miR-145对卵巢癌SKOV3细胞侵袭功能的影响及作用机制的探讨
摘要
目的：
探讨microRNA-145对卵巢癌SKOV3细胞侵袭转移功能的影响及可能的作用机制，为临床卵巢癌的治疗提供新的有效靶点。
方法：
采用qRT-PCR方法，验证包括miR-145在内的7种microRNA在卵巢癌组织及卵巢良性肿瘤组织中的表达情况；针对miR-145做进一步研究，利用miR-145mimic转染SKOV3细胞，通过transwell侵袭及迁移实验、划痕实验等方法研究miR-145对卵巢癌SKOV3细胞侵袭迁移等细胞功能的影响；构建双荧光素酶报告载体验证miR-145与靶基因MUC1的互补作用机制，包装病毒载体，使SKOV3细胞稳定表达miR-145，最后利用western blot、PCR等方法，探讨miR-145对MUC1以及EMT关键蛋白E-cad的作用从而影响卵巢癌细胞侵袭转移作用机制。
结果：
PCR结果显示，与良性卵巢瘤组织相比，miR-200a，miR-93，miR-18，miR-146a在卵巢癌组织中的表达明显增高，miR-143，miR-145，miR-29在卵巢癌组织中表达明显下降；根据生物信息学软件预测，miR-145序列与MUC13’UTR端序列互补，是其预测靶基因；western blot检测显示，MUC1蛋白在卵巢癌组织中表达明显增高；将miR-145-5p mimic转染SKOV3细胞48h后，Transwell侵袭实验表明，穿过matrige膜的细胞数与NC组、MOCK组和Blank组相比较，显著减少。Transwell迁移实验表明，miR-145mimic组迁移出Transwell小室并种植到6孔板的细胞数明显少于NC组、MOCK组和Blank组；划痕实验显示划痕后12h、24h、48h，miR-145-5p mimic（0.074、0.148、0.220）组细胞的迁移率明显低于NC阴性对照组（0.087、0.242、0.276）、mock组（0.125、0.269、0.301）及Blank组（0.152、0.260、0.339），且有统计学意义（P<0.05）；DAPI染色测定SKOV3细胞凋亡后，miR-145-5p mimics组细胞的凋亡率与Blank组、mock组及NC阴性对照组无明显差异（P＞0.05）；以MTT实验检测细胞增殖活性，细胞接种96孔板24h、48h和72h后，转染miR-145-5p mimics组与NC阴性对照组、Mock组、及Blank组比较，OD490值明显下降，增殖活性明显下降；以紫杉醇处理SKOV3细胞，用药72h后，转染miR-145-5p mimic组与NC阴性对照组、Mock组及Blank组比较OD490值明显下降，同时，MUC1mRNA和蛋白的表达明显下调。双荧光素酶报告实验显示，将含MUC13’UTR结合序列的荧光素酶报告载体和miR-145-5p mimic共转染293T细胞后，检测荧光表达显著下降；将突变型载体与miR-145-5p mimic共转染293T细胞后，荧光表达无显著差异。成功构建稳定表达miR-145的卵巢癌细胞系miR-145-CMV-RFP-SKOV3，检测MUC1的蛋白表达量明显减少，而E-cad的表达量增加；与之相反，卵巢癌细胞系miR-145-CMV-RFP-SKOV3转染MUC1表达质粒后，E-cad的表达量减少，SKOV3细胞侵袭转移能力增强；
结论：
miR-145是卵巢癌的抑癌基因，miR-145的上调能够抑制卵巢癌细胞的侵袭、转移以及增殖，并能够增强由紫杉醇诱导的细胞抑制效应。MUC1是miR-145的直接靶基因，后者通过与其互补结合抑制其蛋白表达，引起EMT标志性蛋白E-cad表达增强，说明miR-145通过抑制MUC1/E-cad信号通路阻滞了EMT过程导致的SKOV3细胞侵袭转移；而miR-145的这一作用能够被MUC1上调解除，说明MUC1是miR-145作用于EMT的桥梁因子。
Objective:
To investigate roles of high expression of miR-145in ovarian carcinoma, and toelucidate its possible mechanisms.
Methods:
Western blotting and immunochemistry were used to determine the expression oftumor suppressor gene miR-145in ovarian carcinoma; miR-145expression vector,together with transwell method analysis or MTT analysis, was used to study theeffects of miR-145upregulation on ovarian carcinoma cell invasion，metastasis，apoptosis and cell proliferation ability, respectively; MUC1expression vector,together with q-RT PCR and western blotting, was used to investigate the effects ofmiR-145on E-cad induced cell invasion and the related molecular mechanisms.
Results:
miR-145expression levels were significantly decreased in ovarian carcinomatissues compared with the non-neoplastic ovarian samples. After transfection withmiR-145mimics, invasion and metastasis induced by MUC1was reducedsignificantly, and cell proliferation ability was reduced, which indicated inhibition ofMUC1led to invasion resistance, and promotion of cell sensitive to Taxon; Aftertreating with miR-145, MUC1preotein expression level was inhibited efficiently inSKOV3cells,E-cad erpression levels in SKOV3cells were promoted; It showed thatpromotion of E-cad signaling induced by miR-145was released by MUC1inhibition.The results of western blotting revealed that E-cad erpression levels were increasedinSKOV3cells with decreased MUC1expression.
Conclusion:
miR-145/MUC1signaling pathway was constitutively inactivated in ovariancarcinoma tissues. Increased expression of miR-145in ovarian carcinoma cellsresulted in the inhibition of invasion and metastasis and proliferation, as well as theenhanced sensitive ability to Taxon. Meanwhile, upregulation of miR-145inhibited MUC1expression and the level of E-cad protein was increased, which inhibited theactivity of EMT. All in all, high expression of miR-145inhibits MUC1/E-cadsignaling pathway in ovarian carcinoma, and contributes to inhibition of invasionthrough inhibiting EMT process.
探讨microRNA-145对卵巢癌SKOV3细胞侵袭转移功能的影响及可能的作用机制，为临床卵巢癌的治疗提供新的有效靶点。
方法：
采用qRT-PCR方法，验证包括miR-145在内的7种microRNA在卵巢癌组织及卵巢良性肿瘤组织中的表达情况；针对miR-145做进一步研究，利用miR-145mimic转染SKOV3细胞，通过transwell侵袭及迁移实验、划痕实验等方法研究miR-145对卵巢癌SKOV3细胞侵袭迁移等细胞功能的影响；构建双荧光素酶报告载体验证miR-145与靶基因MUC1的互补作用机制，包装病毒载体，使SKOV3细胞稳定表达miR-145，最后利用western blot、PCR等方法，探讨miR-145对MUC1以及EMT关键蛋白E-cad的作用从而影响卵巢癌细胞侵袭转移作用机制。
结果：
PCR结果显示，与良性卵巢瘤组织相比，miR-200a，miR-93，miR-18，miR-146a在卵巢癌组织中的表达明显增高，miR-143，miR-145，miR-29在卵巢癌组织中表达明显下降；根据生物信息学软件预测，miR-145序列与MUC13’UTR端序列互补，是其预测靶基因；western blot检测显示，MUC1蛋白在卵巢癌组织中表达明显增高；将miR-145-5p mimic转染SKOV3细胞48h后，Transwell侵袭实验表明，穿过matrige膜的细胞数与NC组、MOCK组和Blank组相比较，显著减少。Transwell迁移实验表明，miR-145mimic组迁移出Transwell小室并种植到6孔板的细胞数明显少于NC组、MOCK组和Blank组；划痕实验显示划痕后12h、24h、48h，miR-145-5p mimic（0.074、0.148、0.220）组细胞的迁移率明显低于NC阴性对照组（0.087、0.242、0.276）、mock组（0.125、0.269、0.301）及Blank组（0.152、0.260、0.339），且有统计学意义（P<0.05）；DAPI染色测定SKOV3细胞凋亡后，miR-145-5p mimics组细胞的凋亡率与Blank组、mock组及NC阴性对照组无明显差异（P＞0.05）；以MTT实验检测细胞增殖活性，细胞接种96孔板24h、48h和72h后，转染miR-145-5p mimics组与NC阴性对照组、Mock组、及Blank组比较，OD490值明显下降，增殖活性明显下降；以紫杉醇处理SKOV3细胞，用药72h后，转染miR-145-5p mimic组与NC阴性对照组、Mock组及Blank组比较OD490值明显下降，同时，MUC1mRNA和蛋白的表达明显下调。双荧光素酶报告实验显示，将含MUC13’UTR结合序列的荧光素酶报告载体和miR-145-5p mimic共转染293T细胞后，检测荧光表达显著下降；将突变型载体与miR-145-5p mimic共转染293T细胞后，荧光表达无显著差异。成功构建稳定表达miR-145的卵巢癌细胞系miR-145-CMV-RFP-SKOV3，检测MUC1的蛋白表达量明显减少，而E-cad的表达量增加；与之相反，卵巢癌细胞系miR-145-CMV-RFP-SKOV3转染MUC1表达质粒后，E-cad的表达量减少，SKOV3细胞侵袭转移能力增强；
结论：
miR-145是卵巢癌的抑癌基因，miR-145的上调能够抑制卵巢癌细胞的侵袭、转移以及增殖，并能够增强由紫杉醇诱导的细胞抑制效应。MUC1是miR-145的直接靶基因，后者通过与其互补结合抑制其蛋白表达，引起EMT标志性蛋白E-cad表达增强，说明miR-145通过抑制MUC1/E-cad信号通路阻滞了EMT过程导致的SKOV3细胞侵袭转移；而miR-145的这一作用能够被MUC1上调解除，说明MUC1是miR-145作用于EMT的桥梁因子。
Objective:
To investigate roles of high expression of miR-145in ovarian carcinoma, and toelucidate its possible mechanisms.
Methods:
Western blotting and immunochemistry were used to determine the expression oftumor suppressor gene miR-145in ovarian carcinoma; miR-145expression vector,together with transwell method analysis or MTT analysis, was used to study theeffects of miR-145upregulation on ovarian carcinoma cell invasion，metastasis，apoptosis and cell proliferation ability, respectively; MUC1expression vector,together with q-RT PCR and western blotting, was used to investigate the effects ofmiR-145on E-cad induced cell invasion and the related molecular mechanisms.
Results:
miR-145expression levels were significantly decreased in ovarian carcinomatissues compared with the non-neoplastic ovarian samples. After transfection withmiR-145mimics, invasion and metastasis induced by MUC1was reducedsignificantly, and cell proliferation ability was reduced, which indicated inhibition ofMUC1led to invasion resistance, and promotion of cell sensitive to Taxon; Aftertreating with miR-145, MUC1preotein expression level was inhibited efficiently inSKOV3cells,E-cad erpression levels in SKOV3cells were promoted; It showed thatpromotion of E-cad signaling induced by miR-145was released by MUC1inhibition.The results of western blotting revealed that E-cad erpression levels were increasedinSKOV3cells with decreased MUC1expression.
Conclusion:
miR-145/MUC1signaling pathway was constitutively inactivated in ovariancarcinoma tissues. Increased expression of miR-145in ovarian carcinoma cellsresulted in the inhibition of invasion and metastasis and proliferation, as well as theenhanced sensitive ability to Taxon. Meanwhile, upregulation of miR-145inhibited MUC1expression and the level of E-cad protein was increased, which inhibited theactivity of EMT. All in all, high expression of miR-145inhibits MUC1/E-cadsignaling pathway in ovarian carcinoma, and contributes to inhibition of invasionthrough inhibiting EMT process.
引文
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