miR-21对宫颈癌Siha细胞增殖侵袭及调控SPRY2表达的影响
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摘要
目的探讨微小RNA-21(miR-21)对人宫颈癌Siha细胞增殖、凋亡、侵袭及调控快速发育生长因子同源蛋白2抗体(SPRY2)表达的影响。方法收集2016年1月至2017年12月广州市番禺区中心医院收治患者的宫颈癌组织和正常宫颈组织标本各22例,采用实时荧光定量聚合酶链反应(QPCR)检测宫颈癌组织中miR-21和SPRY2的表达水平。采用Lipofectamine~(TM)2000脂质体法将miR-21模拟物(minic-21组)、模拟物对照寡核苷酸(minic-nc组)、抑制剂(inhibitor-21组)和抑制剂对照寡核苷酸(inhibitor-nc组)转染到宫颈癌Siha细胞。转染48h后,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖,流式细胞仪检测细胞凋亡,Transwell检测细胞侵袭,QPCR和Western blot检测各组细胞中SPRY2mRNA和蛋白水平。结果与正常宫颈组织比较,宫颈癌组织中miR-21表达明显增高,SPRY2表达明显降低(P<0.05),二者呈负相关(P<0.01)。与相应对照组相比,minic-21组Siha细胞增殖率升高,凋亡率降低,侵袭能力提高(P<0.05);inhibitor-21组Siha细胞增殖率降低,凋亡率升高,侵袭能力减弱(P<0.05)。与相应对照组相比,minic-21组SPRY2蛋白水平明显降低(P<0.05),SPRY2mRNA水平无明显差异(P>0.05);inhibitor-21组SPRY2mRNA和蛋白水平均明显升高(P<0.01)。结论 miR-21在宫颈癌中可能通过调控肿瘤抑制基因SPRY2发挥作用,有望成为宫颈癌基因治疗的一个新靶点。
Objective To investigate the effect of micro RNA-21(miR-21)on proliferation,invasion,apoptosis and regulating expression of Sprouty2(SPRY2)in cervical cancer Siha cells.Methods The samples from 22 pairs of cervical cancer patients and normal cervical tissue in Panyu District Central Hospital from January 2016 to December 2017 were collected.Real-time fluorescence quantitative PCR(QPCR)was used to examine the level of mi R-21 and SPRY2 in cervical cancer tissues.Lipofectamine~(TM)2000 liposome was used to transfect miR-21 minic(minic-21 group),minic oligonucleotides(minic-nc group),miR-21 inhibitor(inhibitor-21 group)and inhibitor oligonucleotides(inhibitor-nc group)into human cervical cancer Siha cells.At the time of 48 hafter transfection,the cell proliferation was checked with MMT,the cell apoptosis was examined by flow cytometry and the cell invasion was tested with Trans well.The mRNA and protein levels of SPRY2 in each group were determined by QPCR and Western blot.Results Compared with the normal cervical tissues,the expression of miR-21 was significantly increased,the expression of SPRY2 was significantly decreased in cervical cancer tissues(P<0.05).Both were negatively correlated(P<0.01).Compared with the corresponding control group,the proliferation rate and invasion ability were increased with the apoptotic rate decreased in the minic-21 group.However,the proliferation rate and invasion ability were decreased with apoptotic rate was increased in the inhibitor-21 group(P<0.05).Compared with the corresponding control group,the SPRY2 protein level in the minic-21 group was significantly decreased(P<0.05),while the mRNA level of SPRY2 had no statistically significant difference(P>0.05).Both mRNA and protein levels of SPRY2 in the inhibitor-21 group were significantly increaed(P<0.05).Conclusion miR-21 may play the cancer gene role by regulating tumor inhibiting gene APRY2,and is expected to become a new target point in cervical cancer genetic therapy.
Objective To investigate the effect of micro RNA-21(miR-21)on proliferation,invasion,apoptosis and regulating expression of Sprouty2(SPRY2)in cervical cancer Siha cells.Methods The samples from 22 pairs of cervical cancer patients and normal cervical tissue in Panyu District Central Hospital from January 2016 to December 2017 were collected.Real-time fluorescence quantitative PCR(QPCR)was used to examine the level of mi R-21 and SPRY2 in cervical cancer tissues.Lipofectamine~(TM)2000 liposome was used to transfect miR-21 minic(minic-21 group),minic oligonucleotides(minic-nc group),miR-21 inhibitor(inhibitor-21 group)and inhibitor oligonucleotides(inhibitor-nc group)into human cervical cancer Siha cells.At the time of 48 hafter transfection,the cell proliferation was checked with MMT,the cell apoptosis was examined by flow cytometry and the cell invasion was tested with Trans well.The mRNA and protein levels of SPRY2 in each group were determined by QPCR and Western blot.Results Compared with the normal cervical tissues,the expression of miR-21 was significantly increased,the expression of SPRY2 was significantly decreased in cervical cancer tissues(P<0.05).Both were negatively correlated(P<0.01).Compared with the corresponding control group,the proliferation rate and invasion ability were increased with the apoptotic rate decreased in the minic-21 group.However,the proliferation rate and invasion ability were decreased with apoptotic rate was increased in the inhibitor-21 group(P<0.05).Compared with the corresponding control group,the SPRY2 protein level in the minic-21 group was significantly decreased(P<0.05),while the mRNA level of SPRY2 had no statistically significant difference(P>0.05).Both mRNA and protein levels of SPRY2 in the inhibitor-21 group were significantly increaed(P<0.05).Conclusion miR-21 may play the cancer gene role by regulating tumor inhibiting gene APRY2,and is expected to become a new target point in cervical cancer genetic therapy.
引文
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[19]BARBACHANO A,FERNANDEZ-BARRAL A,PEREI-RA F,et al.SPROUTY-2represses the epithelial phenotype of colon carcinoma cells via up regulation of ZEB1mediated by ETS1and miR-200/miR-150[J].Oncogene,2016,35(23):2991-3003.
[2]IRANI S.miRNAs signature in head and neck squamous cell carcinoma metastasis:a literature review[J].Dent(Shiraz),2016,17(2):71-83.
[3]MELNIK B C.MiR-21:an environmental driver of malignant melanoma?[J].J Transl Med,2015,13(1):202.
[4]WANG J H,ZHOU W W,CHENG S T,et al.Down regulation of Sprouty homolog2by micro RNA-21inhibits proliferation,metastasis and invasion,however promotes the apoptosis of multiple myeloma cells[J].Mol Med Rep,2015,12(2):1810-1816.
[5]BALDU-FELSKOV B,MUNK C,NIELSEN T S,et al.Trends in the incidence of cervical cancer and severe precancerous lesions in Denmark,1997-2012[J].Cancer Causes Control,2015,26(8):1105-1116.
[6]Ol IVETO S,MANCINO M,MANFRINI N,et al.Role of micro RNAs in translation regulation and cancer[J].World J Biol Chem,2017,8(1):45-56.
[7]TIAN L,SHAN W,ZHANG Y,et al.Erratum to:Upregulation of miR-21expression predicate advanced clinicopathological features and poor prognosis in patients with non-small cell lung cancer[J].Pathol Oncol Res,2016,22(2):439.
[8]MERCADO-PIMENTE L,ONYEAGUCHA B C,LI Q,et al.The S100P/RAGE signaling pathway regulates expression of micro RNA-21in colon cancer cells[J].FEBS Lett,2015,589(18):2388-2393.
[9]WANG W,LI J,ZHU W,et al.Micro RNA-21and the clinical outcomes of various carcinomas:a systematic review and meta-analysis[J].BMC Cancer,2014,1(4):819.
[10]WANG N,XU Z,WANG K,et al.Construction and analysis of regulatory genetic networks in cervical cancer based on involved micro RNAs,target genes,transcription factors and host genes[J].Oncol Lett,2014,7(4):1279-1283.
[11]GONZALEZ-QUINTANA V,PALMA-BERRE L,CAM-POS-PARRA A D,et al.MicroRNAs are involved in cervical cancer development,progression,clinical outcome and improvement treatment response[J].Oncol Rep,2016,35(1):3-12.
[12]FUJITA S,ITO T,MIZUTANI T,et al.MiR-21gene expression triggered by AP-1is sustained through a doublenegative feedback mechanism[J].J Mol Biol,2008,37(8):492-504.
[13]YAO T,LIN Z.MiR-21is involved in cervical squamous cell tumor genesis and regulates CCL20[J].Biochem Biophys Acta,2012,1822(2):248-260.
[14]TSUJITA Y,MITSUI-SEKINAKA K,IMAI K,et al.Phosphatase and tension homolog(PTEN)mutation can cause activated phosphatidylinositol 3-kinase delta syndrome-like immunodeficiency[J].J Allergy Clin Immunol,2016,138(6):1672-1680.
[15]YANG Y,GUO J X,SHAO Z Q.MiR-21targets and inhibits tumor suppressor gene PTEN to promote prostate cancer cell proliferation and invasion:An experimental study[J].Asian Pac J Trop Med,2017,10(1):87-91.
[16]LUO G,LUO W,SUN X,et al.Micro RNA21promotes migration and invasion of glioma cells via activation of Sox2and beta catenin signaling[J].Mol Med Rep,2017,15(1):187-193.
[17]MASOUMI-MOGHADDAM S,AMINI A,MORRIS DL.The developing story of Sprouty and cancer[J].Cancer Metastasis Rev,2014,33(2/3):695-720.
[18]SHUKLA A,RAI K,SHUKLA V,et al.Sprouty 2:a novel attenuator of B-cell receptor and MAPK-Erk signaling in CLL[J].Blood,2016,127(19):2310-2321.
[19]BARBACHANO A,FERNANDEZ-BARRAL A,PEREI-RA F,et al.SPROUTY-2represses the epithelial phenotype of colon carcinoma cells via up regulation of ZEB1mediated by ETS1and miR-200/miR-150[J].Oncogene,2016,35(23):2991-3003.