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 intermediate-high 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 ability-associated 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.