慢病毒介导的hTERT-shRNA联合光动力治疗对宫颈癌细胞SiHa的影响

doi:10.6040/j.issn.1671-7554.0.2014.576

摘要/Abstract

摘要： 目的探讨慢病毒介导的人端粒酶逆转录酶(hTERT)联合四甲基吡啶卟啉(TMPyP4)及光动力疗法对宫颈癌细胞SiHa的影响。方法实验分4组:空白对照组、单纯基因治疗组、单纯光动力治疗组、联合治疗组。体外化学合成Lenti-hTERT-shRNA并转染SiHa细胞,确定最适感染复数(MOI)值,成功稳定转染后给予浓度为7.5 μmol/L的TMPyP4 行光动力治疗。RT-PCR法检测hTERT、p53、HPV-16 E6、Caspase-8 mRNA的表达;免疫荧光检测hTERT蛋白的表达;细胞增殖及细胞毒性(CCK-8)检测各组中人宫颈癌细胞SiHa的抑制作用;AnnexinV-PE/7-AAD双染试剂盒检测各组细胞的凋亡率;Transwell实验检测各组细胞侵袭能力的改变。结果与空白对照组比较,其他3组hTERT、HPV-16 E6、Caspase-8 mRNA的表达水平均下降,p53mRNA的表达水平升高,hTERT蛋白表达水平相应下降,其中联合治疗组最明显(P<0.05);联合治疗组较其他组明显抑制SiHa细胞的增殖(P<0.05);流式细胞学AnnexinV-PE/7-AAD和Transwell实验检测结果显示,联合治疗组较单纯基因治疗组和单纯光动力治疗组的凋亡率明显升高,细胞侵袭能力明显减弱(P<0.05)。结论慢病毒介导的hTERT-shRNA联合光动力治疗明显抑制宫颈癌SiHa细胞的增殖和侵袭能力,并促进细胞凋亡。

关键词: 人端粒酶逆转录酶, 宫颈肿瘤, SiHa细胞, 光动力治疗, 四甲基吡啶卟啉

Abstract: Objective To investigate the effects of lenti-hTERT-shRNA combined with photodynamic therapy mediated by meso-5,10,15,20-Tetrakis-(Nmethyl-4-pyridyl) (TMPyP4) on cervical cancer SiHa cells in vitro. Methods Four groups were enrolled in the experiment, including the blank control group, gene therapy group, PDT group and combined treatment group. Lenti-hTERT-shRNA was synthesized and SiHa cells were transfected, then the optimum multiplicity of infection (MOI) was confirmed. After that, the cells were treated with 7.5 μmol/L TMPyP4 and irradiation (blue laser, 4 J/cm²). The proliferative activity of SiHa cells was assessed by Cell Counting Kit (CCK-8) assay; the expressions of hTERT, p53, HPV-16 E6, and Caspase-8 mRNA were detected by real-time PCR, and the hTERT protein was tested by immunofluorescence (IF); the apoptosis was examined by AnnexinV-PE/7-AAD double dye kit; the ability of cellular invasion was assessed by Transwell experiment. Results Compared with the blank control group, the other three groups had decreased the expressions of hTERT, HPV-16 E6, and Caspase-8 mRNA, but increased the expression of p53 mRNA. The down-regulation of hTERT protein was the most significant in the combined treatment group (P<0.05). In the combined treatment group, the cellular survivability and invasion ability of SiHa cells were inhibited more markedly than in the other groups (P<0.05), while the apoptosis rate increased more significantly (P<0.05). Conclusion Lenti virus-mediated hTERT RNA interference combined with photodynamic therapy of TMPyP4can suppress the abilities of proliferation and invasion, and enhance the apoptosis rate of cervix cancer SiHa cells.

Key words: Photodynamic therapy, SiHa cells, Cervix neoplasms, Human telomerase reverse transcriptase, Meso-5,10,15, 20-Tetrakis- (Nmethyl-4-pyridyl) porphine

中图分类号:

R454.2

王颉, 张友忠, 冯进波, 刘洪丽, 宗丽菊. 慢病毒介导的hTERT-shRNA联合光动力治疗对宫颈癌细胞SiHa的影响[J]. 山东大学学报（医学版）, 2015, 53(1): 27-33.

WANG Jie, ZHANG Youzhong, FENG Jinbo, LIU Hongli, ZONG Liju. Inhibitive effects of lenti virus-mediated hTERT RNA interference and TMPyP4-PDT on cervix cancer SiHa cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2015, 53(1): 27-33.

参考文献

[1] Baskaran P, Subramanian P, Rahman RA, et al. Perceived susceptibility, and cervical cancer screening benefits and barriers in malaysian women visiting outpatient clinics[J]. Asian Pac J Cancer Prev, 2013, 14(12): 7693-7699.
[2] Friedberg JS. Radical pleurectomy and photodynamic therapy for malignant pleural mesothelioma [J]. Ann Cardiothorac Surg, 2012, 1(4): 472-480.
[3] Anbil S, Rizvi I, Celli JP, et al. Impact of treatment response metrics on photodynamic therapy planning and outcomes in a three-dimensional model of ovarian cancer[J]. J Biomed Opt, 2013, 18(9): 1-13.
[4] Nagesh N, Buscaglia R, Dettler JM, et al. Studies on the site and mode of TMPyP4 interactions with Bcl-2 promoter sequence G-Quadruplexes[J]. Biophys J, 2010, 98(11): 2628-2633.
[5] Andrew EJ, Merchan S, Lawless C, et al. Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4[J]. PLoS One, 2013, 8(6): 1-10.
[6] Zvereva MI, Shcherbakova DM, Dontsova OA. Telomerase: structure, functions, and activity regulation[J]. in Biochemistry (Mosc), 2010, 75(13):1563-1583.
[7] Liang Y, Li X, Lin R, et al. Combinatorial gene targeting hTERT and BI-1 in CNE-2 nasopharyngeal carcinoma cell line[J]. Biomed Rep, 2013, 1(2): 285-293.
[8] Zhang W, Xing L. RNAi gene therapy of SiHa cells via targeting human TERT induces growth inhibition and enhances radiosensitivity[J]. Int J Oncol, 2013, 43(4): 1228-1234.
[9] Wang L, Xiao H, Zhang X, et al. The role of telomeres and telomerase in hematologic malignancies and hematopoietic stem cell transplantation[J]. J Hematol Oncol, 2014, 7(1): 61.
[10] Gül I, Dündar O, Bodur S, et al. The status of telomerase enzyme activity in benign and malignant gynaecologic pathologies[J]. Balkan Med J, 2013, 30(3): 287-292.
[11] Sharma NK, Reyes A, Green P, et al. Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria[J]. Nucleic Acids Res, 2012, 40(2): 712-725.
[12] Chung SS, Aroh C, Vadgama JV. Constitutive activation of STAT3 signaling regulates hTERT and promotes stem cell-like traits in human breast cancer cells[J]. PLoS One, 2013, 8(12): 1-10.
[13] Zeng H, Sun M, Zhou C, et al. Hematoporphyrin monomethyl ether-mediated photodynamic therapy selectively kills sarcomas by inducing apoptosis[J]. PLoS One, 2013, 8(10): 1-11.
[14] Le HT, Miller MC, Buscaglia R, et al. Not all G-quadruplexes are created equally: an investigation of the structural polymorphism of the c-Myc G-quadruplex-forming sequence and its interaction with the porphyrin TMPyP4[J]. Org Biomol Chem, 2012, 10(47): 9393-9404.
[15] Xu Y, Wang L, Zheng X, et al. Positive expression of p53, c-erbB2 and MRP proteins is correlated with survival rates of NSCLC patients[J]. Mol Clin Oncol, 2013, 1(3): 487-492.
[16] Madhumati G, Kavita S, Anju M, et al. Immunohistochemical Expression of Cell Proliferating Nuclear Antigen (PCNA) and p53 Protein in Cervical Cancer[J]. J Obstet Gynaecol India, 2012, 62(5): 557-561.
[17] Tan J, Chen B, He L, et al. Anacardic acid (6-pentadecylsalicylic acid) induces apoptosis of prostate cancer cells through inhibition of androgen receptor and activation of p53 signaling[J]. Chin J Cancer Res, 2012, 24(4): 275-283.
[18] Mehrkens A, Karim MZ, Kim S, et al. Canine notochordal cell-secreted factors protect murine and human nucleus pulposus cells from apoptosis by inhibition of activated caspase-9 and caspase-3/7[J]. Evid Based Spine Care J, 2013, 4(2): 154-156.
[19] Laukens B, Jennewein C, Schenk B, et al. Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor α-induced necroptosis[J]. Neoplasia, 2011, 13(10): 971-979.
[20] Cornet I, Gheit T, Iannacone MR, et al. HPV16 genetic variation and the development of cervical cancer worldwide[J]. Br J Cancer, 2013, 108(1): 240-244.
[21] Qmichou Z, Khyatti M, Berraho M, et al. Analysis of mutations in the E6 oncogene of human papillomavirus 16 in cervical cancer isolates from Moroccan women[J]. BMC Infect Dis, 2013, 13(1): 378.
[22] Guo Y, Meng X, Wang Q, et al. The ING4 Binding with p53 and Induced p53 Acetylation were Attenuated by Human Papillomavirus 16 E6[J]. PLoS One, 2013, 8(8): 1-9.
[23] 丁佰娟, 张友忠, 吕昌帅, 等. TMPyP4-PDT对人宫颈癌细胞的杀伤效应[J]. 山东大学学报: 医学版, 2012, 50(11): 1-5. DING Baijuan, ZHANG Youzhong, LV Changshuai, et al. Effects of TMPyP4-PDT on human cervical cancer cells[J]. Journal of Shandong University: Health Sciences, 2012, 50(11): 1-5.

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