Background and Aims: Recent investigations demonstrated that microRNA-671-5p (miR-671-5p) participates in the occurrence and development of several types of cancer, and is associated with the liver injury induced by various types of virus. However, the role of miR-671-5p in hepatocellular carcinoma (HCC) has not yet been reported to date. This study was conducted to observe the miR-671-5p expression in HCC, analyze its function and its association with the biological behaviors and clinicopathologic features of HCC, and preliminarily investigate the possible mechanism. 
Methods: The miR-671-5p expressions in 80 paired specimens of HCC and tumor adjacent tissue as well as different HCC cell lines (Hep3B, MHCC-97H, HepG2 and SMMC-7721) and normal human hepatic cell line (L02) were determined by qRT-PCR method, and meanwhile, the expression difference in miR-671-5p between HCC tissue and tumor adjacent tissue were analyzed in TCGA database. The relations of miR-671-5p expression level with the clinicopathologic factors were analyzed. In MHCC-97H cells after knockdown of miR-671-5p expression by transfection with miR-671-5p inhibitors, the changes in proliferative and invasion/migration abilities were examined by CCK-8 assay and Transwell assay, respectively. The target genes of miR-671-5p were predicted by using the TargetScan and Starbase online websites, and then verified by Western blot, dual luciferase assay and TCGA database analysis, respectively. The influences of miR-671-5p knockdown on the expressions of target gene of miR-671-5p and the proteins (E-cadherin, N-cadherin and vimentin) associated with epithelial-mesenchymal transition (EMT) in MHCC-97H cells, as well as the changes in expressions of above proteins upon the same condition with the synchronous knockdown of the target gene were detected by Western blot.
Results: The miR-671-5p expressions in HCC tissue and all studied HCC cell lines were significantly higher than that in tumor adjacent tissue or the normal hepatic cell line, and was increased with the elevation of the tumor stage of the sample and invasion ability of the HCC cells (all P<0.05); the TCGA database analysis also showed that the miR-671-5p expression level in HCC tissue was significantly higher than that in tumor adjacent tissue (P<0.05). The miR-671-5p expression level was significantly related to AFP level, tumor number, venous invasion, Edmondson-Steiner grade and TNM stage (all P<0.05). In MHCC-97H cells after transfected with miR-671-5p inhibitors, the proliferative, invasion and migration abilities were all significantly reduced (all P<0.05). Bio-informatics analysis and dual luciferase assay suggested that cofilin 2 (CFL2) was the potential target gene of miR-671-5p, and the TCGA database analysis also showed that there was a negative correlation between miR-671-5p expression and CFL2 expression (P<0.05). In MHCC-97H cells with down-regulated miR-671-5p expression, the CFL2 expression was significantly increased and the expressions of EMT-associated proteins were significantly decreased (all P<0.05), but all these changes were reversed to significant extents by synchronous interference of the CFL2 expression (all P<0.05).
Conclusion: The miR-671-5p expression is up-regulated in HCC, which is closely associated with the unfavorable clinicopathologic features of HCC. MiR-671-5p can promote the proliferation, invasion and migration of HCC cells, and the mechanism may be probably related to its down-regulating CFL2 expression and thereby promote EMT process.