The Study Of Noxa And Puma Gene Induced Apoptosis Of Hepatocellular Carcinoma Cell Induced By HTERT Promoter

As a malignant tumor,primary liver cancer(PLC)is a serious threat to people’s health and quality of life.Hepatocellular carcinoma is the sixth largest cancer in the world,and is the second most common cancer in China,with more than 50% of the world’s liver cancer occurring in our country.Surgical treatment、chemical treatment、radiation therapy、biological therapy、Chinese medicine therapy and so on are main clinical methods in the treatment of liver cancer,but liver cancer as a kind of cancer which has high invasive character,early symptoms is not obvious,so now the therapeutic effect of hepatocellular carcinoma and the overall prognosis is still poor.In recent years,with the continuous development of molecular biology and oncology,the molecular targeted therapy for tumor has become a new ideal strategy.Apoptosis is a process of programmed cell death which is regulated by a variety of genes.By activating the apoptosis of tumor cells can be a method to treat tumor.The apoptosis process of cells can be regulated by Bcl-2 protein family.In recent years,Noxa and Puma protein were found to be two kinds of apoptotic proteins of the BH3-only protein family with strong apoptotic effect.Telomerase is known of the broad spectrum of tumor markers,human telomerase reverse transcriptase(human telomerase reverse transcriptase,hTERT)is one of the components of telomerase,and is a key limiting step of telomerase activation.hTERT expression level in normal cell is very low,in contrast,with large amounts of expression in tumor cells.The upstream of hTERT transcription start site has multiple transcription factor binding sites,prompt hTERT promoter not only has a tumor targeting character,but also has strong translation initiation activity.Therefore,our study is intended to study the effects of Noxa and Puma gene regulated by hTERT promoter on the proliferation、apoptosis 、 migration and invasion of HepG2 cells and their potential molecular mechanisms.Objective:To explore the effects of pcTERT-Noxa and pcTERT-Puma recombinant plasmid on the proliferation 、apoptosis、 migration and invasion of HepG2 cells and their potential molecular mechanisms.Methods:(1)Cell proliferation analysis: two kinds of recombinant eukaryotic expression plasmid pcTERT-Noxa and pcTERT-Puma respectively transfection HepG2 cells after24 h,48h,72 h,the cell proliferation were analysised by following experiments:(1)Cell morphological observation;(2)The survival rate of HepG2 cells after transfection was analyzed by CCK-8.(2)Apoptosis analysis: two kinds of recombinant eukaryotic expression plasmid pcTERT-Noxa and pcTERT-Puma respectively transfection HepG2 cell,cell apoptosis were analysised by the following experiments:(1)Hoechst33258 dye analysis;(2)Annexin V-FITC analyzed the apoptosis rate of HepG2 cells after transfection;(3)The mRNA expression level of bcl-2、bax、mcl-1 and A1 were detected by real-time PCR;(4)The expression level of apoptosis related proteins Bcl-2 、 Bax 、cleaved-Caspase3 、 cleaved-Caspase8 and cleaved-Caspase9 were analyzed by Western blot.(3)Cell migration and invasion analysis: two kinds of recombinant eukaryotic expression plasmid pcTERT-Noxa and pcTERT-Puma respectively transfection HepG2 cell,the cell migration and invasion ability were analysised by the following experiments :(1)The clone forming experiments;(2)The experiment of cell scratches;(3)Transwell assay;(4)The mRNA expression level of mmp-9 was detected by real-time PCR.(4)Tumor cell targeting character analysis: two kinds of recombinant eukaryotic expression plasmid transfection human liver cancer cell HepG2 and human normal hepatic cell HL7702,using the method of CCK-8 compared the two kinds of cell’s survival rate.Results:(1)Recombinant plasmid pcTERT-Noxa and pcTERT-Puma significantly inhibited proliferation of HepG2 cells.First under the optics microscope,pcTERT-Noxa,pcTERT-Puma group compared with the blank group and the negative control group,showing a more typical character of apoptotic cells,such as cell shrinkage,turn round,internal particles increased,the number of exfoliated cells increased,and these phenomenon gradually increased with the extension of time.The results of the detection of cell survival rate by CCK-8 method showed that compared with the pcTERT group,pcTERT-Noxa,pcTERT-Puma group’s cell survival rate significantly decreased and were time-dependent.After 72 h transfection,the survival rate of both groups decreased by about 30%.Meanwhile,the cell survival rate of the pcTERT-Puma group was slightly lower than that of the pc TERT-Noxa group at all time points.(2)Recombinant plasmid pcTERT-Noxa and pcTERT-Puma significantly promote the apoptosis of HepG2 cells:(1)After Hoechst33258 dye and observe in fluorescence microscope,pcTERT-Noxa and pcTERT-Puma group appeared more apoptosis cells.After preliminary calculation of the cell apoptosis rate,the results showed that the apoptosis rates of the pcTERT-Noxa 、 pcTERT-Puma group and pcTERT group were(31.09±3.87)%,(34.87±3.23)% and %(15.92±2.9)%(P<0.05),respectively;(2)The Annexin V-FITC method analysis the cell apoptosis rate,according to the results of pcTERT-Noxa、pcTERT-Puma plasmid transfection after72 h,HepG2 cell’s apoptosis rate were(29.35±0.64)% and(32.9±0.71)% respectively,were significantly higher than negative control pcTERT group(18.67±1.21)%(P<0.01),and pcTERT-Puma group’s cell apoptosis rate is higher than pcTERT-Noxa group;(3)The results of real-time PCR experiment showed that after transfection HepG2 cells 72 h,pcTERT-Noxa,pcTERT Puma group compared with pc TERT group,the resistance to apoptosis related gene bcl-2,mcl-1,bcl2A1 mRNA expression level decreased significantly(P<0.05),and promote apoptosis gene bax mRNA expression level was significantly increased(P<0.05);(4)The results of protein immunoblot analysis showed that after the transfection HepG2 cells 72 h,pcTERT-Noxa,pcTERT-Puma group compared with pcTERT group,the expression of apoptosis related proteins the Bcl-2 levels decreased significantly(P < 0.05),Bax 、cleaved-Caspase3、cleaved-Caspase8、cleaved-Caspase9 expression levels increased significantly(P<0.05).(3)Recombinant plasmid pcTERT-Noxa and pcTERT-Puma obviously inhibit the migration and invasion of HepG2 cells:(1)The results of clony formation experiment showed that the number of cell clone of the blank group and the control group is more,and a single clone is big compared with the pcTERT-Noxa and pcTERT-Puma group,and the clone formation rate has significant difference;(2)The results of cell scratch experimental showed that after making the cell wound model,pcTERT-Noxa,pcTERT-Puma group compared with pcTERT group,with the extension of time,the spacing decrease trend is very weak,and had more exfoliated cells.After 24、48、72h of making the cell wound model,pcTERT-Noxa,pcTERT-Puma scratches healing rate were significantly lower than pcTERT group;(3)As shown by the results of Transwell small chamber,when two recombinant plasmids were transfected into HepG2 cells,the cells attached to the subcompartment polycarbonate membrane of the pcTERT-Noxa and pcTERT-Puma groups were significantly lower than those in the pcTERT group and the blank group;(4)The results of real-time PCR analysis showed that,after transfection of HepG2 cells for 72 h,the mRNA expression level of mmp-9 mRNA was significantly decreased in the pc TERT-Noxa and pcTERT-Puma group compared with the pcTERT group(P<0.05).(4)To preliminarily study the targeted effect of recombinant plasmid on livercancer cells.The two recombinant plasmid transfection HepG2 cells and normal hepatocyte HL7702 cells,according to the results of HL7702 cell survival rate with time extend downward trend is not obvious,and the extension of HepG2 cell survival over time showed obvious downward trend,and at all time points HL7702 cell survival rate compared with HepG2 cells have significant difference(P<0.05).After transfection,the survival rate of HL7702 cells in the pcTERT-Noxa and pcTERT-Puma group decreased by only about 20% at 72 h,while the survival rate of HepG2 cells decreased by about 50% after transfection.It is suggested that the two recombinant plasmids have different inhibitory effects on the proliferation of HepG2 cells and normal cells.Conclusions:(1)pcTERT-Noxa and pcTERT-Puma plasmid could induce the apoptosis of human liver cancer cell HepG2,the two plasmids could inhibit the migration and invasion of human liver cancer cell HepG2.(2)The proliferation inhibition effect of pcTERT-Noxa and pcTERT-Puma on HepG2 cells and normal HL7702 cells was shown to be different.