The Expression Of MiR-32 And Targets ADAMTS9 In Hepatocellular Carcinoma Cell Lines

Objective: Many microRNAs(miRNAs)have been found in hepatocellylar carcinoma(HCC),it's abnormal expression suggested that mi RNAs might participate in the progression of HCC,but the mechanism is still not exact clear.Reasently,A disintegrin and metallo-proteinase with thrombospondin motifs-9(ADAMTS9)has been found as a tumor-inhibiting factor.ADAMTS9 plays an important role in tumorigenesis and progression of many kinds of cancer.In this study,we used quantitative real-time fluorescent quantitative PCR(qRT-PCR)to find the expression of miR-32 in 3 HCC cell lines-SMMC-7721,Huh7 and HepG2,then Dual-Luciferase Report Gene System was used to explor the relationgship between miR-32 and ADAMTS9 in HCC.We hoped that it would provide a new idea to study the mechanism of HCC deeply and a broad application prospects in the early diagnosis and treatment and biological treatment of HCC.Methods:1 cell culture: HCC cell lines SMMC-7721 was purchased from Cell Bank of Chinese Academy of Science(Shang Hai),HCC cell lines Huh7 and HepG2,human renal epithelial cells 293 T were from Hebei Medical University.SMMC-7721,Huh7 and HepG2 were cultured in DMEM medium(Hyclone)and 293 T was cultured in RPMI-1640 medium(Gibco),they were all supplemented with 10 % fetal bovine serum and 1% penicillin-streptomycin solution(Solarbio).Cells were incubated at 37 °C under a 5 % CO2 condition.2 RNA extraction and quantitative PCR: Total RNA containing miRNAs and mRNAs from cells were extracted with TRIzol reagent(Jie Rui,Shang hai)according to the manufacturer's protocol.RNA was resuspended in DEPC-treated H2 O.The relative levels of miR-32 was examined by qRT-PCR using U6 as a control.The primer of miR-32 for reverse transcription(RT)was: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA TACGACTGCAACTT-3',The primer of ADAMTS9 for RT was random primers in the kit.The primer of mi R-32 for PCR was 5'-GTGCAGGGTCCGAGGTATT-3'(forward primer);5'-GCCGCTATTGCA CATTACTAAGTT-3'(reverse primer),The primer of ADAMTS9 for PCR was 5'-CATAATGAACAGGATGGGCCT-3'(forward primer);5'-TTGACCACATCCAGGGGTTG-3'(reverse primer).The expression of ADAMTS9 mRNA was detected by quantitative PCR and ?-actin gene was used as a control.Quantitative PCR was performed on MX3005 P Real-time PCR Instrument(Agilent)using Real-time PCR Mixture Reagent(Promega)according to the manufacturer's instructions.The relative expression levels of mi R-32 was calculated by the 2-??CT method.3 Cell transfection: The cells of SMMC-7721 were grown in 6-well plate(1×106 per well)when the amount about 70%,then transfected with 150 nm/well of miR-32 inhibitor or control microRNAs(Rui bo,Guang zhou)using Lipofectamine 2000(Invitrogen)according to the protocol.The plasmid of miR-32 or miR-32-NC(Genechem,Shang Hai)were transfected with 2ug/well by using Lipofectamine 2000 and RNA was extracted after 24 h of transfection.4 Luciferase reporter assay: For luciferase reporter experiments,Pmi R-ADAMTS9-WT and PmiR-ADAMTS9-Mut were constructed by Genechem,Shang Hai.293 T cells were co-transfected in 24-well plates with 0.5ug of the firefly luciferase and renilla luciferase report vector,as well as with 0.5ug of miR-32 or miR-32-NC.Firefly and renilla luciferase activities were measured consecutively using dual-luciferase assays.5 Statistical analysis: Data are expressed as the mean±SD from at least there separate experiments.Differences between groups were analyzed with Student's t test or one-way ANOVA using SPSS software version 17.0.A value of P<0.05 was considered statistically significant.1 The expression of miR-32 in HCC cell lines SMMC-7721,Huh7 and HepG2The purity of total RNA was high and with no obvious degradation by using an ultraviolet spectrophotometer.The melting curves of miR-32 and U6 were “S” which had a singlet at 80? and 82?,indicating that the primers were specific.The cDNA could have an effective amplification according to the amplification curve of miR-32 and U6.The expression of miR-32 in HCC cell lines SMMC-7721,Huh7 and HepG2 has no statistical significance(P>0.05).2 The expression of mi R-32 and ADAMTS9 mRNA after transfected with group of miR-32 over-expressing plasmid or group of miR-32-5p inhibitor in HCC cell lines SMMC-7721The purity of total RNA was high and with no obvious degradation by using an ultraviolet spectrophotometer.The method to analyze the melting curves and amplification curve was the same as above.The expression of miR-32 after transfected with mi R-32 over-expressing plasmid in SMMC-7721 cells was up-regulated(P<0.05)and transfected with miR-32-NC plasmid had no obvious change(P>0.05).The expression of miR-32 after transfected with miR-32-5p inhibitor was down-regulated(P<0.05)and transfected with mi R-32-5p NC had no obvious change(P>0.05).The expression of ADAMTS9 mRNA after transfected with miR-32 over-expressing plasmid in SMMC-7721 cells was down-regulated(P<0.05),when transfected with miR-32-5p inhibitor,the expression of ADAMTS9 mRNA was up-regulated(P<0.05).3 dual-luciferase assays293T cell line co-transfected with PmiR-ADAMTS9-WT and miR-32 plasmid showed a decrease of reporter activity in comparison with those co-transfected with the PmiR-ADAMTS9-WT and miR-32-NC plasmid(P<0.05),when co-transfected with PmiR-ADAMTS9-WT and mi R-32 plasmid showed a decrease of reporter activity in comparison with those co-transfected with the PmiR-ADAMTS9-Mut and mi R-32 Results: plasmid(P<0.05).miR-32 could suppress the reporter activity of ADAMTS9 by binding to the ADAMTS9 3'UTR.Conclusion: miR-32 could suppress the transcribe of ADAMTS9 by binding to the ADAMTS9 3'UTR and targets ADAMTS9.