Targeting Knockdown AKT1 Inhibits The Proliferation And Migration In Hepatocellular Carcinoma Cells

Objects: HCC represents more than 90% of primary liver cancer,the prognosis for liver cancer is very poor.Although the operative treatment is still the most effective treatment so far,most HCC patients are diagnosed is too late and cannot receive surgical treatments and the 5-year survival rate is only 5 % without treatment.Therefore,it is of crucial significance to investigate novel therapeutic targets of HCC in order to explore a new effective feasible treatment for HCC.Increasing evidence has shown that PI3K/AKT/m TOR signaling pathway plays critical roles in tumorigenesis and development of hepatocellular carcinoma cells(HCC).AKT is the important point of the PI3K/AKT/m TOR signaling pathway which has been shown to play important role of regulating cell viability signal and closely relating to a variety of proliferation diseases.There are many data demonstrated that single AKT isoforms(i.e.AKT1,AKT2 and AKT3)have different or even opposing functions in the modulation of cancer cell proliferation,migration and invasion,Finding the hypothesis that AKT isoforms might have adverse effects on cancer dissemination.The AKT family of serine/threonine protein kinases,especially AKT1 isoform has been identified abnormally expressed in hepatocellular carcinoma(HCC)cells,and highly associated with cell behaviour,including proliferation,survival,metabolism,and tumorigenesis.However,the specific mechanism of the AKT1 needs further studying.The purpose of our study is to reveal the effects of AKT1 on the proliferation and migration of HCC cells,and to investigate the mechanisms involved.Methods: Total RNA was extracted from HCC cells with TRIzol Reagent and c DNA sample were subjected to q PCR using the SYBR Premix Ex Taq kit.We used reverse transcription-PCR(RT-PCR)to evaluate the the expression levels of the AKT1 in two HCC cell lines(SMMC-7721 and Hep G2).Blank plasmid and AKT1-RNAi plasmid extracted by plasmid extraction kit instructions and separately transfected cells with Effectene Transfection Reagent.After transfected for 48 h,the transfection efficiency was detected by PT-PCR.Molecular mechanisms and the influences of different regulation the expression were determined by Western blot.MTT assay was used to detect the effect of AKT1 on the proliferation of HCC cells.The wound healing assays explored HCC cells migration ability.All experiments were carried out three times.Results: As compared to the expression level in normal liver cell line HL-7702,AKT1 was significantly up-regulated in HCC cell lines,including Hep G2 and SMMC-7721(**p<0.01).We then assessed transfection efficiency of down regulation plasmid(AKT1-RNAi plasmid).The corresponding is down regulation plasmid inhibiting the expression of AKT1.MTT assay was performed to assess the role of AKT1 in HCC cell proliferation.The observation was showed that downregulation of AKT1 expression by AKT1-RNAi plasmid could suppress cell proliferation(p<0.05).The AKT1-RNAi plasmid-transfected cells SMMC-7721 and Hep G2 cells showed about the ability of wound healing significant decrease than control(**p<0.01).Down-regulation of AKT1 could inhibit proliferation and migration of HCC cell lines.Furthermore,it made down-regulation the activities and expression of MMP-2 and MMP-9 via knockdowning the transcriptional activation of NF-?B(**p<0.01).In the Western blotting assay,knockdown the AKT1 protein had obviously effect on the NF-?B,MMP-2 and MMP-9expression.MMP-2 and MMP-9 activities were lower in AKT1-RNAi plasmid-transfected relatived to Blank plasmid-transfected cells(**p<0.01).In addition,we found that down-regulation AKT1 inhibited the AKT1-mediated expression and secretion of NF-?B in HCC cells(**p<0.01).Conclusion:Our findings revealed that AKT1 activation plays an important role in the proliferation and migration of HCC cell lines.Blocking AKT1 treatment inhibited NF-?B nuclear expression levels and declined HCC development.We confirm that down-regulate AKT1 expression might inhibit MMP-2,MMP-9 expression level and catalytic activity via deducing NF-?B activation to inhibit the proliferation and migration in hepatocellular carcinoma cells.Targeting knockdown AKT1 inhibits the proliferation and migration in hepatocellular carcinoma cells.Molecular targeting of AKT1 could has the potential application value as a novel therapeutic approach to HCC.