下调SUZ12抑制人宫颈癌细胞的增殖及作用机制
|
摘要
目的探讨下调SUZ12表达对人宫颈癌细胞增殖的影响及其作用机制。方法通过RNAi(RNA干扰技术)下调SUZ12和Cdc25C蛋白的表达,通过慢病毒转染法对Hela细胞构建Cdc25C蛋白过表达及其对照细胞株,并用含嘌呤霉素培养基进行为期14 d的筛选,荧光拍照检测绿色荧光蛋白以及Western Blot法进行验证;MTT法检测下调SUZ12和Cdc25C蛋白对细胞增殖的影响;Western Blot法检测下调SUZ12表达后蛋白表达量的改变。结果 MTT结果显示,siSUZ12组中Hela及SiHa的增殖率远低于对照组,细胞增殖明显受到抑制,Hela的生存率为(60.65±7.64)%(P<0.01),SiHa的生存率为(73.81±9.30)%(P<0.01)。时效研究结果显示,转染siSUZ12后,Hela细胞在转染第2天后开始出现抑制,其中第5天最为明显,生存率为(56.13±7.78)%(P<0.01);SiHa细胞在转染第1天后开始出现抑制,其中第5天最为明显,生存率为(70.50±3.15)%(P<0.01)。细胞周期结果显示,Hela细胞在siSUZ12作用48 h后,G0/G1期的比例明显增加(P<0.05),细胞发生G1/S期阻滞。SiHa细胞在siSUZ12作用60 h后,G2/M期细胞数比例显著升高(P<0.01),细胞发生G2/M期阻滞。Western Blot结果显示,下调SUZ12表达后,细胞内Cdc25C的表达明显被抑制。阻断Cdc25C的表达后能显著拮抗siSUZ12对Hela、SiHa细胞的抑制作用,而过表达Cdc25C能降低siSUZ12对细胞的抑制作用。结论下调SUZ12能通过干扰Cdc25C的表达,产生抑制人宫颈癌细胞增殖的作用。
Objective To investigate whether down-regulation of SUZ12 influences the proliferation and cell cycle of human cervical cancer cell lines Hela and SiHa,and to research its mechanism. Methods RNA interference technology was used to down-regulate the expression of protein SUZ12 and Cdc25 C. The lentiviral transfection method was used to construct Cdc25 C over-expression cell lines and its negative control cell lines in Hela,which was screened by complete medium containing puromycin for 14 days. Fluorescence Photography detected Green fluorescent protein and Western Blot for verifying.MTT assay was used to detect the effect on cell proliferation after decreasing the express of SUZ12 and Cdc25 C. Western Blot method was used to detect the expression level of protein after down regulating of SUZ12. Results MTT results showed that the proliferation of Hela and SiHa cells in siSUZ12 group was significantly lower than control group. The proliferation of cells was significantly inhibited. The survival rate of Hela was(60.65±7.64)%(P<0.01), and SiHa was(73.81±9.30)%(P<0.01).The results of the time courses showed that after siSUZ12 transfection, Hela cells began to show inhibition after the second day of transfection, of which the survival rate was the most obvious at the fifth day was(56.13±7.78)%(P<0.01). The inhibition of SiHa cells began after the first day of transfection, with the most obvious survival rate at the fifth day was(70.50±3.15)%(P<0.01). As the cell circle analysis showed, the proportion of G0/G1 phase cell increased significantly(P<0.05)in Hela after using siSUZ12 for 48 h. G1/S phase arrest of cells appeared. The proportion of G2/M cells increased significantly in SiHa after using siSUZ12 for 60 h(P<0.01). G2/M phase arrest of cells occurred. Western Blot results showed that the expression of Cdc25 C in Hela and SiHa cells were significantly decreased by siSUZ12. Blocking Cdc25 C expression significantly antagonized the inhibition of siSUZ12 on Hela and SiHa cells. Over expression of Cdc25 C reduced the inhibitory effect of siSUZ12 on cells.Conclusion By inhibiting the expression of Cdc25 C, down-regulation of SUZ12 can inhibit the proliferation of human cervical cancer cells.
Objective To investigate whether down-regulation of SUZ12 influences the proliferation and cell cycle of human cervical cancer cell lines Hela and SiHa,and to research its mechanism. Methods RNA interference technology was used to down-regulate the expression of protein SUZ12 and Cdc25 C. The lentiviral transfection method was used to construct Cdc25 C over-expression cell lines and its negative control cell lines in Hela,which was screened by complete medium containing puromycin for 14 days. Fluorescence Photography detected Green fluorescent protein and Western Blot for verifying.MTT assay was used to detect the effect on cell proliferation after decreasing the express of SUZ12 and Cdc25 C. Western Blot method was used to detect the expression level of protein after down regulating of SUZ12. Results MTT results showed that the proliferation of Hela and SiHa cells in siSUZ12 group was significantly lower than control group. The proliferation of cells was significantly inhibited. The survival rate of Hela was(60.65±7.64)%(P<0.01), and SiHa was(73.81±9.30)%(P<0.01).The results of the time courses showed that after siSUZ12 transfection, Hela cells began to show inhibition after the second day of transfection, of which the survival rate was the most obvious at the fifth day was(56.13±7.78)%(P<0.01). The inhibition of SiHa cells began after the first day of transfection, with the most obvious survival rate at the fifth day was(70.50±3.15)%(P<0.01). As the cell circle analysis showed, the proportion of G0/G1 phase cell increased significantly(P<0.05)in Hela after using siSUZ12 for 48 h. G1/S phase arrest of cells appeared. The proportion of G2/M cells increased significantly in SiHa after using siSUZ12 for 60 h(P<0.01). G2/M phase arrest of cells occurred. Western Blot results showed that the expression of Cdc25 C in Hela and SiHa cells were significantly decreased by siSUZ12. Blocking Cdc25 C expression significantly antagonized the inhibition of siSUZ12 on Hela and SiHa cells. Over expression of Cdc25 C reduced the inhibitory effect of siSUZ12 on cells.Conclusion By inhibiting the expression of Cdc25 C, down-regulation of SUZ12 can inhibit the proliferation of human cervical cancer cells.
引文
[1]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2017[J].CA Cancer J Clin,2017,67(1):7-30.
[2]GOLBABAPOUR S,MAJID N A,HASSANDARVISH P,et al.Gene silencing and Polycomb group proteins:An overview of their structure,mechanisms and phylogenetics[J].OMICS,2013,17(6):283-296.
[3]SIEGEL R,NAISHADHAM D,JEMAL A.Cancer statistics,2012[J].CA:A Cancer Journal for Clinicians,2012,62(1):10-29.
[4]WU Y P,HU H J,ZHANG W,et al.SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma[J].J Cell Mol Med,2018,22(7):3582-3594.
[5]苏永辉,侯冰宗,徐家琪,等.PRC2成分SUZ12在结直肠癌组织中的表达及其临床意义[J].消化肿瘤杂志(电子版),2014,6(1):31-34.
[6]LI H,CAI Q,WU H,et al.SUZ12 promotes human epithelial ovarian cancer by suppressing apoptosis via silencing HRK[J].Mol Cancer Res,2012,10(11):1462-1472.
[7]毛楠.沉默SUZ12表达对人非小细胞肺癌增殖转移的抑制作用及其机制研究[D].广州:南方医科大学,2014.
[8]LIU C H,SHI X F,WANG L,et al.SUZ12 is involved in progression of non-small cell lung cancer by promoting cell proliferation and metastasis[J].Tumour Biol,2014,35(6):6073-6082.
[9]LEE S R,ROH Y G,KIM S K,et al.Activation of EZH2and SUZ12regulated by E2F1predicts the disease progression and aggressive characteristics of bladder cancer[J].Clin Cancer Res,2015,21(23):5391-5403.
[10]XU X T,TAO Z Z,SONG Q B,et al.SUZ12 RNA interference inhibits the invasion of gastric carcinoma cells[J].Hepatogastroenterology,2014,61(136):2416-2420.
[11]CONWAY E,HEALY E,BRACKEN A P.PRC2mediated H3K27methylations in cellular identity and cancer[J].Curr Opin Cell Biol,2015,37:42-48.
[12]WANG J Y.Regulation of cell death by the Abl tyrosine kinase[J].Oncogene,2000,19(49):5643-5650.
[13]LIU Y L,GAO X,JIANG Y,et al.Expression and clinicopathological significance of EED,SUZ12 and EZH2mRNA in colorectal cancer[J].J Cancer Res Clin Oncol,2015,141(4):661-669.
[14]HU H J,WANG Y,LI Z W,et al.Overexpression of suppressor of zest 12 is associated with cervical node metastasis and unfavorable prognosis in tongue squamous cell carcinoma[J].Cancer Cell Int,2017,17:26.
[15]KIRMIZIS A,BARTLEY S M,FARNHAM P J.Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy[J].Mol Cancer Ther,2003,2(1):113-121.
[16]JUNG J W,LEE S,SEO M S,et al.Histone deacetylase controls adult stem cell aging by balancing the expression of polycomb genes and jumonji domain containing 3[J].Cell Mol Life Sci,2010,67(7):1165-1176.
[2]GOLBABAPOUR S,MAJID N A,HASSANDARVISH P,et al.Gene silencing and Polycomb group proteins:An overview of their structure,mechanisms and phylogenetics[J].OMICS,2013,17(6):283-296.
[3]SIEGEL R,NAISHADHAM D,JEMAL A.Cancer statistics,2012[J].CA:A Cancer Journal for Clinicians,2012,62(1):10-29.
[4]WU Y P,HU H J,ZHANG W,et al.SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma[J].J Cell Mol Med,2018,22(7):3582-3594.
[5]苏永辉,侯冰宗,徐家琪,等.PRC2成分SUZ12在结直肠癌组织中的表达及其临床意义[J].消化肿瘤杂志(电子版),2014,6(1):31-34.
[6]LI H,CAI Q,WU H,et al.SUZ12 promotes human epithelial ovarian cancer by suppressing apoptosis via silencing HRK[J].Mol Cancer Res,2012,10(11):1462-1472.
[7]毛楠.沉默SUZ12表达对人非小细胞肺癌增殖转移的抑制作用及其机制研究[D].广州:南方医科大学,2014.
[8]LIU C H,SHI X F,WANG L,et al.SUZ12 is involved in progression of non-small cell lung cancer by promoting cell proliferation and metastasis[J].Tumour Biol,2014,35(6):6073-6082.
[9]LEE S R,ROH Y G,KIM S K,et al.Activation of EZH2and SUZ12regulated by E2F1predicts the disease progression and aggressive characteristics of bladder cancer[J].Clin Cancer Res,2015,21(23):5391-5403.
[10]XU X T,TAO Z Z,SONG Q B,et al.SUZ12 RNA interference inhibits the invasion of gastric carcinoma cells[J].Hepatogastroenterology,2014,61(136):2416-2420.
[11]CONWAY E,HEALY E,BRACKEN A P.PRC2mediated H3K27methylations in cellular identity and cancer[J].Curr Opin Cell Biol,2015,37:42-48.
[12]WANG J Y.Regulation of cell death by the Abl tyrosine kinase[J].Oncogene,2000,19(49):5643-5650.
[13]LIU Y L,GAO X,JIANG Y,et al.Expression and clinicopathological significance of EED,SUZ12 and EZH2mRNA in colorectal cancer[J].J Cancer Res Clin Oncol,2015,141(4):661-669.
[14]HU H J,WANG Y,LI Z W,et al.Overexpression of suppressor of zest 12 is associated with cervical node metastasis and unfavorable prognosis in tongue squamous cell carcinoma[J].Cancer Cell Int,2017,17:26.
[15]KIRMIZIS A,BARTLEY S M,FARNHAM P J.Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy[J].Mol Cancer Ther,2003,2(1):113-121.
[16]JUNG J W,LEE S,SEO M S,et al.Histone deacetylase controls adult stem cell aging by balancing the expression of polycomb genes and jumonji domain containing 3[J].Cell Mol Life Sci,2010,67(7):1165-1176.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |