【摘要】：目的:探討人微小RNA-218(Homo sapiens microRNA-218,hsa-miR-218)對(duì)宮頸癌HeLa細(xì)胞生長(zhǎng)的影響及分子機(jī)制。方法:構(gòu)建hsa-miR-218的重組慢病毒表達(dá)載體pmiR-218,并將pmiR-218感染至HeLa細(xì)胞,臺(tái)盼藍(lán)拒染法檢測(cè)細(xì)胞數(shù)量的變化,WST-8法檢測(cè)細(xì)胞活力,構(gòu)建螢光素酶報(bào)告基因載體驗(yàn)證miR-218與LIM和SH3蛋白1(LASP1)的相互結(jié)合作用,real-time PCR檢測(cè)LASP1的mRNA相對(duì)表達(dá)水平,Western blot檢測(cè)LASP1蛋白的表達(dá)水平。結(jié)果:經(jīng)DNA測(cè)序證實(shí)與設(shè)計(jì)完全一致,測(cè)序結(jié)果顯示成功構(gòu)建了重組慢病毒表達(dá)載體pmiR-218。pmiR-218轉(zhuǎn)染HeLa細(xì)胞72 h后HeLa細(xì)胞存活數(shù)量為176±9,對(duì)HeLa細(xì)胞活力的抑制率具有時(shí)間效應(yīng)關(guān)系,較空白對(duì)照組比較,差異顯著(P0.05);螢光素酶活性結(jié)果顯示,克隆LASP1-3’UTR的質(zhì)粒與miR-218 mimics共轉(zhuǎn)染293T細(xì)胞,引起螢光素酶活性的減低;real-time PCR結(jié)果顯示轉(zhuǎn)染pmiR-218后,miR-218表達(dá)水平增加;過表達(dá)miR-128能下調(diào)LASP1 mRNA及蛋白的相對(duì)表達(dá)水平,與空白對(duì)照組及陰性對(duì)照組比較,差異顯著(P0.01)。結(jié)論:pmiR-218有效抑制HeLa細(xì)胞的生長(zhǎng),并具有時(shí)間-效應(yīng)關(guān)系,其機(jī)制可能是miR-218通過靶向結(jié)合LASP1的3’UTR,從而下調(diào)其在HeLa細(xì)胞的表達(dá)有關(guān)。
[Abstract]:Objective: to investigate the effect of human minimal RNA-218 (Homo sapiens microRNA-218,hsa-miR-218 on the growth of cervical cancer HeLa cells and its molecular mechanism. Methods: the recombinant lentivirus expression vector pmiR-218, of hsa-miR-218 was constructed and pmiR-218 was infected into HeLa cells. Trypan blue exclusion assay was used to detect cell number and WST-8 method was used to detect cell viability. The luciferase reporter gene vector was constructed to verify the interaction of miR-218 with LIM and SH3 protein 1 (LASP1). The mRNA relative expression level of LASP1 was detected by real-time PCR and the expression level of LASP1 protein was detected by, Western blot. Results: the results of DNA sequencing showed that the recombinant lentivirus expression vector pmiR-218.pmiR-218 was successfully constructed, and the survival number of HeLa cells was 176 鹵9 after 72 h transfection. There was a time-effect relationship on the inhibition rate of HeLa cell viability, compared with the blank control group, the difference was significant (P0.05). The results of luciferase activity showed that the plasmids of LASP1-3'UTR cloned into 293T cells were cotransfected with miR-218 mimics, which resulted in the decrease of luciferase activity, and real-time PCR showed that the expression level of miR-218 increased after transfection of pmiR-218. Overexpression of miR-128 could down-regulate the relative expression of LASP1 mRNA and protein, compared with the control group and negative control group, the difference was significant (P0.01). Conclusion: pmiR-218 can effectively inhibit the growth of HeLa cells and has a time-effect relationship. The mechanism may be that miR-218 can down-regulate the expression of miR-218 in HeLa cells by targeting LASP1.
【作者單位】： 吉安市婦幼保健院婦產(chǎn)科;廣州市南吉生物技術(shù)有限公司;廣州市番禺區(qū)中心醫(yī)院檢驗(yàn)科;
【基金】：廣州市番禺區(qū)科技局珠江科技新星項(xiàng)目(No.2013-專-15-6.09)
【分類號(hào)】：R737.33

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9 徐t，

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