Mechemism Of HTERT RNAi Inducing Hepatocarcinoma HepG2 Cell Apoptosis

Background & Aims:Hepatocellular carcinogenesis is a multifactorial,multistep,and complex process in which activation of oncogenes,inactivation of tumor-suppressor genes, genetic instability and reactivation of telomerase are involved.It has been found that telomerase becomes a important tumor biological characteristics owing to its exist in extensive tumor cells.Human telomerase reverse transciptase(hTERT)is the catalytic subunit and rate-limiting enzyme of telomerase and plays a key role to maintain telomerase activity.It has been reported that 80%to 90%of hepatocellular carcinomas(HCCs) overexpress hTERT.From our previous researchs,we draw a conclusion that hTERT RNA interfere(RNAi) may inhibit the telomerase activity to induce hepatocarcinoma HepG2 cells apoptosis and senescence.However the molecular mechanism is unclear.Mitochondria is central of adenosine triphosphate(ATP) production in cells,and have been described to be involved in apoptotic process.Mitochondrial control of apoptosis has been described at two levels:(1) maintenance of ATP production and(2) mitochondrial membrane potential (△Ψm) and mitochondrial membrane permeability for the release of certain apoptogenic factors from the intermembrane space(IMS)into the cytosol,but its mechanism is not fully understood.therefore,We attempt to explore primarily probable mechemism that hTERT RNAi induces cell apoptosis by mitochonrial pathway via observing changes of△Ψm, telomerase,apoptosis-associated protein and mitochondrial release of four IMS pro-apoptotic proteins in the HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT(small hairpin RNA hTERT).Methods:MTT assay and flow cytometry were used to determine cell growth,△Ψm and cell apoptosis;△Ψm and morphological feature of karyon was observed by confocal microscope;Telomerase activity was detected by telomeric repeat amplification protocol (TRAP);Expressions of caspase-3,X-linked inhibitor of apoptosis protein(XIAP), caspase-9,hTERT,Bcl-2,Bax and Bcl-xl,mitochondrial and cytoplasmic cytochrome C (cyt C),second mitochondria-derived activator of caspase(Smac),apoptosis inducing factor(AIF) and endonuclease G(Endo G),and heat shock protein(Hsp60) in HepG2 cells transfected with pSliencer 3.1-H1 neo-shTERT(transfected cells),control cells and untransfected cells were detected by western-blot or immunocytochemistry(ICC).Results:(1) Compared to that in the untransfected cells,proliferation in three groups of control cells,the eleventh and thirtieth generation transfected cells was slightly decreased,and the twentieth transfected cells were obviously reduced;There are no significant difference in proliferation rates among three groups of control cells; Proliferation in the thirtieth generation cells is the rapidest,the twentieth generation cells is midest and the eleventh generation cells is slowest among three group of transfected cells; (2) The percentage of apoptotic cells in the eleventh transfected cells has no significant change compared to that in relevant control cells;A increased percentage of apoptotic cells in the twentieth and thirtieth generation transfected cells was observed compared to that in relevant control cells(P＜0.05)and compared to that in the eleventh generation transfected cells(P＜0.05);There is no significant difference in percentage of apopototic cells between the twentieth generation and the thirtieth generation transfected cells(P=0.051),and between untransfected cells and three groups of control cells(P＞0.05);(3) In comparison with that in relevant control cells,the percentages of depolarized△Ψm in three groups of transfected cells were obviously increased in a post- transfected generation-dependent manner(P＜0.05),however no significant differences were found among untransfected cells and three groups of control cells(P＞0.05);(4) The changes of△Ψm was observed by confocal microscope.A lot of red fluorescence was found in untransfected cells and three groups of control cells under confocal microscope and a little of green fluorescence was also identified in the eleventh generation transfected cells.A repression of red fluorescence and an increase of green fluorescence were observed in the twentieth and the thirtieth transfected cells.An increase of green fluorescence was found in the thirtieth transfected cells compared to that in the twentieth transfected cells;(5) Compared with relevant control cells,three groups of tansfected cells showed a significant decrease in expression of mitochondrial cyt C and a significant increase in cytoplasmic cyt C(P＜0.05).There are no significant differences in expression of mitochondrial and cytoplasmic cyt C amomng three groups of tansfected cells(P＞0.05)and amomng untransfected cells and three groups of control cells(P＞0.05);(6) Expressions of mitochondrial Smac in the twentieth and the thirtieth transfected cells were significantly decreased and expression of cytoplasmic Smac were obviously increased compared to that in relevant control cells(P＜0.05)and expression of mitochondrial Smac in the thirtieth transfected cells were significantly decreased and expression of cytoplasmic Smac were obviously increased compared to the twentieth transfected cells(P＜0.05)and no singnificant differences were found in expression of mitochondrial and cytoplasmic Smac between the eleventh transfected cells and the control cells,and between the untransfected cells and three groups of control cells(P＞0.05);(7) Expressions of AIF and Endo were detected by ICC.Expression of AIF and Endo was positive only in cytoplast in the untransfected cells,the eleventh transfected cells and three groups of control cells;Expressions of AIF and Endo were positive not only in cytoplast but also in partial karyon in twentieth and the thirtieth transfected cells;(8) Expression of mitochondrial AIF and Endo G showed no significant change in the eleventh transfected cells compared to that in relevant control cells;Expression of mitochondrial AIF and Endo G were significantly decreased(P＜0.05)and expression of cytoplasmic AIF and Endo G have no significant change(P＞0.05)and expression of nuclear AIF and Endo G were obviously increased(P＜0.05)in the twentieth and the thirtieth transfected cells compared to that in relevant control cells;There was no signicant difference found in the each other groups(P＞0.05);(9) Expression of mitochondrial Hsp60 in the twentieth and the thirtieth transfected cells was significantly decreased and expression of cytoplasmic Hsp60 were obviously increased compared to that in relevant control cells(P＜0.05);There was no significant different expression of mitochondrial and cytoplasmic Hsp60 between the twentieth and the thirtieth transfected cells(P＞0.05);There were also no significant different expression found between the eleventh transfected cells and control cells and between the untransfected cells and three groups of control cells(P＞0.05);(10) Expression of hTERT and Bcl-2 in three groups of tansfected cells were significantly decreased compared to that in relevant control cells(P＜0.05);No significant difference expression of hTERT and Bcl-2 was found among three groups of tansfected cells(P＞0.05)and untransfected cells and three groups of control cells(P＞0.05);(11) Relative telomerase activity in the twentieth and the thirtieth transfected cells was significantly lower than that in relevant control cells and telomerase activity in the thirtieth transfected cells were significantly decreased than the twentieth transfected cells;No significant difference was found in telomerase activity among the untransfected cells,the eleventh transfected cells and three groups of control cells(P＞0.05);(12) No significant different expression of Bcl-xl was found among untransfected cells,three groups of control cells and three groups of tansfected cells(P＞0.05);(13) Expression of Bax and Caspase-3 in the twentieth and the thirtieth transfected cells were significantly increased compared to that in relevant control cells(P＜0.05),but no significant different expression was found between in the twentiethand and in the thirtieth transfected cells(P＞0.05);No significant different expression of Bax and Caspase-3 was found among the eleventh transfected cells,the eleventh control cells,the untransfected cells and three groups of control cells(P＞0.05);(14) Expression of XIAP in the twentieth and the thirtieth transfected Cells were significantly decreased compared with that in relevant control cells(P＜0.05) and significantly lower in the thirtieth transfected cells compared to the twentieth transfected cells(P＜0.05);No significant different expression of XIAP was found in the eleventh transfected cells comprared to that in the eleventh control cells and in the untransfected cells comprared with that in three groups of control cells(P＞0.05);(15) Untransfected cells,the eleventh transfected cells and the eleventh,the twentieth and the thirtieth control cells showed only procaspase-9 expression,while the twentieth and the thirtieth transfected cells showed not only procaspase-9 expression but also caspase-9 p35 expression;(16) The the eleventh transfected cells,and the eleventh,the twentieth and thirtieth control cells showed a round or oval nucleus with irregular clumping chromatin and evident nucleoli;In portion of the twentieth and thirtieth transfected cells,condensation of nucleus,compaction of chromatin, nucleoli shrinkage or disappear and nucleus fragment could be found.Conclusion:(1)Hepatocarcinoma HepG2 cell apoptosis induced by hTERT RNAi is a slow and gradual progress in which△Ψm decline is an early event,and apopototic cells gradually increase with△Ψm decrease;(2) a△Ψm decline may promote mitochondrial IMS proteins release from mitochondria to cytoplasm,and cyt C release is earlier than Smac, AIF,Endo G release;(3)△Ψm decline is earlier than telomerse activity inhibition,thereby we presume that hTERT may adjust cell apopotosis by a pathway independent on telomerase activity;(4) In hepatocarcinoma HepG2 cell apoptosis induced by hTERT RNAi, Bcl-2 and Bax regulate mitochondrial IMS proteins release together,and Bcl-2 may be initial factor to adjust mitochondrial pathway in Bcl-2 family protein;Bcl-xl may be involved in apoptosis induced by hTERT RNAi;(5) Mitochondrial matrix protein-Hsp60 and IMS proteins release from mitochondria to cytoplasm,suggesting that IMS proteins may be released by mitochondrial permeability transition pore;(6)Cytoplasmic cyt C and Smac activate caspase-3 by different pathways:first cyt C activate caspase-9 by Apaf-1/ procaspase-9 pathway,then caspase-3;cytoplasmic Smac inhibit the expression of XIAP and activate caspase-3 in direct or indirect manner;(7) Cytoplasmic AIF and Endo G release from mitochondria to cytoplasm,then enter nuclei and led to nuclear condensation and fragmentation.