miRNA-21对乳腺癌细胞放射敏感性的影响及其机制
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摘要
目的:观察miRNA-21对乳腺癌细胞放射敏感性的影响,并初步探讨其作用机制。方法:乳腺癌T47D和MDA-MB-361细胞分别给予0.0、2.5和5.0Gyγ射线照射,CCK8法检测细胞存活率,流式细胞术分析细胞周期进程,实时定量PCR法检测72h内细胞中miRNA-21表达水平。T47D和MDA-MB-361细胞分别转染anti-miRNA-21序列(anti-miRNA-21组)和阴性对照序列(阴性对照组),并设置未转染细胞为空白对照组,实时定量PCR法检测各组细胞中miRNA-21表达水平;经0.0和5.0Gyγ射线照射后,CCK8法检测细胞存活率,流式细胞术分析细胞周期进程。结果:经5.0Gyγ射线照射后,T47D细胞存活率明显高于MDA-MB-361细胞(P<0.05);与0.0Gy照射组比较,5.0Gy照射组T47D和MDA-MB-361细胞G2/M期细胞百分率均明显升高(P<0.05),miRNA-21表达水平均明显升高(P<0.05)。与空白对照组比较,anti-miRNA-21组T47D和MDA-MB-361细胞中miRNA-21表达水平均明显降低(P<0.05或P<0.01);给予5.0Gyγ射线照射后,anti-miRNA-21组T47D和MDA-MB-361细胞存活率均明显降低(P<0.05或P<0.01),T47D细胞G2/M期细胞百分率明显降低(P<0.05),而sub G1期细胞百分率明显升高(P<0.05)。结论:miRNA-21表达下调能够增强乳腺癌细胞的放射敏感性,其机制可能与抑制细胞周期G2/M期阻滞有关。
Objective:To observe the influence of miRNA-21 in the radiosensitivity of the breast cancer cells,and to preliminarily explore its mechanism.Methods:The breast cancer T47 D and MDA-MB-361 cells were irradiated with 0.0,2.5,and 5.0 Gyγ-ray,respectively;CCK8 assay was used to detect the survival rates of T47 Dand MDA-MB-361 cells and flow cytometry was used to analyze the cell cycle;real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells during 72 h.The T47 Dand MDA-MB-361 cells were transfected with anti-miRNA-21 sequence(anti-miRNA-21 group)and negative control sequence(negative control group),respectively;the untransfected cells were set as blank control group.Real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells in various groups.After irradiation with 0.0 and 5.0 Gyγ-ray,CCK8 assay was used to detect the survival rates,and flow cytometry was used to analyze the cell cycle.Results:After irradiation with 5.0 Gyγ-ray,the survival rate of T47 Dcells was significantly higher than that of the MDA-MB-361 cells(P<0.05).Compared with 0.0 Gy radiation group,the percentages of T47 Dand MDA-MB-361 cells in G2/M phase in 5.0 Gy radiation group were significantly increased(P <0.05);the miRNA-21 expression levels in the T47 Dand MDA-MB-361 cells were significantly increased(P<0.05).The miRNA-21 expression levels in the T47 Dand MDA-MB-361 cells in anti-miRNA-21 group were lower than those in blank control group(P<0.05).After irradiation with 5.0 Gyγ-ray,the survival rates of T47 Dand MDA-MB-361 cells in anti-miRNA-21 group were significantly lower than those in blank control group(P<0.05 or P<0.01),and the percentage of T47 Dcells in G2/M phase in anti-miRNA-21 group was significantly lower than that in blank control group(P<0.05),and the percentage of T47 Dcells in subG1 phase was significantly higher than that in blank control group(P<0.05).Conclusion:The down-regulation of miRNA-21 may enhance the radiosensitivity of breast cancer cells,and its mechanism may be related to the inhibition of G2/M phase arrest.
Objective:To observe the influence of miRNA-21 in the radiosensitivity of the breast cancer cells,and to preliminarily explore its mechanism.Methods:The breast cancer T47 D and MDA-MB-361 cells were irradiated with 0.0,2.5,and 5.0 Gyγ-ray,respectively;CCK8 assay was used to detect the survival rates of T47 Dand MDA-MB-361 cells and flow cytometry was used to analyze the cell cycle;real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells during 72 h.The T47 Dand MDA-MB-361 cells were transfected with anti-miRNA-21 sequence(anti-miRNA-21 group)and negative control sequence(negative control group),respectively;the untransfected cells were set as blank control group.Real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells in various groups.After irradiation with 0.0 and 5.0 Gyγ-ray,CCK8 assay was used to detect the survival rates,and flow cytometry was used to analyze the cell cycle.Results:After irradiation with 5.0 Gyγ-ray,the survival rate of T47 Dcells was significantly higher than that of the MDA-MB-361 cells(P<0.05).Compared with 0.0 Gy radiation group,the percentages of T47 Dand MDA-MB-361 cells in G2/M phase in 5.0 Gy radiation group were significantly increased(P <0.05);the miRNA-21 expression levels in the T47 Dand MDA-MB-361 cells were significantly increased(P<0.05).The miRNA-21 expression levels in the T47 Dand MDA-MB-361 cells in anti-miRNA-21 group were lower than those in blank control group(P<0.05).After irradiation with 5.0 Gyγ-ray,the survival rates of T47 Dand MDA-MB-361 cells in anti-miRNA-21 group were significantly lower than those in blank control group(P<0.05 or P<0.01),and the percentage of T47 Dcells in G2/M phase in anti-miRNA-21 group was significantly lower than that in blank control group(P<0.05),and the percentage of T47 Dcells in subG1 phase was significantly higher than that in blank control group(P<0.05).Conclusion:The down-regulation of miRNA-21 may enhance the radiosensitivity of breast cancer cells,and its mechanism may be related to the inhibition of G2/M phase arrest.
引文
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[15]狄英波,孙颖,张桂英,等.MicroRNA-21与乳腺癌细胞MDA-MB-231放射敏感性之间关系的研究[J].中国妇幼保健,2012,27(34):5571-5573.
[16]GABRIELY G,WURDINGER T,KESARI S,et al.MicroRNA 21promotes glioma invasion by targeting matrix metalloproteinase regulators[J].Mol Cell Biol,2008,28(17):5369-5380.
[17]CARLETTI M Z,FIEDLER S D,CHRISTENSON L K.MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells[J].Biol Reprod,2010,83(2):286-295.
[18]ZHANG L,KANG W Q,LU X L,et al.LncRNA CASC11promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway[J].Cell Cycle,2018,17(15):1886-1900.
[19]ZHANG W F,XIONG Y W,ZHU T T,et al.MicroRNAlet-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1cell cycle arrest by targeting c-myc[J].Life Sci,2017,170:9-15.
[20]ANASTASOV N,HFIG I,VASCONCELLOS I G,et al.Radiation resistance due to high expression of miR-21 and G2/M checkpoint arrest in breast cancer cells[J].Radiat Oncol,2012,7:206.
[2]CROKER A K,ALLAN A L.Inhibition of aldehyde dehydrogenase(ALDH)activity reduces chemotherapy and radiation resistance of stem-like ALDHhiCD44+human breast cancer cells[J].Breast Cancer Res Treat,2012,133(1):75-87.
[3]KUMAR S,KEERTHANA R,PAZHANIMUTHU A,et al.Overexpression of circulating miRNA-21and miRNA-146ain plasma samples of breast cancer patients[J].Indian JBiochem Biophys,2013,50(3):210-214.
[4]ZHANG H L,YANG L F,ZHU Y,et al.Serum miRNA-21:Elevated levels in patients with metastatic hormone-refractory prostate cancer and potential predictive factor for the efficacy of docetaxel-based chemotherapy[J].Prostate,2011,71(3):326-331.
[5]COTE G A,GORE A J,MCELYEA S D,et al.A pilot study to develop a diagnostic test for pancreatic ductal adenocarcinoma based on differential expression of select miRNA in plasma and bile[J].Am J Gastroenterol,2014,109(12):1942-1952.
[6]BARANWAL S,ALAHARI S K.miRNA control of tumor cell invasion and metastasis[J].Int J Cancer,2010,126(6):1283-1290.
[7]WANG X C,WANG W,ZHANG Z B,et al.Overexpression of miRNA-21promotes radiation-resistance of non-small cell lung cancer[J].Radiat Oncol,2013,8:146.
[8]WONG S T,ZHANG X Q,ZHUANG J T,et al.MicroRNA-21inhibition enhances in vitro chemosensitivity of temozolomide-resistant glioblastoma cells[J].Anticancer Res,2012,32(7):2835-2841.
[9]KUMARSWAMY R,VOLKMANN I,THUM T.Regulation and function of miRNA-21in health and disease[J].RNABiol,2011,8(5):706-713.
[10]钟莉莉,赵银龙,李炳锦,等.MicroRNA-210对卵巢癌细胞生长及放射敏感性的影响[J].吉林大学学报:医学版,2018,44(2):265-269.
[11]MARTIN S L,KALA R,TOLLEFSBOL T O.Mechanisms for the inhibition of colon cancer cells by sulforaphane through epigenetic modulation of MicroRNA-21and human telomerase reverse transcriptase(hTERT)down-regulation[J].Curr Cancer Drug Targets,2018,18(1):97-106.
[12]KUMAR S,KEERTHANA R,PAZHANIMUTHU A,et al.Overexpression of circulating miRNA-21 and miRNA-146ain plasma samples of breast cancer patients[J].Indian J Biochem Biophys,2013,50(3):210-214.
[13]SHENOUDA S K,ALAHARI S K.MicroRNA function in cancer:oncogene or a tumor suppressor?[J].Cancer Metastasis Rev,2009,28(3/4):369-378.
[14]WANG Z X,LU B B,WANG H,et al.MicroRNA-21modulates chemosensitivity of breast cancer cells to doxorubicin by targeting PTEN[J].Arch Med Res,2011,42(4):281-290.
[15]狄英波,孙颖,张桂英,等.MicroRNA-21与乳腺癌细胞MDA-MB-231放射敏感性之间关系的研究[J].中国妇幼保健,2012,27(34):5571-5573.
[16]GABRIELY G,WURDINGER T,KESARI S,et al.MicroRNA 21promotes glioma invasion by targeting matrix metalloproteinase regulators[J].Mol Cell Biol,2008,28(17):5369-5380.
[17]CARLETTI M Z,FIEDLER S D,CHRISTENSON L K.MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells[J].Biol Reprod,2010,83(2):286-295.
[18]ZHANG L,KANG W Q,LU X L,et al.LncRNA CASC11promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway[J].Cell Cycle,2018,17(15):1886-1900.
[19]ZHANG W F,XIONG Y W,ZHU T T,et al.MicroRNAlet-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1cell cycle arrest by targeting c-myc[J].Life Sci,2017,170:9-15.
[20]ANASTASOV N,HFIG I,VASCONCELLOS I G,et al.Radiation resistance due to high expression of miR-21 and G2/M checkpoint arrest in breast cancer cells[J].Radiat Oncol,2012,7:206.