肺腺癌miR-195的表达及靶基因筛选

Expression and target gene screening of miR-195 in lung adenocarcinoma

目的研究miR-195在肺腺癌中的表达差异,筛选miR-195靶基因,分析靶基因作用网络及其对患者生存预后的影响。方法利用TCGA数据库,比较miR-195在肺腺癌癌组织与癌旁正常组织间的表达差异;采用实时荧光定量PCR检测miR-195在临床标本中的相对表达水平。设计miR-195的Hybrid PCR引物,扩增出靶基因的mRNA 3′-非翻译区(3′UTR),菌落PCR筛选靶基因的3′UTR片段进行测序。美国国家生物信息中心网站比对测序结果,得到靶基因的基因注释和全长序列,构建靶基因作用网络并进行生存分析。结果miR-195在肺腺癌癌组织中的相对表达水平低于癌旁正常组织,差异有统计学意义(P<0.01)。Hybrid PCR筛选miR-195候选靶基因显示多个条带。miR-195与靶基因的结合位点在基因3′UTR上有19个。靶基因GCLC、NOB1的低表达与肺腺癌患者的良好预后相关。结论肺腺癌癌组织中miR-195呈低表达。miR-195可能通过直接抑制靶基因GCLC、NOB1的功能,进而影响肺腺癌的发生、发展。

Objective To study the expression difference of miR-195 in lung adenocarcinoma,screen the target gene of miR-195,analyze the interaction network of target gene and its influence on survival and prognosis of patients.Methods TCGA database was used to compare the expression of miR-195 between lung adenocarcinoma tissues and normal tissues,and real-time fluorescent quantitative PCR was used to detect the relative expression level of miR-195 in clinical specimens.A Hybrid PCR primer for miR-195 was designed to amplify the 3′UTR of the target gene,the 3′UTR fragment of target gene was screened by colony PCR and sequenced.NCBI website was used compare the sequencing results and obtain the target gene annotation and full-length sequence.The target gene action network was constructed and survival analysis was carried out.Results The relative expression level of miR-195 in lung adenocarcinoma tissues was lower than that in adjacent normal tissues(P<0.01).Hybrid PCR was used to screen miR-195 candidate target gene to display multiple bands.There was 19 binding sites of miR-195 to target gene in 3′UTR.Low expression of target genes GCLC and NOB1 was associated with good prognosis of lung adenocarcinoma.Conclusion The expression of miR-195 was down regulated in lung adenocarcinoma.miR-195 may affect the occurrence and development of lung adenocarcinoma by directly inhibiting the function of GCLC and NOB1.