腺病毒载体介导的PTEN基因对黑色素瘤SK-MEL-3细胞化疗敏感性的影响
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摘要
目的探讨腺病毒载体介导的PTEN基因对黑色素瘤SK-MEL-3细胞化疗敏感性的影响。方法以pcDNA3.0-PTEN重组质粒为模板,加入PTEN引物,构建重组腺病毒载体PTEN(Ad-PTEN),并用其感染QBI-293A细胞,进行病毒扩增和效价测定。将SK-MEL-3细胞分为对照组(PBS组)、空载体腺病毒组(ADDP处理组)、Ad-PTEN基因治疗组(Ad-PTEN处理组)、顺铂对照组(DDP处理组)、Ad-PTEN+DDP联合处理组。采用RT-PCR和Western blot法检测PTEN、Bax、Bcl-2、P21、P53和Caspase-3的表达水平。分别用划痕实验、侵袭实验、MTT法、流式细胞仪检测各组细胞的迁移、侵袭能力及生长、凋亡情况。结果本研究成功构建了Ad-PTEN,且感染QBI-293细胞后可进行增殖,病毒效价为108 pfu·mL~(-1)。与PBS处理组和ADDP处理组比较,Ad-PTEN处理组、DDP处理组、Ad-PTEN+DDP联合处理组细胞活力减弱,凋亡率上升,Bax、P21、P53及Caspase-3的转录和表达水平上调,Bcl-2下调,差异均具有统计学意义(P<0.0001)。Ad-PTEN+DDP联合处理组与Ad-PTEN处理组、DDP处理组比较,细胞活力减弱,凋亡率上升,Bax、P21、P53及Caspase-3的转录和表达水平上调,Bcl-2下调,差异均具有统计学意义(P<0.0001)。结论 Ad-PTEN可抑制SK-MEL-3细胞增殖并诱导其凋亡,呈现明显的抑癌作用;Ad-PTEN与DDP联合使用对SK-MEL-3细胞的增殖抑制作用和诱导凋亡作用显著优于单用Ad-PTEN或DDP,具有化疗增敏作用。
Objective To explore the effects of adenovirus mediated PTEN gene on chemosensitivity of melanoma SK-MEL-3 cells.Methods A recombinant adenovirus vector PTEN(Ad-PTEN) was constructed by using pcDNA3.0-PTEN recombinant plasmid as template and adding PTEN primer. After infection of QBI-293A cells with Ad-PTEN, virus amplification and titer were measured. Then, SK-MEL-3 cells were divided into control group(PBS group), empty vector adenovirus group(ADDP group), Ad-PTEN gene therapy group(Ad-PTEN group), cisplatin control group(DDP group), and Ad-PTEN + DDP group. RT-qPCR and Western blot analysis were used to detect expressions of PTEN, Bax, Bcl-2, P21, P53 and Caspase-3. Scratch test, Transwell assay, MTT assay and flow cytometry were applied to measure migration, invasion, proliferation, and apoptosis of SK-MEL-3 cells. Results Initially, it was observed that Ad-PTEN was successfully constructed, and that after infection with QBI-293 cells, cells proliferated and the viral titer was 10~8 pfu·mL~(-1). Compared with the PBS and ADDP groups, the Ad-PTEN, DDP and Ad-PTEN + DDP groups showed weakened viability, increased apoptosis rate, upregulated Bax,P21, P53 and Caspase-3, and downregulated Bcl-2(P<0.0001). Compared with the Ad-PTEN and DDP groups, weakened viability, increased apoptosis rate, upregulated Bax, P21, P53 and Caspase-3, and downregulated Bcl-2 were observed in the Ad-PTEN + DDP group(P<0.0001). Conclusion Ad-PTEN could inhibit the proliferation and induce apoptosis of SK-MEL-3 cells, presenting obvious anticancer effect. AD-PTEN and DDP in combination had better efficacy in inhibiting cell proliferation and inducing apoptosis than AD-PTEN or DDP alone. The combination of Ad-PTEN and DDP could increase the chemotherapy sensitivity.
Objective To explore the effects of adenovirus mediated PTEN gene on chemosensitivity of melanoma SK-MEL-3 cells.Methods A recombinant adenovirus vector PTEN(Ad-PTEN) was constructed by using pcDNA3.0-PTEN recombinant plasmid as template and adding PTEN primer. After infection of QBI-293A cells with Ad-PTEN, virus amplification and titer were measured. Then, SK-MEL-3 cells were divided into control group(PBS group), empty vector adenovirus group(ADDP group), Ad-PTEN gene therapy group(Ad-PTEN group), cisplatin control group(DDP group), and Ad-PTEN + DDP group. RT-qPCR and Western blot analysis were used to detect expressions of PTEN, Bax, Bcl-2, P21, P53 and Caspase-3. Scratch test, Transwell assay, MTT assay and flow cytometry were applied to measure migration, invasion, proliferation, and apoptosis of SK-MEL-3 cells. Results Initially, it was observed that Ad-PTEN was successfully constructed, and that after infection with QBI-293 cells, cells proliferated and the viral titer was 10~8 pfu·mL~(-1). Compared with the PBS and ADDP groups, the Ad-PTEN, DDP and Ad-PTEN + DDP groups showed weakened viability, increased apoptosis rate, upregulated Bax,P21, P53 and Caspase-3, and downregulated Bcl-2(P<0.0001). Compared with the Ad-PTEN and DDP groups, weakened viability, increased apoptosis rate, upregulated Bax, P21, P53 and Caspase-3, and downregulated Bcl-2 were observed in the Ad-PTEN + DDP group(P<0.0001). Conclusion Ad-PTEN could inhibit the proliferation and induce apoptosis of SK-MEL-3 cells, presenting obvious anticancer effect. AD-PTEN and DDP in combination had better efficacy in inhibiting cell proliferation and inducing apoptosis than AD-PTEN or DDP alone. The combination of Ad-PTEN and DDP could increase the chemotherapy sensitivity.
引文
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[17]Zhao C, Tao T, Yang L, et al. Loss of PDZK1 expression activates PI3K/AKT signaling via PTEN phosphorylation in gastric cancer[J]. Cancer Lett, 2019, 453:107-121. doi:10.1016/j. canlet.2019.03.043.
[18]Cui C, Li S, Wu D. Znhit1 inhibits breast cancer by up-regulating PTEN to deactivate the PI3K/Akt/mTOR pathway[J].Life Sci, 2019, 224:204-211. doi:10.1016/j. lfs.2019.03.067.
[19]杜跃耀,殷文瑾,陆劲松. DNA拓扑异构酶Ⅱα(topoⅡα)与乳腺癌蒽环类药物化疗敏感性的研究进展[J].复旦学报(医学版), 2012, 39(2):203-206. doi:10.3969/j.issn.1672-8467.2012.02.019.
[20]傅玉峰,陈培,李雪甫.抑癌基因PTEN提高耐药食管癌化疗敏感性的作用及其机制研究[J].中国临床研究, 2016,29(2):214-217. doi:10.13429/j. cnki. cjcr.2016.02.018.
[21]Yang H, Kong W, He L, et al. MicroRNA expression profiling in human ovarian cancer:miR-214 induces cell survival and cisplatin resistance by targeting PTEN[J]. Cancer Res, 2008,68(2):425-433. doi:10.1158/0008-5472. CAN-07-2488.
[22]Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence[J]. J Natl Cancer Inst, 1997, 89(14):1027-1036. doi:10.1093/jnci/89.14.1027.
[23]Jin SJ, Yang Y, Ma L, et al. In vivo and in vitro induction of the apoptotic effects of oxysophoridine on colorectal cancer cells via the Bcl-2/Bax/caspase-3 signaling pathway[J]. Oncol Lett, 2017, 14(6):8000-8006. doi:10.3892/ol.2017.7227.
[2]Kudchadkar RR, Smalley KS, Glass LF, et al. Targeted therapy in melanoma[J]. Clin Dermatol, 2013, 31(2):200-208. doi:10.1016/j. clindermatol.2012.08.013.
[3]Sanlorenzo M, Vujic I, Posch C, et al. The risk of melanoma in pilots and cabin crew:UV measurements in flying airplanes[J]. JAMA Dermatol, 2015, 151(4):450-452. doi:10.1001/jamadermatol.2014.4643.
[4]Akman T, Oztop I, Unek IT, et al. Long-term outcomes and prognostic factors of high-risk malignant melanoma patients after surgery and adjuvant high-dose interferon treatment:a single-center experience[J]. Chemotherapy, 2014, 60(4):228-238. doi:10.1159/000371838.
[5]沈存思,范方田,陶丽,等.抑癌基因PTEN与肿瘤血管生成研究进展[J].中国药理学通报, 2013, 29(5):597-600. doi:10.3969/j. issn.1001-1978.2013.05.002.
[6]陈昆仑,刘莹,李明利,等. PTEN通过AKT介导的细胞代谢途径抑制宫颈癌细胞的增殖[J].西安交通大学学报:医学版, 2013(05):563-567. doi:10.7652/jdyxb201305002.
[7]Parsons R. Human cancer, PTEN and the PI-3 kinase pathway[J]. Semin Cell Dev Biol, 2004, 15(2):171-176.
[8]何利珍,钟雪云. PTEN基因在胶质瘤细胞SWO38及其亚系中表达的异质性研究[J].实用医院临床杂志, 2017,14(5):27-30. doi:CNKI:SUN:YYLC.0.2017-05-010.
[9]李航,张杰,张风华,等.三阴性乳腺癌中PTEN基因启动子甲基化的临床意义[J].肿瘤, 2016, 36(11):1218-1224.
[10]梁媛,冯洋洋,李琳琳,等. MicroRNA-29a靶向抑制PTEN基因诱导非小细胞肺癌细胞上皮间质转化的机制研究[J].现代肿瘤医学, 2018, 20(5):653-659. doi:10.3969/j. issn.1672-4992.2018.05.002.
[11]O'Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J]. Nature, 2007, 445(7123):106-110. doi:10.1038/nature05372.
[12]周含煜,孙语舒,李姗珊,等. PIPP与PTEN对黑色素瘤的抑制作用[J].解剖科学进展, 2017, 24(4):433-435. doi:CNKI:SUN:JPKX.0.2017-04-027.
[13]陈健华,孙颖,段友容.静脉递送腺病毒载体在肿瘤基因治疗中的研究进展[J].肿瘤, 2017, 37(12):1339-1343.
[14]陈苗苗,王颖,张文文,等.重组腺病毒介导的S100A9基因表达对宫颈鳞癌细胞增殖和凋亡的影响[J].温州医科大学学报, 2017, 47(4):235-238. doi:10.3969/j.issn.2095-9400.2017.04.001.
[15]高磊,潘铁军,武国军,等.腺病毒介导的PTEN基因对前列腺癌细胞株PC-3增殖及cyclinD1、P21表达的影响[J].中华男科学杂志, 2014, 20(3):207-212. doi:10.13263/j. cnki. nja.2014.03.003.
[16]吴达军,许峰.晚期恶性黑色素瘤的新药治疗进展[J].华西医学, 2016, 31(12):2079-2083.
[17]Zhao C, Tao T, Yang L, et al. Loss of PDZK1 expression activates PI3K/AKT signaling via PTEN phosphorylation in gastric cancer[J]. Cancer Lett, 2019, 453:107-121. doi:10.1016/j. canlet.2019.03.043.
[18]Cui C, Li S, Wu D. Znhit1 inhibits breast cancer by up-regulating PTEN to deactivate the PI3K/Akt/mTOR pathway[J].Life Sci, 2019, 224:204-211. doi:10.1016/j. lfs.2019.03.067.
[19]杜跃耀,殷文瑾,陆劲松. DNA拓扑异构酶Ⅱα(topoⅡα)与乳腺癌蒽环类药物化疗敏感性的研究进展[J].复旦学报(医学版), 2012, 39(2):203-206. doi:10.3969/j.issn.1672-8467.2012.02.019.
[20]傅玉峰,陈培,李雪甫.抑癌基因PTEN提高耐药食管癌化疗敏感性的作用及其机制研究[J].中国临床研究, 2016,29(2):214-217. doi:10.13429/j. cnki. cjcr.2016.02.018.
[21]Yang H, Kong W, He L, et al. MicroRNA expression profiling in human ovarian cancer:miR-214 induces cell survival and cisplatin resistance by targeting PTEN[J]. Cancer Res, 2008,68(2):425-433. doi:10.1158/0008-5472. CAN-07-2488.
[22]Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence[J]. J Natl Cancer Inst, 1997, 89(14):1027-1036. doi:10.1093/jnci/89.14.1027.
[23]Jin SJ, Yang Y, Ma L, et al. In vivo and in vitro induction of the apoptotic effects of oxysophoridine on colorectal cancer cells via the Bcl-2/Bax/caspase-3 signaling pathway[J]. Oncol Lett, 2017, 14(6):8000-8006. doi:10.3892/ol.2017.7227.