The Mechanisms Of Growth Regulation Of Cancer Cells By Human Telomerase Catalytic Subunit (hTERT)

[Background and Object]The abnormal high expression of telomerase is closely correlated with pathogenisis and prognosis of cancer.The telomerase catalytic subunit of telomerase (hTERT) is a speed-limited factor of telomerase synthesis.Many studies have showed that inhibition of telomerase activity can depress the growth of cancer cells,and accompanyed with cell cycle arrest and apoptosis.Some reports claimed that the growth regulation of cancer cells by telomerase not only resulted from the sustain of telomere by telomerase,but also from the telomere-independent function of telomerase.RNAi(RNA interference) can efficiently inhibit the expression of target mRNA,and had been widely used to study the functions of genes and it had introduced the possibility of enzyme inhibition as an exciting prospect for cancer therapy.In this study,we aimed to use the chemically synthesized hTERT-siRNA to inhibit the expression of hTERT,and to observe whether growth inhibition and telomere-independent apoptosis can occur after telomerase activity depressed.Using the microarray,we investigated the changes of gene expression and microRNA related to the apoptosis.[Methods]The hTERT-siRNA synthesized chemically was transfected into the HeLa cells. The RT-PCR,Western blotting,RQ TRAP were used to investigate the inhibiting effect of telomerase activity and gene expression of hTERT by hTERT-siRNA. Through the soft agar colony forming assay and injecting the hTERT-siRNA into transplanted tumor of nude mice,we investigated the repression of cancer growth by hTERT-siRNA in vitro and in vivo,respectively.The apoptosis was detected in HeLa cells transfected hTERT-siRNA by TUNEL assay.Finally,using the cDNA microarray and miRNA microarray,we analyzed the differential expression of gene expression and microRNA.[Results]The result of RT-PCR showed that the relative expression level of hTERT mRNA in hTERT-siRNA group was smaller compared with the two control groups(hTERT-siRNA group:0.20;β-gal-siRNA group:0.73;LipofectAmine only group:0.78).The result of Western blotting showed the expression level of the fusion protein hTERT·myc in hTERT-siRNA group was significantly lower than those in the two control groups.The result of real-time quantitive TRAP showed the telomerase activity of hTERT-siRNA group was only 38%that of the two control groups.So it proved that the hTERT-siRNA designed is effective.In colony forming assay,the value abtained from the hTERT-siRNA group,1.00±1.00,was lower than that obtained from theβ-gal-siRNA group,4.33±0.58,or LipofectAmine only group,4.00±1.00.In animal experiment,the mice in hTERT-siRNA group developed smaller tumor size of 940.12±244.2mm~3 and lower tumor weight of 0.71±0.23g,compared with 2137.9±403.72 mm~3 and 1.46±0.32g forβ-gal-siRNA, and 1896.92±291.23 mm~3 and 1.34±0.23g for LipofectAmine only group.The results showed hTERT-siRNA can remarkably depressed tumorigenicity of HeLa cells in vitro and in vivo.TUNEL assay showed,48 hours after transfected,the apoptosis rate in hTERT-siRNA group was 86.9%±3.5%,and it was higher than that inβ-gal-siRNA group,2.6%±2.3%,or that in LipofectAmine only group,9.0%±2.3%.It indicated that hTERT-siRNA can induce the apoptosis in some telomere-independent manners.Through cDNA microarray screening,we found 54 genes that up-regulated more than 4 folds or down-regulated more than 4 folds between the hTERT-siRNA group and theβ-gal-siRNA group.Among them,16 genes may relate to apoptosis.We validated 6 of them using semi-quantitive RT-PCR. MicroRNA chip showed there were 18 microRNAs expression in hTERT-siRNA group and non-expression inβ-gal-siRNA group,and only one microRNA expression inβ-gal-siRNA group and non-expression in hTERT-siRNA group..[Conclusions]hTERT can inhibit the apoptosis of cancer cells in some telomere-independent manners.hTERT-siRNA can induce the apoptosis of cancer cells and depress the energy for growth in vivo and in vitro.Through cDNA microarray and microRNA microarray screening,we found some genes related to hTERT-induced apoptosis and the differential expression of microRNA.