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

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.

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

PLoS ONE ◽

10.1371/journal.pone.0242179 ◽

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.

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MiR-466 Inhibits the Progression of Severe Hepatocellular Carcinoma via Regulating FMNL2-Mediated Activation of NF-κB and Wnt/β-Catenin Pathways

Journal of Oncology ◽

10.1155/2021/3554219 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Jianwei Li ◽

Su Yan

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Luciferase Reporter ◽

Regulation Mechanism ◽

Transwell Assay ◽

Flow Cytometry Assay ◽

Qrt Pcr ◽

Hcc Cells ◽

Dual Luciferase Reporter Assay

Hepatocellular carcinoma (HCC) has threatened the health of humans, and some evidence has indicated that miR-466 involves the progressions of some cancers. This study focused on the role of miR-466 in the formation and development of HCC. The expression levels of miR-466 in the tissues of patients and HCC cell lines were measured by qRT-PCR, and CCK-8, transwell assay, and flow cytometry assay were used to observe the functions of miR-466 on the HCC cells. Moreover, the miRNA databases, dual-luciferase reporter assay, and Western blot were used for the investigation of the regulation mechanism of miR-466 on HCC cells. The results showed that miR-466 was significantly downregulated in HCC tissues and cell lines, and inhibited proliferation, invasion, and high apoptosis were found in HCC cells when miR-466 was overexpressed. The results confirmed that FMNL2 was a target of miR-466, and increased FMNL2 could reverse the effects of miR-466 on the phenotype of HCC cells. Besides, it was also found that miR-466 was involved in the regulation of NF-κB and Wnt/β-catenin pathways in HCC cells via targeting FMNL2. In conclusion, the results of this study suggest that miR-466 regulates the activities of NF-κB and Wnt/β-catenin pathways to inhibit the progression of HCC cells via targeting FMNL2.

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lncRNA CRNDE promotes the proliferation and metastasis by acting as sponge miR-539-p to regulate POU2F1 expression in HCC

10.21203/rs.3.rs-15899/v2 ◽

2020 ◽

Author(s):

Zhixi Li ◽

Gang Wu ◽

Jie Li ◽

Youyu Wang ◽

Xueming Ju ◽

...

Keyword(s):

Western Blot ◽

Luciferase Reporter ◽

Akt Pathway ◽

Migration And Invasion ◽

Reporter Assay ◽

Normal Cells ◽

Normal Tissues ◽

Qrt Pcr ◽

Hcc Cells ◽

Dual Luciferase Reporter Assay

Abstract Background This article focuses on the roles and mechanism of lncRNA CRNDE on the progression of HCC. Methods We used qRT-PCR to detect the expression of lncRNA CRNDE in HCC cells, normal cells and clinical tissues. MTT assay, FCM analysis, Transwell migration and invasion assay were used to detect the effects of lncRNA CRNDE on cell viability, apoptosis, migration and invasion of HCC cells. The expression of apoptosis-related proteins Bcl-2, Bax, Cleaved Caspase 3, Cleaved Caspase 9, EMT epithelial marker E-cadherin and mesothelial marker Vimentin were analyzed by Western blot. Online prediction software was used to predict the binding sites between lncRNA CRNDE and miR-539-5p, or miR-539-5p and POU2F1 3’UTR. Dual luciferase reporter assay, qRT-PCR and RNA pulldown were used to detect target-relationship between lncRNA CRNDE and miR-539-5p. Dual luciferase reporter assay, qRT-PCR, Western blot and Immunofluorescence were used to detect target-relationship between miR-539-5p and POU2F1. qRT-PCR was used to detect the expression of miR-539-5p and POU2F1 in clinical tissues. Rescue experiments was used to evaluate the association among lncRNA CRNDE, miR-539-5p and POU2F1. Finally, we used Western blot to detect the effects of lncRNA CRNDE, miR-539-5p and POU2F1 on NF-κB and AKT pathway. Results lncRNA CRNDE was highly expressed in HCC cells and HCC tissues compared with normal cells and the corresponding adjacent normal tissues. lncRNA CRNDE promoted the cell viability, migration and invasion of HCC cells, while inhibited the apoptosis and promoted the EMT process of HCC cells. lncRNA CRNDE adsorbed miR-539-5p acts as a competitive endogenous RNA to regulate POU2F1 expression indirectly. In HCC clinical tissues, miR-539-5p expression decreased and POU2F1 increased compared with the corresponding adjacent normal tissues. lncRNA CRNDE/miR-539-5p/POU2- F1 participated the NF-κB and AKT pathway in HCC. Conclusion lncRNA CRNDE promotes the expression of POU2F1 by adsorbing miR-539-5p, thus promoting the progression of HCC. Keywords: HCC, lncRNA CRNDE, miR-539-5p, POU2F1, ceRNA

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Down-regulation of miR-206 is associated with Hirschsprung disease and suppresses cell migration and proliferation in cell models

Scientific Reports ◽

10.1038/srep09302 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 21

Author(s):

Ankur Sharan ◽

Hairong Zhu ◽

Hua Xie ◽

Hongxing Li ◽

Junwei Tang ◽

...

Keyword(s):

Cell Migration ◽

Hirschsprung Disease ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Down Regulation ◽

Tissue Samples ◽

Qrt Pcr ◽

Cell Migration And Proliferation ◽

Dual Luciferase Reporter Assay

Abstract Hirschsprung disease (HSCR) is a well-known congenital digestive disease that originates due to the developmental disorder of neural crest cells. MiR-206 is kown to have a relationship with digestive malfunctions. Therefore, we investigated whether or not miR-206 was involved in the pathogenesis of HSCR. qRT-PCR and Western blot assays were used to detect the expression levels of miRNA and mRNAs and proteins in case and control tissue samples and two cell lines (293T and SH-SY5Y). The functions of miR-206 in vitro were measured by transwell assay, CCK8 assay and flow cytometry. Finally, we conducted dual-luciferase reporter assay to verify the connections between miR-206 and the target mRNA SDPR. Down-regulation of miR-206 was found in HSCR case tissue samples compared with controls, which was validated to be connected with the increased level of mRNA and protein of SDPR by qRT-PCR and dual-luciferase reporter assay. Moreover, miR-206 suppressed the cell migration and proliferation and silencing of SDPR could rescue the extent of the suppressing effects by miR-206 inhibitor. The findings suggest that miR-206 may play a significant role in the pathogenesis of HSCR, as well as inhibiting the cell migration and proliferation by targeting SDPR in disease models.

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

10.21203/rs.3.rs-42530/v1 ◽

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.

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

Cellular Physiology and Biochemistry ◽

10.1159/000445600 ◽

2016 ◽

Vol 38 (6) ◽

pp. 2500-2508 ◽

Cited By ~ 9

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.

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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

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2950 ◽

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.

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MiR-877-5p down-regulation of PDK-1 involving in aspirin-induced gastric epithelial cells damage

10.21203/rs.2.23284/v1 ◽

2020 ◽

Author(s):

zongdan jiang ◽

Ran Dan ◽

Zou Jianjun ◽

Wang Zhibing ◽

Wang Zhi ◽

...

Keyword(s):

Epithelial Cells ◽

Target Gene ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Gastric Epithelial Cells ◽

Reporter Assay ◽

Qrt Pcr ◽

Proliferation And Apoptosis ◽

Mucosal Erosion ◽

Dual Luciferase Reporter Assay

Abstract It has been found that the expression of miR-877-5p is increased in serum of patients taking NSAIDs drugs. However, whether miR-877-5p play a role in aspirin-induced gastrointestinal mucosal erosion remains largely unknown. In this study, we investigated the effects of miR-877-5p on gastric epithelial cells (GES-1) proliferation and apoptosis in vitro. MiR-877-5p mimic/inhibitor and their oligonucleotides were transfected into GES-1 cells, then GES-1 cells were treated with different concentrations of aspirin (1.25, 2.5, 5 and 10 mmol/L). The bioinformatics software and dual-luciferase reporter assay were used to predict and verify the target gene of miR-877-5p. qRT-PCR and Western Blotting were employed to assess gene and protein expression, and CCK-8 assay and flow cytometry analysis were used to detect cell proliferation and apoptosis, respectively. qRT-PCR data showed that miR-877-5p level was significantly increased in aspirin incubated GES-1 cells. The proliferation of GES-1 cells were markedly inhibited and apoptosis was significantly induced in the miR-877-5p mimic groups compared to control groups. Using PITA, Targetscan and miRWalk database, the three databases indicated that PDK1 was a target gene of miR-miR-877-5p. Dual luciferase reporter assay confirmed that the existence of a direct interaction between miR-877-5p and PDK1 mRNA. Importantly, miR-877-5p knockdown resulted in a significant upregulation of PDK1 mRNA and its encoded protein in GES-1 cells. miR-877-5p plays a role in aspirin-induced gastrointestinal mucosal erosion, which may via down-regulation of targeting PDK1 gene.

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MicroRNA-449b-5p suppresses cell proliferation, migration and invasion by targeting TPD52 in nasopharyngeal carcinoma

The Journal of Biochemistry ◽

10.1093/jb/mvz057 ◽

2019 ◽

Vol 166 (5) ◽

pp. 433-440 ◽

Cited By ~ 6

Author(s):

Wei Yin ◽

Lei Shi ◽

Yanjiao Mao

Keyword(s):

Cell Proliferation ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Western Blot Assay ◽

Qrt Pcr ◽

Dual Luciferase Reporter Assay

Abstract Nasopharyngeal carcinoma (NPC) is an important type of head and neck malignant cancer with geographical distribution. MicroRNA-449b-5p (miR-449b-5p) is related to the development of various cancers, while its function in NPC remains unknown. The present study aimed to investigate the role and target gene of miR-449b-5p in NPC. Expressions of miR-449b-5p in NPC cell lines and clinical tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by MTT and colony formation assays. Migration and invasion abilities after different treatment were evaluated by wound healing and Transwell assays, respectively. Dual-luciferase reporter assay was performed to explore the relationship between miR-449b-5p and tumour protein D52 (TPD52). TPD52 expression was determined by qRT-PCR and western blot assay. miR-449b-5p was significantly downregulated in NPC cell lines and clinical tissues than the matched control. Overexpression of miR-449b-5p inhibited proliferation, migration and invasion of NPC cells. Dual-luciferase reporter assay indicated that miR-449b-5p directly targeted TPD52. Furthermore, shRNA-mediated downregulation of TPD52 rectified the promotion of cell migration and invasion by miR-449b-5p inhibition. In conclusion, the present study suggests that miR-449b-5p, as a novel tumour-suppressive miRNA against NPC, inhibits proliferation, migration and invasion of NPC cells via inhibiting TPD52 expression.

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

Archives of Medical Science ◽

10.5114/aoms/112694 ◽

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.

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