 downregulation reduced the migratory capacity of hepatocellular carcinoma
ACCEPTED MANUSCRIPT cells (Figure 1C, 1D). No change was observed in apoptotic rate of hepatocellular carcinoma cells influenced by SNHG1 (Figure 1E, 1F). Taken together, these results revealed that SNHG1 may exert regulatory effects on migratory and proliferative potentials of hepatocellular carcinoma cells. Subcellular distribution of SNHG1
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The biological function is determined by subcellular distribution of lncRNA. To confirm the cellular localization of SNHG1, we isolated hepatocellular carcinoma cells
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into cytoplasmic and nuclear fractions, with GAPDH and U6 as controls, respectively. QRT-PCR results showed that 68% and 72% of SNHG1 was distributed in the
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cytoplasmic fraction of H-97 and HuH7 cells, respectively (Figure 2A). It may be
through post-transcriptional regulation. SNHG1 is targeted by miR-195-5p
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concluded that SNHG1 participated in the development of hepatocellular carcinoma
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Given that SNHG1 was primarily located in the cytoplasmic fraction, SNHG1 was assumed to act as a ceRNA in the development of hepatocellular carcinoma. QRT-PCR
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data revealed that as contrary to the expression trend of SNHG1, hepatocellular
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carcinoma cells showed lower miR-195-5p expression (Figure 2B). RegRNA and Starbase prediction showed that sequences in miR-195-5p were highly matched to SNHG1 3'UTR. Based on these binding sequences, pGL3-SNHG1-WT and pGL3-
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SNHG1-MUT were constructed (Figure 2C). Luciferase activity was obviously downregulated in H-97 and HuH7 cells co-transfected with SNHG1 WT and miR-195-
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5p mimics, while it remained unchanged after transfection with SNHG1 MUT (Figure 2D). RIP analysis was carried out to elucidate whether SNHG1 was involved in RNAcontaining ribonucleoprotein complex. QRT-PCR results showed that SNHG1 was enriched in anti-AGO2 antibody than controls. Similar results were yielded in miR195-5p (Figure 2E). This result suggesting that miR-195-5p may bind SNHG1 in vitro. SNHG1 regulates PDCD4, the target gene of miR-195-5p To investigate the potential role of miR-195-5p in the development of hepatocellular carcinoma, the target genes of miR-195-5p were screened out by bioinformatics prediction (TargetScan, Starbase, RegRNA). PDCD4 got high scores on TargetScan
ACCEPTED MANUSCRIPT (http://www.targetscan.org/), Starbase (http://starbase.sysu.edu.cn/) and RegRNA (http://regrna2.mbc.nctu.edu.tw/). So, PDCD4 was selected for further analyses. After construction of luciferase plasmids pGL3-PDCD4-WT and pGL3-PDCD4-MUT, they were co-transfected through miR-195-5p mimics or NC in H-97 and HuH7 cells, respectively (Figure 3A). Luciferase activity of the WT reporter was inhibited, while
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MUT reporter group remained unchanged (Figure 3B). The above results indicated that PDCD4 was a potential target gene of miR-195-5p. Subsequently, PDCD4 expression
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in hepatocellular carcinoma cell line was determined by qRT-PCR. The mRNA level of PDCD4 was remarkably increased in hepatocellular carcinoma cells compared with
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HL-7702 cells (Figure 3C). Western blot analysis revealed the same result at the protein level (Figure 3D).
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To elucidate whether SNHG1 regulated PDCD4 expression via targeting miR-1955p, we detected expression levels of PDCD4 in hepatocellular carcinoma cells after
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altering SNHG1 or miR-195-5p expressions. Transfection efficiency of miR-195-5p mimics or inhibitor were shown in Supplementary Figure 1B. PDCD4 expression was
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upregulated by transfection of miR-195-5p inhibitor in H-97 cells and reversed by co-
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transfection of miR-195-5p inhibitor and SNHG1 siRNA (Figure 4A, 4B). Furthermore, PDCD4 expression was inhibited by transfection of miR-195-5p mimics in HuH7 cells and reversed by co-transfection of miR-195-5p mimics and SNHG1 overexpression
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plasmid (Figure 4C, 4D). Subsequently, HuH7 cells were transfected with SNHG1 overexpression plasmid and its mutant overexpression plasmid, followed by
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determination of PDCD4 expression. As shown by qRT-PCR and Western blot, overexpression of wild-type SNHG1 upregulated PDCD4 expression in hepatocellular carcinoma cells, whereas mutant-type SNHG1 did not disrupt base pairing between SNHG1 and miR-195-5p (Figure 4E, 4F). To sum up, our findings confirmed that SNHG1 positively regulated the expression of PDCD4 by directly binding to miR-1955p. SNHG1/miR-195-5p axis regulates behaviors of hepatocellular carcinoma cells We next explored whether miR-195-5p could affect proliferative and migratory potentials of H-97 and HuH7 cells. Downregulation of miR-195-5p in H-97 cells
ACCEPTED MANUSCRIPT markedly promoted proliferative and migratory potentials compared to controls, which were partially reversed by co-transfection of miR-195-5p inhibitor and SNHG1 siRNA (Figure 5A, 5B). In addition, overexpressed miR-195-5p inhibited proliferative and migratory potentials of HuH7 cells, and were partially reversed by SNHG1 overexpression (Figure 5A, 5C). Overexpression of mutant-type SNHG1 in HuH7 cells
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had no influence on migratory potentials (Figure 5D). Based on the above results, SNHG1/miR-195-5p/PDCD4 axis showed great effects on regulating behaviors of
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hepatocellular carcinoma cells.
SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo
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To explore the role of SNHG1 in hepatocellular carcinoma in vivo, H-97 cells transfected with negative control or SNHG1 siRNA were subcutaneously injected into
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nude mice. Our results showed that down-regulation of SNHG1 decreased the tumor volume (Figure 6A, B) and tumor weight (Figure 6C) after subcutaneous inoculation.
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Furthermore, immunohistochemistry demonstrated that mice treated with SNHG1 siRNA appeared to have lower level of PCNA, the proliferation-specific gene (Figure
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6D). Interestingly, after removing the lung tissue of nude mice, we found that the
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destruction of lung tissue was more pronounced in control group compared to the
Discussion
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SNHG1 siRNA group (Figure 6E).
Since SNHG1 had been identified to participate in cell proliferation and migration,
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SNHG1 was assumed to involve in the pathogenesis of hepatocellular carcinoma(Tian et al., 2018). Our study showed higher expression of SNHG1 in hepatocellular carcinoma cells relative to normal liver cells. In addition, downregulation of SNHG1 expression considerably reduced proliferative and migratory capacities in vitro, suggesting that SNHG1 was an important growth regulator for hepatocellular carcinoma cells as an oncogene. To further verified the function of SNHG1 in hepatocellular carcinoma, tumor xenograft model was used. And, the in vivo experiments showed that SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo. Therefore, explorations on the effect of SNHG1 on accelerating the growth of
ACCEPTED MANUSCRIPT hepatocellular carcinoma cells are of great significance for in-depth studies of the occurrence, development and metastasis of hepatocellular carcinoma. Through separation of cytoplasm and nucleus, we confirmed that SNHG1 was mainly distributed in cell cytoplasm, indicating that SNHG1 may serve as a ceRNA. Subsequently, RIP and dual-luciferase reporter gene assay clarified that SNHG1 bound
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to miR-195-5p. So far, lowly expressed miR-195-5p has been proved in gliomas and thyroid cancer(Zhao et al., 2017; Zhen et al., 2018). Our study demonstrated
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downregulated miR-195-5p in hepatocellular carcinoma cells. Transfection of miR195-5p mimics suppressed migratory and proliferative capacities of hepatocellular
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carcinoma cells, which could be reversed by SNHG1 overexpression. We concluded that both SNHG1 and miR-195-5p may involve in the development and progression of
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hepatocellular carcinoma.
PDCD4 gene encoded a protein localized to the nucleus in proliferating cells(Azzoni
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et al., 1998). The cytokines in natural killer and T cells modulated the expression of this gene. The gene product was considered important in apoptosis; however, its specific
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role remained undetermined(Vikhreva et al., 2017). Chen Z et al declared that
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upregulatedPDCD4 resulted in aromatase inhibitor resistance and a poor prognosis in estrogen receptor-positive breast cancer(Chen et al., 2015b). Wigington CP et al indicated that PDCD4 mRNA was subject to post-transcriptional regulation by the
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RNA-binding proteins human antigen R (HuR) and T-cell intracellular antigen 1 (TIA1)(Wigington et al., 2015). Accordingly, our study confirmed that upregulated
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SNHG1 increased expression of PDCD4, the target gene of miR-195-5p, further leading to abnormal proliferation and migration of hepatocellular carcinoma cells. Nevertheless, Other alternative or additional mechanisms could be also involved in SNHG1 induced enhanced malignant phenotype of hepatocellular carcinoma cells. We would further explore this point in follow up research. To sum up, SNHG1 is a competitive endogenous RNA for the regulation of PDCD4 expression by sponging miR-195-5p, thus regulating the development of hepatocellular carcinoma.
ACCEPTED MANUSCRIPT Abbreviations lncRNA:
Long
non-coding
RNA;
PI:
propidium
iodide;
RIP:
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immunoprecipitation; SNHG1: Small Nucleolar RNA Host Gene 1; PDCD4: programmed cell death 4; HuR: human antigen R; TIA1: T-cell intracellular antigen 1
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Acknowledgements
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We are grateful to Dr. Xing Gao and Dr. Jian Gao for modifying the article.
Funding
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Availability of data and materials
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None.
Data sharing not applicable to this article as no datasets were generated or analysed
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during the current study.
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Authors’ contributions
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All authors have participated in the study and manuscript preparation. HDL and WYQ performed all experiments, HDL and ZJX performed the statistical analysis and drafted the manuscript. WFY designed the work and revised the manuscript. All authors have
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approved the final article.
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Ethics approval and consent to participate The animal experiments were approved by the Animal Care and Use Committee of Changyi People's Hospital of Shandong Province.
Consent for publication Not applicable.
Competing interests The authors declare that they have no competing interests.
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ACCEPTED MANUSCRIPT Table 1: Sequences of primers for qRT-PCR
PDCD4 GAPDH U6
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miR-195-5p
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5’-TTATTGGGCTCCTGTCTGCA-3’ 5’-GCCCTGACATTTGTTGCGTA-3’ 5’-GCAAAAAGGCGACTAAGGAAAAA-3’ 5’-TAAGGGCGTCACTCCCACT-3’ 5’-GCACCGTCAAGGCTGAGAAC-3’ 5’-GGATCTCGCTCCTGGAAGATG-3’ 5’-CTCGCTTCGGCAGCACA-3’ 5’-AACGCTTCACGAATTTGCGT-3’ 5’-ACACTCCAGCTGGGCTAGCAGCACAGAAAT-3’ 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAATAT-3’
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Name lncRNA-SNHG1
ACCEPTED MANUSCRIPT Figures Figure 1. Regulatory effect of SNHG1 on proliferation, migration and apoptosis of
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hepatocellular carcinoma cells.
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(A) SNHG1 expression in hepatocellular carcinoma cell lines (Li-7, HuH7, HHCC, H97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702 detected by qRT-
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PCR. (B) CCK-8 assay showed proliferation of H-97 transfected with SNHG1 siRNA and HuH7 cells transfected with SNHG1 overexpression vector. (C, D) Transwell assay showed migration of H-97 transfected with SNHG1 siRNA and HuH7 cells transfected with SNHG1 overexpression vector. Images were captured under a light microscope with the magnification of ×200. (E, F) Cell apoptosis was detected by BD Biosciences FACS Calibur Flow Cytometry. Data were presented as mean ± s.d. *P<0.05, ns, no significantly difference.
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Figure 2. SNHG1 directly interacts with miR-195-5p.
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(A) Cytoplasmic and nuclear levels of SNHG1 in H-97 and HuH7 cells analyzed by qRT-PCR. (B) miR-195-5p expression in hepatocellular carcinoma cell lines (Li-7,
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HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702
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detected by qRT-PCR. (C) A schematic representation of the miR-195-5p binding site sequence with SNHG1. (D) Dual-luciferase reporter gene assay in H-97 and HuH7 cells after transfection with negative control or miR-195-5p mimics, renilla luciferase vector
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pRL-SV40 and the reporter constructs. (E) RIP experiments for the amount of SNHG1
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and miR-195-5p in H-97 and HuH7 cells. Data were presented as mean ± s.d. *P<0.05.
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Figure 3. PDCD4 is the direct target of miR-195-5p.
(A) The putative miRNA binding sites in the PDCD4 sequence. (B) Dual-luciferase
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reporter gene assay was performed to confirm the direct target sites. (C) PDCD4
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expression in hepatocellular carcinoma cell lines (Li-7, HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702 detected by qRT-PCR. (D)
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Protein level of PDCD4 in HL-7702, H-97 and HuH7 cell lines were detected by
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Western Blot. Data were presented as mean ± s.d. *P<0.05.
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Figure 4. SNHG1/miR-195-5p axis is critical for the expression of PDCD4.
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(A) miR-195-5p inhibitor with or without SNHG1 siRNA was transfected into H-97 cells and the mRNA level of PDCD4 was evaluated by qRT-PCR. (B) Western blot
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analysis of PDCD4 protein level following treatment of H-97 cells with miR-195-5p inhibitor or SNHG1 siRNA. GAPDH was used as control. (C) HuH7 cells were transfected with miR-195-5p with or without SNHG1 overexpress plasmid and qRTPCR was used to detect the relative mRNA levels of PDCD4 compared with control. (D) Relative protein level of PDCD4 when transfected with miR-195-5p mimics and reversed by SNHG1 expression plasmid. (E) Relative mRNA level of PDCD4 when transfected with SNHG1-WT overexpression plasmid or SNHG1-MUT overexpression plasmid. (F) Relative protein level of PDCD4 when transfected with SNHG1-WT overexpression plasmid or SNHG1-MUT overexpression plasmid. Data were presented
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as mean ± s.d. *P<0.05, ns, no significantly difference.
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Figure 5. SNHG1 regulates cell function through miR-195-5p.
(A) CCK-8 assay was performed to determine the proliferation of H-97 and HuH7 cells.
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(B, C) The migration ability with respect to changes of H-97 and HuH7 cell lines after different transfection. (D) Transwell assay was performed to determine the migration
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of HuH7 cells treated with SNHG1-MUT overexpression plasmid. Data were presented
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as mean ± s.d. *P<0.05, ns, no significantly difference.
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Figure 6. SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo.
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(A) Representative images of xenografts tumor in nude mice. (B) Tumor volume was monitored. (C) Tumor weight was monitored. (D) Representative images of IHC
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stained PCNA is shown. (E) Representative images of HE stained Lung tissues are
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shown.
ACCEPTED MANUSCRIPT Highlights
Our results indicated a high expression of SNHG1 in HCC cells.
SNHG1 promotes cell proliferation and migration in vitro and in vivo.
SNHG1 acts as a ceRNA to promote HCC progression by sponging miR-
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195-5p.
Dongli Huang, Yuying Wei, Juxia Zhu, Fengyong Wang PII: DOI: Article Number: Reference:
S0378-1119(19)30653-5 https://doi.org/10.1016/j.gene.2019.143994 143994 GENE 143994
To appear in:
Gene
Received date: Revised date: Accepted date:
5 February 2019 17 July 2019 18 July 2019
Please cite this article as: D. Huang, Y. Wei, J. Zhu, et al., Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma, Gene, https://doi.org/10.1016/ j.gene.2019.143994
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma Dongli Huang1, Yuying Wei2, Juxia Zhu2, Fengyong Wang 3* Department of Hepatobiliary Surgery, Changyi People's Hospital of Shandong Province
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Department of Infectious Disease, Changyi People's Hospital of Shandong Province
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Department of general surgery, Tongde Hospital of Zhejiang Province
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*Correspondence to: Fengyong Wang; email: [email protected]
ACCEPTED MANUSCRIPT Abstract Long non-coding RNA (lncRNA) potentially regulates tumorigenesis. LncRNA small nucleolar RNA host gene 1 (SNHG1) expression remains high in hepatocellular carcinoma cells; however, its biological mechanism in hepatocellular carcinoma remains unknown. In this study, SNHG1 expression in hepatocellular carcinoma cells
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was detected by qRT-PCR. Proliferative and migratory potentials of hepatocellular carcinoma cells were determined by CCK-8 and Transwell assay, respectively. Then,
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the nude mice model of xenograft was employed to verify the effect of SNHG1 on tumor formation in vivo. We identified the potential target of SNHG1 through
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bioinformatics and dual-luciferase reporter gene. Furthermore, Western blot and RIP assay was used for clarifying their interaction and functions in regulating the
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development of hepatocellular carcinoma. Our results indicated a high expression of SNHG1 in hepatocellular carcinoma cells. Downregulation of SNHG1 inhibited
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proliferative and migratory potentials of hepatocellular carcinoma cells in vitro and in vivo. Moreover, the expression of programmed cell death 4 (PDCD4) was positively
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regulated by SNHG1 through competing with miR-195-5p. These results indicated that
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SNHG1 participated in the development of hepatocellular carcinoma as a ceRNA to competitively bind to miR-195-5p and thus mediate PDCD4 expression.
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Keywords: SNHG1; cell proliferation; miRNA; hepatocellular carcinoma
ACCEPTED MANUSCRIPT Introduction Hepatocellular carcinoma is the fifth most common tumor in patients worldwide and the third most common cause of cancer-related death next to lung and stomach cancer(Coskun, 2017). The 5-year survival rate of hepatocellular carcinoma is only about 60%, with the incidence increasing year by year in recent years(Omura, 2014;
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Gharat et al., 2016). Hepatocellular carcinoma related mortality accounted for the highest proportion among all cancers. Moreover, 90% of all hepatocellular carcinomas
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developed from liver cirrhosis(Costentin, 2017). The hepatocellular carcinoma may be diagnosed frequently and exclusively by cross-sectional imaging based on
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characteristic multiphase contrast instead of strict need for tissue collection. In spite of the progress in medical, locoregional and surgical therapies, hepatocellular carcinoma
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remained one of the most common causes of cancer-related death in the world (Hartke et al., 2017). The current studies on hepatocellular carcinoma have been extensive and
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gradually deepened to the level of genes, molecules and proteins. For example, Xiao J et al found that lncRNA HANR promoted tumorigenesis and increased the
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chemoresistance in hepatocellular carcinoma(Xiao et al., 2017). Wang F et al
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discovered that upregulated lncRNA-UCA1 was associated with the progression of hepatocellular carcinoma through inhibiting miR-216b and activating FGFR1/ERK signaling pathway(Chen et al., 2015a). To further explore the pathogenesis of
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hepatocellular carcinoma, we aim to elucidate mechanisms of the occurrence, progression, invasion and metastasis of hepatocellular carcinoma at the epigenetic level.
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Our results are of great significance to improve the diagnostic and therapeutic approaches of hepatocellular carcinoma. Long non-coding RNAs (lncRNAs) are non-coding RNAs with over 200 nucleotides that are capable of regulating gene expressions(Lorenzen and Thum, 2016; Sun et al., 2017a). They have been widely considered in recent years due to their complex biological functions. It is reported that certain lncRNAs exert their crucial role in proliferation, apoptosis, invasion and infiltration of various tumor cells(Min et al., 2016; Chen et al., 2017; Mao et al., 2017; Wang et al., 2017). LncRNA SNHG1 participates in the pathogenesis of many diseases, such as nervous system diseases, cardiovascular
ACCEPTED MANUSCRIPT diseases and various tumors(Cui et al., 2017; Hu et al., 2017; Sun et al., 2017b; Xu et al., 2017; Wang et al., 2018). It has been identified that SNHG1 plays promotive role in colorectal cancer(Xiao et al., 2018) and laryngeal cancer(Zheng et al., 2018). The biological effect of SNHG1 in the pathological process of hepatocellular carcinoma has been pointed out(Zhang et al., 2016b). Zhang H et al reported that expression of
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lncRNA small nucleolar RNA host gene 1 (SNHG1) exacerbated hepatocellular carcinoma by virtue of miR-195 suppression. They concluded that SNHG1 may be a
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potential oncogene in hepatocellular carcinoma(Zhang et al., 2016a). However, the specific effect of SNHG1 in the development of hepatocellular carcinoma remains
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unclear. So, further studies are warranted to explore the specific mechanism of SNHG1 involvement in hepatocellular carcinoma development.
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Our results showed that SNHG1 was highly expressed in hepatocellular carcinoma cell lines. SNHG1 overexpression accelerated proliferative and migratory potentials of
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H-97 and HuH7 cells. To sum up, we verified that SNHG1 participated in the development of hepatocellular carcinoma by competitively binding to miR-195-5p to
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mediate PDCD4 expression, which creates a new perspective for studying the
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pathogenesis of hepatocellular carcinoma.
Materials and Methods
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Cell culture and transfection
Human normal liver cell line HL-7702 and hepatocellular carcinoma cell lines Li-7,
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HuH7, HHCC, H-97, Hep3b and SMMC-7721 were purchased from ATCC (Manassas VA, USA). Cells were cultured in DMEM containing 10% FBS (Beyotime, Nantong, China), 100 μg/ml streptomycin and 100 IU/ml penicillin (Invitrogen, USA), and maintained at 37 °C 5% CO2. SNHG1 overexpression plasmid, SNHG1 siRNA, miR195-5p mimics and miR-195-5p inhibitor were all constructed by GenePharma (Shanghai, China). The transfection experiments were performed using Lipofectamine 2000 Reagent (Invitrogen, CA, USA) following the manufacturers’ instruction. All cells, which were cultured to about 50%–60% view of cell-culture dish, were transfected with in serum-free medium for 24 hours.
ACCEPTED MANUSCRIPT RNA extraction and qRT-PCR LncRNA and mRNA were reversely transcribed to cDNA with the Reverse Transcription Kit (Takara, Tokyo, Japan). For miRNA, cDNA was synthesized using a miRNA First Strand cDNA Synthesis Kit (Sangon Biotech, China). Subsequently, the cDNA was subjected to real-time PCR on a Quantstudio™ DX system (Applied Biosystems, Singapore). LncRNA and mRNA were quantified through normalizing to
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GAPDH using 2−ΔΔCT method. The expression of miRNA was normalized to small
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nuclear U6. The 2-ΔΔCT method was used to calculate relative expression. Each experiment was independently conducted in triplicate. All the PCR primers were listed
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in Table 1. Cell proliferation assays
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Approximately 1.0 × 103 HuH7 and H-97 cells that were transfected were cultured in 96-well plates and incubated with CCK-8 reagent (Beyotime, Nantong, China) for one
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hour. The absorbance at 450 nm was recorded using a TECAN infinite M200 Multimode microplate reader (Tecan, Mechelen, Belgium).
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Cell migration determination
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Migratory potential was measured with Transwell chamber (Millipore Corporation, Billerica, MA). 100 μL suspension containing 5×105 transfected cells was added to the apical chamber, whereas 600 μL medium containing 10% FBS was added to the
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basolateral chamber. After 24 hours of incubation, 10-minute fixation in 4% polymethanol and 20-minute dyeing using 0.1% crystal violet (Beyotime, Nantong,
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China) were performed. Cells were captured with ten fields in each well (magnification 200×). Metastatic cells were calculated by Image-pro Plus 6.0 (Media Cybernetics, USA).
Cell apoptosis assays Two hole of full view HuH7 and H-97 cells in 6-well plates that were transfected were collected. For the cell apoptosis, annexin-V mixed with PI (KeyGEN, Nanjing, China) was used to stain the treated cells. All assays were conducted and analysed with a flow cytometer (FACScan; BD Biosciences, USA) equipped with Cell Quest software (BD Biosciences).
ACCEPTED MANUSCRIPT Subcellular distribution RNA extraction was performed in cytoplasm and nucleus using the PARIS Kit (Life Technologies, USA). Total RNA in each fraction was quantified by qRT-RCR, with GAPDH and U6 utilized as cytoplasm and nucleus internal references, respectively. Dual-luciferase reporter gene assay
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We constructed wild-type plasmids SNHG1-WT and PDCD4-WT, as well as mutanttype plasmids SNHG1-MUT and PDCD4-MUT. H-97 and HuH7 cells seeded into 24-
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well plates were co-transfected with 50 nM miR-195-5p mimics or a negative control and wild-type or mutant-type plasmid using Lipofectamine 2000. 5 ng of pRL-SV40
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was added per 80 ng of plasmid. Dual-luciferase reporter assay kit (Promega, Madison, WI, USA) was used for determining luciferase intensity on a microplate reader.
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RNA immunoprecipitation (RIP)
RIP assay was conducted with Magna Nuclear RIP™ (Native) Nuclear RNA-Binding
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Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Cell lysis was performed in complete RIPA buffer containing protease inhibitor cocktail and an RNase
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inhibitor. Cell extract was subject to incubation with RIP buffer containing magnetic
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beads conjugated to human anti-AGO2 antibody (Millipore) or IgG control. Immunoprecipitated RNA was obtained from protein digestion. Finally, the purified RNA was quantified by qRT-PCR. Anti-SNHG1 used for RIP assay was purchased from
Western blot
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Abcam (Cambridge, MA, USA).
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Cells were lysed in RIPA buffer (CWBIO, China) with protease and phosphatase inhibitors (CWBIO, China). Identical quantities of proteins were electrophoresed by SDS-PAGE, transferred onto PVDF membranes and incubated with primary antibodies specific for PDCD4 (1:1000 dilution, Proteintech, USA), GAPDH (1:1000 dilution, ABclonal, China) at 4 °C overnight, followed by incubation with appropriate HRPconjugated secondary antibodies at room temperature for 1 h. Signals were detected by Immobilon ECL substrate (Millipore, Germany), and the images were acquired using an Optimax X-ray Film Processor (Protec, Germany). Construction of xenograft models
ACCEPTED MANUSCRIPT BALB/c nude mice (male, 4–5-week-old, 18-20g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and randomly divided into two groups (n=3 per group). H97 cells (1×106 per injection) that were transfected with si-SNHG1 or si-NC, respectively, were implanted into the right flank of the mice via subcutaneous injection. Tumor volumes were measured every 7 days after being apparently observed
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and calculated with the following formula: Volume= (length × width2)/2. After 4 weeks, all mice were sacrificed under anesthesia. Tumor tissues were harvested for further
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analysis. The animal experiments were approved by the Animal Care and Use Committee of Changyi People's Hospital of Shandong Province.
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Statistical processing
SPSS 20.0 software and GraphPad Prism 6.0 were utilized for statistical analyses.
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Quantitative data were represented as mean ± SD. Two-tailed Student’s t-test, Wilcoxon rank-sum test, or Mann-Whitney U-test were used to determine statistically
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considered statistically significant.
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significant differences between two groups, as appropriate. P value<0.05 was
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Results
SNHG1 expression and function in hepatocellular carcinoma cell lines qRT-PCR was used for detecting the SNHG1 expression in hepatocellular carcinoma
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cells (Li-7, HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cells HL-7702. As with the results of Zhang H et al, SNHG1 was highly expressed in
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hepatocellular carcinoma cells. In particular, the SNHG1 expression was the highest in H-97 cells and the lowest in HuH7 cells among the selected hepatocellular carcinoma cell lines, which were chosen for the subsequent experiments (Figure 1A). Transfection efficiency of SNHG1 siRNA or overexpression plasmid were shown in Supplementary Figure 1A. CCK-8 assay indicated that SNHG1 downregulation markedly decreased the proliferative ability of hepatocellular carcinoma cells. SNHG1 overexpression accelerated the proliferative rate of hepatocellular carcinoma cells (Figure 1B). In addition, cell migration experiment showed that as opposed to SNHG1 overexpression, SNHG1 downregulation reduced the migratory capacity of hepatocellular carcinoma
ACCEPTED MANUSCRIPT cells (Figure 1C, 1D). No change was observed in apoptotic rate of hepatocellular carcinoma cells influenced by SNHG1 (Figure 1E, 1F). Taken together, these results revealed that SNHG1 may exert regulatory effects on migratory and proliferative potentials of hepatocellular carcinoma cells. Subcellular distribution of SNHG1
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The biological function is determined by subcellular distribution of lncRNA. To confirm the cellular localization of SNHG1, we isolated hepatocellular carcinoma cells
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into cytoplasmic and nuclear fractions, with GAPDH and U6 as controls, respectively. QRT-PCR results showed that 68% and 72% of SNHG1 was distributed in the
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cytoplasmic fraction of H-97 and HuH7 cells, respectively (Figure 2A). It may be
through post-transcriptional regulation. SNHG1 is targeted by miR-195-5p
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concluded that SNHG1 participated in the development of hepatocellular carcinoma
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Given that SNHG1 was primarily located in the cytoplasmic fraction, SNHG1 was assumed to act as a ceRNA in the development of hepatocellular carcinoma. QRT-PCR
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data revealed that as contrary to the expression trend of SNHG1, hepatocellular
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carcinoma cells showed lower miR-195-5p expression (Figure 2B). RegRNA and Starbase prediction showed that sequences in miR-195-5p were highly matched to SNHG1 3'UTR. Based on these binding sequences, pGL3-SNHG1-WT and pGL3-
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SNHG1-MUT were constructed (Figure 2C). Luciferase activity was obviously downregulated in H-97 and HuH7 cells co-transfected with SNHG1 WT and miR-195-
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5p mimics, while it remained unchanged after transfection with SNHG1 MUT (Figure 2D). RIP analysis was carried out to elucidate whether SNHG1 was involved in RNAcontaining ribonucleoprotein complex. QRT-PCR results showed that SNHG1 was enriched in anti-AGO2 antibody than controls. Similar results were yielded in miR195-5p (Figure 2E). This result suggesting that miR-195-5p may bind SNHG1 in vitro. SNHG1 regulates PDCD4, the target gene of miR-195-5p To investigate the potential role of miR-195-5p in the development of hepatocellular carcinoma, the target genes of miR-195-5p were screened out by bioinformatics prediction (TargetScan, Starbase, RegRNA). PDCD4 got high scores on TargetScan
ACCEPTED MANUSCRIPT (http://www.targetscan.org/), Starbase (http://starbase.sysu.edu.cn/) and RegRNA (http://regrna2.mbc.nctu.edu.tw/). So, PDCD4 was selected for further analyses. After construction of luciferase plasmids pGL3-PDCD4-WT and pGL3-PDCD4-MUT, they were co-transfected through miR-195-5p mimics or NC in H-97 and HuH7 cells, respectively (Figure 3A). Luciferase activity of the WT reporter was inhibited, while
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MUT reporter group remained unchanged (Figure 3B). The above results indicated that PDCD4 was a potential target gene of miR-195-5p. Subsequently, PDCD4 expression
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in hepatocellular carcinoma cell line was determined by qRT-PCR. The mRNA level of PDCD4 was remarkably increased in hepatocellular carcinoma cells compared with
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HL-7702 cells (Figure 3C). Western blot analysis revealed the same result at the protein level (Figure 3D).
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To elucidate whether SNHG1 regulated PDCD4 expression via targeting miR-1955p, we detected expression levels of PDCD4 in hepatocellular carcinoma cells after
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altering SNHG1 or miR-195-5p expressions. Transfection efficiency of miR-195-5p mimics or inhibitor were shown in Supplementary Figure 1B. PDCD4 expression was
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upregulated by transfection of miR-195-5p inhibitor in H-97 cells and reversed by co-
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transfection of miR-195-5p inhibitor and SNHG1 siRNA (Figure 4A, 4B). Furthermore, PDCD4 expression was inhibited by transfection of miR-195-5p mimics in HuH7 cells and reversed by co-transfection of miR-195-5p mimics and SNHG1 overexpression
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plasmid (Figure 4C, 4D). Subsequently, HuH7 cells were transfected with SNHG1 overexpression plasmid and its mutant overexpression plasmid, followed by
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determination of PDCD4 expression. As shown by qRT-PCR and Western blot, overexpression of wild-type SNHG1 upregulated PDCD4 expression in hepatocellular carcinoma cells, whereas mutant-type SNHG1 did not disrupt base pairing between SNHG1 and miR-195-5p (Figure 4E, 4F). To sum up, our findings confirmed that SNHG1 positively regulated the expression of PDCD4 by directly binding to miR-1955p. SNHG1/miR-195-5p axis regulates behaviors of hepatocellular carcinoma cells We next explored whether miR-195-5p could affect proliferative and migratory potentials of H-97 and HuH7 cells. Downregulation of miR-195-5p in H-97 cells
ACCEPTED MANUSCRIPT markedly promoted proliferative and migratory potentials compared to controls, which were partially reversed by co-transfection of miR-195-5p inhibitor and SNHG1 siRNA (Figure 5A, 5B). In addition, overexpressed miR-195-5p inhibited proliferative and migratory potentials of HuH7 cells, and were partially reversed by SNHG1 overexpression (Figure 5A, 5C). Overexpression of mutant-type SNHG1 in HuH7 cells
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had no influence on migratory potentials (Figure 5D). Based on the above results, SNHG1/miR-195-5p/PDCD4 axis showed great effects on regulating behaviors of
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hepatocellular carcinoma cells.
SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo
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To explore the role of SNHG1 in hepatocellular carcinoma in vivo, H-97 cells transfected with negative control or SNHG1 siRNA were subcutaneously injected into
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nude mice. Our results showed that down-regulation of SNHG1 decreased the tumor volume (Figure 6A, B) and tumor weight (Figure 6C) after subcutaneous inoculation.
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Furthermore, immunohistochemistry demonstrated that mice treated with SNHG1 siRNA appeared to have lower level of PCNA, the proliferation-specific gene (Figure
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6D). Interestingly, after removing the lung tissue of nude mice, we found that the
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destruction of lung tissue was more pronounced in control group compared to the
Discussion
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SNHG1 siRNA group (Figure 6E).
Since SNHG1 had been identified to participate in cell proliferation and migration,
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SNHG1 was assumed to involve in the pathogenesis of hepatocellular carcinoma(Tian et al., 2018). Our study showed higher expression of SNHG1 in hepatocellular carcinoma cells relative to normal liver cells. In addition, downregulation of SNHG1 expression considerably reduced proliferative and migratory capacities in vitro, suggesting that SNHG1 was an important growth regulator for hepatocellular carcinoma cells as an oncogene. To further verified the function of SNHG1 in hepatocellular carcinoma, tumor xenograft model was used. And, the in vivo experiments showed that SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo. Therefore, explorations on the effect of SNHG1 on accelerating the growth of
ACCEPTED MANUSCRIPT hepatocellular carcinoma cells are of great significance for in-depth studies of the occurrence, development and metastasis of hepatocellular carcinoma. Through separation of cytoplasm and nucleus, we confirmed that SNHG1 was mainly distributed in cell cytoplasm, indicating that SNHG1 may serve as a ceRNA. Subsequently, RIP and dual-luciferase reporter gene assay clarified that SNHG1 bound
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to miR-195-5p. So far, lowly expressed miR-195-5p has been proved in gliomas and thyroid cancer(Zhao et al., 2017; Zhen et al., 2018). Our study demonstrated
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downregulated miR-195-5p in hepatocellular carcinoma cells. Transfection of miR195-5p mimics suppressed migratory and proliferative capacities of hepatocellular
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carcinoma cells, which could be reversed by SNHG1 overexpression. We concluded that both SNHG1 and miR-195-5p may involve in the development and progression of
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hepatocellular carcinoma.
PDCD4 gene encoded a protein localized to the nucleus in proliferating cells(Azzoni
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et al., 1998). The cytokines in natural killer and T cells modulated the expression of this gene. The gene product was considered important in apoptosis; however, its specific
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role remained undetermined(Vikhreva et al., 2017). Chen Z et al declared that
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upregulatedPDCD4 resulted in aromatase inhibitor resistance and a poor prognosis in estrogen receptor-positive breast cancer(Chen et al., 2015b). Wigington CP et al indicated that PDCD4 mRNA was subject to post-transcriptional regulation by the
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RNA-binding proteins human antigen R (HuR) and T-cell intracellular antigen 1 (TIA1)(Wigington et al., 2015). Accordingly, our study confirmed that upregulated
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SNHG1 increased expression of PDCD4, the target gene of miR-195-5p, further leading to abnormal proliferation and migration of hepatocellular carcinoma cells. Nevertheless, Other alternative or additional mechanisms could be also involved in SNHG1 induced enhanced malignant phenotype of hepatocellular carcinoma cells. We would further explore this point in follow up research. To sum up, SNHG1 is a competitive endogenous RNA for the regulation of PDCD4 expression by sponging miR-195-5p, thus regulating the development of hepatocellular carcinoma.
ACCEPTED MANUSCRIPT Abbreviations lncRNA:
Long
non-coding
RNA;
PI:
propidium
iodide;
RIP:
RNA
immunoprecipitation; SNHG1: Small Nucleolar RNA Host Gene 1; PDCD4: programmed cell death 4; HuR: human antigen R; TIA1: T-cell intracellular antigen 1
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Acknowledgements
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We are grateful to Dr. Xing Gao and Dr. Jian Gao for modifying the article.
Funding
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Availability of data and materials
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None.
Data sharing not applicable to this article as no datasets were generated or analysed
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during the current study.
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Authors’ contributions
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All authors have participated in the study and manuscript preparation. HDL and WYQ performed all experiments, HDL and ZJX performed the statistical analysis and drafted the manuscript. WFY designed the work and revised the manuscript. All authors have
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approved the final article.
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Ethics approval and consent to participate The animal experiments were approved by the Animal Care and Use Committee of Changyi People's Hospital of Shandong Province.
Consent for publication Not applicable.
Competing interests The authors declare that they have no competing interests.
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ACCEPTED MANUSCRIPT Table 1: Sequences of primers for qRT-PCR
PDCD4 GAPDH U6
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miR-195-5p
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5’-TTATTGGGCTCCTGTCTGCA-3’ 5’-GCCCTGACATTTGTTGCGTA-3’ 5’-GCAAAAAGGCGACTAAGGAAAAA-3’ 5’-TAAGGGCGTCACTCCCACT-3’ 5’-GCACCGTCAAGGCTGAGAAC-3’ 5’-GGATCTCGCTCCTGGAAGATG-3’ 5’-CTCGCTTCGGCAGCACA-3’ 5’-AACGCTTCACGAATTTGCGT-3’ 5’-ACACTCCAGCTGGGCTAGCAGCACAGAAAT-3’ 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAATAT-3’
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Name lncRNA-SNHG1
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hepatocellular carcinoma cells.
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(A) SNHG1 expression in hepatocellular carcinoma cell lines (Li-7, HuH7, HHCC, H97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702 detected by qRT-
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PCR. (B) CCK-8 assay showed proliferation of H-97 transfected with SNHG1 siRNA and HuH7 cells transfected with SNHG1 overexpression vector. (C, D) Transwell assay showed migration of H-97 transfected with SNHG1 siRNA and HuH7 cells transfected with SNHG1 overexpression vector. Images were captured under a light microscope with the magnification of ×200. (E, F) Cell apoptosis was detected by BD Biosciences FACS Calibur Flow Cytometry. Data were presented as mean ± s.d. *P<0.05, ns, no significantly difference.
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Figure 2. SNHG1 directly interacts with miR-195-5p.
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(A) Cytoplasmic and nuclear levels of SNHG1 in H-97 and HuH7 cells analyzed by qRT-PCR. (B) miR-195-5p expression in hepatocellular carcinoma cell lines (Li-7,
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HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702
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detected by qRT-PCR. (C) A schematic representation of the miR-195-5p binding site sequence with SNHG1. (D) Dual-luciferase reporter gene assay in H-97 and HuH7 cells after transfection with negative control or miR-195-5p mimics, renilla luciferase vector
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pRL-SV40 and the reporter constructs. (E) RIP experiments for the amount of SNHG1
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and miR-195-5p in H-97 and HuH7 cells. Data were presented as mean ± s.d. *P<0.05.
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Figure 3. PDCD4 is the direct target of miR-195-5p.
(A) The putative miRNA binding sites in the PDCD4 sequence. (B) Dual-luciferase
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reporter gene assay was performed to confirm the direct target sites. (C) PDCD4
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expression in hepatocellular carcinoma cell lines (Li-7, HuH7, HHCC, H-97, Hep3b, SMMC-7721) and human normal liver cell line HL-7702 detected by qRT-PCR. (D)
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Protein level of PDCD4 in HL-7702, H-97 and HuH7 cell lines were detected by
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Western Blot. Data were presented as mean ± s.d. *P<0.05.
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Figure 4. SNHG1/miR-195-5p axis is critical for the expression of PDCD4.
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(A) miR-195-5p inhibitor with or without SNHG1 siRNA was transfected into H-97 cells and the mRNA level of PDCD4 was evaluated by qRT-PCR. (B) Western blot
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analysis of PDCD4 protein level following treatment of H-97 cells with miR-195-5p inhibitor or SNHG1 siRNA. GAPDH was used as control. (C) HuH7 cells were transfected with miR-195-5p with or without SNHG1 overexpress plasmid and qRTPCR was used to detect the relative mRNA levels of PDCD4 compared with control. (D) Relative protein level of PDCD4 when transfected with miR-195-5p mimics and reversed by SNHG1 expression plasmid. (E) Relative mRNA level of PDCD4 when transfected with SNHG1-WT overexpression plasmid or SNHG1-MUT overexpression plasmid. (F) Relative protein level of PDCD4 when transfected with SNHG1-WT overexpression plasmid or SNHG1-MUT overexpression plasmid. Data were presented
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as mean ± s.d. *P<0.05, ns, no significantly difference.
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Figure 5. SNHG1 regulates cell function through miR-195-5p.
(A) CCK-8 assay was performed to determine the proliferation of H-97 and HuH7 cells.
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(B, C) The migration ability with respect to changes of H-97 and HuH7 cell lines after different transfection. (D) Transwell assay was performed to determine the migration
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of HuH7 cells treated with SNHG1-MUT overexpression plasmid. Data were presented
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Figure 6. SNHG1 knockdown in H-97 cells suppresses tumor growth in vivo.
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(A) Representative images of xenografts tumor in nude mice. (B) Tumor volume was monitored. (C) Tumor weight was monitored. (D) Representative images of IHC
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ACCEPTED MANUSCRIPT Highlights
Our results indicated a high expression of SNHG1 in HCC cells.
SNHG1 promotes cell proliferation and migration in vitro and in vivo.
SNHG1 acts as a ceRNA to promote HCC progression by sponging miR-
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195-5p.