The Inhibitory Effect Of MiR-199a On Biological Behavior And Its Possible Mechanism In Human Hepatic Carcinoma Cell

microRNAs (miRNAs) are small,evolutionarily conserved,endogenous and noncod- ing RNA molecules of about 22 nuleotides in length that function as post- transcriptional gene regulators. MiRNAs inhibit the expression of target genes by affecting the translation or stability of target miRNA by binding to a target site in the 3'-UTR of target mRNAs. Researches indicate that miRNAs are involved in various biological processes,including development,differentiation,cell proliferation and its death. Accumulating evidence suggests that alterations of their expression may play a role in the development of human cancers. A miRNA,usually down-regulated in a particular human cancer (a"tumor suppressor"miRNA),can have tumor suppressor- like effects if the main targets for that specific cell types are oncogenes.miR-199a are highly conserved miRNA,always deregulated in numerous human tumors. In this study, to validate the down-modulation of miR-199a in HCC compared with normal matched tissue, we determined miR-199a expression in human HCC and its normal adjacent tissue (NAT) by quantitative reverse transcription PCR (qRT-PCR) assay. The results show that miR-199a was significantly reduced in hepatocellular carcinoma when compared with normal tissues. Consequently we hypothesized that down-regulated of miR-199a may be associated with hepatocarcinogenesis and the restoration of miR-199a may inhibit tumorigenic growth of HCC. Methods1.The expression level of miR-199a in 11 pairs of matched HCC neoplastic and adjacent non-neoplastic tissues were examined by stem-loop quantitative real-time PCR analysis .2. A search was conducted to locate putative targets of miR-199a through silico analysis. We looked for the target genes of miR-199a with the application of bioinformatics tools. Potential target genes expression were assessed by western blot for proteins, real-time PCR for miRNAs. Luciferase repot gene assays were carried out using HIF-1α-3'-UTR reporter constructs or its miR-199a binding site mutant construct co-transfected with miR-199a precusor mimics.3. Establishment and characterization of miR-199a high expression stable clones The Lentivial vector Lenti-miR-199a or Lenti-scramble and Lentiviral packaging plasmids Arrest-In co-transfected 293FT packaging cells, 48 hours later, the Lentivirus in supernatant were collected and filtered,used to infect cancer cells with polybreen. The stable clones were selected by antibiotic resistance and strong red fluorescence,validated by Real-time PCR for miR-199a expression,western blot for proteins. Luciferase repot gene assays were carried out in the stable clones.4.To gain an insight into the biological effect of miR-199a on HCC tumorigenesis For the miR-199a mimics transfected cancer cells and miR-199a stable clones,the following assays were employed to examine the biological effects:(1) Cells were counted to generate cell growth curves;(2) MTT assay for cell proliferation;(3) Colony formation assay for clonogenic growth;(4) Cell cycle analysis by flow cytometry;(5) Transwell invasion assay for cell invasion.The miR-199a stable clones inoculated in nude mice is treated with miR-199a and the growth rate and volume of tumors in vivo are compared with the controls.The tumors and animal body weight were investigated for tumor growth.Results1.MiR-199a was significantly down-regulated in 11 primary hepatocellular carcinomacarcinoma tissues compared with pair-matched adjacent non-tumor tissues.2.The expression of miR-199a was also found to be substantially reduced in several human hepatocellular carcinoma cell lines, including HepG2, SMMC-7721, BEL-7402, MHCC-97H, and BEL-7401. Bioinformatics analysis deduced a key transcription factor, HIF-1α, as the target of miR-199a. Western blot results showed that cancer cells transfected with miR-199a mimics have reduced expression of target genes HIF-1αin normoxia and hypoxia, Real-time PCR confirmed that there were no changes in the target genes mRNA. Further investigation revealed that miR-199a significantly repressed the activity of luciferase carrying the 3'-untranslated region of HIF-1αand reduced the endogenous protein level of HIF-1α.3. The Lentivial vector Lenti-miR-199a or Lenti-scramble and Lentiviral packaging plasmids arrest-in co-transfected 293FT packaging cells,48 hours later,the Lentivirus in supernatant were collected and filtered,used to infect cancer cells with polybreen. After antibiotic selection for two weeks, the stable clones were obtained. The expression of miR-199a was also found to be increased in the stable clones. Western blot results showed that the miR-199a stable clones have reduced expression of HIF-1αand P-AKT. Real-time PCR confirmed that there are no changes in the target genes mRNAs; HIF-1α-3'-UTR reporter assay showed that miR-199a in the cells are functional.4. In addition, significant decreases in cell growth were observed after transfection of miR-199a precusors into human hepatocellular carcinoma HepG2 cells.(1) Cell count experiments showed that miR-199a could significantly inhibit the cell proliferation of HepG2 cells.(2) MTT assay indicated that miR-199a could inhibit HepG2 cells growth when compared with negative control .(3) MiR-199a reduced HepG2 cells colony formation.(4) MiR-199a could arrest HepG2 cells at G1/G0 as indicated by flow cytometric analysis.(5) MiR-199a could reduce cell invasion.ConclusionsThese findings suggest the involvement of miR-199a miRNA in the growth of hepatoma cells and carcinogenesis of HCC partly by down-regulating HIF-1αexpression. Thus, miRNA may be useful as a novel therapeutic target in HCC.