HIF-1α基因沉默對人脈絡膜黑色素瘤細胞上皮—間質轉化的影響.pdf

1、HIF1a基因沉默對人脈絡膜黑色素瘤細胞上皮一間質轉化的影響摘要目的：以特異性小干擾RNA(siRNA)沉默缺氧培養(yǎng)的人眼脈絡膜黑色瘤細胞(OCM一1)中缺氧誘導因子一10【(HIF—la)基因，觀察其對細胞骨架分子波形蛋白(Vimentin)表達的影響，以初步探討HIF1a對人眼脈絡膜黑色瘤上皮一間質轉化(EMT)的調控作用。方法：在正常培養(yǎng)基qbJJll21009mol／L的氯化鈷(CoCl2)以模擬腫瘤內部缺氧微環(huán)境，構建靶向HI

2、F—ls的siRNA干擾載體，同時設立6一actinljH性對照、無義寡核苷酸陰性對照及空載脂質體對照組，以LipofectamineTM2000為載體介導siRNA轉染缺氧培養(yǎng)的OCM1細胞，以RTPCR和Westernblot方法檢測缺氧培養(yǎng)前后及轉染前后HIF1、VimentinmRNA和蛋白的表達情況。結果：OCM一1細胞經缺氧培養(yǎng)后可由上皮細胞樣形態(tài)轉變成間質細胞樣形態(tài)。與常氧組相比，單純缺氧組HIF1amRNA表達無明顯差異

3、(PO05)，蛋白表達顯著升高(PO05)。結論：1缺氧培養(yǎng)可使OCM1細胞由上皮細胞樣形態(tài)轉變成間質細胞樣形態(tài)，提示缺氧微環(huán)境可促使OCM一1細胞發(fā)生EMT。2缺氧可在蛋白水平上調OCM一1細胞中HIF一10【的表達，提示缺氧對HIF一10【的調控主要發(fā)生在轉錄后水平。3HIF—ls可通過轉錄激活Vimentin，使其表達上調，提示HIFla可能參與調控OCM1細胞的EMT，進而影響其侵襲、轉移能力。4可成功利用Lipofectami

4、neTM2000為載體將siRNA轉入OCM一1細胞內。EffectofHIFlaGeneSilencingonEMTInMalignantMelanomaoftheHumanChoroidCellAbstractObjectives：Toinvestigatetheeffectofspecificsilencinghypoxiainduciblefactor1alpha(HIFla)bysmallinterferenceRNA(siR

5、NA)onexpressionofVimentininmalignantmelanomaofthehumanchoroidcelllines(OCM1)underhypoxia，inordertodiscusstheroleofHIF一1Qonepithelial—mesenchymaltransition(EMT)inmalignantmelanomaofthehumanchoroidMethods：Chemicalhypoxiain

6、ducerCobaltchloride(COCl2)wereaddedintonutrientmediumattheconcentrationoflOOgmol／LtosimulatehypoxiamicroenvironmentinsidetumorHIF一1Q—siRNAweredesignedandsynthetizedinvitroLipofectamineTM2000wastakentotransfectsiRNAintoOC

7、M1cellsunderhypoxiaAndhypoxicgroupcellsweredividedintofivegroups：simplehypoxicgroup，HIF1Qinterferencegroup，13actinpositivecontrolgroup，negativecontrolgroupandliposomegroupRTPCRandWesternblotwereusedtochecktheexpressionst

8、atusofHIF—laandVimentinonmRNAandproteinlevelsbeforeandafterhypoxiacultureandcelltransfectionResults：Afterhypoxiaculture，OCM1cellscouldtransformfromepitheliallikeintomesenchymallikeinmorphologyComparedwithnormoxiagroup，th

9、erewasnoobviouschangeontheexpressionlevelofmRNAofHIF一1Qinsimplehypoxiagroup伊005)，whiletheexpressionlevelofitsproteinincreasedobviously(P005)Conelusion：1Afterhypoxiaculture，OCM一1cellscouldtransformfromepitheliallikeintome