Expression Of Human Telomerase Reverse Transciptase In Cancer Stem-like Cells

Objective：1. To detect telomerase activity by RQ-TRAP and TRAP-ELISA in different cells inthe sake of proving the superiority of RQ-TRAP.2. To investigate the regulative mechanism oftelomere length in cancer stem-like cells through comparing the difference in telomere lengthbetween cancer stem-like cells and common cancer cells.3.To compared the difference oftelomerase activity, hTERT mRNA and hTERT protein levels between cancer stem-like cells andcommon cancer cells.Methods：1.To detect telomerase activity by RQ-TRAP and TRAP-ELISA in12kinds of cellsincluded9tumor cell lines and3normal cell lines. The results between2kinds of lines werecompared.2. Serum-free suspension culture was used to culture sphere forming cells-SFCs. Todetect the changes of cancer stem-like cells percentage by flow cytometry. RT-PCR were used toexplore the express of Oct-4,SOX-2and CK-18.3. Telomere length in cancer stem-like cells andnormal cancer cells were detected by PCR（Q-PCR）.4. To detect telomerase activity in cancerstem-like cells and normal cancer cells by TRAP-ELISA.5. RT-PCR were used to detect theexpress of hTERT mRNA levels and Western blot were used to detect the express of protein levelsin cancer stem-like cells and normal cancer cells.Results：1.The RQ-TRAP method was both accurate and specifical in measuring the telomeraseactivity of a dilution series of protein extracts from293T cells. The sensitivity of this method was8cells, and the amplification efficiency was99%. Telomerase activity was not detected innegative control group. Statistical analysis revealed a strong correlation between the two assays(r2=0.7625).2. Serum-free suspension culture got a higher proportion of cancer stem-like cells.Compared with normal tumor cells, cancer stem-like cells got a higher expression in the level ofOct-4、SOX-2and a lower expression in CK-18(P＜0.05).3. The telomere length of MCF7-SFCs（1.201±0.05212）was longer than MCF7（1.019±0.1005），the telomere length of Hela-SFCs （1.602±0.1355） was longer than Hela (1.024±0.0977), but there was no statisticallysignificant difference(P>0.05).4.Both cancer stem-like cells and normal cancer cells are positivein telomerase activity, but the cancer stem-like cells was weaker than normal tumor cells inrelative telomerase activity(RTA),and there was a statistically significant difference(P<0.05).5.The expression of hTERT mRNA of MCF7-SFCs was weaker than MCF7，The expression ofhTERT mRNA of Hela-SFCs was weaker than Hela, and there was a statistically significantdifference (P<0.05). The hTERT protein levels express in cancer stem-like cells were weakerthan cancer cell. Conclusion:1.The feasibility of RQ-TRAP was proved in this article. Compared with TRAP-ELISA, the RQ-TRAP method has many advantages. Other than sample extraction and real-timecycling, no additional time-consuming steps have to be performed for telomerase quantification；the costs are less and supports high throughput.RQ-TRAP provides a new tool for the rapid andreliable quantification of telomerase activity.2.The telomere length of human cancer stem-likecells was longer than cancer cells in MCF7cell lines and Hela cell lines, but telomerase activityand the levels of hTERT expression in cancer stem-like cells were weaker than cancer cell.