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Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression.

ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. To elucidate new regulatory mechanisms for heptocarcinogenesis, we investigated the regulation of p21, a cyclin-dependent kinase (CDK) inhibitor encoded by CDKN1A, in HCC. The expression level of p21 is decreased with the progression of HCC. Luciferase assays with a luciferase-p21-3' UTR reporter and its serial deletions identified a 15-bp repressor element at the 3'-UTR of CDKN1A, which contains a binding site for miR-95-3p. Mutation of the binding site eliminated the regulatory effect of miR-95-3p on p21 expression. Posttranscriptional regulation of p21 expression by miR-95-3p is mainly on the protein level (suppression of translation). Overexpression of miR-95-3p in two different HCC cell lines, HepG2 and SMMC7721, significantly promoted cell proliferation, cell cycle progression and cell migration, whereas a miR-95-3p specific inhibitor decreased cell proliferation, cell cycle progression and cell migration. The effects of miR-95-3p on cellular functions were rescued by overexpression of p21. Overexpression of miR-95-3p promoted cell proliferation and tumor growth in HCC xenograft mouse models. Expression of miR-95-3p was significantly higher in HCC samples than in adjacent non-cancerous samples. These results demonstrate that miR-95-3p is a potential new marker for HCC and regulates hepatocarcinogenesis by directly targeting CDKN1A/p21 expression.

SUBMITTER: Ye J

PROVIDER: S-EPMC5048429 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression.

Ye Jian J Yao Yufeng Y Song Qixue Q Li Sisi S Hu Zhenkun Z Yu Yubing Y Hu Changqing C Da Xingwen X Li Hui H Chen Qiuyun Q Wang Qing K QK

Scientific reports 20161004

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. To elucidate new regulatory mechanisms for heptocarcinogenesis, we investigated the regulation of p21, a cyclin-dependent kinase (CDK) inhibitor encoded by CDKN1A, in HCC. The expression level of p21 is decreased with the progression of HCC. Luciferase assays with a luciferase-p21-3' UTR reporter and its serial deletions identified a 15-bp repressor element at the 3'-UTR of CDKN1A, which contains a binding site for miR-9 ...[more]

PMID: 27698442

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Project description:BackgroundHepatocellular carcinoma (HCC) remains a major global health burden due to its high prevalence and mortality. Emerging evidence reveals that microRNA (miRNA) plays a vital role in cancer pathogenesis and is widely involved in the regulation of signaling pathways via their targeting of downstream genes. MiR-21-3p, a liver-enriched miRNA, and SMAD7, the negative regulator of the TGF-β signaling pathway, likely exert a vital influence on HCC progression.AimsHere, we explore the role of the miR-21-3p-SMAD7/YAP1 axis on HCC pathogenesis.MethodsMiRNA microarray analysis was performed for miRNA screening. The dual-luciferase assay was adopted for target verification. Expression of miRNA and related genes were quantified via qRT-PCR, western blotting, and immunohistochemical staining. Flow cytometry and the transwell migration assay were used to detail cell apoptosis, invasion and metastases. Rat models were established to explore the role of the miR-21-3p-SMAD7/YAP1 axis in hepatocarcinogenesis. Bioinformatics analysis was conducted for exploring genes of clinical significance.ResultsMiR-21-3p levels were found to be significantly elevated in hepatocellular carcinoma and indicate poor overall survival. High miR-21-3p levels were associated with advanced tumor stages (P = 0.029), in particular T staging (P = 0.026). Low SMAD7/high YAP1 levels were confirmed in both HCC and rat models with advanced liver fibrosis and cirrhosis. Besides, SMAD7 was demonstrated to be the direct target of miR-21-3p. The effect of MiR-21-3p on tumor phenotypes and YAP1 upregulation could be partly reversed via the restoration of SMAD7 expression in HCC cell lines. Overexpression of YAP1 after miR-21-3p upregulation promoted expression of nuclear transcription effector connective tissue growth factor. Co-survival analysis indicated that lower miR-21-3p/higher SMAD7 (P = 0.0494) and lower miR-21-3p/lower YAP1 (P = 0.0379) group patients had better overall survival rates. Gene Set Variation Analysis revealed that gene sets related to miR-21-3p and SMAD7 were significantly associated with the TGF-β signaling pathway in HCC.ConclusionMiR-21-3p promotes migration and invasion of HCC cells and upregulation of YAP1 expression via direct inhibition of SMAD7, underscoring a major epigenetic mechanism in the pathogenesis of HCC.

Project description:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide with a low 5-year survival rate. Long non-coding RNA (lncRNA) double homeobox A pseudogene 8 (DUXAP8) is an oncogene and a potential biomarker in various tumors, such as ovarian, colorectal and non-small-cell lung cancer. However, the function and molecular mechanism underlying DUXAP8 in HCC progression is not completely understood. The expression of DUXAP8, microRNA (miR)-9-3p and insulin-like growth factor 1 receptor (IGF1R) in HCC tissues and cells was detected via reverse transcription-quantitative PCR. The expression levels of IGF1R and epithelial-mesenchymal transition-associated proteins (Snail, Slug, E-cadherin, N-cadherin and vimentin) were assessed via western blotting. The effects of DUXAP8, miR-9-3p and IGF1R on proliferation, migration and invasion were examined by conducting Cell Counting Kit-8 and Transwell assays, respectively. The interaction between miR-9-3p and DUXAP8 or IGF1R was predicted using StarBase or TargetScan, and further assessed using dual luciferase reporter and RNA immunoprecipitation assays. DUXAP8 and IGF1R were upregulated and miR-9-3p was downregulated in HCC tissues and cells compared with adjacent healthy tissues and a normal liver cell line, respectively. miR-9-3p overexpression decreased the protein expression level of IGF1R, and miR-9-3p knockdown enhanced the protein expression level of IGF1R in HCC cells compared with the corresponding control groups. Moreover, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression increased E-cadherin protein expression levels, and decreased Snail, Slug, N-cadherin and vimentin protein expression levels. However, miR-9-3p inhibitor and IGF1R overexpression reversed DUXAP8 knockdown- and miR-9-3p overexpression-induced effects, respectively. In addition, compared with the corresponding control groups, DUXAP8 knockdown and miR-9-3p overexpression suppressed proliferation, migration and invasion, which was reversed by miR-9-3p inhibitor and IGF1R overexpression, respectively. Moreover, miR-9-3p as the target of DUXAP8 and IGF1R as the target of miR-9-3p were verified in HCC cells. lncRNA DUXAP8 contributed to HCC tumorigenesis via the miR-9-3p/IGF1R axis, providing a novel therapeutic approach for HCC diagnosis and treatment.

Project description:BackgroundEVA1A (Eva-1 homolog A), a novel protein involved in autophagy and apoptosis, functions as a tumor suppressor in some human primary cancers, including hepatocellular carcinoma (HCC). While it is consistently downregulated in several cancers, its involvement in hepatocarcinogenesis is still largely unknown.MethodsWe first detected the expression of EVA1A in HCC tissues and cell lines using RT‒qPCR, immunohistochemistry and western blotting and detected the expression of miR-103a-3p by RT‒qPCR. Then, bioinformatics prediction, dual-luciferase reporter gene assays and western blotting were used to screen and identify the upstream microRNA of EVA1A. After manipulating the expression of miR-103a-3p or EVA1A, wound healing, invasion, proliferation, colony formation, apoptosis, autophagy, mitosis and mitochondrial function assays, including mitochondrial membrane potential, ROS and ATP production assays, were performed to investigate the functions of miR-103a-3p targeting EVA1A in HCC cells. Apoptosis-related proteins were assessed by RT‒qPCR (TP53) or western blotting (TP53, BAX, Bcl-2 and caspase-3). Autophagy level was evaluated by observing LC3 puncta and examining the protein levels of p62, Beclin1 and LC3-II/I.ResultsWe found that EVA1A expression was decreased while miR-103a-3p expression was increased in HCC tissues and cell lines and that their expression was inversely correlated in HCC patients. The expression of miR-103a-3p was associated with HCC tumor stage and poor prognosis. miR-103a-3p could target EVA1A through direct binding to its 3'-UTR and suppress its expression. Overexpression of miR-103a-3p significantly downregulated the expression of EVA1A, TP53 and BAX, upregulated the JAK2/STAT3 pathway and promoted HCC cell migration, invasion and proliferation, while repression of miR-103a-3p dramatically upregulated the expression of EVA1A, TP53, BAX and cleaved-caspase-3, inhibited HCC cell migration, invasion and proliferation, and caused mitochondrial dysfunction and apoptosis. Overexpression of EVA1A significantly attenuated the cancer-promoting effects of miR-103a-3p in HCC cells, while knockdown of EVA1A alleviated the mitochondrial dysfunction and apoptosis caused by miR-103a-3p inhibition. Overexpression of EVA1A did not induce significant changes in autophagy levels, nor did it affect G2/M transition or mitosis.ConclusionThese findings indicate that the downregulation of the tumor suppressor EVA1A by miR-103a-3p potentially acts as a key mediator in HCC progression, mainly by inhibiting apoptosis and promoting metastasis. The miR-103a/EVA1A/TP53 axis provides a new potential diagnostic and therapeutic target for HCC treatment.

Dataset Information

Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression.

Publications

Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression.

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