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1、第33卷第4期南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版)2013年4月ACTAUNIVERSITAnSMEDICINALISNANJING(NaturalScience)·449·miR．135a／b逆轉(zhuǎn)肺癌A549／CDDP細(xì)胞對(duì)順鉑耐藥性及相關(guān)機(jī)制研究周麗，邱天竹，陳雯姣，朱偉，束永前，劉平(南京醫(yī)科大學(xué)第一附屬醫(yī)院腫瘤科，江蘇南京210029)[摘要]目的：研究miR．135a／b對(duì)肺癌耐順鉑細(xì)胞株A549／CDDP順鉑耐藥的影響。方法：運(yùn)用實(shí)時(shí)熒光定量PCR檢測(cè)miR一135a／b在A549和A549／CDDP細(xì)胞株中的差異表達(dá)；MTF法檢測(cè)轉(zhuǎn)染后A549及A549／CDDP細(xì)
2、胞對(duì)CDDP的敏感性；構(gòu)建MCL1．3'-UTR熒光素酶報(bào)告質(zhì)粒驗(yàn)證miR一135a／b的靶基因：Westernblot檢測(cè)轉(zhuǎn)染前后細(xì)胞MCL1蛋白的表達(dá)差異；流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染后耐藥細(xì)胞對(duì)順鉑誘導(dǎo)凋亡的影響。結(jié)果：miR一135a／b在A549／CDDP細(xì)胞中表達(dá)量降低；在耐藥株中上調(diào)miR一135afb后顯著增加細(xì)胞對(duì)順鉑的敏感性；熒光素酶實(shí)驗(yàn)證實(shí)MCL1是miR．135a／b的靶基因；抗凋亡蛋白MCL1在A549／CDDP細(xì)胞中呈高表達(dá)，上調(diào)miR．135a／b明顯抑制耐藥細(xì)胞中MCL1蛋白的表達(dá)：miR一135a／b顯著增加A549／CDDP細(xì)胞對(duì)順鉑誘導(dǎo)的凋亡。結(jié)
3、論：miR—I35a／b通過靶向調(diào)控MCL1蛋白表達(dá)增加NSCLC細(xì)胞對(duì)順鉑的敏感性和凋亡。[關(guān)鍵詞]miR一135a／b；順鉑耐藥；凋亡；MCL1；肺癌[中圖分類號(hào)]R734．2[文獻(xiàn)標(biāo)志碼]A[文章編號(hào)]1007—4368(2013)04—449—07doi：10．7655／NYDXBN$20130406MechanismofmiR-135a／bonregulatingcisplatinresistanceofhumanlungcancercelllineA549／CDDPZhouLi，QiuTianzhu，ChenWenjiao，ZhuWei，ShuYongqian，
4、LiuPing(DepartmentofOncology，theFirstAffiliatedHospitalofNJMU，Nanjing210029，China)[Abstract]Objective：ToinvestigatewhethermiR一135a／bcouldmodulatethedrugresistanceofthehumanlungcancercelllineA549／CDDPtocisplatin(CDDP)，andexplorethemechanismofmiR一135a／bontheCDDPsensitivityofA549／CDDPcells．Me
5、tbods：MiR一135a／bexpressionwasmeasuredbyquantitativerea1．timePCR．TransienttransfectionwasusedinA549andA549／CDDPcelllines．CellviabilitywasdetectedbyMT'Fassay．MCL13’．untranslatedregion．basedluciferasereporterplasmidswasconstructedtotestifythetargetgeneofmiR一135a／b．Proteinexpressionsweremeasur
6、edbywesternblot．FlowcytometrywasusedtodetectCDDP-inducedapoptosis．Results：WefoundthatmiR一135a／bweredownregulatedwhileMCL1wasupregulatedinA549／CDDPcells．comparedwiththeparentalA549cells．Invitrodrugsensitivityassaydemonstratedthattheover-expressionofmiR一135a／bsensitizedA549／CDDPcellstocispla
7、tin．TheluciferaseactivityofMCL13’一untranslatedregion—basedreporterconstructedinA549／CDDPcellssuggestedthatMCL1wasthedirecttargetgeneofmiR-135a／b．EnforcedmiR一135a／hexpressionreducedMCL1proteinlevelandsensitizedA549／CDDPcellstoCDDP—inducedapoptosis．Conclusion：Th
