microRNA-200家族调控AP-2α基因表达的研究

发布时间：2018-07-04 19:47

本文选题：AP-2α + miRNA-200家族　；参考：《湖南师范大学》2012年硕士论文

【摘要】：AP-2α是转录因子AP-2家族的成员之一,通过调控细胞增殖和凋亡过程中的多种基因起到肿瘤抑制的作用。已有研究表明：包括顺铂在内的多种化疗药物可诱导内源性AP-2a的表达,从而促进癌细胞凋亡、增强细胞对化疗药物的敏感性。与此相反,microRNA-200(miR-200)家族(包括miR-200b、miR-200c和miR-429)在子宫内膜癌与食道癌中高表达,而它们的过度表达与细胞对顺铂的抗性相关。因此,本文研究miR-200家族对AP-2α基因的调控,以及与子宫内膜癌细胞对顺铂敏感性的关系。我们通过生物信息学软件Target Scan、Microcosm和miRanda来预测AP-2a基因上的miRNA结合位点,发现在AP-2a基因的3'端非翻译区(UTR)有一个miR-200b、miR-200c和miR-429共有的miRNA应答元件(MRE),而且在这个应答元件中具有一个单核苷酸多态性位点(SNP) rs1045385。为了验证这个预测的MRE是否正确,我们分别将AP-2a基因的野生型3'UTR、MRE缺失的3'UTR以及MRE克隆进双荧光素酶报告基因载体pmirGLO中,并且分别与miR-200b, miR-200c和miR-429共转染进入HEK293细胞中,转染后24小时测定荧光素酶活性。结果表明：过表达miR-200b、miR-200c和miR-429都降低了MRE以及包含有MRE的野生型3'UTR的荧光素酶表达,而缺失了MRE的3'UTR的荧光素酶活性不受影响。这些结果表明miR-200b, miR-200c和miR-429确实能结合到AP-2α基因3'UTR的MRE上。为了研究miR-200家族对内源的AP-2a蛋白表达的影响,我们在AP-2a高表达的宫颈癌细胞系HeLa细胞中分别转染miR-200b, miR-200c和miR-429；在miR-200表达高的子宫内膜癌细胞系HEC-1-A细胞中转染miR-200b, miR-200c和miR-429的抑制剂。转染后36小时用Western blot检测内源AP-2α的表达。结果显示在AP-2α高表达的宫颈癌细胞系HeLa中过表达miR-200b, miR-200c和miR-429中的任一个miRNA都能够抑制内源的AP-2α蛋白的表达,而在miR-200表达高的子宫内膜癌细胞系HEC-1-A细胞中抑制miR-200b, miR-200c和miR-429中的任一个miRNA的表达都能增强AP-2α的表达。这些结果说明miR-200b, miR-200c和miR-429都能负调控细胞内源性的AP-2α的表达。为了研究SNP rs1045385AC变化对miRNA结合的影响,我们将完整的3'UTR以及突变了SNP位点的3'UTR克隆进入pmirGLO中,并且与miRNA共转染进入HEK293细胞中,结果显示当等位位点C替换为A后抑制了miR-200b, miR-200c和miR-429结合到3'UTR。为了进一步证明这个结论,我们构建了两种基因型的全长AP-2α(包含完整的编码区和3'UTR)表达质粒pMyc-AP-2a(A）和pMyc-AP-2a(C),然后将其分别与miR-200b, miR-200c和miR-429共转染进HEK293细胞中。Western结果分析也表明AP-2α的3'UTR中rs1045385位点A改变为C后,干扰了miR-200b, miR-200c和miR-429与AP-2α基因3'UTR的结合来,导致AP-2α的表达上调。我们接着测试了SNP rs1045385AC变化是否影响子宫内膜细胞对顺铂的敏感性。我们将两种基因型的AP-2α表达质粒pMyc-AP-2α(A)和pMyc-AP-2α(C)分别转染进入HEC-1-A细胞中,并用不同浓度的顺铂处理6小时。克隆形成实验等结果表明,转染pMyc-AP-2α(C)的HEC-1-A细胞对顺铂更加敏感,而转染pMyc-AP-2α(A)的HEC-1-A细胞相对于对照组细胞则无太大影响。总之,我们发现miR-200b, miR-200c和miR-429在子宫内癌细胞中通过抑制AP-2α的表达来降低细胞对顺铂的敏感性,且发现了AP-2a的一个SNP位点(rs1045385)通过影响miR-200、miR-200c和miR-429与AP-2α mRNA的结合,从而影响HEC-1-A细胞对顺铂的敏感性。我们的结果表明,SNP位点rs1045385很可能是一个顺铂治疗的预后标志。
[Abstract]:In contrast , the microRNA - 200 ( miR - 200 ) family ( including miR - 200b , miR - 200 and miR - 429 ) is highly expressed in endometrial cancer and esophageal cancer . In contrast , the microRNA - 200 ( miR - 200 ) family ( including miR - 200b , miR - 200 and miR - 429 ) is highly expressed in endometrial cancer and esophageal cancer , and therefore , the expression of the microRNA - 200 ( miR - 200 ) family ( including miR - 200b , miR - 200 and miR - 429 ) is related to the resistance of cells to cisplatin .

We identified miRNA binding sites on the AP - 2a gene by bioinformatics software , Target Scan , MicroRNAs and miRanda . It was found that there was a miRNA response element in the 3 ' - untranslated region of the AP - 2a gene and a single nucleotide polymorphism site ( SNP ) rs1045385 .

In order to investigate the effect of the miR - 200 family on the expression of AP - 2a protein in endogenous AP - 2a , we transfected the miR - 200b , miR - 200 and miR - 429 respectively in HeLa cells of cervical cancer cell line with high expression of AP - 2a ;
The results showed that overexpression of miR - 200b , miR - 200 and miR - 429 in human cervical cancer cell line HeLa expressing high miR - 200 expression can inhibit the expression of AP - 2alpha in endogenous AP - 2 alpha , while miR - 200b , miR - 200 and miR - 429 can be used for inhibiting the expression of endogenous AP - 2alpha in human uterine endometrial cancer cell line HEC - 1 - A cell with high expression of miR - 200 .

In order to study the effect of SNP rs1045385AC on miRNA binding , we cloned the complete 3 ' untranslated region into pmirGLO and co - transfected with miRNA into pmirGLO . The results showed that the expression plasmid pMyc - AP - 2a ( A ) and pMyc - AP - 2a ( C ) were co - transfected with miRNA .

We then tested whether SNP rs1045385AC changed the sensitivity of endometrial cells to cisplatin . We transfected two genotypes of AP - 2伪 expression plasmid pMyc - AP - 2.alpha ( A ) and pMyc - AP - 2.alpha ( C ) into HEC - 1 - A cells , respectively , and treated with cisplatin at different concentrations for 6 hours . The results showed that HEC - 1 - A cells transfected with pMyc - AP - 2.alpha . ( C ) were more sensitive to cisplatin , whereas the HEC - 1 - A cells transfected with pMyc - AP - 2.alpha . ( A ) had no significant effect on cisplatin .

In conclusion , we have found that miR - 200b , miR - 200 and miR - 429 decrease the sensitivity of cells to cisplatin by inhibiting the expression of AP - 2alpha in cancer cells in the uterus , and a SNP site ( rs1045385 ) of AP - 2a has been found to affect the sensitivity of HEC - 1 - A cells to cisplatin . Our findings suggest that SNP site rs1045385 is likely to be a prognostic marker for cisplatin treatment .
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【相似文献】