CAJ | 학술논문

目的探究微小RNA-155(miR-155)在结肠炎相关结肠癌发生过程中的作用并初步筛选其下游的靶基因.方法联合应用氧化偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)诱导C57BL/6小鼠发生结肠炎相关结肠癌,得到从结肠炎到结肠癌的3个不同阶段的小鼠模型(分别命名为AD1、AD2和AD3),同时设立无任何处理的对照组和只给予DSS来模拟慢性炎症的DSS组,取各组小鼠的结肠组织提取总RNA,逆转录成cDNA后应用实时荧光定量PCR检测miR-155的表达.结合前期的全基因组表达谱芯片数据和miRNA靶基因预测软件TargetScan和PicTar预测出来的潜在靶基因,初步筛选miR-155的下游靶基因,并用PCR进一步验证潜在靶基因的表达变化.结果 ADM和DSS联合使用能有效地模拟人类结肠炎相关结肠癌,结肠炎相关结肠癌的演变顺序为正常、轻度不典型增生、中度不典型增生、重度不典型增生和腺癌.DSS组小鼠结肠miR-155的表达水平(0.005 6±0.003 7)与对照组(0.0120±0.005 1)比较,差异无统计学意义(P＞0.05),而AD3组小鼠的miR-155表达水平(0.054 4±0.027 0)高于DSS组(0.005 6±0.003 7),差异有统计学意义(P＜0.01).结合全基因组表达谱芯片和miRNA靶基因预测软件,初步筛选出miR-155可能的下游靶基因为Tie4、Kcna1、Itk、Bcorl1、Cacna1c、Rspo2和Foxo3,其中Kcna1和Cacna1c经PCR验证与芯片检测结果一致.结论 miR-155的表达升高与结肠炎相关结肠癌有关,而与慢性炎症无关.
목적 탐구미소RNA-155(miR-155)재결장염상관결장암발생과정중적작용병초보사선기하유적파기인.방법 연합응용양화우담갑완(AOM)화포취당류산납(DSS)유도C57BL/6소서발생결장염상관결장암,득도종결장염도결장암적3개불동계단적소서모형(분별명명위AD1、AD2화AD3),동시설립무임하처리적대조조화지급여DSS래모의만성염증적DSS조,취각조소서적결장조직제취총RNA,역전록성cDNA후응용실시형광정량PCR검측miR-155적표체.결합전기적전기인조표체보심편수거화miRNA파기인예측연건TargetScan화PicTar예측출래적잠재파기인,초보사선miR-155적하유파기인,병용PCR진일보험증잠재파기인적표체변화.결과 ADM화DSS연합사용능유효지모의인류결장염상관결장암,결장염상관결장암적연변순서위정상、경도불전형증생、중도불전형증생、중도불전형증생화선암.DSS조소서결장miR-155적표체수평(0.005 6±0.003 7)여대조조(0.0120±0.005 1)비교,차이무통계학의의(P＞0.05),이AD3조소서적miR-155표체수평(0.054 4±0.027 0)고우DSS조(0.005 6±0.003 7),차이유통계학의의(P＜0.01).결합전기인조표체보심편화miRNA파기인예측연건,초보사선출miR-155가능적하유파기인위Tie4、Kcna1、Itk、Bcorl1、Cacna1c、Rspo2화Foxo3,기중Kcna1화Cacna1c경PCR험증여심편검측결과일치.결론 miR-155적표체승고여결장염상관결장암유관,이여만성염증무관.
Objective To investigate the changes of miR-155 and its target genes in colitisassociated carcinogenesis.Methods Colitis-associated colon cancer was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice.Mice of three different stages during the development of colon cancer were obtained,named AD1,AD2 and AD3,respectively.A control group of mice without any treatment and a DSS only group representing chronic inflammation without cancer were set up as well.Colon tissue was collected and expression of miR-155 in the colon tissues was measured by real-time fluorescent quantitative PCR.TargetScan and PicTar were used to predict potential target genes of miR-155,which were then preliminarily screened with our gene expression microarray database of AOM-DSS mouse model.Regular PCR was used to confirm the changes of the expression of these potential target genes in AOM-DSS mouse model.Results Colitis-associated colon cancer was effectively induced by azoxymethane and dextran sulfate sodium in C57BL/6 mice.Histological examination revealed that the evolution process was sequentially from normal,mild dysplasia,moderate dysplasia,and severe dysplasia to adenocarcinoma in the AOM-DSS mouse model.The level of miR-155 was gradually elevated with the formation of colitisassociated colon cancer.There was no significant difference between the levels of miR-155 expression in the DSS group (0.005 6 ± 0.003 7) and control group (0.012 0 ± 0.005 1) (P ＞ 0.05),but the level of miR-155 in the AD3 group (0.054 4 ±0.027 0) was significantly higher than that of the DSS group (0.005 6 ± 0.003 7)(P ＜0.01).No significant change of miR-155 expression was found in the DSS only group.The relative expression levels of miR-155 in the control group,DSS only group and AD3 group were 0.012 0 ± 0.005 1,0.005 6 ± 0.003 7,0.054 4 ± 0.027 0,respectively.Data analysis with the gene expression microarray showed that Tle4,Kcna1,Itk,Bcorl1,Cacna1c,Rspo2 and Foxo3 were potential target genes of miR-155 in the AOM-DSS mouse model.Changes of Kcna1 and Cacna1c in the AOM-DSS mouse model were validated to be consistent withthe changes obtained with the gene expression microarray.Conclusion The upregulation of miR-155 is related to colitis-associated carcinogenesis,but is irrelevant to chronic inflammation in the mouse model.