miR-34a-5p通过靶向IMP3、PD-L1对乳腺癌细胞生物学行为的影响  

Effect of miR-34a-5p on biological behavior of breast cancer cells by targeting insulin-like growth factor Ⅱ mRNA-binding protein 3 and programmed death ligand-1

作　　者：蒋莉萍[1] 王霞 彭心华[3] JIANG Liping;WANG Xia;PENG Xinhua(Department of Pathology,Maternal and Child Health Hospital of Ji’an,Ji’an 343000,China.;Medical Science Center of Jinggangshan University,Ji’an 343000.;Clinical Laboratory,Maternal and Child Health Hospital of Ji’an,Ji’an 343000)

机构地区：[1]江西省吉安市妇幼保健院病理科,江西吉安343000 [2]井冈山大学医学部,江西吉安343000 [3]江西省吉安市妇幼保健院检验科,江西吉安343000

出　　处：《中国比较医学杂志》2023年第7期67-77,共11页Chinese Journal of Comparative Medicine

基　　金：江西省卫生健康委员会科技计划项目(20204844)。

摘　　要：目的探究miR-34a-5p通过靶向胰岛素样生长因子ⅡmRNA结合蛋白3(IMP3)、程序性死亡配体-1(PD-L1)表达对乳腺癌细胞生物学行为的影响。方法利用Human Protein Atlas和GEPIA在线分析TCGA数据库和GTEx项目中乳腺癌及正常组织IMP3、PD-L1的表达水平、患者的预后生存期;qRT-PCR、Western blot检测160例患者乳腺癌组织及癌旁正常组织中miR-34a-5p、IMP3、PD-L1 mRNA与蛋白表达;Pearson检验分析miR-34a-5p分别与IMP3 mRNA、PD-L1 mRNA的相关性;免疫组化检测患者乳腺癌及正常组织IMP3、PD-L1、白细胞分化抗原44变异体6(CD44v6)的表达,分析IMP3、PD-L1表达水平与患者临床病理参数的关系;靶基因预测、双荧光素酶报告实验验证miR-34a-5p对IMP3、PD-L1的靶向调控作用。MCF7细胞分为control组、miR-NC组、miR-34a-5p组、miR-NC+pcDNA-NC组、miR-NC+IMP3组、miR-NC+PD-L1组、miR-34a-5p+pcDNA-NC组、miR-34a-5p+IMP3组和miR-34a-5p+PD-L1组,采用MTT法、克隆形成、划痕、Transwell小室实验测定细胞增殖活力、迁移、侵袭能力;Western blot检测IMP3、PD-L1、CD44v6、基质金属蛋白酶2(MMP-2)、MMP-9、E-钙黏蛋白(E-cadherin)表达水平。结果生物数据库显示乳腺癌组织IMP3、PD-L1表达水平越高,患者生存期越短(P<0.05);qRT-PCR、Western blot结果显示,乳腺癌组织miR-34a-5p表达水平明显低于正常组织,IMP3、PD-L1 mRNA与蛋白表达水平明显高于正常组织,miR-34a-5p表达分别与IMP3 mRNA、PD-L1 mRNA呈明显负相关(P<0.05);免疫组化结果显示,乳腺癌组织IMP3、PD-L1、CD44v6呈阳性表达,且IMP3、PD-L1表达越高,TNM分期越晚,肿瘤分化程度越低、越容易发生淋巴结和远处转移(P<0.05);双荧光素酶实验验证了miR-34a-5p对IMP3、PD-L1的靶向调控作用;与control组和miR-NC组相比,miR-34a-5p组的细胞活力、单克隆形成数目、划痕愈合率、细胞侵袭数目、IMP3、PD-L1、CD44v6、MMP-2、MMP-9蛋白表达明显降低,E-cadherin蛋白表达明显升�Objective To explore how miR-34a-5p regulates insulin-like growth factorⅡmRNA-binding protein 3(IMP3)and programmed death ligand-1(PD-L1)expression and its influence on the biological behavior of breast cancer cells.Methods We analyzed IMP3 and PD-L1 expression in breast cancer and normal tissues using the Human Protein Atlas and GEPIA,and patient prognosis and survival using The Cancer Genome Atlas and GTEx project.miR-34a-5p,IMP3,and PD-L1 mRNA levels,IMP3 and PD-L1 protein levels were detected in breast cancer and adjacent normal tissues by quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot.Correlations between miR-34a-5p and IMP3 and PD-L1 mRNAs were analyzed by Pearson’s test.Immunohistochemistry was used to detect IMP3,PD-L1,and leukocyte differentiation antigen 44 variant 6(CD44v6)expression in breast cancer and normal tissues,and the relationships between IMP3 and PD-L1 expression and clinicopathological parameters were analyzed.Target gene prediction and dual luciferase reporter assay were used to verify the targeted regulation of miR-34a-5p on IMP3 and PD-L1.MCF7 cells were divided into control group,miR-NC group,miR-34a-5p group,miR-NC+pcDNA-NC group,miR-NC+IMP3 group,miR-NC+PD-L1 group,miR-34a-5p+pcDNA-NC group,miR-34a-5p+IMP3 group,and miR-34a-5p+PD-L1 group.Cell proliferation,migration,and invasion were determined by MTT,colony-formation,scratch,and Transwell chamber tests,respectively.Expression levels of IMP3,PD-L1,CD44v6,matrix metalloproteinase-2(MMP-2),MMP-9,and E-cadherin were detected by Western blot.Results The biological databases showed that higher expression levels of IMP3 and PD-L1 in breast cancer tissues were associated with shorter survival(P<0.05).Expression levels of miR-34a-5p were significantly lower while expression levels of IMP3 and PD-L1 mRNAs and proteins were significantly higher in breast cancer tissues than in normal tissues.miR-34a-5p expression was negatively correlated with IMP3 and PD-L1 mRNAs,respectively(P<0.05).Immunohistochemi

关 键 词：miR-34a-5p 胰岛素样生长因子ⅡmRNA结合蛋白3 程序性死亡配体-1 乳腺癌 增殖 侵袭 

分 类 号：R-33[医药卫生]