本文選題：AP-2α + miRNA-200家族��； 參考：《湖南師范大學(xué)》2012年碩士論文

【摘要】：AP-2α是轉(zhuǎn)錄因子AP-2家族的成員之一,通過(guò)調(diào)控細(xì)胞增殖和凋亡過(guò)程中的多種基因起到腫瘤抑制的作用。已有研究表明：包括順鉑在內(nèi)的多種化療藥物可誘導(dǎo)內(nèi)源性AP-2a的表達(dá),從而促進(jìn)癌細(xì)胞凋亡、增強(qiáng)細(xì)胞對(duì)化療藥物的敏感性。與此相反,microRNA-200(miR-200)家族(包括miR-200b、miR-200c和miR-429)在子宮內(nèi)膜癌與食道癌中高表達(dá),而它們的過(guò)度表達(dá)與細(xì)胞對(duì)順鉑的抗性相關(guān)。因此,本文研究miR-200家族對(duì)AP-2α基因的調(diào)控,以及與子宮內(nèi)膜癌細(xì)胞對(duì)順鉑敏感性的關(guān)系。 我們通過(guò)生物信息學(xué)軟件Target Scan、Microcosm和miRanda來(lái)預(yù)測(cè)AP-2a基因上的miRNA結(jié)合位點(diǎn),發(fā)現(xiàn)在AP-2a基因的3'端非翻譯區(qū)(UTR)有一個(gè)miR-200b、miR-200c和miR-429共有的miRNA應(yīng)答元件(MRE),而且在這個(gè)應(yīng)答元件中具有一個(gè)單核苷酸多態(tài)性位點(diǎn)(SNP) rs1045385。為了驗(yàn)證這個(gè)預(yù)測(cè)的MRE是否正確,我們分別將AP-2a基因的野生型3'UTR、MRE缺失的3'UTR以及MRE克隆進(jìn)雙熒光素酶報(bào)告基因載體pmirGLO中,并且分別與miR-200b, miR-200c和miR-429共轉(zhuǎn)染進(jìn)入HEK293細(xì)胞中,轉(zhuǎn)染后24小時(shí)測(cè)定熒光素酶活性。結(jié)果表明：過(guò)表達(dá)miR-200b、miR-200c和miR-429都降低了MRE以及包含有MRE的野生型3'UTR的熒光素酶表達(dá),而缺失了MRE的3'UTR的熒光素酶活性不受影響。這些結(jié)果表明miR-200b, miR-200c和miR-429確實(shí)能結(jié)合到AP-2α基因3'UTR的MRE上。 為了研究miR-200家族對(duì)內(nèi)源的AP-2a蛋白表達(dá)的影響,我們?cè)贏P-2a高表達(dá)的宮頸癌細(xì)胞系HeLa細(xì)胞中分別轉(zhuǎn)染miR-200b, miR-200c和miR-429；在miR-200表達(dá)高的子宮內(nèi)膜癌細(xì)胞系HEC-1-A細(xì)胞中轉(zhuǎn)染miR-200b, miR-200c和miR-429的抑制劑。轉(zhuǎn)染后36小時(shí)用Western blot檢測(cè)內(nèi)源AP-2α的表達(dá)。結(jié)果顯示在AP-2α高表達(dá)的宮頸癌細(xì)胞系HeLa中過(guò)表達(dá)miR-200b, miR-200c和miR-429中的任一個(gè)miRNA都能夠抑制內(nèi)源的AP-2α蛋白的表達(dá),而在miR-200表達(dá)高的子宮內(nèi)膜癌細(xì)胞系HEC-1-A細(xì)胞中抑制miR-200b, miR-200c和miR-429中的任一個(gè)miRNA的表達(dá)都能增強(qiáng)AP-2α的表達(dá)。這些結(jié)果說(shuō)明miR-200b, miR-200c和miR-429都能負(fù)調(diào)控細(xì)胞內(nèi)源性的AP-2α的表達(dá)。 為了研究SNP rs1045385AC變化對(duì)miRNA結(jié)合的影響,我們將完整的3'UTR以及突變了SNP位點(diǎn)的3'UTR克隆進(jìn)入pmirGLO中,并且與miRNA共轉(zhuǎn)染進(jìn)入HEK293細(xì)胞中,結(jié)果顯示當(dāng)?shù)任晃稽c(diǎn)C替換為A后抑制了miR-200b, miR-200c和miR-429結(jié)合到3'UTR。為了進(jìn)一步證明這個(gè)結(jié)論,我們構(gòu)建了兩種基因型的全長(zhǎng)AP-2α(包含完整的編碼區(qū)和3'UTR)表達(dá)質(zhì)粒pMyc-AP-2a(A）和pMyc-AP-2a(C),然后將其分別與miR-200b, miR-200c和miR-429共轉(zhuǎn)染進(jìn)HEK293細(xì)胞中。Western結(jié)果分析也表明AP-2α的3'UTR中rs1045385位點(diǎn)A改變?yōu)镃后,干擾了miR-200b, miR-200c和miR-429與AP-2α基因3'UTR的結(jié)合來(lái),導(dǎo)致AP-2α的表達(dá)上調(diào)。 我們接著測(cè)試了SNP rs1045385AC變化是否影響子宮內(nèi)膜細(xì)胞對(duì)順鉑的敏感性。我們將兩種基因型的AP-2α表達(dá)質(zhì)粒pMyc-AP-2α(A)和pMyc-AP-2α(C)分別轉(zhuǎn)染進(jìn)入HEC-1-A細(xì)胞中,并用不同濃度的順鉑處理6小時(shí)�？寺⌒纬蓪�(shí)驗(yàn)等結(jié)果表明,轉(zhuǎn)染pMyc-AP-2α(C)的HEC-1-A細(xì)胞對(duì)順鉑更加敏感,而轉(zhuǎn)染pMyc-AP-2α(A)的HEC-1-A細(xì)胞相對(duì)于對(duì)照組細(xì)胞則無(wú)太大影響。 總之,我們發(fā)現(xiàn)miR-200b, miR-200c和miR-429在子宮內(nèi)癌細(xì)胞中通過(guò)抑制AP-2α的表達(dá)來(lái)降低細(xì)胞對(duì)順鉑的敏感性,且發(fā)現(xiàn)了AP-2a的一個(gè)SNP位點(diǎn)(rs1045385)通過(guò)影響miR-200、miR-200c和miR-429與AP-2α mRNA的結(jié)合,從而影響HEC-1-A細(xì)胞對(duì)順鉑的敏感性。我們的結(jié)果表明,SNP位點(diǎn)rs1045385很可能是一個(gè)順鉑治療的預(yù)后標(biāo)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偽 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 .
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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