Long Non-coding RNA GAS5 Suppresses The Migration And Invasion Of Hepatocellular Carcinoma Cells Via MiR-21

ObjectiveTo demonstrate the expression of lncRNA(Long non-coding RNA)GAS5(growth arrest-specific transcript 5)in hepatocellular carcinoma(HCC)tissues and HCC cells,as well as the molecular regulation mechanism of lncRNA GAS5 on HCCs migration and invasion,which have provided new approaches and research strategies for HCC development,diagnosis,targeted therapy and prognosis prediction.Methods(1)The levels of GAS5 and miRNA-21 in HCC tissues and in paired adjacent normal tissues and the levels of miRNA-21 in HCCs were determined by qRT-PCR assays;GAS5 levels were determined by RT-PCR for HCC cell lines with different aggressive characteristics.(2)Kaplan-Meier curves for survival time for patients with HCC cancers divided according to the levels of GAS5 and miR-21.(3)After Bel-7402 and HCCLM3 cells were respectively transfected with GAS5-siRNA and pCDNA-GAS5,the expression of its PDCD4 and PTEN were determined by Western blots;Putative binding sites of GAS5 within the miR-21,as identified by RNA22 program;Luciferase Reporter Assay confirmed this.(4)After Bel-7402 and HCCLM3 cells were respectively co-transfected,the expressions of PDCD4 and PTEN were determined by Western blots.(5)Through down-expression or up-expression of GAS 5 in Bel-7402 and HCCLM3 cells,representative images of cell invasion and cell migration were determined by Transwell assays;After Bel-7402 and HCCLM3 cells were respectively co-transfected,representative images of cell invasion and cell migration were determined by Transwell assays and Wound healing analyses assays.Results(1)The levels of GAS5 are down-regulated and that miR-21 is over-expressed in HCC specimens and HCCs,correlating with the clinicopathological characteristics of HCC and the aggressive characteristics of liver cancer cells.(2)The longer survival for HCC patients with higher levels of GAS5 or lower levels of miR-21.(3)After down-expression or up-expression of GAS5 in Bel-7402 and HCCLM3 cells,the levels of miRNA-21 were higher or lower;Putative binding sites of GAS5 within the miR-21,as identified by RNA22 program,where the putative binding site is in exon 4;GAS5 can directly regulate miR-21 in HCC cells by Luciferase Reporter Assay.(4)Then we found that GAS5-siRNA-3 suppressed PDCD4 and PTEN expression in Bel-7402 cells;The results were opposite in HCCLM3 cells transfected with pCDNA-GAS5;Down-regulation of GAS5 suppressed PDCD4 and PTEN expression of Bel-7402 cells transfected with GAS5-siRNA-3,but these processes were reversed in Bel-7402 cells co-transfected with anti-miR-21;The results were opposite in HCCLM3 cells co-transfected with pCDNA-GAS5 and miR-21-mimic.(5)After down-expression or up-expression of GAS5 in Bel-7402 and HCCLM3 cells,the invasion and migration of HCC cells were greater or lower;Down-regulation of GAS5 promoted the migration and invasion of Bel-7402 cells transfected with GAS5-siRNA-3,but these processes were inhibited in Bel-7402 cells co-transfected with GAS5-siRNA-3 or anti-miR-21 by Transwell assays and Wound healing analyses assays;The results were opposite in HCCLM3 cells co-transfected with pCDNA-GAS5 and miR-21-mimic.ConclusionOur results demonstrated that GAS5,as a tumor suppressor,is a prognostic indicator in HCC patients,further,it can suppress migration and invasion of HCC cells through regulating miR-21 and its targets,which have provided new diagnosis and targeted therapy for HCC developmen.