Overexpreesion Of Mitochondrial Telomerase On Drug Resistance In Human Hepatocellular Carcinoma Cells

Background and objective:Multidrug resistance (MDR) remains the major obstacle in clinical chemotherapy inpatients with Hepatocellular Carcinoma (HCC). This study focused on the role ofmitochondrial hTERT in multidrug resistance of HCC.Methods:Lentivirus(PLeno-mito-hTERT-GTP) expressing hTERT targetting to mitochondria wasconstructed by molecular cloning technology,and was verified by DNA sequencing. HepG2and SK-Hep1cells were transfected with Lentivirus PLeno-mito-hTERT-GTP,andexpression of mitochondrial hTERT was detected by laser confocal microscope and Westernblot; The resistance to multi-drugs was assessed by CCK-8;The level of activated caspase-3, mitochondrial membrane potential (MMP) were determined by laser confocalmicroscope; apoptosis was detected by flow cytometry;Mitochondrial DNA copy numberwas caculated by Q-PCR.Results:1. DNA sequencing and mitochondrial hTERT detection showed the PLeno-mito-hTERT-GTP lentivirus were constructed perfectly.2. HepG2cells transfected with PLeno-mito-hTERT-GTP lentivirus (HepG2–mito-hTERT) were insensitive to the chemotherapeutic drugs in contrary to HepG2cellsand HepG2-PLE cells (negative control) at the same treatment group(P <0.05).Similarly,compared with SK-Hep1and SK-Hep1-PLE cells at the same treatment group, SK-Hep1-mito-hTERT cells showed a significant difference in chemothearpeutic resistance tomulti-drugs(P <0.05).3． Compared with the cells transfected with PLeno-GTP lentivirus (negative control)cells, the reduced level of activated caspase-3in HepG2-mito-hTERT and SK-Hep1-mito-hTERT cells were found significantly(P <0.05); 4. In contrary to the parental cells and the negative cells,the obvious decreased levelof apoptosis were detected in mitochondrial hTERT over-expression HepG2cells after drugtreatment (P <0.05).5. Contrasted with the parental cells and the negative cells, the obvious increased levelof MMP were detected in mitochondrial hTERT over-expression HepG2cells after drugtreatment (P <0.05)5Mitochondrial hTERT over-expression HepG2cells had a significant increasedmitochondrial DNA copy number compared with negative control cells and the parentalcells before and after drug treatment (P <0.05);Conclusion：Mitochondrial hTERT overexpression may induce HCC cells resistant tochemotherapeutic drugs. Through increasing mtDNA copy number, and reducingactivation of the mitochondrial apoptotic pathway,mitochondrial hTERT contribute tomulti-drug resistance in HCC cells.