敲除CDC20可明显抑制宫颈癌细胞的增殖及侵袭转移

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Abstract：ObjectiveTostudytheefectofCDC20knockdownonproliferation，migrationandinvasionofcervicalcaceels andits underlying mechanism.Methods CDC20 expresion incervicalcancer tisues was analyzed using theTCGA database， and the protein expressionsof CDC20 and β -Catenininclinical specimensofcervicalcancerandadjacenttissuesweredetected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown incervical cancer C33A celsusing CRISPR/Cas9 technology，and CDC20 mRNA and protein expression levels in the transfectedcellsweredetectedusingqRT-PCRand Westernblotting.Thechanges inproliferation，cellcycle，apoptosis， migrationandinvasivenessofthetransfectedcelswereevaluatedusingcolony-formingassayfluorescenceactivatedcel sorting（FACS）andTranswellassay.Intheanimal experiment，naive C33Acellsand thecellswith CDC20knockdown were injectedsubcutaneouslyintotheleftandrightaxillaeofnudemice（n=5）toobservetumorgrowth.TheexpressonsofCDC20 and β -Catenin proteinsintransfectedcelsandthexenograft wereanalyzedusing Westernblotting，andtheir interaction was confirmedbyco-immunoprecipitation （CoIP）andimmunofluorescenceco-localizationassays.ResultsCervicalcancertissues expressed significantly higher CDC20 and β -Cateninlevels thantheadjacent tissues. C33A cellswith CDC20knockdown showedreduced proliferation，increased apoptosis，andloweredmigrationandinvasionabilities.CDC20 knockdown significantlysuppresedthegrowthofC33Acellxenograft innudemice，andthetumor-bearingmicedidnotexhibitobvious body mass changes. CDC20 and β -Catenin levels were both significantly lowered in C33A cels with CDC20 knockdown. Coimmunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β -Catenin. Conclusion CDC20 is highlyexpreed incevicalcancertisuesandC20nockdowncansuppressproliferationivasionandastais whileenhancingapoptosisof C33Acels，whichiscloselyrelated withtheregulationof theWnt/β-Cateninsignalingpathway. Keywords： CRISPR/Cas9; CDC20; cervical cancer; Wnt/β-Catenin

宫颈癌（CC）是最常见的妇科恶性肿瘤之一，据最新统计分析，每年约有50万新发病例和27万死亡病例，虽然随着人类乳头瘤病毒（HPV）疫苗的普及和宫颈癌筛查技术的不断更新有助于及早发现和预防，但对于中晚期患者的治疗效果不佳且病死率呈年轻化增长趋势[1-3]。（剩余20062字）