【作者】 杨曦；

【导师】 王娅兰；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2015， 硕士

【摘要】 目的：探究精氨酸特异性单腺苷二磷酸核糖基化转移酶-1(Arginine-specific mono-ADP-ribosytransferases-1, ART1)对uPA基因启动子甲基化的影响及其在小鼠结肠癌CT26细胞侵袭转移中的作用。方法：本实验以CT26细胞ART1高表达组(GFP-ART1)、ART1沉默组(GFP-shARTl)为实验组,CT26细胞空载体组(GFP-Vector组)、未转染组(Un-transfection组)为对照组,western blot测定不同组别DNA甲基转移酶1(DNMT1)表达变化,同时,ART1高表达组、ART1沉默组、空载体组及未转染组CT26细胞分别接种于BALB/C小鼠,构建各组别BALB/C小鼠脾脏移植瘤模型并提取各组移植瘤组织蛋白,Western blot测定不同组别DNA甲基转移酶1(DNMT1)表达变化。DNA甲基化特异性PCR测序法检测不同组别uPA基因启动子甲基化状态变化；免疫共沉淀方法观察CT26细胞ART1高表达组、空载体组及未转染组ART1与DNMT1之间的关系。应用DNA甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-aza-2-dc)处理ART1沉默组CT26细胞,划痕实验和Transwell侵袭实验观察细胞迁移及侵袭能力的变化。BALB/C小鼠脾脏接种ART1基因沉默CT26细胞,腹腔注射5-aza-2-dc,与对照组比较小鼠脾脏移植瘤细胞肝转移情况。采用western blot检测GFP-shARTl、GFP-ART1、Un-transfection及GFP-Vector各组肿瘤细胞uPA表达变化,同时检测各组细胞脾脏移植瘤uPA表达变化,并应用DNA甲基转移酶抑制剂5-aza-2-dc处理ART1沉默组CT26细胞,western blot方法检测其uPA表达变化,同时检测5-aza-2-dc处理沉默组与未处理沉默组脾脏移植瘤uPA表达变化。结果：(1)ARTl对小鼠结肠癌CT26细胞uPA基因启动子甲基化水平的影响：western blot方法检测ART1高表达组、ART1沉默组、未转染组、空载体组CT26细胞DNMT1表达变化,结果显示,与对照组相比,ART1高表达组DNMT1表达减少,而ART1沉默组DNMT1表达增加(P<0.01)；同时检测各组小鼠脾脏移植瘤蛋白中DNMT1表达变化,结果显示同样的趋势(P<0.01)；采用DNA甲基化特异性PCR测序方法测定uPA启动子甲基化水平在各组的变化,结果显示与对照组相比,ART1高表达组uPA基因启动子甲基化水平降低,而ART1沉默组uPA基因启动子甲基化水平升高。免疫共沉淀结果显示ART1与DNMT1之间尚未发生直接的结合。(2)ARTl通过对uPA基因启动子甲基化状态的影响导致其表达变化及其在小鼠结肠癌CT26细胞侵袭、转移能力的作用：细胞划痕实验和Transwell侵袭实验检测5-aza-2-dc处理组和未处理组CT26沉默细胞迁移及侵袭能力,结果显示与未处理组相比,5-aza-2-dc处理组细胞24h迁移距离明显增高(P<0.01),且降解Matrigel基质胶穿过小室的细胞数明显升高(P<0.01)。采用5-aza-dc处理沉默组CT26细胞构建小鼠脾脏移植瘤模型,观察小鼠肝转移情况,结果显示与未处理组相比,处理组小鼠肝转移模型的肝脏转移结节数(P<0.05)和肝脏重量显著增加(P<0.05)。采用western blot方法检测ARTl高表达组,ART1沉默组,未转染组,空载体组CT26细胞uPA表达变化,结果显示与对照组相比,GFP-ART1组uPA表达增加,而GFP-shART1组uPA表达减少(P<0.01)；同时检测各组小鼠脾脏移植瘤蛋白中uPA表达变化,结果显示同样的趋势(P<0.01)；采用5-aza-2-dc处理沉默组细胞后,显示uPA表达增加(P<0.01)；同时western blot方法检测5-aza-2-dc处理组与未处理组CT26沉默细胞移植瘤蛋白结果显示,与未处理组相比,uPA表达显著增加(P<0.01)。结论：ART1沉默能够促进DNMT1表达,促进侵袭转移相关基因uPA启动子高甲基化,引起uPA蛋白表达减少,相反ART1高表达,则使DNMT1表达降低,uPA启动子甲基化程度降低,其蛋白表达增加,同时,抑制DNMT1, uPA基因蛋白表达增加,CT26细胞侵袭、转移能力增强,提示ARTl可通过抑制uPA基因启动子甲基化,而促进其表达,从而在小鼠结肠癌CT26细胞侵袭转移中发挥促进作用。其详细的机制仍有待深入探究。更多还原

【Abstract】 Objective:To search for the effect of ART1 on DNA methylation status and probe the effect and possible mechanism of ART1 on metastasis and metastasis of mouse colon CT26 cells.Methods:In this experiment, we set up GFP-ART1 group and GFP-shARTl group as experimental group, GFP-Vector group and un-transfection group as control group.Expression of DNA methyltransferase1(DNMT1) was detected by western blotting in vitro.The methylation status of uPA gene was detected by bisulfite sequencing PCR.The combination in GFP-ART1,GFP-Vector and un-transfection group between ART1 and DNMT1 was assessed by co-immunoprecipitation.BALB/C mouse model of liver metastasis was successfully established through injecting the mentioned group CT26 cells.Expression of DNA methyltransferasel(DNMT1) was detected by western blotting in vivo.GFP-shART1 CT26 cells were treated with 5-aza-dc,abilities of metastasis and invasion were determined by Wound healing assay and Transwell invasion assay respectively between treated and untreated group.5-aza-dc treated and untreated GFP-shART1 CT26 cells were injected into BALB/C mouse respectively,and we have successfully built BALB/C mouse model of liver metastasis.Liver metastasis was observed.Expression of uPA in GFP-shART1,ART1 GFP-ARTl,Un-transfection,GFP-Vector group was detected by western blot in vitro and in vivo.Meanwhile,the uPA expression between 5-aza-2-dc treated and untreated group in vitro and in vivo.Results:(1) Effect and possible mechanism of ART1 on uPA promoter methylaytion status:It was demonstrated by western blot that the expression of DNMT1 significantly increased in GFP-shARTl group,while decreased in GFP-ART1 group compared with the control group(p<0.01) in vivo and in vitro.BSP showed that a significant increase of uPA promoter methylastion status appeared in GFP-shARTl group,and a relative decrease in GFP-ART1 group.CO-IP revealed that there was no combination between ART1 and DNMT1 in GFP-ART1,GFP-Vector and un-transfection group.meanwhile,(2) Effect of ART 1 on methylation status of uPA promoter thus its expression and on CT26 cells metastatic and invasion potential:We treated GFP-shARTl group with 5-aza-2-dc,the treated group promoted CT26 cells abilities of invasion and metastasis in vitro(p<0.05) compared with the untreated group.We successfully built the BALB/C mouse model of liver metastasis through under the splenic capsule with GFP-shARTl group CT26 cells with or without 5-aza-2-dc injection.The weight of liver was much heavier in treated group compared with untreated group(p<0.05),and the numer of metastasis nodules showed an increase in treated group compared with untreated group in vivo(p<0.05).Western blot was taken to test the expression of uPA in GFP-shART1, ART 1 GFP-ART1,Un-transfection GFP-Vector group,it significantly increased in GFP-ART1 group in vivo and in vitro,while decreased in GFP-shART1 group(p<0.01).Meanwhile,we treated GFP-shARTl group with 5-aza-2-dc,it was illustrated that expression of uPA increased in treated group in vivo and in vitro compared with the untreated group(p<0.01).Conclusion:This study reveals that ART1 silencing induces hypermethylation of uPA promoter thus causing the downregulation of its expression and ART1 over-expression causes hypomethylation of uPA promoter thus causing its overexpression.It probably relates to the feedback mechanism of NF-κB to PARP1 thus mediating the expression and activity of DNMT1.To some extent,the uPA gene could be a certain factor of invasion and metastasis in colorectal carcinoma.Meanwhile,inhibition of DNMT1 results in upregulation of uPA expression in order to promote the metastatic and invasive potential.It reveals that ART1 could inhibit uPA promoter methylation status thus increasing its expression,so as to play a role in CT26 cell metastasis and invasion.In a word,the relationship between ART1 and DNA methlation might offer a new perspective of the therapeutic target for the enhanced treatment of this illustrious world-wide cancer.However,the exact mechanism need to be taken into research in the near future.更多还原