摘要
目的探讨环状RNA circCSPP1通过吸附miR-431而促进结直肠癌增殖和转移的作用及其机制。方法通过生物信息学方法分析circCSPP1在人结直肠癌组织中的表达;实时定量PCR法检测结直肠癌临床样本中circCSPP1的表达;以结肠癌细胞株SW620和LOVO为研究对象,MTT检测circCSPP1对细胞增殖活性的影响;克隆形成实验探究circCSPP1对结直肠癌细胞克隆形成的效应;Transwell实验检测circCSPP1在肿瘤细胞迁移/侵袭中的作用;生物信息学方法预测circCSPP1可能的miRNA结合位点,通过双荧光素酶实验验证cricCSPP1与miR-431之间的联系,敲减和过表达circCSPP1后检测miR-431的表达,验证二者的相关性;荧光原位杂交实验验证circCSPP1与miR-431在胞浆中共表达,分析miR-431表达与结直肠癌临床特征的关系。结果生物信息学分析表明circCSPP1在结直肠癌组织中高表达,这一现象在临床样本中得到证实;MTT实验结果表明,circCSPP1受到抑制后,结直肠癌细胞的增殖活性下降;克隆形成实验的结果提示沉默circCSPP1可以减少结直肠癌细胞的克隆。Transwell迁移实验中,抑制circCSPP1可以降低肿瘤细胞的迁移率。Transwell侵袭实验表明,结直肠癌细胞株的circCSPP1受到抑制后,侵袭率显著下降。在探究circCSPP1影响癌细胞的机制时,对预测的5种miRNA逐一验证,发现miR-431的荧光素酶活性降低,表明cricCSPP1与miR-431之间存在结合位点。通过正反验证,敲减circCSPP1后,细胞中miR-431水平升高,过表达circCSPP1后,miR-431表达水平降低。FISH实验中,circCSPP1及miR-431均被证实主要存在于胞浆中。结论circCSPP1在结直肠癌组织中显著高表达;circCSPP1正向调控结直肠癌的进展。circCSPP1通过海绵样吸附作用抑制了相关细胞中的miR-431表达水平,并最终影响结直肠癌患者的生存及预后。
Objective To investigate the role and mechanism of circular RNA circCSPP1 in promoting colorectal cancer proliferation and metastasis by sponging miR-431.Methods The bioinformatics approach was used to analyze the expression of circCSPP1 in human colorectal cancer tissues.qRT-PCR was used to detect the expression of circCSPP1 in colorectal cancer clinical samples.The MTT assay was used to detect the effect of circCSPP1 on cell proliferation activity with the colon cancer cell lines SW620 and LOVO.Clone formation assay was used to investigate the effect of circCSPP1 on clone formation in colorectal cancer cells.Transwell assay detects the effect of circCSPP1 in tumor cell migration/invasion.Bioinformatic approach was used to predict possible miRNA binding sites for circCSPP1.Double luciferase assay was used to verify the correlation between cricCSPP1 and miR-431,and miR-431 expression was detected after knockdown and overexpression of circCSPP1.Fluorescence in situ hybridization assay was used to verify the co-expression of circCSPP1 with miR431 in the cytoplasm and to analyze the correlation between miR-431 expression and clinical features of colorectal cancer.Results Bioinformatics analysis showed that circCSPP1 was highly expressed in colorectal cancer tissues,which was confirmed in clinical samples.MTT assay results showed that the proliferative activity of colorectal cancer cells declined after circCSPP1 being inhibited.The results of clonogenic assays suggested that silencing circCSPP1 reduces the cloning of colorectal cancer cells.Transwell migration assay showed that inhibition of circCSPP1 reduced the migration rate of tumor cells.Transwell invasion assay showed that colorectal cancer cell lines with circCSPP1 inhibition exhibited a significant decrease in invasion rate.Five predicted miRNAs were verified individually to investigate the mechanism by which circCSPP1 affected cancer cells,and the luciferase activity of miR-431 was found to be reduced,indicating the existence of a binding site between cricCS
作者
袁晨磊
张姚
关杰文
窦唯
张鹏
赵鑫
YUAN Chen-lei;ZHANG Yao;GUAN Jie-wen;DOU Wei;ZHANG Peng;ZHAO Xin(School of Medicine,Soochow University,Suzhou,Jiangsu,215123,China;Department of GastrointestinalSurgery,the First Affiliated Hospital of Soochow University,Suzhou,Jiangsu,215006,China)
出处
《中国血液流变学杂志》
CAS
2021年第3期298-306,332,F0002,共11页
Chinese Journal of Hemorheology
基金
国家自然科学基金面上项目(31770985,82073180),江苏省社会发展项目(BE2019665),江苏省“333工程”科研项目(BRA2019327),江苏省青年医学人才(QNRC2016732),苏州市“姑苏卫生人才”计划青年拔尖人才(2018-057),姑苏卫生人才培养项目(GSWS2019028),苏州大学校级大学生创新创业训练计划项目(2021xj061)。
