摘要目的 研究不同细胞和肺癌组织中miR-30c侧翼区单核苷酸多态性rs928508(A/G)对初级miR-30c(pri-miR-30c)、前体miR-30c(pre-miR-30c)、成熟miR-30c的表达水平以及miR-30c成熟进程的影响.方法 构建含有不同基因型miR-30c侧翼区的报告基因表达载体pGL3-promoter-miR-30c-A和pGL3-promoter-miR-30c-G,分别与内参质粒pRL-SV40共转染于人肺癌A549细胞、人胚肾293A细胞和中国仓鼠卵巢细胞(CHO细胞)后,进行荧光素酶活性分析.采用Taqman基因分型技术对50例肺癌组织进行基因分型,以实时定量PCR法检测不同基因型组织标本中pri-miR-30c、pre-miR-30c、成熟miR-30c以及miR-30c宿主基因核转录因子Y(NFYC)的转录水平.结果 在A549、293A和CHO细胞中,pGL3-promoter-miR-30c-A组荧光素酶的活性与pGL3-promoter-miR-30c-G组比较,差异均无统计学意义(均P＞0.05).基因分型的结果显示,50例肺癌组织中,AA基因型17例,AG基因型21例,GG基因型12例.在rs928508 AG/GG基因型肺癌组织中,pre-miR-30c和成熟miR-30c的相对表达水平分别为1.306±0.878和0.384±0.227,均显著低于rs928508 AA基因型组织的表达水平(分别为2.103±1.173和0.588±0.307,P=0.009和P=0.011).而pri-miR-30c和NFYC基因在各基因型肺癌组织中的相对表达量差异均无统计学意义(P =0.335和P=0.393).结论 在肺癌组织中,miR-30c侧翼区的单核苷酸多态性rs928508(A/G)可以影响p6-miR40c→pre-miR-30c→成熟miR-30c的转化进程,但不能影响pri-miR-30c合成的转录进程.

abstractsObjective To investigate the effect of a common polymorphism rs928508 (A/G) in flanking region of miR-30c on the expression of pri,pre and mature miR-30c,and discuss the effect of this polymorphism on the maturing process of miR-30c in lung carcinoma.Methods The pGL3-promoter-miR-30c-A and pGL3-promoter-miR-30c-G lueiferase plasmids were created containing A or G allele of miR-30c flanking region.Taqman assay was used to genotype rs928508 polymorphism in 50 lung cancer tissues.RTPCR was performed to determine the expression of pri-miR-30c,pre-miR-30c,mature miR-30c and miR-30c host gene NFYC in the 50 lung cancer tissues.Results The luciferase expression level of the pGL3-promoter-miR-30c-A construct group was not significantly different compared with that in the the pGL3-promoter-miR-30c-G construct group (A549 cells,P =0.758 ; 293A cells,P =0.554 ; CHO cells,P =0.175).The results demonstrated that rs928508 (A/G) variant had no effect on the transcriptional regulation of pri-miR-30c.In the genotype-phenotype collection analysis of the 50 lung cancer tissues,the expression of pre-miR-30c and mature miR-30c for rs928508 AG/GG genotypes showed significantly lower levels compared with those in the AA genotype (P =0.009,P =0.011).However,the expression of primiR-30c showed no significant difference between AG/GG genotypes and AA genotype.Similarly,the expression of host NFYC gene was correlated with pri-miR-30c,showed no significant difference between AG/GG genotypes and AA genotype.Conclusion The rs928508(A/G) polymorphism in flanking region of miR-30c could influence the processing from pri-miR-30c to mature miR-30c,but does not influence the transcription of pri-miR-30c.