The Role And Mechanism Of MiR-338-3p In The Thyroid Cancer Progression

A growing body of evidence suggests that microRNAs have been recognized as key regulators in cancer development and progression.Mi R-338-3p,one of microRNAs,has been reported to be downregulated and function as tumor suppressor in many types of cancers,such as non-small lung cancer and glioma.However,the role and the molecular mechanisms of miR-338-3p in thyroid cancer remain unclear.Therefore,the purpose of this study was to investigate the clinical significance of miR-338-3p expression in thyroid cancer,and to evaluate its role and underlying mechanisms in thyroid cancer.Our results were divided into six parts as follows.1.miR-338-3p expression is downregulated in thyroid cancer tissues and cell lines.The expression levels of miR-338-3p were detected thyroid cancer tissues and their matched adjacent noncancerous tissues(ANT)from 48 patients using quantitative reverse transcription-PCR(q-PCR).As expected,miR-338-3p was downregulated in thyroid cancer samples compared with the adjacent noncancerous tissues.To further investigate clinical significance of miR-338-3p in thyroid cancer,48 patients divided into two groups: high expression group(>0.34,n=22)and low expression group(<0.34,n=26),according to the median value(0.34)of the miR-383-3p in thyroid cancer tissues.Statistical analysis showed that the level of miR-383-3p expression was significantly related to lymph node metastasis and clinical stage,which are both indicators of poor prognosis.In addition,we further evaluated the expression of miR-338-3p in different thyroid cancer cell lines(8505C,TPC-1,SW1736),and human thyroid follicular epithelial cells(Nthy-ori3-1).Consistently,the expression of miR-338-3p was decreased to varying degrees in all thyroid cancer cell lines compared with the expression in Nthy-ori 3-1 cells.2.miR-338-3p inhibits thyroid cancer growth in vitro and in vivo.To assess the biological effects of miR-338-3p in thyroid cancer cells,miR-338-3p mimic was transfected into TPC-1 cells which has lowest expression in three thyroid cancer cell lines.The transfections of miR-338-3p were successful,and significantly increased miR-338-3p expression level in TPC-1 cells.Result of MTT assay revealed that miR-338-3p overexpression in TPC-1 cells led to a significantly reduction in proliferation.To further assess the effect of miR-338-3p on cell proliferation,a colony formation assay was used.Consistently,miR-338-3p overexpression also significantly decreased colony formation of TPC-1 cells.To confirm the above data in vivo,we created tumor xenograft mouse models.Consistently,miR-338-3p overexpression significantly inhibited tumor growth in vivo)The size of subcutaneous tumors and weight derived from miR-338-3p expressing TPC-1 cells were dramatically smaller than that of miR-NC expressing cells.These data suggest that miR-338-3p suppressed thyroid cancer growth in vitro and in vivo.3.miR-338-3p inhibits thyroid cancer cell migration and invasion.To study the effect of miR-338-3p on metastasis ability of thyroid cancer cells,migration and invasion assays were performed in TPC-1 cells transfected with miR-338-3p or miR-NC by wound healing and invasion chamber assays,respectively.It was found that restoration of miR-338-3p could significantly reduce migration and invasion capacity in TPC-1 cells.4.AKT3 is a direct target of miR-338-3p.To understand the mechanisms by which miR-338-3p suppressed thyroid cancer growth,we searched for candidate targets of miR-338-3p that might play a role in thyroid cancer pathogenesis using miRanda,Target Scan and Pic Tar algorithms.We identified the 3'-UTR of AKT3 that were able to bind to the “seed region” of miR-338-3p.To further confirm that miR-338-3p might bind to the 3'-UTR of AKT3,we performed a luciferase assay.miR-338-3p was bound to AKT3 3'-UTR,resulting in markedly reduced luciferase activities.In addition,miR-338-3p overexpression significantly inhibited AKT3 expression and its downstream protein,p-AKT expression,which caused AKT pathway inhibition.Importantly,we also found that the AKT3 m RNA level was greatly increased in thyroid cancer tissues comparing to adjacent non-cancerous tissues,and that AKT3 m RNA expression level was inversely corrected with the expression of miR-338-3p in thyroid cancer tissues(r=-0.614,p<0.0001).These data suggested that AKT3 was a target of miR-338-3p in thyroid cancer.5.Silencing AKT3 exhibits similar effect with miR-338-3p overexpression in thyroid cancer cells.To investigate the biological functions of AKT3 in thyroid cancer cells,AKT3 expression was silenced in TPC-1 cells by transfection with si-AKT3 or si-NC.qRT-PCR and Western blot assay confirmed that AKT3 expression was significantly downregulated in TPC-1 cells transfection with si-AKT3 compared to cells transfection with miR-NC.Functional assays showed that downregulation of AKT3 significantly inhibited cell proliferation and colony formation,migration and invasion in TPC-1 cells,which mimicked the inhibition effect with miR-338-3p overexpression in TPC-1 cells.6.AKT3 overexpression rescues the miR-338-3p-mediated inhibition effect on thyroid cancer cells.To further elucidate whether AKT3 is a functional target of miR-338-3p in thyroid cancer cells,we performed a rescue experiment by transfection of AKT3 plasmids into miR-338-3p-expressing TPC-1 cells.It was found that TPC-1transfected with AKT3 overexpression plasmid could restore AKT3 expression that caused reduction by miR-338-3p overexpression.In addition,overexpression of AKT3 in TPC-1 cells could rescue the inhibition effect of miR-338-3p on cell proliferation,colony formation,migration and invasion.Taken together,these results indicated that miR-338-3p exerted it suppressive roles in thyroid cancer by repressing AKT3.Conclusion and innovation pointThe result of our study showed that miR-338-3p expression was downregulated in thyroid cancer tissues and cell lines.Overexpression of miR-338 in thyroid cancer cells inhibited cell growth in vitro and tumor growth in nude mice.Furthermore,we showed that AKT3 was a direct target of miR-338-3p in thyroid cancer.Our findings showed miR-338-3p in thyroid cancer constitutes a potential biomarker of sensitivity or response and target miR-338-3p represented a new approach to sensitize thyroid cancer to treatment.