MiR-21-3p Promotes Hepatocellular Carcinoma Progression via SMAD7/YAP1 Regulation

Abstract

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.

Mir-21 Suppression Promotes Mouse Hepatocarcinogenesis

“…MiR-21 was also previously reported to regulate pro-oncogenic pathways (MAPK, STAT3, PI3K/AKT, and HiPPO) [ 28 , 55 , 61 , 62 , 63 ], in agreement with our in silico analyses showing that genes upregulated in the absence of miR-21 were involved for example in MAPK, STAT3, and PI3K/AKT signaling ( Figure 1 F and Figure S3 ). We thus investigated the expression and activation by phosphorylation of key effectors of these pro-oncogenic pathways by Western blot.…”

“…Finally, genetic deletion of the MiR-21a gene in our miR21KO, LmiR21KO, and LPTENmiR21KO mice also prevents expression of the passenger strand miR-21*, which is not inhibited by antagomiRs against the miR-21-5p. Interestingly, miR-21* (or miR-21-3p) is now reported as a functionally relevant passenger strand, as shown in human HepG2 hepatoblastoma cells where it inhibited growth and triggered apoptosis [ 77 ], but promoted, in another study, migration and invasion of the same cells [ 63 ]. MiR-21* was also reported to play a significant role in other processes such as cardiac and renal fibrosis [ 78 , 79 ], hypertension [ 80 ], or inflammation [ 81 , 82 , 83 ].…”

Hsa-miR-21-3p associates with breast cancer patient survival and targets genes in tumor suppressive pathways

“…Previous studies identified miR-21-3p target genes: in ovarian cells it targets NAV3 [73], a known tumor suppressor; in hepatocellular carcinoma, it targets SMAD7, an inhibitor of the TGFβ pathway [74]; and in ESCC, it targets TRAF4 [58]. Conversely, miR-21-3p expression upregulates L1CAM, which promotes cell motility, invasion, metastasis, and chemoresistance [75].…”

Functional and Clinical Significance of Dysregulated microRNAs in Liver Cancer

“…Additionally, miR-21 is reported to induce hepatic inflammation through promotion of inflammatory gene expression via the STAT3 signaling pathways, leading to liver disease [38]. High miR-21-3p levels are positively associated with advanced tumor stages [39]. MiR-21-3p promotes metastasis of HCC cells and upregulation of Yes-Associated Protein 1 (YAP1) expression via direct inhibition of SMAD7, which represents a major epigenetic mechanism in the pathogenesis of HCC [39].…”

“…High miR-21-3p levels are positively associated with advanced tumor stages [39]. MiR-21-3p promotes metastasis of HCC cells and upregulation of Yes-Associated Protein 1 (YAP1) expression via direct inhibition of SMAD7, which represents a major epigenetic mechanism in the pathogenesis of HCC [39]. High-Mobility Group Box 1 (HMGB1) and Cluster Differentiation 44 (CD44) additionally play critical roles in HCC progression.…”