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Zou D.-L.,Chongqing Institute of Oncology | Zhou Q.,Chongqing Institute of Oncology | Wang D.,Chongqing Institute of Oncology
Tumor | Year: 2014

Objective: To establish an adenovirus expression vector containing human telomerase reverse transcriptase (hTERT) promoter-driven p53 up-regulated modulator of apoptosis (Puma), and further to achieve the directional expression of Puma in ovarian carcinoma COC1 cells. Methods: The full-length open reading frame (ORF) of Puma and the promoter of hTERT were amplified by RT-PCR and cloned into the adenovirus shuttle vector pDC316. Recombinant adenovirus pDC316-hTERT-Puma was obtained and infected into human normal ovarian epithelial HUM-CELL-0088 cells and ovarian carcinoma COC1 cells. The expression levels of Puma mRNA and protein were detected by real-time fluorogenic quantitative-PCR (RFQ-PCR) and Western blotting, respectively. The cell proliferation was detected by cell counting kit-8 (CCK-8) assay. The apoptosis was detected by flow cytometry (FCM). Results: The adenovirus expression vector pDC316-hTERT-Puma containing hTERT promoter-driven Puma was successfully constructed, and the corresponding adenovirus could achieve the specific and high expression of Puma in ovarian carcinoma cells but not in the normal ovarian cells. The successful expression of Puma could inhibit the proliferation of COC1 cells evidently (P 〈 0.05) and induce apoptosis (P 〈 0.05). Conclusion: The tumor-targeted expression of Puma gene in ovarian carcinoma cells is achieved through adenovirus vector, and it will be beneficial for the use of Puma in targated therapy of ovarian carcinoma. Copyright© 2014 by TUMOR. Source


Zou D.-L.,Chongqing Institute of Oncology | Wang D.,Chongqing Institute of Oncology | Zhou Q.,Chongqing Institute of Oncology
Tumor | Year: 2014

Objective: To analyze the expression level of microRNA-29b (miR-29b) in human cervical cancer tissues, and to investigate the function of miR-29b in regulating the genesis of human cervical cancer and its mechanism. Methods: The expressions of miR-29b in 50 tissue specimens of cervical cancer and the corresponding adjacent paracancerous tissues were detected by real-time fluorescent quantitative PCR. The relationship of miR-29b expression and clinicopathological features of patients with cervical cancer was analyzed. After the transfection of miR-29b mimics into cervival cancer SiHa cells, the effects of miR-29b overexpression on proliferation, cell cycle distribution and migration of SiHa cells were detected by cell count kit-8 (CCK-8), flow cytometry, wound-healing and Transwell assays, respectively. The target gene of miR-29b was predicted by Targetscan software. Then the interaction between miR-29b and its target gene in human cervical cancer cells was confirmed by dual-luciferase reporter gene system and Western blotting. The expression of target gene of miR-29b in cervical cancer tissues was detected by real-time fluorescent quantitative PCR. Results: Compared with the corresponding adjacent paracancerous tissues, the expression of miR-29b was down-regulated in cervical cancer tissues (P < 0.05). For SiHa cells transfected with miR-29b mimics, overexpression of miR-29b could significantly inhibit the cell proliferation and migration of SiHa cells, and also significantly increased the percentage of SiHa cells in G1 phase (all P < 0.05). MiR-29b interacted with its target gene cyclin D2(CCND2), meanwhile they presented a negative correlation (r2 = 0.225, P < 0.05) in cervical cancer SiHa cells. The expression level of CCND2 protein in cervical cancer tissues was higher than that in the corresponding adjacent paracancerous tissues (P < 0.05). Conclusion: MiR-29b is down-regulated in human cervical cancer tissues. MiR-29b may be served as a tumor-suppressor through down-regulating CCND2 expression, so it may be used as a new target for therapy and diagnosis of human cervical cancer. Copyright © 2014 by TUMOR. Source

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