RNA结合蛋白RBMS2通过上调p21抑制人乳腺癌细胞增殖的机制研究

Foundation：TCGA data analysis showed that the expression of RBMS2 was positively related with the survival rate of patients with breast cancer. Lentivirus was used to over-express and knock-down RBMS2 in the MCF-7 and SUM-1315 breast cancer cells. With the analysis of CCK-8 and colony formation, we found that over expression of RBMS2 could inhibit the proliferation of breast cancer cells. The flow cytometer assay showed that RBMS2 could suppress the transformation of tumor cells from G1 to S phase. The analysis of RNA high throughput sequencing (RNA-seq) showed, after over-expression of RBMS2, the expression of p21 was up-regulated. RNA immunoprecipitation (RIP) assay followed by RT-PCR and qRT-PCR determined that RBMS2 could bind to p21 mRNA directly ..Hypothesis: In breast cancer, RBMS2 can inhibit the proliferation of cancer cells by up regulation the expression of p21..Objective: Nude mice experiments will be applied to verify the effect of RBMS2 on the proliferation of breast cancer cells in vivo. REMSA and dual-luciferase reporter assays will be used to investigate the mechanism of RNA binding protein RBMS2 to increase the expression of p21. We will use the small interfering (siRNA) of p21 to further study whether the increased expression of p21 by RBM38 affect RBM38-induced growth suppression.

研究基础:TCGA数据库分析发现乳腺癌患者的生存率与RBMS2的表达呈正相关；构建RBMS2慢病毒稳转细胞系，进行CCK-8、克隆形试验发现：RBMS2可以抑制乳腺癌细胞的增殖；流式检测发现：RBMS2可以通过抑制肿瘤细胞由G1期向S期转换；通过RNA高通量测序（RNA-seq）发现：RBMS2过表达后，p21的表达被上调。RNA免疫共沉淀证实，RBMS2可以与p21的mRNA直接结合。假设：在乳腺癌中，RBMS2可以通过上调P21的表达抑制乳腺肿瘤细胞的增殖，进而发挥RBMS2的抑癌作用。研究目的：通过裸鼠成瘤实验，验证RBMS2在体内对乳腺肿瘤的影响；应用RNA稳定性实验、RNA凝胶电泳迁移率实验以及双荧光素酶报告基因实验，探索RBMS2通过p21发挥生物学功能的机制；应用小干扰沉默p21的表达，进一步验证p21对RBMS2表达量的影响，及其对RBMS2生物学功能的影响。

背景：RNA结合蛋白在乳腺肿瘤的发生发展中起着重要的作用，乳腺肿瘤的增殖速度和乳腺肿瘤对化疗药物的反应对乳腺癌患者的预后有重大意义。.主要研究内容：RBMS2是RNA结合蛋白的一员，RBMS2过表达可以抑制乳腺癌细胞的增殖并且促进细胞凋亡。体内、外实验均证实， RBMS2过表达可以抑制细胞增殖，并且促进阿霉素的化疗药物敏感性。细胞周期流式检测发现，RBSM2可以抑制肿瘤细胞G1期向S期的转换，在阿霉素的刺激下，流式分析发现RBMS2过表达可以促进阿霉素诱导的细胞凋亡。RNA高通量测序发现，BMS2过表达后，周期调节基因p21和促进凋亡相关基因BMF的表达被明显上调。Western Blot和RT-PCR实验发现，RBMS2的表达与p21和BMF的表达呈现正相关。应用siRNA沉默p21发现，RBMS2在乳腺癌中发挥的抑制肿瘤细胞增殖的作用依赖于p21的表达。应用siRNA沉默BMF的表达发现，RBMS2发挥的促细胞凋亡作用依赖于BMF的表达。.重要结果：RBMS2可以通过增强p21的mRNA稳定性抑制乳腺癌细胞的增殖。RBMS2可以通过促进BMF的表达促进阿霉素的化疗敏感性。.科学意义：首次证实了RNA结合蛋白RBMS2在乳腺癌中发挥着抑癌基因的作用， 并且发现RBMS2的表达可以影响化疗药物阿霉素的敏感性。