EB病毒相關(guān)胃癌組織及胃癌細(xì)胞系中LOXL1基因甲基化狀態(tài)與表達(dá)的研究.pdf

1、EB病毒相關(guān)胃癌組織及胃癌細(xì)胞系中LOxLl基因甲基化狀態(tài)與表達(dá)的研究摘要EB病毒(EpsteinBarrvirus，EBV)是公認(rèn)的DNA致瘤病毒，現(xiàn)己確認(rèn)EBV感染與部分胃癌發(fā)生密切相關(guān)，但發(fā)病機(jī)制有待進(jìn)一步研究。EBV感染可誘導(dǎo)相關(guān)抑癌基因啟動(dòng)子區(qū)呈高甲基化狀態(tài)，這被認(rèn)為是引起EBV相關(guān)腫瘤抑制基因功能失活的重要機(jī)制之一。目的檢測(cè)并比較分析部分胃癌細(xì)胞系及EBV相關(guān)胃癌(EBVassociatedgastriccarcinomaE

2、BVaGC)和EBV陰性胃癌(EBVnegativedgastriccarcinomaEBVnGC)組織中LOXLl基因甲基化狀態(tài)及其轉(zhuǎn)錄表達(dá)，探討EBV感染對(duì)LOXLl基因啟動(dòng)子區(qū)甲基化狀態(tài)及其轉(zhuǎn)錄表達(dá)的影響，為進(jìn)一步研究LOXLl基因在EBVaGC發(fā)生中的作用機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。方法采用甲基化特異性PCR(Methylation—SpecificPCR，MSP)和亞硫酸氫鹽基因組測(cè)序法(Bisulfitegenomicsequenci

3、ng，BGS)檢測(cè)EBV陰性5fnISH性胃癌細(xì)胞系以及EBVaGC和EBVnGC組織中LOXLl基因啟動(dòng)子區(qū)的甲基化狀態(tài)。用5氮雜2’脫氧胞苷(5Aza—CdR)對(duì)EBV陽(yáng)性胃癌細(xì)胞系進(jìn)行去甲基化處理，MSP及BGS檢測(cè)LOXLl基因啟動(dòng)子區(qū)甲基化狀態(tài)的變化。采用實(shí)時(shí)熒光定量PCR(RealtimequantitativePCR，RealtimeqPCR)檢測(cè)比較EBV陰性胃癌細(xì)胞系、EBV陽(yáng)性胃癌細(xì)胞系之間以及5AzaCdR作用前后

4、EBV陽(yáng)性胃癌細(xì)胞系之間LOXLl基因轉(zhuǎn)錄表達(dá)水平的差異。結(jié)果①胃癌細(xì)胞系MSP結(jié)果顯示：3種EBV陽(yáng)性胃癌細(xì)胞系SNU719，GT38，GT39和4種EBV陰性胃癌細(xì)胞系MKN45，SGC7901，HGC27，BGC823中LOXLl基因啟動(dòng)子區(qū)均呈Mu型。②胃癌細(xì)胞系BGS結(jié)果顯示：3種EBV陽(yáng)性胃癌細(xì)胞系中LOXLl基因啟動(dòng)子區(qū)有大多數(shù)CpG位點(diǎn)呈現(xiàn)甲基化狀態(tài)，EBV陰性胃癌細(xì)胞系中未發(fā)現(xiàn)甲基化位點(diǎn)，EBV陽(yáng)性胃癌細(xì)胞系甲基化程度

5、明顯高于EBV陰性胃癌細(xì)胞系。③胃癌組織MSP結(jié)果顯示：EBVaGC和EBVnGC組織中LOXLl基因啟動(dòng)子區(qū)也均表現(xiàn)為MU型，但EBVaGC組織中LOXLl基因啟動(dòng)子的甲基化程度較EBVnGC組織高。④部分胃癌組織BGS測(cè)序結(jié)果顯示：不同胃癌組織標(biāo)本中LOXLl基因啟動(dòng)子區(qū)的20個(gè)CpG位點(diǎn)呈現(xiàn)不同的甲基化狀態(tài)，甲基化程度存在一定的差異；其中EBVaGC組織中LOXLl基因啟動(dòng)子區(qū)有更多的CpG位點(diǎn)呈現(xiàn)甲基化狀態(tài)，總體甲基化程度高于E

6、BVnGC組織。⑤5AzaCdR處理后EBV陽(yáng)性胃癌細(xì)胞系MSPStudyofmethylationstatusandexpressionononLOXL1geneinEpsteinBarrvirus—associatedgastriccarcinomatiussesandgastriccarcinomacelllinesAbstractEpsteinBarrvirus(EBV)isknownasanimportantDNAtumorv

7、irusEBVinfectionISrelatedtogastriccarcinoma，butpathogenicmechanismofthatisnotwellunderstoodMethylationoftumorsuppressorgeneinducedbyEBVinfectionisknownasanimportantmechanismforthescilenceoftumorsuppressorgeneObjectiveThe

8、studyaimedtoexploretheinfluenceofEBVinfectiononLOXLlgenepromotermethylationstatusandtranscriptionalexpression，andfurthertoresearchtherolesofLOXL1playedintheoccurrenceofEBVassociatedcarcinomaMethodsThemethylationstausofLO

9、XLlgenepromoterinEBVpositiveand—negativegastriccancereelllinesandEBVaGCsandEBVnGCstissuewasdetectedbymethylation‘specificPCRandbisulfitegenomicsequencingThedemethylationlevelsofLOXL1genepromoterswasdetectedbybisulfitegen

10、omicsequencingandMSPandLOXLlgeneexpressionwasdetectedbytherealtimequantitativePCRbeforeandaftertreatmentwith5AzaCdRinEBVpositivecelllinesGT38andRajiResults@MSPresultshowedthemethylationstausofLOXLlgenepromoterwasallMUtyp

11、einEBVpositiveand—negativegastriccarcinomacelllines@Therewere20sitesofCpGislandsinLOXL1genepromoterBGSresultsshowedthatmostofCpGsitesonLOXL1genepromoterweremethylationinEBVpositivegastriccarcinomacelllines，andnomethylati

12、oninEBVnegativegastriccarcinomacelllines@MSPresultsshowedthatmethylationstausofLOXL1genepromoterwasMUtypeinEBVaGCsandEBVnGCsMethylationstatusofLOXLlgeneinEBVaGCtissuewassignificantlyhigherevidentthanEBVnGC④BGSresultsshow

13、edthatdifferentgastriccarcinomaspecimensrepresenteddifferentmethylationstausAveragedegreeofLOXL1genepromotermethylationinEBVaGCwassignificantlyhigherthanEBVnGCs⑤Aftertreatedwith5AzaCdR，theLOXL1genepromotermethylationinEB

14、Vpositivegastriccarcinomacelllineswaslowerthanbefore@LOXL1geneexpressionwasdifferentbetweenEBVnegativeandEBVpositivegastriccarcinomacelllines，itsexpressioninEBVnegativegastriccarcinomacelllinesis435timeshigherthaninEBVpo