Identification of hTERT gene regulator：RNAi apporach

• Main Authors: Sheng Chin Wang, 王聖智
• Other Authors: T. C. Wang
• Format: Others
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/39405814439728171143

碩士 === 長庚大學 === 生物醫學研究所 === 98 === Human telomerase is a specialized reverse transcriptase that contains a template-containing RNA component (hTR), a human telomerase reverse transcriptase (hTERT) and many associated proteins. Telomerase directs the synthesis of telomeric repeats (TTAGGG) at chromosomal ends and is thought to play an important role in the maintenance of telomere length and cellular senescence. Telomerase activity is repressed in most human somatic cells, but is activated in most cancers and immortal cells. Exactly how telomerase activity is regulated in human cell remains poorly understood. Studies on the regulation of telomerase activity in human cells indicate that transcriptional regulation of hTERT is the primary determining factor governing the expression of telomerase activity. In this work, the involvement of signaling pathways in the regulation of hTERT expression was examined by using an RNAi approach with a pooled RNAi library against kinases and phosphatases. The NPC- pRevh3.4k GFP cells, which contain an hTERT promoter-driven GFP reporter gene, were infected with a library of lentivirus shRNA pool for knocking-down kinases and phosphatases, and the infected cells with increased or decreased GFP expression were sorted by FACS. The DNAs from the sorted cells were screened for shRNA sequence to identify the putative genes affected by shRNA. A total of 119 shRNA sequences corresponding to 76 putative candidate genes were identified that may regulate hTERT expression. Seven of these putative genes (ERCC8, NF2, AKT3, TRIM24, DGKZ, PPP6C and PIK3C3) were further tested, and knocking-down TRIM24 was confirmed to increase the hTERT promoter activity, whereas knocking-down AKT3 was found to decrease the hTERT promoter activity. In addition, overexpression of AKT3 in telomerase-negative U2OS cells appears to reactivate the expression of endogeneous hTERT. These results suggest that TRIM24 is negative regulator and AKT3 is positive regulator for hTERT gene expression.