miR-30b-5p通过HELLS抑制肺腺癌细胞增殖

miR-30b-5p inhibits the proliferation of lung adenocarcinoma cells by targeting HELLS

为探讨miR-30b-5p靶向淋巴样特异性解旋酶(helicase lymphoid-specific, HELLS)对肺腺癌细胞增殖、凋亡的影响及其机制,通过生物信息学分析miR-30b-5p和HELLS在肺腺癌组织中的表达及其与临床表型的关系;将体外培养的A549和Calu-3细胞分为miR-NC组(转染miR-NC)、miR-30b-5p组(转染miR-30b-5p)、miR-30b-5p+HELLS组(转染miR-30b-5p和HELLS),采用RT-PCR检测各组细胞miR-30b-5p和HELLS mRNA的表达,Western blotting检测各组细胞核蛋白抗原Ki67、HELLS蛋白的表达,CCK-8检测各组细胞增殖情况,克隆形成试验检测各组细胞克隆形成数,FACS检测各组细胞周期及凋亡率,双荧光素酶报告试验验证miR-30b-5p与HELLS的靶向关系;建立肺腺癌裸鼠移植瘤模型,分为miR-NC组、miR-30b-5p组,记录裸鼠肿瘤体积、质量,免疫组化分析2组移植瘤Ki67蛋白表达。结果显示,GEO2R和TCGA数据库分析表明,miR-30b-5p在肺腺癌组织中表达下调,且其表达水平与患者生存期、N分期、复发和预后有关(P<0.05)。HELLS在肺腺癌组织中的表达水平高于癌旁组织(P<0.05),且其表达水平与患者肿瘤TNM分期、肿瘤分级、预后和生存期有关(P<0.05)。在A549和Calu-3细胞中,与miR-NC组比较,miR-30b-5p组miR-30b-5p表达量、G0/G1期细胞数、细胞凋亡率增加(P<0.05),细胞活力、克隆形成数、S期细胞数、HELLS mRNA水平及Ki67、HELLS蛋白水平显著降低(P<0.05);与miR-30b-5p组比较,miR-30b-5p+HELLS组G0/G1期细胞数、细胞凋亡率降低(P<0.05),细胞活力、克隆形成数、S期细胞数及Ki67、HELLS蛋白水平增加(P<0.05)。双荧光素酶报告试验表明,miR-30b-5p可靶向调控HELLS。与miR-NC组比较,miR-30b-5p组裸鼠移植瘤体积、质量及Ki67水平降低(P<0.05)。该研究提示,miR-30b-5p可靶向负调控HELLS的表达,抑制肺腺癌细胞增殖并诱导细胞凋亡。

The study aims to explore the effects and mechanism of miR-30b-5p on the proliferation and apoptosis of lung adenocarcinoma cells by targeting helicase lymphoid-specific(HELLS).To this end,the expressions of miR-30b-5p and HELLS in lung adenocarcinoma tissues and their relationship with clinical phenotypes were analyzed by bioinformatics.Next,A549 and Calu-3 cells cultured in vitro were divided into the miR-NC group(transfected with miR-NC),miR-30b-5p group(transfected with miR-30b-5p),and miR-30b-5p+HELLS group(transfected with miR-30b-5p and HELLS).The mRNA expressions of miR-30b-5p and HELLS in each group were detected by RT-PCR.The expressions of nucleoprotein antigen Ki67 and HELLS proteins in each group were detected by Western blotting.The cell proliferation was detected by CCK-8.The cell survival ability in each group was detected by colony formation assay.The cell cycle and apoptosis rate in each group were detected by FACS.The targeted relationship between miR-30b-5p and HELLS was verified by dual-luciferase assay.Then the xenograft models of nude mice with lung adenocarcinoma were established and divided into miR-NC group and miR-30b-5p group.The volume and weight of tumors were measured.The expression of Ki67 protein in xenograft tumors was analyzed by immunohistochemistry.The results showed that based on GEO2R and TCGA,the expression of miR-30b-5p in lung adenocarcinoma tissues was down-regulated,and correlated with survival period,N staging,recurrence,and prognosis(P<0.05).The expression of HELLS in lung adenocarcinoma tissues was significantly higher than that in para-cancerous tissues(P<0.05)and correlated with the staging of TNM,tumor grading,prognosis,and survival period(P<0.05).Compared to the miR-NC group,the expression level of miR-30b-5p,the number of cells in G0/G1 phase,and the apoptosis rate in A549 and Calu-3 cells were all significantly increased in the miR-30b-5p group(P<0.05),while the cells activity,number of clone cells,number of cells in S phase,levels of HELLS mRNA,Ki67,and HELLS protein were significantly decreased(P<0.05).Compared to the miR-30b-5p group,the number of cells in G0/G1 phase and apoptosis rate were significantly decreased in the miR-30b-5p+HELLS group(P<0.05),while the cells activity,number of clone cells,number of cells in S phase,levels of Ki67 and HELLS proteins were significantly increased(P<0.05).The dual-luciferase assay confirmed that miR-30b-5p could target HELLS.Compared to the miR-NC group,the volume and weight of xenograft tumors,and the cellular Ki67 level were significantly decreased in the miR-30b-5p group(P<0.05).The results conclude that miR-30b-5p inhibits the proliferation and induces apoptosis of lung adenocarcinoma cells by negatively regulating the expression of HELLS.