抑制mir-223表达增强雷公藤红素抗mcf-7和pc3增殖作用-inhibiting mir - 223 expression enhances tripterines anti-proliferation effects on mcf - 7 and pc3.docxVIP

抑制miR-223的表达增强雷公藤红素抗MCF-7和PC3增殖作用摘要目的雷公藤红素是一种新型抗肿瘤药物，深入研究其作用机制，从而提升其抗肿瘤效果，近来受到广泛关注。本项目观察在肿瘤发生和发展过程中起重要作用的分子miR-223，对雷公藤红素抑制肿瘤细胞增殖作用是否存在影响及可能机制。方法通过qRT-PCR、Westernblot检测人乳腺癌MCF-7细胞和人前列腺癌PC3细胞中miR-223的表达及蛋白的表达；采用流式细胞术法检测两种癌细胞的增殖情况。结果我们发现，雷公藤红素处理人乳腺癌MCF-7细胞和人前列腺癌PC3细胞6h后，两种肿瘤细胞miR-223表达明显升高。下调或者过表达miR-223分别可促进或者抑制雷公藤红素在两种细胞的抗增殖作用。除诱导miR-223表达外，雷公藤红素还能影响两种细胞mTOR活性（加入雷公藤红素后5minmTOR活化升高，1h后mTOR活性转向被抑制）及促进HSP70表达增加（通过激活HSF-1）。有意思的是，mTOR活化、HSP70水平和miR-223表达三者之间有相互依赖的关系，降低（或升高）其中的任何一项，另外两项也降低（或升高）。最后，抑制mTOR或者降低HSP70表达，无论雷公藤红素存在与否，均可抑制细胞增殖；表明mTOR活化和HSP70表达对细胞增殖很重要。结论本项目首次发现，在遭遇具有抗增殖作用的雷公藤红素时，细胞至少可启用三种“武器”（上调miR-223，早期活化mTOR以及诱导HSP70表达），对抗雷公藤红素的抗增殖作用，以维持生存和增殖。与此对应，下调miR-223、抑制mTOR活化或阻止热休克蛋白活化，这三种方法均能促进雷公藤红素的抗肿瘤作用。关键字miR-223，雷公藤红素，肿瘤，HSF-1，mTORIInhibitingInduciblemiR-223EnhancesAnti-ProliferativeEffectsofCelastrolonHumanCancerCellLinesMCF-7andPC3AbstractPurpose：Celastrolisanovelanti-tumoragent.Furtherunderstandingthemechanismsandenhancementofthiseffectofcelastrolhasattractedmuchresearchattention.Methods：TheexpressionlevelofmiR-223andproteininMCF-7andPC3cellsweredeterminedbyqRT-PCRandWesternblot.Theproliferationofcancercellsweredetectedbyflowcytometryassay.Results:HerewereporttheinteractionbetweenmiR-223,mTORactivationandHSP70elevationincelastrol-treatedcancercellsandtheeffectsoftheseeventsoncelastrol’santi-proliferation.Wefoundthatafter6hcelastroltreatmenthumanbreastcancercelllineMCF-7andprostatecancercelllinePC3showedelevatedmiR-223.Down-regulatingorover-expressingmiR-223couldenhanceorreducecelastrol’santi-proliferationeffects,respectively.Inaddition,celastrolcouldcauseearlyactivationandlaterinhibitionofmTOR,aswellasraiseHSP70(precededbyHSF-1activation).Interestingly,thesethreeevents,i.e.,mTORactivation,HSF-1-relatedHSP70elevationandmiR-223inductionshowedmutuallydependentrelations,eachactionpromotingtheothertwo.Moreover,inhibitingmTORordown-regulatingHSP70couldreducecellproliferationinbothcelastrol-treatedanduntreatedcells,indicatingmTORactivationandHSP70elevationarealsoimport