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Inhibitory effect of antisense human telomerase RNA(hTR) gene on hepatocellular carcinoma cells
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Abstract:
To study the inhibitory effect of retrovirusmediated antisense human telomerase RNA (hTR) gene on hepatocelluar carcinoma, so as to explore an effective way to inhibit telomrerase activity in the treatment of hepatocellular carcinoma. Methods: Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into HepG2 cells. After G418 selection, PCR was used to verify the integration of the hTR gene. Cell growth curves were drawn using MTT assay and the expression of PCNA was determined by immunofluorescence. TRAPPCRELISA was adopted to detect the telomerase activity; cell cycle and apoptosis were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2hTREcoRⅠ and HepG2hTRBamHⅠ cells, but not in untransfected HepG2 cells. The antisense hTR complementary to the template region of telomerase inhibited growth and proliferation of HepG2 cells. The expression of neutrophil proliferating cell nuclear antigen (PCNA) was decreased. Telomerase activity in the antisense hTRtreated group was (2.31±0.16),which was significantly lower than those of the other 2 groups(P<001). FCM showed that the apoptosis rate of the experimental group was (958±138)%, which was significantly different from those of the other 2 groups(P<0.01). G2/M phase blockage was detected in the HepG2hTRBamHⅠ cells. Conclusion: Telomerase RNA is an important component of telomerase; downregulation of its expression through antitechnology can lead to the growth inhibition and apoptosis of hepatocellular carcinoma cells.
Keywords:
telomerase ; retroviral vector ; antisense RNA ; hepatocellular carcinoma
