宫颈癌组织和Hela细胞中miR-664的表达及其对顺铂化疗敏感性的影响
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摘要
目的:探讨miR-664在宫颈癌组织和Hela细胞中的表达及其对顺铂化疗敏感性的影响。方法:采用RTPCR法检测142例宫颈癌组织及相应癌旁组织标本miR-664的表达,分析其表达与患者临床病理参数的关系。将宫颈癌Hela-229细胞分为3组:对照组、顺铂组、顺铂+转染组。其中,顺铂+转染组细胞转染miR-664拟似物。细胞培养结束后,采用MTT法检测各组细胞活力、镜下观察单克隆形成数目,流式细胞术检测细胞凋亡率、G_1期细胞比例,Transwell细胞迁移实验测定穿膜细胞数,RT-PCR法检测细胞miR-664表达水平,ELISA法检测细胞培养液中的Caspase-3、Caspase-9浓度。结果:(1)宫颈癌组织miR-664表达水平明显低于癌旁组织(P <0.01); TNM分期越高、病理学分期越高、有复发、有淋巴结转移的病例,miR-664阳性表达率明显下降(均P <0.01);(2)体外细胞实验中,顺铂组、顺铂+转染组细胞存活率、单克隆形成数目、G_1期细胞数、细胞穿膜数均明显低于对照组(均P <0.01),细胞凋亡率、Caspase-3、Caspase-9浓度、miR-664表达显著高于对照组(P <0.01);顺铂+转染组存活率、单克隆形成数目、G_1期细胞比率、细胞穿膜数明显低于顺铂组(均P <0.01),细胞凋亡率、Caspase-3、Caspase-9浓度、miR-664表达显著高于顺铂组(均P <0.01)。结论:miR-664在宫颈癌组织中表达降低; miR-664通过诱导Caspase-3、Caspase-9过表达,进而抑制宫颈癌细胞株的增殖、迁移、浸润,增强宫颈癌细胞对顺铂化疗的敏感性。
Objective: To investigate the expression levels of miR-664 in cervical cancer tissues and its relationship with chemosensitivity to cisplatin in Hela cells. Methods: The expression of miR-664 in 142 cervical cancer tissues and corresponding adjacent tissues was detected by RT-PCR. The relationship between the expression and clinicopathological parameters was analyzed. Cervical cancer Hela-229 cells were divided into three groups: control group,cisplatin group,cisplatin + transfection group. Among them,cisplatin + transfection group cells were transfected with miR-664 mimics. After the end of cell culture,the viability of each group was detected by MTT assay,the number of monoclonal formation was observed under microscope,the apoptosis rate and cell ratio of G_1 phase were detected by flow cytometry,and the number of transmembrane cells was determined by Transwell cell migration assay. The level of miR-664 was detected by RT-PCR method,and the concentration of Caspase-3 and Caspase-9 in cell culture medium was detected by ELISA method. Results: The expression level of miR-664 in cervical cancer tissues was significantly lower than that in adjacent tissues(P < 0.01). The higher the TNM stage,the higher the pathological stage,the recurrence and lymph node metastasis,the lower the positive expression rate of miR-664(P < 0.01). In vitro cell experiments,cisplatin group,cisplatin + transfection group cell survival rate,number of monoclonal formation,number of G_1 cells,cell permeation number were significantly lower than the control group(P < 0.01); apoptosis rate,Caspase-3,Caspase-9 concentration,miR-664 expression was significantly higher than the control group(P < 0.01); cisplatin + transfection group survival rate,number of monoclonal formation,G_1 phase cell ratio,the number of transmembrane cells was significantly lower than that of cisplatin group(P < 0.01). The apoptosis rate,Caspase-3,Caspase-9 concentration and miR-664 expression in cisplatin + transfection group were significantly higher than those in cisplatin group(P < 0.01). Conclusion: miR-664 expression is down-regulated in cervical cancer tissues,while the miR-664 can enhance chemosensitivity to cisplatin,and its mechanism may be related to miR-664 induced overexpression of Caspase-3 and Caspase-9,as well as the inhibition of the proliferation,migration and infiltration of cervical cancer cells.
Objective: To investigate the expression levels of miR-664 in cervical cancer tissues and its relationship with chemosensitivity to cisplatin in Hela cells. Methods: The expression of miR-664 in 142 cervical cancer tissues and corresponding adjacent tissues was detected by RT-PCR. The relationship between the expression and clinicopathological parameters was analyzed. Cervical cancer Hela-229 cells were divided into three groups: control group,cisplatin group,cisplatin + transfection group. Among them,cisplatin + transfection group cells were transfected with miR-664 mimics. After the end of cell culture,the viability of each group was detected by MTT assay,the number of monoclonal formation was observed under microscope,the apoptosis rate and cell ratio of G_1 phase were detected by flow cytometry,and the number of transmembrane cells was determined by Transwell cell migration assay. The level of miR-664 was detected by RT-PCR method,and the concentration of Caspase-3 and Caspase-9 in cell culture medium was detected by ELISA method. Results: The expression level of miR-664 in cervical cancer tissues was significantly lower than that in adjacent tissues(P < 0.01). The higher the TNM stage,the higher the pathological stage,the recurrence and lymph node metastasis,the lower the positive expression rate of miR-664(P < 0.01). In vitro cell experiments,cisplatin group,cisplatin + transfection group cell survival rate,number of monoclonal formation,number of G_1 cells,cell permeation number were significantly lower than the control group(P < 0.01); apoptosis rate,Caspase-3,Caspase-9 concentration,miR-664 expression was significantly higher than the control group(P < 0.01); cisplatin + transfection group survival rate,number of monoclonal formation,G_1 phase cell ratio,the number of transmembrane cells was significantly lower than that of cisplatin group(P < 0.01). The apoptosis rate,Caspase-3,Caspase-9 concentration and miR-664 expression in cisplatin + transfection group were significantly higher than those in cisplatin group(P < 0.01). Conclusion: miR-664 expression is down-regulated in cervical cancer tissues,while the miR-664 can enhance chemosensitivity to cisplatin,and its mechanism may be related to miR-664 induced overexpression of Caspase-3 and Caspase-9,as well as the inhibition of the proliferation,migration and infiltration of cervical cancer cells.
引文
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[13]Lee BP,Buric'I,George-Pandeth A,et al.MicroRNAs miR-203-3p,miR-664-3p and miR-708-5p are associated with median strain lifespan in mice[J].Sci Rep,2017,7:44620.
[14]王同帅,张颖,黄金凤,等.MiRNA-664在肿瘤中的研究进展[J].现代生物医学进展,2017,17(9):1776-1779.
[2]Song W,Tang L,Xu Y,et al.PARP inhibitor increases chemosensitivity by upregulating miR-664b-5p in BRCA1-mutated triple-negative breast cancer[J].Sci Rep,2017,7:42319.
[3]Yang Y,Liu H,Wang X,et al.Up-regulation of microRNA-664 inhibits cell growth and increases cisplatin sensitivity in cervical cancer[J].Int J Clin Exp Med,2015,8(10):18123-18129.
[4]Massad LS,Einstein MH,Huh WK,et al.2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors[J].Obstet Gynecol,2013,121(4):829-846.
[5]颜黎栩,黄马燕,吴秋良,等.miR-21表达异常与乳腺癌临床病理特征及预后的关系[J].中国病理生理杂志,2009,25(4):676-681.
[6]Ding Z,Jian S,Peng X,et al.Loss of miR-664 expression enhances cutaneous malignant melanoma proliferation by upregulating PLP2[J].Medicine(Baltimore),2015,94(33):e1327.
[7]Sahin Y,Altan Z,Arman K,et al.Inhibition of miR-664a interferes with the migration of osteosarcoma cells via modulation of MEG3[J].Biochem Biophys Res Commun,2017,490(3):1100-1105.
[8]Chanyshev MD,Ushakov DS,Gulyaeva LF.Expression of miR-21 and its Acat1,Armcx1,and Pten target genes in liver of female rats treated with DDT and Benzo[a]pyrene[J].Mol Biol(Mosk),2017,51(4):664-670.
[9]Chen B,Bao Y,Chen X,et al.miR-664 promotes osteosarcoma cells proliferation via downregulating of FOXO4[J].Biomed Pharmacother,2015,75:1-7.
[10]Fiala O,Pitule P,Hosek P,et al.The association of miR-126-3p,miR-126-5p and miR-664-3p expression profiles with outcomes of patients with metastatic colorectal cancer treated with bevacizumab[J].Tumour Biol,2017,39(7):1010428317709283.
[11]Liu H,Zhou Y,Liu Q,et al.Association of miR-608rs4919510 polymorphism and cancer risk:a meta-analysis based on 13,664 subjects[J].Oncotarget,2016,8(23):37023-37031.
[12]Zhu X,Ju S,Yuan F,et al.microRNA-664 enhances proliferation,migration and invasion of lung cancer cells[J].Exp Ther Med,2017,13(6):3555-3562.
[13]Lee BP,Buric'I,George-Pandeth A,et al.MicroRNAs miR-203-3p,miR-664-3p and miR-708-5p are associated with median strain lifespan in mice[J].Sci Rep,2017,7:44620.
[14]王同帅,张颖,黄金凤,等.MiRNA-664在肿瘤中的研究进展[J].现代生物医学进展,2017,17(9):1776-1779.