scholarly journals MiR-424-5p regulates cell cycle and inhibits proliferation of hepatocellular carcinoma cells by targeting E2F7

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242179
Author(s):  
Yichao Zhao ◽  
Chaoqian Zhu ◽  
Qing Chang ◽  
Peng Peng ◽  
Jie Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Ectopic Expression ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
G1 Phase ◽  
Reporter Assay ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Objective This study aims to explore the mechanism of the miR-424-5p/E2F7 axis in hepatocellular carcinoma (HCC) and provide new ideas for targeted therapy of HCC. Methods Bioinformatics analysis was used to identify the target differentially expressed miRNA in HCC and predict its target gene. qRT-PCR was employed to verify the expression of miR-424-5p and E2F7 mRNA in HCC cells. Western blot was performed to detect the effect of miR-424-5p ectopic expression on the protein expression of E2F7. CCK-8 was used to detect proliferative activity of HCC cells and flow cytometry was carried out for analyzing cell cycle distribution. Dual luciferase reporter assay was conducted to verify the direct targeting relationship between miR-424-5p and E2F7. Results We observed that miR-424-5p was down-regulated in HCC cells. CCK-8 showed that overexpression of miR-424-5p inhibited cell proliferation, and flow cytometry showed that miR-424-5p could block cells in G0/G1 phase. E2F7 was up-regulated in HCC cells, and E2F7 overexpression could facilitate the proliferative ability of HCC cells and promote the cell cycle progressing from G0/G1 to S phase. Furthermore, dual-luciferase reporter assay indicated that miR-424-5p could directly down-regulate E2F7 expression. Analysis on cell function demonstrated that miR-424-5p inhibited the proliferation of HCC cells and blocked cell cycle at G0/G1 phase by targeting E2F7. Conclusion Our results proved that E2F7 was a direct target of miR-424-5p, and miR-424-5p could regulate cell cycle and further inhibit the proliferation of HCC cells by targeting E2F7.

scholarly journals LncRNA ZEB1-AS1 regulates hepatocellular carcinoma progression by targeting miR-23c

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shuai Xue ◽  
Fengqin Lu ◽  
Chunhui Sun ◽  
Jingjing Zhao ◽  
Honghua Zhen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Correlation Analysis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Proliferation And Invasion ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background It has been reported that long-chain non-coding RNA (lncRNA) zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is an oncogene in various cancers, including hepatocellular carcinoma (HCC). We investigated the role and mechanism of ZEB1-AS1 as a competitive endogenous RNA (ceRNA) combined with miR-23c in HCC cell proliferation and invasion. Methods QRT-PCR was used to detect ZEB1-AS1 and miR-23c expressions in HCC tissues and cells. The dual luciferase reporter assay detected the targeted regulation of miR-23c and ZEB1-AS1. We also performed the correlation analysis of their expression in HCC tissues by the Spearman’s correlation analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of hepatoma cells. Cell invasion was assessed by the Transwell assay. Results QRT-PCR results indicated ZEB1-AS1 was upregulated and miR-23c was downregulated in HCC tissues and cell lines. ZEB1-AS1 knockdown hampered the proliferation and invasion of HCC cells. Dual luciferase reporter assay showed that miR-23c is a target of ZEB1-AS1, and ZEB1-AS1 was significantly negatively correlated with the miR-23c expression in HCC tissues. The results of MTT and Transwell assay showed that miR-23c inhibition restored the inhibitory effect of ZEB1-AS1 knockdown on HCC cells proliferation and invasion. Conclusions As a ceRNA, lncRNA ZEB1-AS1 may play a vital role in inhibiting HCC progression through miR-23c, which will provide new clues and theoretical basis for the HCC diagnosis and treatment.

scholarly journals LncRNA ZFPM2-AS1 Promotes Metastasis and Epithelial-mesenchymal Transition of Hepatocellular Carcinoma via Targeting miR-3612/ADAM15 Axis

2020 ◽  
Author(s):  
Nan Yang ◽  
Tianxiang Chen ◽  
Bowen Yao ◽  
Liang Wang ◽  
Runkun Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Biological Effects ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Mesenchymal Transition ◽  
Hcc Cells

Abstract Background: Long non-coding RNAs (lncRNAs) have obtained growing attention due to their potential effects as novel regulators in various tumors. This study aimed to investigate the expression and roles of lncRNA ZFPM2-AS1 in the progression of hepatocellular carcinoma (HCC). Methods: Transwell was used to determine migration and invasion of HCC cells in vitro. The lung metastasis mouse model was established to detect tumor metastasis of HCC in vivo. The direct binding of miR-3612 to 3'UTR of DAM15 was confirmed by luciferase reporter assay. The expression of ZFPM2-AS1 and miR-3612 in HCC specimens and cell lines were detected by real-time PCR. The correlation among ZFPM2-AS1 and miR-3612 were disclosed by a dual-luciferase reporter assay, RIP assay and biotin pull-down assay.Results: In present study, we found that ZFPM2-AS1 was up-regulated in HCC tissues and cells and its upregulation was associated with TNM stage, vascular invasion, and poor prognosis of HCC patients. Functionally, gain- and loss-of-function experiments indicated that ZFPM2-AS1 promoted cell migration, invasion and EMT progress in vitro and in vivo. ZFPM2-AS1 could function as a competing endogenous RNA (ceRNA) by sponging miR-3612 in HCC cells. Mechanically, miR-3612 inhibited HCC metastasis and alternation of miR-3612 reversed the promotive effects of ZFPM2-AS1 on HCC cells. In addition, we confirmed that ADAM15 was a direct target of miR-3612 in HCC and mediated the biological effects of miR-3612 and ZFPM2-AS1 in HCC. Curcumin, an active derivative from turmeric, exerts its anticancer effects through ZFPM2-AS1/miR-3612/ADAM15 pathway. Our data identified ZFPM2-AS1 as a novel oncogenic lncRNA and correlated malignant clinical outcomes in HCC patients. Conclusions: ZFPM2-AS1 performed as oncogenic role via targeting miR-3612 and subsequently promoted ADAM15 expression in HCC. Our results revealed that ZFPM2-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

scholarly journals MiR-196b-5p regulates the proliferation of drug-resistant hepatocellular carcinoma cell lines by activating NFκB/ABCB1 signaling pathway

2020 ◽  
Vol 19 (1) ◽  
pp. 39-44
Author(s):  
Bangming Pu ◽  
Yong Cao ◽  
Yan Li ◽  
Li Tang ◽  
Jiyi Xia ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Hepg2 Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Drug Resistant ◽  
Reporter Assay

Purpose: To explore the molecular function of miR-196b-5p in hepatocellular carcinoma (HCC).Methods: MiR-196b-5p expression levels in HCC tissue samples were assessed by qRT-PCR. MiR-196b-5p was knocked-down or over-expressed in HepG2 cells by transfecting the cells with plasmids expressing either a miR-196b-5p inhibitor or mimic, respectively, while cell proliferation was  assessed by MTT assay. The interaction of miR-196b-5p with target molecules was confirmed using luciferase reporter assay. Cell cycle was investigated by flow cytometry, while NFκBIA expression was assessed by western blotting.Results: MiR-196b-5p was over-expressed in HCC, and miR-196b-5p expression levels in patients with HCC were related to tumor grade. MiR-196b-5p over-expression promoted cell proliferation and colony formation and suppressed cell cycle arrest and apoptosis. The results of luciferase reporter assay showed that miR-196b-5p reduced NFκBIA expression in HepG2 cells by binding to a response element in the 3′ UTR of NFκBIA. Further investigation showed that NFκBIA interacts with NFκB1 and reduces the concentration of NFκB1 in HepG2 cells. The promoter of ATP-binding cassette sub-family B member 1 (ABCB1) was also targeted and bound by NFκB1, which altered the expression of ABCB1 in HepG2 cells.Conclusion: MiR-196b-5p regulates cell proliferation in drug-resistant HCC cell lines via activation of the NFκB/ABCB1 signaling pathway. Keywords: Hepatocellular carcinoma, miR-196b-5p, NFκBIA, NFκB1, ABCB1

scholarly journals BTB and CNC homology 1 (Bach1) induces lung cancer stem cell phenotypes by stimulating CD44 expression

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Pan Jiang ◽  
Fan Li ◽  
Zilong Liu ◽  
Shengyu Hao ◽  
Jian Gao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Flow Cytometry ◽  
Mapk Signaling ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Flow Cytometry Analysis ◽  
Tumor Sphere ◽  
Sphere Formation ◽  
Dual Luciferase Reporter Assay

Abstract Background Growing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently. Methods The expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67. Results The ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44− CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression. Conclusion In summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

scholarly journals Brucine Inhibits Proliferation of U87 Glioblastoma Cells by Targeting the G-quadruplexes in the c-Myb Promoter

2020 ◽  
Author(s):  
Qiaochu Liu ◽  
Di Huo ◽  
Qunhui Wang ◽  
Chuanqi Lv ◽  
Ziqiang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Promoter Region ◽  
Luciferase Reporter Assay ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Ionization Mass ◽  
Reporter Assay ◽  
U87 Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background: The proto-oncogene c-Myb plays an important role in the proliferation of cells and its upregulation affects the development of glioblastomas. G-quadruplexes are secondary structures of DNA or RNA that usually form in the promoter region of oncogenes, including c-Myb, and regulate the expression of these genes. The traditional Chinese medicine brucine is a ligand of G-quadruplexes located in the promoter region of c-Myb. In this study, the U87 cell line was used both in vitro and in vivo to investigate the therapeutic effect and mechanism of action of brucine. Methods: MTT assay and flow cytometry were used to determine the effect of brucine on the cell cycle, viability, and apoptosis of U87 cells. The effects of brucine on transcription and expression of c-Myb were determined through RT-PCR and western blotting. Dual-luciferase reporter assay and electrospray ionization-mass spectrometry were used to investigate whether brucine acts directly and binds G-quadruplexes in the promoter region of c-Myb, respectively. Results: The results showed that brucine suppressed the growth of U87 cells in vitro by arresting the cell cycle and reducing the expression of c-Myb. Through the dual luciferase reporter assay, brucine was found to inhibit the expression of c-Myb by targeting the guanine-rich sequence that forms G-quadruplexes in the c-Myb promoter. Moreover, U87 tumors were suppressed by brucine in a tumor xenograft nude mice model. Conclusion: The findings of the study indicate that brucine is a potentially effective medicine for treatment of glioblastomas.

scholarly journals A Function Variant at miR-501 Alters Susceptibility to Hepatocellular Carcinoma in a Chinese Han Population

2016 ◽  
Vol 38 (6) ◽  
pp. 2500-2508 ◽  
Author(s):  
Yang Liu ◽  
Yi Chai ◽  
Jian Zhang ◽  
Junwei Tang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Case Control Study ◽  
Case Control ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Study Results ◽  
Control Study ◽  
Dual Luciferase Reporter Assay

Backgroud/Aims: Previous studies have shown that miR-501 is involved in the development of hepatocellular carcinoma (HCC) by promoting cell proliferation through CYLD. From the published MirSNP database that enrolls all single nucleotide polymorphisms(SNPs) of microRNA (miRNA), we found an interesting SNP (rs112489955, G>A) located in the mature region of miR-501. Methods: We performed a case-control study focusing on the predicted SNP located in miRNA-501 to investigate the further relationship of the SNPs with miRNAs among HCC patients. Genotyping, real time PCR assay, cell transfection and the dual luciferase reporter assay were used in our study. Results: Bioinformatic analysis indicated that this SNP would inhibit the binding of miR-501 to CYLD. In a case-control study, subjects with the variant genotypes (AG, GG) showed a significantly increased risk of HCC relative to AA carriers. A significant association of miR-501 variant genotypes with enhanced tumor growth was also observed. Further functional analyses indicated that patients with the AA genotype might attenuate the level of CYLD compared to that regulated by miR-501 with the GG genotype. A dual luciferase reporter assay also confirmed that miR-501 with the A allele had reduced binding to CYLD. We further confirmed a suppression of cell proliferation and promotion of apoptosis in SMMC-7721 and Hep3B cell lines treated with the AA genotype. Conclusions: We identified a novel SNP located in miR-501 acting as an important factor of the HCC susceptibility by modulating miR-501 and CYLD levels.

Long Non-Coding RNA CASC7 Promotes Proliferation and Inhibits Apoptosis of Hepatocellular Carcinoma Cells via Downregulating miR-340-5p CASC7/miR-340-5p Axis in Hepatocellular Carcinoma

2022 ◽  
Vol 12 (4) ◽  
pp. 747-755
Author(s):  
Shengyong Liu ◽  
Xiangcheng Li
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Nrf2 Pathway ◽  
Non Coding Rna ◽  
Tumorigenesis And Progression ◽  
Long Non Coding Rna ◽  
Dual Luciferase Reporter Assay

Background: Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide with a poor prognosis. Amounting studies revealed that long non-coding RNAs (lncRNAs) show important roles in various biological processes. The purpose of this study was to explore the biological function and potential molecular mechanism of CASC7 in HCC. Methods: CASC7 expression in HCC cell lines was detected by qRT-PCR. The expressions of CASC7 and miR-340-5p were changed by transfection of miR-340-5p mimic, the CASC7 overexpression and knockdown plasmids. The interaction between CASC7 and miR-340-5p was assessed by a Dual-Luciferase reporter assay. The biological functions of CASC7 were evaluated by CCK-8, colony formation assay, ROS assay kit, immunofluorescence and flow cytometry (FCM). Results: CASC7 was upregulated in HCC cell lines. CASC7 overexpression significantly promoted cell proliferation, as well as inhibited apoptosis and oxidative stress. In contrast, CASC7 knockdown could reverse these above changes. The result of the Dual-luciferase reporter assay revealed that CASC7 directly targeted miR-340-5p and negatively regulated its expression. In addition, CASC7 promoted proliferation and inhibited apoptosis of HCC cells through activating Nrf2 pathway by downregulating miR-340-5p. Conclusions: In summary, CASC7 promotes HCC tumorigenesis and progression through the Nrf2 pathway by targeting miR-340-5p, which may provide a new target for therapy of HCC.

scholarly journals Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

2020 ◽  
Author(s):  
Yang Gu ◽  
Shulan Zhang
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter Assay ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Abstract Background: The molecular mechanisms of ovarian cancer (OC) remain unclear. We sought to comprehensively identify microRNAs (miRNAs) that are aberrantly expressed in metastatic OC. Methods: Differentially expressed miRNAs were screened from six pairs of primary and metastatic OC tissues; their possible functions were analyzed and target genes were predicted by bioinformatics. Then gene expression profiling results were established by reverse transcription quantitative polymerase chain reaction and western blot. Targeting relationship between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. CCK-8, Transwell assay, scratch test and flow cytometry were used for cell function detection after miR-7-5p overexpression. Results: Twelve miRNAs and 10 target mRNAs were differentially expressed in primary and metastatic OC tissues. ITGB3, TGFβ2 and TNC correlated to miRNA function in metastatic OC. Among all 7 miRNAs, expression of hsa-miR-141-3p, hsa-miR-7-5p, hsa-miR-187-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p in metastatic OC tissues was obviously lower than that in primary OC tissues ( p < 0.05). Moreover, there was a significant correlation between hsa-miR-7-5p and TGFβ2 in OC tissues. Dual-luciferase reporter assay confirmed that hsa-miR-7-5p negatively targeted TGFβ2. After miR-7-5p overexpression, the OC cell viability and invasion were reduced, the cell cycle was blocked ( p < 0.05). Conclusions: Hsa-miR-141-3p, hsa-miR-187-5p, hsa-miR-7-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p expression was prominently lower in metastatic OC than in primary OC, while TGFβ2 expression was markedly increased in metastatic OC tissues. Hsa-miR-7-5p bound to TGFβ2 3’-UTR to inhibit its expression. Hsa-miR-7-5p targeted TGFβ2 to inhibit cell proliferation, invasion and cell cycle entry.

scholarly journals MicroRNA miR-212-3p targets NFIA to suppress HBV replication and tumor progression in hepatocellular carcinoma via repressing Enhancer I activity

2021 ◽  
Author(s):  
Hui Tian ◽  
Zhenkun He
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B ◽  
Cell Viability ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Hbv Replication ◽  
Dual Luciferase Reporter Assay

IntroductionEmerging evidence identifies that microRNAs (miRNAs) are associated with hepatitis B virus (HBV) infection. In the current study, we mainly focus on the functions and underlying mechanisms of miR-212-3p in HBV replication in hepatocellular carcinoma (HCC).Material and methodsThe levels of miR-212-3p, nuclear factor I A (NFIA) and HBV DNA copies were measured by qRT-PCR. The level of core particle-associated HBV DNA, the productions of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg), and the expression of NFIA were detected via southern blot assay, ELISA and western blot assay, respectively. The putative target of miR-212-3p was predicted by TargetScan and Pictar, followed by the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay to validate the interaction. The interaction between miR-212-3p and Enhancer I/X promoter (EnI/Xp) reporter was also verified by dual luciferase reporter assay. In addition, the cell viability and apoptotic rate were detected by MTT and flow cytometry, respectively.ResultsmiR-212-3p mimics or NFIA knockdown inhibited HBV expression and replication in HepG2.2.15 cells, while miR-212-3p inhibitor or NFIA overexpression showed the opposite trend. NFIA was confirmed as a direct target of miR-212-3p. Furthermore, miR-212-3p impeded HBV expression and replication by suppressing NFIA. Besides, miR-212-3p lowered EnI/Xp activity by regulating NFIA. In addition, miR-212-3p retarded cell viability and induced apoptosis through targeting NFIA.ConclusionsmiR-212-3p targets NFIA to down-regulate its expression, thereby inhibiting HBV replication and tumorigenesis in HCC. Our finding might provide a promising therapeutic target for HBV infection.

scholarly journals HOXA-AS2 Promotes Proliferation and Induces Epithelial-Mesenchymal Transition via the miR-520c-3p/GPC3 Axis in Hepatocellular Carcinoma

2018 ◽  
Vol 50 (6) ◽  
pp. 2124-2138 ◽  
Author(s):  
Ying Zhang ◽  
Jianliang Xu ◽  
Shaoquan Zhang ◽  
Jun An ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Online Programs ◽  
Hcc Cells

Background/Aims: Previous studies have demonstrated that long non-coding RNAs (lncRNAs) may play critical roles in cancer biology, including Hepatocellular carcinoma (HCC). The HOXA cluster antisense RNA2 (HOXA-AS2) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOXA-AS2 in HCC remains unknown. The present study examined the effects of HOXA-AS2 on the progression of HCC, and explored the underlying molecular mechanisms. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in HCC tissues and cell lines. Furthermore, the effects of HOXA-AS2 silencing and overexpression on cell proliferation, cell cycle, apoptosis, migration, and invasion were assessed in HCC in vitro and in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in HCC cells. Results: We observed that HOXA-AS2 was up-regulated in HCC tissues and cell lines. In vitro experiments revealed that HOXA-AS2 knockdown significantly inhibited HCC cells proliferation by causing G1 arrest and promoting apoptosis, whereas HOXA-AS2 overexpression promoted cell growth. Further functional assays indicated that HOXA-AS2 significantly promoted HCC cell migration and invasion by promoting EMT. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in HCC cells. MiR-520c-3p was down-regulated and inversely correlated with HOXA-AS2 expression in HCC tissues. miR-520c-3p suppressed cell proliferation, invasion and migration in HCC cells, and enforced expression of miR-520c-3p attenuated the oncogenic effects of HOXA-AS2 in HCC cells. By bioinformatic analysis and dual-luciferase reporter assay, we found that miR-223-3p directly targeted the 3’-untranslated region (UTR) of Glypican-3 (GPC3), one of the key players in HCC. GPC3 was up-regulated in HCC tissues, and was negatively correlated with miR-520c-3p expression and positively correlated with HOXA-AS2 expression. Conclusion: In summary, our results suggested that the HOXA-AS2/miR-520c-3p/GPC3 axis may play an important role in the regulation of PTC progression, which could serve as a biomarker and therapeutic target for HCC.

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