microRNA-186-5p及其靶基因CLDN18与甲状腺乳头状癌复发风险的关系

Relationship between microRNA-186-5p with its target gene CLDN18 and recurrence risk of papillary thyroid carcinoma

背景与目的:microRNA(miRNA)在肿瘤的发生、发展中发挥重要作用,而且不同的miRNA表达状态与肿瘤的不同生物学特征紧密关联。本研究通过分析不同复发风险甲状腺乳头状癌(PTC)患者差异表达的miRNA,筛选与PTC复发风险相关的miRNA,并分析其作用机制。方法:用基因芯片技术分析低危复发风险与中高危复发风险PTC患者血清外泌体中差异表达的miRNA,然后用qRT-PCR方法PTC患者肿瘤组织中验证;用Transwell实验筛选出与PTC细胞侵袭能力有关的差异表达miRNA。通过OncomiR在线数据库对筛选的细胞侵袭力相关miRNA的靶基因进行预测,随后采用过表达与敲低策略,分析PTC细胞相关蛋白表达(Western blot)与PTC细胞侵袭能力(Transwell)的变化,明确细胞侵袭力相关miRNA与预测靶基因的关系。最后,通过GEPIA在线网站对TCGA数据库中PTC临床样本的分析进一步确证。结果:基因芯片分析结果显示,与低危复发风险PTC患者比较,中高危复发风险PTC患者血清外泌体中4个miRNA(miR-186-5p、miR-532-3p、miR-199b-3p、miR-3158-5p)表达上调,1个miRNA(miR-3605-5p)表达下调(均P<0.05);组织标本qRT-PCR验证结果显示,中高危复发风险PTC患者癌组织中miR-186-5p和miR-3158-5p表达上调(均P<0.05);Transwell实验结果显示,过表达miR-186-5p后PTC细胞侵袭能力明显增强,敲低则明显减弱(均P<0.05),但改变miR-3158-5p的表达水平对PTC细胞的侵袭能力无明显影响(均P>0.05)。OncomiR在线数据库预测PRDX6、S100PBP、CLDN18、MAP2可能是miR-186-5p的靶基因;Western blot结果显示,过表达miR-186-5p后PTC细胞中CLDN18的蛋白表达明显降低,敲低则相反,但改变miR-3158-5p的表达水平对其他3个基因的蛋白表达无明显影响;Transwell实验结果显示,过表达CLDN18表达后PTC细胞的侵袭能力明显减弱,敲低则明显增强,而过表达或敲低miR-186-5p对PTC细胞的作用被同时过表达或敲低CLDN18所逆转(均P<0.05)。TCGA数据库分析结果显示,PTC组织中CLDN18表达较正常甲状腺组织明显降低。结论:miR-186-5p表达的增高可能与PTC的复发风险密切相关,机制可能与其通过调节下游CLDN18基因的表达而影响PTC细胞的侵袭能力有关。

Background and Aims:MicroRNAs(miRNAs)play important roles in the process of occurrence and development of tumors,and different miRNA expression profiles may be closely connected to the different biological characteristics of tumors.Therefore,this study was conducted to screen the miRNAs associated with the risk of recurrence of papillary thyroid carcinoma(PTC)by analyzing the differentially expressed miRNAs between PTC patients with different recurrence risks,and investigate their action mechanisms.Methods:The differentially expressed miRNAs in serum exosomes from PTC patients with low or intermediatehigh recurrence risk were determined by microarray analysis,and then the differentially expressed miRNAs were verified in the PTC tissues by qRT-PCR method.The differentially expressed miRNAs associated with the invasion ability of PTC cells were picked up by Transwell assay.The potential target genes of the selected miRNAs were predicted using OncomiR online database.Subsequently,the relationship between the cell invasion abilityassociated miRNAs and the predicted target genes were revealed through analyzing the changes in the relevant protein expressions(Western blot analysis)and invasion ability(Transwell assay)of the PTC cells using both overexpression and knock-down strategies.Finally,further confirmation was performed by analyzing the clinical samples of PTC in the TCGA database through GEPIA website.Results:The results of microarray analysis showed that the expressions of 4 miRNAs(miR-186-5p,miR-532-3p,miR-199b-3p,and miR-3158-5p)were up-regulated and one miRNA(miR-3605-5p)was down-regulated in serum exosomes from PTC patients with intermediate-high recurrence risk compared with PTC patients with PTC patients with low recurrence risk(all P<0.05).The verification by qRT-PCR analysis of the tissue specimens revealed that the expressions of miR-186-5p and miR-3158-5p were up-regulated in the tumor tissues of PTC tissues with intermediate-high recurrence risk(both P<0.05).The results of Transwell assay demonstrated that the invasion ability of PTC cells was significantly enhanced by miR-186-5p overexpression,and was significantly weakened by miR-186-5p knock-down(both P<0.05),but the invasion ability PTC cells was not significantly affected by changing the expression level of miR-3158-5p(both P>0.05).PRDX6,S100PBP,CLDN18,and MAP2 were predicted as the potential target genes of miR-186-5p using OncomiR online software.The results of Western blot analysis showed that the protein expression of CLDN18 was significantly decreased after miR-186-5p overexpression,and the opposite was true after miR-186-5p knock-down(both P<0.05),but the protein expressions of the other 3 genes were not significantly affected by changing the expression level of miR-3158-5p.The results of Transwell assay showed that the invasion ability of PTC cells was significantly reduced by CLDN18 overexpression,and was significantly increased by CLDN18 knock-down,moreover,the effects of miR-3158-5p overexpression or knock-down exerted on PTC cells were reversed by simultaneous CLDN18 overexpression or knock-down(all P<0.05).Conclusion:The increased miR-186-5p expression may be closely related to the recurrence risk of PTC,and the mechanism may be probably associated with its regulating the expression of downstream CLDN18 gene and thereby affecting the invasion ability of PTC cells.