| Objective:Chemotherapy is one of the widely used methods of tumor therapy.However,chemotherapy resistance is facing a major problem to be solved,tumor resistance is complex,multi-factor,drug resistance mechanism is still unclear.Human telomerase reverse transcriptase(hTERT)is a kind of ribonuclease with reverse transcriptase activity.hTERT is the most important catalytic subunit of telomerase,which consists of hTERT,telomerase RNA and telomerase-associated protein.Telomeric repeat sequence can be synthesized based on its own template and added to the end of the chromosome,which will maintain the integrity and stability of the chromosome.Telomerase is mainly expressed in germ cells,embryonic stem cells and tumor cells,and its activity is inhibited in normal human cells.Telomerase can fill DNA defects and reduce the loss of telomeres during cell division.Its activity is closely related to the infinite proliferation potential of tumor cells.High expression of hTERT is closely related to the biological behavior of tumor cells,which affects the development and predicts prognosis of the disease.Most previous studies on hTERT mainly focused on the role of its reverse transcriptase activity involved in maintaining telomere length,tumor cell proliferation,invasion,metastasis and chemotherapy resistance,interestingly,accumulating evidence showed the importance of its non-reverse transcriptase activity.Studies have revealed that hTERT can promote anti-apoptosis,DNA damage repair,and participate in stemness maintenance and gene expression regulation even the absence of the reverse transcriptase function.It has also been demonstrated that hTERT can enter mitochondria under mitochondrial localization sequence and bind to mitochondrial DNA(mt DNA),which affects the stability of mt DNA.The expression of mitochondria hTERT was gradually upregulated with the increase of drug resistance of hepatocarcinoma cells,which indicated that mitochondrial hTERT was closely related to drug resistance of hepatocarcinoma cells,but the relevant mechanism was unknown.Therefore,elucidation the role and mechanism of hTERT mitochondria translocation in drug resistance of hepatocarcinoma cells will provide novel strategy for the treatment of liver cancer.Methods:Part Ⅰ:1.CCK-8 assay was applied to test the effect of telomerase activity on hepatocellular cell drug resistance after transfection of wild-type hTERT(WThTERT).2.TRAP and CCK-8 assay were performed to detect the role of dominant negative DNhTERT(D711A and V712I)on drug resistance of hepatocellular cell after transfection of DNhTERT.3.Western blot and immunofluorescence assays were employed to analyze the expression of hTERT in mitochondria in DNhTERT transfected cells.4.Western Blot and immunofluorescence were carried out to further confirm the expression of mitochondrial hTERT in drug-resistant cells.5.Cellular staining,flow cytometry and Western blot were conducted to identify the changes of stemness-related characteristics of in drug-resistant and the corresponding control cells.6.Mitochondrial localization sequence mutations(MPEAPECRAVRSLLRSHYRE)of hTERT(NUChTERT)lentivirus were constructed based on the telomerase activity(D711A and V712I)deletion plasmid DNhTERT,and then it was used to infect hepatocellular cells.Western blot and immunofluorescence were used to investigate the expression of mitochondrial hTERT in NUChTERT transfected cells,and CCK-8 assay was used to assay the effect of NUChTERT on drug resistance.7.Cellular staining,flow cytometry and Western blot were conducted to analyze the effect of NUChTERT on the mitochondrial membrane potential(Δψm),ROS,ATP and OCT-4.Part Ⅱ:1.Colorimetry assay was employed to testify the effect of DNhTERT on the activity of mitochondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ in Hepatocellular Carcinoma Cells.2.Colorimetry assay was employed to detect the influence of NUChTERT on the activity of mitochondrial respiratory chain complexesⅠ in Hepatocellular Carcinoma Cells.Results:Part Ⅰ:1.The chemotherapeutic resistance(cisplatin,CDDP)of Huh7 cells was significantly increased after transfected with WThTERT.2.The resistance to CDDP of Huh7 cells was enhanced after transfected with DNhTERT.3.Western blot and immunofluorescence experiments showed that mitochondrial hTERT expression increased in Huh7 and U2 OS after overexpression of DNhTERT.4.CCK-8 assay indicated the drug resistance index was significantly increased in drug-resistance cells HepG2/CDDP、Huh7/CDDP compared to the corresponding control cells,Western blot and immunofluorescence experiments demonstrated that mitochondrial hTERT expression was elevated in drug-resistance cells.5.Cellular staining with TMRE followed with con-focal demonstrated that mitochondrial membrane potential(MMP,Δψm)was markedly improved in the drug resistance cells,flow cytometry revealed that the ratio of CD133,EpCAM positive cells were augmented and WB illustrated that OCT-4 were up-regulated in the drug-resistant cells compared with parental cells.6.Western blot and immunofluorescence revealed that mitochondrial hTERT expression decreased in NUChTERT overexpression cells,and CCK-8 showed that resistance index(CDDP)of NUChTERT was dramatically reduced compared with DNhTERT.7.Cellular staining with TMRE followed with con-focal demonstrated that mitochondrial membrane potential(MMP,Δψm)was remarkablely increased,while MitoSox staining revealed that ROS was decreased in Huh7-DNhTERT cells.ATP was reduced while OCT-4 expression was elevated in Huh7-DNhTERT cells.However,the above effects were obviously weakened in Huh7-NUChTERT cells.Part Ⅱ:1.Colorimetry assay showed that DNhTERT repressed the activity of respiratory chain complex Ⅰin Huh7 cells without any influence on the activity of respiratory chain complex Ⅱ,Ⅲ,Ⅳ and Ⅴ.2.Colorimetric assay found that NUChTERT can restore the DNhTERT-repressed activity of respiratory chain complex I.Conclusion:1.The non-reverse transcriptase activity of hTERT promoted hepatocarcinoma cell chemoresistance.2.hTERT mitochondrial translocation contributed to chemoresistance in hepatocellular carcinoma cells.3.Mitochondrial hTERT inhibited the activity of respiratory chain complex Ⅰ,reduced ROS,ATP production and promoted cell stemness,which ultimately led to chemoresistance in hepatocellular carcinoma cells.Taken together,hTERT can enter into the mitochondria under the guidance of mitochondrial leader sequence,inhibits the activity of respiratory chain complexesⅠ,reduces the ROS and ATP production and promotes cell stemness leading to the drug resistance of hepatocellular carcinoma cells. |
