The Expression And Function Mechanism Of MiR-299-3P In Papillary Thyroid Cancer

BackgroundThyroid cancer(TC)is one of the most common malignancies in the world.Over the past decades,the incidence rate of thyroid cancer has increased significantly worldwide.In our country,the incidence rate of thyroid cancer reached fourth of the incidence rate of malignant tumors in 2015,accounting for 8.49%of the female malignant tumors.According to the origin of tumor cells,thyroid cancer is usually divided into four pathological types:papillary carcinoma,medullary carcinoma,follicular carcinoma and undifferentiated carcinoma.Among them,papillary carcinoma is the most common pathological type,accounting for 88%of all thyroid cancers.Some studies suggest that the increasing incidence rate of thyroid cancer has been mainly attributed to the increase of papillary carcinoma incidence rate in recent years.The prognosis of papillary thyroid carcinoma(PTC)is good.It is reported that the 20-year survival rate of papillary thyroid carcinoma is 99%.Although the postoperative survival rate of papillary thyroid carcinoma is very high,the long-term follow-up results show that the recurrence rate is as high as 25%.Papillary thyroid cancer not only brings the body trauma of surgical treatment to patients,but also brings heavy psychological burden because of the fear of recurrence.Therefore,it is of great value to study the molecular mechanism of the occurrence and development of papillary thyroid carcinoma.MicroRNA(miRNA)is a kind of noncoding single stranded small RNA molecules with 19-25 nucleotides encoded by endogenous genes.They can modulate post-transcriptional gene expression by translational repression or degradation.Research shows that a miRNA can regulate hundreds of different mRNAs,and a mRNA can also be regulated by multiple miRNAs.MiRNAs have been well studied by numerous researchers,and the physiological roles of miRNAs have been reported.The biological functions of miRNAs are various,including cell differential,proliferation,apoptosis,metastasis,invasion and senescence.Moreover,it has been reported that aberrant expression of miRNAs can lead to multiple different malignant tumors including GC.In recent studies,miR-299-3p has been found to play some roles in the occurrence and development of a variety of tumors.Wang et al.found that miR-299-3p inhibited the proliferation and invasion of human colon cancer cells by down regulating the expression of vascular endothelial growth factor A(VEGFA)in vivo and in vitro.In HCC(hepatocellular carcinoma),miR-299-3p inhibits tumor growth and invasion by acting on sirtuin 5.Yu et al reported that miR-299-3p could inhibit the proliferation and invasion of cervical cancer by acting on TCF4(transcription factor 4).Zhao et al reported that inhibiting the expression of miR-299-3p would reduce the invasion,migration,proliferation and promote the apoptosis of SKOV3 cells.Further study confirmed that the role of miR-299-3p was realized by regulating the expression of OCT-4(organic cation/carnitine transporter4).It can be seen from the above literature that miR-299-3p plays the role of tumor suppressor gene in colon cancer,liver cancer and cervical cancer,while it plays the role of oncogene in ovarian cancer.So far,the biological role of miR-299-3p in papillary thyroid carcinoma remains unknown.Therefore,the aim of our current research is to investigate the expression and role of miR-299-3p in papillary thyroid carcinoma.ObjectiveIn China,the incidence rate of thyroid cancer reached fourth of the incidence rate of female malignant tumors in 2015,accounting for 8.49%of the female malignant tumors.Papillary thyroid cancer not only brings the body trauma of surgical treatment to patients,but also brings heavy psychological burden because of the fear of recurrence.Therefore,it is of great value to study the molecular mechanism of the occurrence and development of papillary thyroid carcinoma.In recent years,it has been found that miRNA can affect cell proliferation,differentiation and apoptosis by regulating different genes in multiple cell signaling pathways,and then participate in a variety of pathophysiological processes of tumor,including the occurrence and development of tumor.MiR-299-3p plays the role of tumor suppressor gene in colon cancer,liver cancer and cervical cancer,while it plays the role of oncogene in ovarian cancer.So far,the biological role of miR-299-3p in papillary thyroid carcinoma remains unknown.Therefore,the aim of our current research is to investigate the expression and role of miR-299-3p in papillary thyroid carcinoma.This study will be carried out from the following aspects:1.To detect the expression of miR-299-3p in papillary thyroid carcinoma;2.To study the function of miR-299-3p in the proliferation,cycle distribution and apoptosis of thyroid papillary carcinoma cells.3.The effect of over expression of miR-299-3p on the growth of transplanted tumor in mice.4.Prediction and verification of miR-299-3p target gene.Methods1.The expression of miR-299-3p in 30 pairs of papillary thyroid carcinoma and matched paracancerous tissues were detected by real-time fluorescence quantitative PCR;the expression of miR-299-3p in four groups of cell lines,BCPAP and TPC-1(thyroid papillary carcinoma cell lines,),Nthy-ori3-1(normal human thyroid follicular epithelial cell line)and 8505C(undifferentiated thyroid carcinoma cell line),were detected by the same method;the difference was analyzed by statistical methods.2.We used mimics to upregulate the expression of miR-299-3p and inhibitor to inhibit the expression of miR-299-3p,CCK-8 assay and EdU cell proliferation experiment were used to observe the effect of up-regulation and down-regulation of miR-299-3p expression on the proliferation of thyroid papillary carcinoma cells,the effect of up-regulation and down-regulation of miR-299-3p expression on cell cycle distribution was observed by cell cycle experiment,the effect of up-regulation and down-regulation of miR-299-3p expression on apoptosis was observed by apoptosis assay.As the previous results showed that the expression of miR-299-3p in BCPAP was relatively high,while that of miR-299-3p in TCP-1 was relatively low,we used miR-299-3p mimics and its negative control to transfect TCP-1 cells,and miR-299-3p inhibitor and its negative control to transfect BCPAP cells.3.We used the mouse orthotopic tumor model,injected with agomir(the expression of miR-299-3p is up-regulated),observe the growth of transplanted tumor and draw the growth curve of transplanted tumor.The expression of miR-299-3p in the transplanted tumor of the non-intervention group,ago-miR-299-3p group and ago-miR-NC group were compared by Real time fluorescence quantitative PCR,the growth curves of the transplanted tumors were compared among the three groups.4.Firstly,we used four public databases:RNA22,TargetScan,miRWalk and MiRanda,to analyze biological information and predict the target gene of miR-299-3p.The target gene was verified by double Luciferase Report System,real-time fluorescence quantitative PCR and Western blot assay.And then we analyzed the relationship between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinoma.Results1.Expression of miR-299-3p in tissues and cellsThirty pairs of papillary thyroid carcinoma and their matched paracancerous tissues were detected by real-time fluorescence quantitative PCR,the results showed that the expression of miR-299-3p in papillary thyroid carcinoma was significantly lower than that in the paracancerous tissues.The results of cell line detection showed that the expression of miR-299-3p in thyroid cancer cell lines TCP-1,BCPAP and undifferentiated cancer cell line 8505C were significantly lower than that in human thyroid follicular epithelial normal cell line Nthy-ori3-1.The differences were statistically significant.2.Upregulation and inhibition of miR-299-3p expression in papillary thyroid carcinoma cellsThe results of real-time fluorescent quantitative PCR showed that the expression of miR-299-3p in TCP-1 cells transfected with miR-299-3p mimics was significantly higher than that in the non transfected cells and the negative control cells.However,the expression of miR-299-3p in BCPAP cells transfected with miR-299-3p inhibitor was significantly lower than that in the non transfected cells and negative control cells.We successfully up-regulated and down-regulated the expression of miR-299-3p in different cell lines of thyroid papillary carcinoma,which provided conditions for our follow-up study.3.Effect of miR-299-3p expression on proliferation of thyroid papillary carcinoma cellsThe comparison of cell proliferation curve showed that the proliferation curve of TPC-1 transfected with miR-299-3p mimics was significantly lower than that of the negative control group,and the proliferation rate was significantly lower than that of the control group.In contrast,after BCPAP transfected with miR-299-3p inhibitor,the proliferation curve of the experimental group was greatly above the negative control group,and the proliferation speed was significantly higher than that of the control group.The results of EdU cell proliferation assay showed that the positive rate of EdU in TPC-1 transfected with miR-299-3p mimics was significantly lower than that in the blank control group.But after BCPAP transfected with miR-299-3p inhibitor,its EdU positive rate was significantly higher than that of the blank control group.4.Effect of miR-299-3p expression on cell cycle distribution of thyroid papillary carcinomaAfter upregulating the expression of miR-299-3p in TPC-1,compared with the negative control group,the proportion of G1/0 phase cells increased greatly,while the proportion of s phase cells decreased significantly,the results showed that over expression of miR-299-3p could inhibit the cell cycle transition from G1/0 to S phase.However,in BCPAP cells,inhibition of miR-299-3p expression significantly reduced the G1/0 phase cell proportion of BCPAP,but significantly increased the S phase cell proportion.It is confirmed that the down-regulation of miR-299-3p can promote the cell cycle transition from G1/0 to S phase.5.Effect of miR-299-3p expression on apoptosis of thyroid papillary carcinoma cellsThe apoptotic rate of TPC-1 cells with upregulated miR-299-3p was significantly higher than that of the control group;the apoptosis rate of BCPAP cells with inhibited miR-299-3p was significantly lower than that of the control group.These results showed that the expression of miR-299-3p could promote the apoptosis of thyroid papillary carcinoma cells.6.Detection of miR-299-3p expression in transplanted tumor and the effect of over expression of miR-299-3p on the growth curve of transplanted tumorAfter injection of ago-miR-299-3p into the transplanted tumor of mice,the results of real-time fluorescent quantitative PCR showed that the expression of miR-299-3p in the group of ago-miR-299-3p was significantly higher than that in the group of untreated and ago-miR-NC.It is confirmed that the expression of miR-299-3p in transplanted tumor can be up-regulated by the injection of ago-miR-299-3p.The results showed that the growth curve and weight of transplanted tumor in ago-miR-299-3p group were significantly lower than those in untreated group and negative control group.7.Screening and verification of miR-299-3p target gene(1)We used four public databases,RNA22,TargetScan,miRWalk and MiRanda,to analyze biological information.There were 176 potential target genes.After screening,SHOC2 was selected as the candidate target gene.(2)It was confirmed that SHOC2 was the target gene of miR-299-3p by double Luciferase Report SystemIn order to verify whether SHOC2 is a direct target gene of miR-299-3p,we first constructed pGL3-SHOC2 3'UTR and pGL3-SHOC2 3'UTR-Mut fluorescence reporting plasmids.TPC-1 was co-transfected with pGL3-SHOC2 3'UTR and miR-299-3p-mimics or blank control,when miR-299-3p-mimics was transfected,the fluorescence intensity decreased by more than 50%.However,when the mutant pGL3-SHOC2 3'UTR-Mut was co-transfected with miR-299-3p-mimics or blank control,there was no obvious difference in fluorescence intensity.In BCPAP cells,we did the same experiment and got similar results.These results indicate that miR-299-3p directly acts on the binding site of SHOC2 3'UTR,and SHOC2 was a direct downstream target of miR-299-3p in PTC.(3)The relationship between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinomaIn order to further study the mechanism of miR-299-3p acting on SHOC2 in papillary thyroid carcinoma,we explored the relationship between the expression of SHOC2 and miR-299-3p.? The difference of expression of SHOC2 between papillary thyroid carcinoma and paracancerous tissueFirst of all,we compared the expression of SHOC2 in papillary thyroid carcinoma and its matched paracarcinoma tissues by real-time fluorescence quantitative PCR.The results showed that the expression of SHOC2 in papillary thyroid carcinoma was significantly higher than that in matched paracancerous tissue.This is the opposite of miR-299-3p.?Correlation between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinomaFurthermore,we extracted RNA from 30 surgical specimens of thyroid papillary carcinoma and explored the relationship between the expression of SHOC2 and miR-299-3p in PTC.The results of correlation analysis showed that the relative expression of SHOC2 was negatively correlated with that of miR-299-3p,and the correlation coefficient was-0.51(P=0.004).? The effect of miR-299-3p expression on the expression of SHOC2In order to further study the effect of miR-299-3p expression on the expression of SHOC2,we upregulated or downregulated miR-299-3p expression in thyroid papillary carcinoma cell line to observe the change of SHOC2 expression.After miR-299-3p-mimics was transfected into TPC-1 cells and miR-299-3p was upregulated,western blot showed that the expression of SHOC2 was significantly lower than that of the control group;However,after transfection of miR-299-3p inhibitor into BCPAP cells and down-regulation of miR-299-3p,western blot showed that the expression of SHOC2 was significantly higher than that in the control group.These results show that miR-299-3p can inhibit the expression of SHOC2 in papillary thyroid carcinoma.(4)Rescue assayTo further determine the role of miR-299-3p and SHOC2 in PTC,we recruited rescue assay.The results showed that when co-transfected with SHOC2 overexpression plasmid and miR-299-3p-mimics,the expression level of SHOC2 was significantly up-regulated.The up-regulated SHOC2 reversed the influence of miR-299-3p in cell proliferation.Similarly,SHOC2 overexpression promoted cell cycle progression and suppressed the cell cycle ar-rest effect of miR-299-3p.Up-regulated SHOC2 abolished the influence of miR-299-3p in cell apoptosis in PTC.In sum,we validated that miR-299-3p functioned as a tumor suppressor in PTC by targeting SHOC2.Conclusions1.The expression level of miR-299-3p in papillary thyroid carcinoma was significantly lower than that in matched paracancerous tissues.The expression of miR-299-3p in thyroid papillary carcinoma cell line TCP-1 and BCPAP was significantly lower than that in human thyroid follicular epithelial normal cell line Nthy-ori3-1.2.The expression of miR-229-3p can inhibit the proliferation of papillary thyroid carcinoma cells.The expression of miR-299-3p blocked the cell cycle transition from G0/0 to S phase.The expression of miR-299-3p can promote the apoptosis of thyroid papillary carcinoma cells.3.In vivo experiments showed that overexpression of miR-299-3p in TPC-1 could inhibit the growth of its corresponding transplanted tumor.4.SHOC2 is a direct target gene of miR-299-3p.MiR-299-3p can inhibit the proliferation and cycle transformation of papillary thyroid cancer cells and promote the apoptosis of the cells through the negative regulation of the expression of SHOC2.Significances1.Our study confirmed that miR-299-3p functioned as a tumor suppressor in thyroid papillary carcinoma by targeting the expression of SHOC2.2.Our research provided novel insights into the molecular mechanism underlying PTC progression,which might afford some new understanding in biomarkers and therapeutic strategies in PTC development.