Down-regulation Of MiR-100-3p Promotes Hepatocellular Carcinogenesis

[Background]Hepatocellular carcinoma?HCC?accounts for more than 90%of primary liver cancer.China is one of the countries with high incidence and mortality of primary liver cancer in the world.At present,the mechanism of causing HCC has not been fully elucidated.A large number of investigations have shown that the tumorigenesis is the result of the interaction of environmental factors and genetic factors.Among genetic factors,microRNA is one of the factors that have been confirmed.MicroRNAs?miRNAs?,discovered in recent years,are small non-coding single-stranded molecule RNAs of 18-24 nt.MiRNAs play regulatory roles by binding to the 3'-untranslated region?3'UTR?of their target genes.MicroRNA-100?miR-100?is a member of the miR-99 family.The disregulation of miR-100 in various tumor tissues is considered to be closely related to the degree of tumor malignancy and prognosis.A large number studies indicate that miR-100 is down-regulated in hepatocellular carcinoma compared with corresponding adjacent tissues.However,it remains unclear whether the down-regulation of miR-100 promotes the genesis of hepatocellular carcinoma and what's the mechanism.Epithelial to mesenchymal transition?EMT?is a process cells undergo a switch from epithelial phenotype to mesenchymal phenotype.EMT plays a key role in tumorigenesis The Wnt/?-catenin pathway is fond to be closely related to EMT.However,it has not been reported whether the down-regulation of miR-100 in normal hepatocytes can activate the Wnt/?-catenin pathway and induce EMT.[Methods]1.In vitro experiments?1?A stable cell line was constructed in HL02 cell line with low expression of miR-100-3p by lentiviral transfection?HL02-miR-100-3p inhibitor?.?2?The effect of down regulation of miR-100-3p on the malignant behavior of HL02cells was examined by cell behavioral experiments?plate clone,soft agar colony formation,MTT,transwell,cell cycle?.?3?The mouse xenograft model was used to investigate the tumorigenesis ability of down regulation of miR-100-3p in HL02 cells through xenograft tumor growth assay.?4?The expression of EMT-related markers and molecules of Wnt/?-catenin pathway were detected by Western blot.2.In vivo experimentsMice homozygous for floxed miR-100 allele that carried the Alb-Cre transgene(miR-100^flox/floxAlb-Cre⁺)were developed by mating miR-100^flox/floxlox/flox mice with Alb-Cre⁺mice.The effects of miR-100 knockout in the liver on the phenotype and spontaneous HCC was investigated.Liver pathological section was used to evaluate liver histology,and transaminase assay was used to evaluate liver function changes.Western blot and qRT-PCR were used to detect the expression of miR-100 target genes IGF1R-?,CDC25A,mTOR and liver cancer related gene SHP-2 in liver tissues.[Results]1.A stable cell line with low expression of miR-100-3p was successfully constructed in HL02 cell line?designated as HL02-miR-100-3p inhibitor?.Down regulation of miR-100-3p promotes anchorage-independent growth in soft agar,migration and invasion of HL02 cells in vitro,and tumorigenesis in vivo.Consistently,the EMT related marker expressions were also significantly increased in HL02-miR-100-3p inhibitor cells.However,down regulation of miR-100-3p has no significant effect on the colony formation,cell viability and cell cycle.2.A miR-100 liver-specific knockout mouse model was successfully established with genotype of miR-100^flox/flox-Alb Cre⁺.The phenotype of miR-100 liver-specific knockout mice is not obviously different from miR-100^flox/flox?WT?mice younger than12 months.However,we found that miR-100 target gene IGFIR-?,and HCC related gene SHP-2,were up-regulated compared with the control mice.While the expression of mir-100 target genes,such as mTOR and CDC25A were unchanged.Surprisingly,liver pathological sections from 15-months-old miR-100 knockout mice showed significant hepatocyte edema;energy metabolism was biased toward the glycolysis phenotype despit that no spontaneous HCC was observed.[Conclusion]1.Downregulation of miR-100-3p induces EMT phenotypes through Wnt/?-catenin pathway in HL02 Cells.2.The knockdown of miR-100 in the liver has no effect on the mouse phenotype in the early stage,but can promote the up-regulation of miR-100 target gene IGFIR-?and liver cancer related gene SHP-2.Hepatocyte edema and metabolic bias in the glycolytic phenotype are observed in miR-100 knockout older mice.It is suggested that HCC genesis is the result of the interaction between genetic and environmental factors.Knockout miR-100 alone is difficult to cause the spontaneous HCC.Reproduction of spontaneous liver cancer model may require the combination with other inducing factors,such as DEN.