| Background: Thyroid cancer is one of the most common malignant tumors.The incidence has increased and doubled in the past 25 years,accounting for 1% of all malignant tumors.The common pathological types of thyroid cancer including papillary carcinoma,follicular carcinoma,undifferentiated carcinoma and medullary carcinoma.The significant increase in the incidence of thyroid cancer is largely due to the increase in papillary thyroid cancer.Papillary thyroid carcinoma(PTC)is the most common pathological classification of thyroid cancer,accounting for 80%-85%.The current main treatments for PTC include thyroidectomy(including neck lymph node dissection),radioactive iodine(RAI)therapy,and TSH suppression therapy.Although the overall prognosis of PTC is good,there are still some cases with a high risk of recurrence.In the long-term follow-up,about 25% of patients have recurrence,which seriously affects the patient’s prognosis and reduces the patient’s quality of life.In addition,lymph node metastasis is very common in PTC patients,and about 80% of cases are accompanied by cervical lymph node metastasis.According to recent studies,PTC patients with lymph node metastasis are closely related to local recurrence and survival.At present,the mechanism of the pathogenesis and progression of PTC is not clear.It is urgent to deeply explore the factors related to its occurrence,development and malignant biological characteristics.That revealing its potential molecular mechanism,will provide a new direction for finding new targets for early diagnosis,treatment and improving prognosis.MicroRNAs is a kind of small-noncoding RNAs,which consists of only 18 ～ 25 nucleotides.It exists widely in eukaryotes with high conservation,tissue specificity and timing.Mature microRNAs are recognized by ribosome and integrated into RNA induced silencing complex(RISC).Its function is to specifically recognize specific mRNA 3 ’ UTR,which is completely or incompletely complementary,induce mRNA degradation or inhibit mRNA translation,thus inhibiting protein synthesis.In recent years,with the in-depth study of miRNAs,miRNAs were reported to play an indispensable role in the process of tumor development,malignant behavior ang resistance to Chemotherapy.Zhang et al.showed that up-regulation of miR-145-5p can significantly suppress the proliferation and migration of bladder cancer cells.In thyroid follicular carcinoma,miR-1 inhibits the proliferation,migration and invasion of thyroid follicular carcinoma cells by silencing the expression of its target gene CCND2.In addition,miRNAs have been found to play an important role as tumor suppressor or promoter in lung cancer,breast cancer,maxillary sinus squamous cell carcinoma,gallbladder cancer and other malignant tumors.Mi R-613 is a newly discovered miRNA in recent years,with only one 5’ terminal mature sequence.According to recent yeas’studies,miR-613 is dysregulated in a variety of malignant tumors,and it can inhibit tumor growth by up-regulating or down-regulating the expression of miR-613.However,the mechanism of miR-613 in the occurrence of papillary thyroid cancer is still unclear.Methods: 1.A total of 107 pairs of PTC tissues and matched adjacent-normal tissues were collected.Real-time quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of miR-613.Analyzed the relevance between the expression of miR-613 and clinicopathological features of PTC patients.2.PTC cell lines(K1,TPC-1,BCPAP)and normal thyroid follicular epithelial cell line(Nthy-ori-3-1,N3)were cultured,and then detected the expression of miR-613 by RT-PCR.Compared the difference of miR-613 expression in PTC cell line and N3 cells.3.PTC cells were transfected with miR-613 mimics to overexpress the level of miR-613.Explored the expression of miR-613 by RT-PCR to evaluate the transfection efficiency and determine the optimal transfection concentration.The experimental groups are as follows: control group(non-transfected group),NC group(negative control group)and mimics group(overexpression miR-613 group),and then MTS assay,wound healing assay and Matrigel-transwell assay were used to explored the effect of up-regulated miR-613 expression on the proliferation,migration and invasion of PTC cells;4.Up-regulated the expression of miR-613 in the PTC cell line.The expression of epithelial to mesenchymal transition(EMT)markers E-cadherin,N-cadherin,vimentin and matrix metalloproteinase 2(MMP2)were detected by western blotting,compared the difference of the above-mentioned protein expression among each group.5.Targetscan,miRDB,RNAInter and miRWalk databases were used to predict the downstream target genes of miR-613,and took the intersection of the four databases to determine the downstream target.Dual-luciferase reporter experiment was used to verify the bindingsite between TAGLN2 mRNA 3 ’ UTR(3 ’ untranslated region)and miR-613;Then,RT-PCR and western blotting were used to detect the expression of TAGLN2 mRNA and protein levels in PTC tissues and matched adjacent-normal tissues;Overexpressed the miR-613 in the PTC cell lines,and the expression levels of TAGLN2 mRNA and protein were detected by RT-PCR and western blotting;6.Rescue experiment was grouped as follows: control group(non-transfected group),mimics group(overexpression miR-613 group)and mimics+TA group(co-transfected with mimics and TAGLN2 plasmid group).MTS assay,wound healing assay and Matrigel-transwell assay were used to explored the effect of up-regulated miR-613 expression on the proliferation,migration and invasion of PTC cells;the EMT markers E-cadherin,Ncadherin,vimentin and matrix metalloproteinase 2(MMP2)expression levels were detected by western blotting,and compare the differences in protein expression between the groups.7.Overexpression or knockdown of TAGLN2 in the PTC cell line respectively.The experimental groups are as follows: control group(non-transfected group),p CMV-TAGLN2(overexpression TAGLN2 group),p CMV-control group(overexpression empty vector group),sh-TAGLN2 group(knockdown TAGLN2 group)and sh-control group(knockdown empty vector group).Western blotting was used to explore the protein expression levels of PI3 K,p-PI3 K,AKT and p-AKT in PTC cells and compared the difference in each group.Results: 1.The results showed that the relative expression of miR-613 in PTC tissues was 1.339 ± 0.523,which was obviously lower than that in adjacent normal tissues(3.756 ± 0.934,p < 0.01);and the abnormal expression of mir-613 was closely related to cervical lymph node metastasis of PTC(p < 0.01).There were no significant differences between the groups of patients’ others clinicopathological features,including age,gender,tumor size,capsule invasion,and multifocality(p > 0.05).2.Compared with Nthy-ori-3-1 cell line,the expression of miR-613 in K1,TPC-1 and BCPAP cell lines was significantly decreased(p < 0.05).The expression of miR-613 in the above four cell lines was 1.074 ± 0.291,0.195 ± 0.043,0.201 ± 0.053 and 0.270 ±0.052,respectively.3.After overexpression of miR-613,miR-613 was significantly increased,compared with the non-transfected group and negative control group.There was no significant difference between non-transfected group and negative control group(p > 0.05).MTS assay results showed that miR-613 could inhibit the proliferation ability of K1,TPC-1and BCPAP cells,compared with non-transfected group and negative control group(p>0.05).Then,wound healing assay was used to estimate the influence of miR-613 on PTC cells’ migration.It said that the wound healing area of K1 cells in three groups(non-transfected group,negative control group and mimics group)were 68.6% ± 1.40%,74.7% ± 1.80%,52.6% ± 3.20%,severally,and 40.7% ± 2.30%,28.1% ± 1.60%,38.4%± 1.50% in TPC-1 cells,and 38.6% ± 3.60%,37.2 % ± 2.80%,28.0% ± 1.90% in BCPAP cells.These results demonstrated that miR-613 impeded the migration ability of cells significantly(p < 0.05).In Matrigel-transwell assay,the average cell invasion number of K1 cells in non-transfected group,negative control group and mimics group were 225.8 ± 14.3,157.3 ± 25.7,268.0 ± 22.5;that of TPC-1 cells were 496.20 ± 73.5,220.2 ± 5.4,498.2 ± 73.7;and that of BCPAP cells were 568.6 ± 25.6,233.8 ± 24.2,532.0 ± 34.1.Overexpression of miR-613 significantly inhibited the invasion of PTC cells(p < 0.05).4.After transfection of miR-613 mimics,the protein expression of E-cadherin was increased compared with the control group(4.433 ± 0.082 vs 1 ± 0.119 in K1 cells,1.959 ± 0.0214 vs 1 ± 0.0184 in TPC-1 cells,1.460 ± 0.014 vs 1.033 ± 0.141 in BCPAP cells).Meanwhile,the protein expression of N-cadherin,vimentin and MMP2 were decreased compared with the control group(0.218 ± 0.004 vs 1 ± 0.121,0.501 ± 0.008 vs 1 ± 0.064,0.545 ± 0.014 vs 1 ± 0.092 in K1 cells,0.395 ± 0.035 vs 1 ± 0.230,0.581± 0.021 vs 1 ± 0.058,0.240 ± 0.009 vs 1 ± 0.016 in TPC-1 cells,0.474 ± 0.034 vs 1 ±0.017,0.544 ± 0.019 vs 1 ± 0.007,0.267 ± 0.009 vs 1 ± 0.017 in BCPAP cells).There was no significant difference between non-transfected group and negative control group.5.Target Scan,miRDB,RNAInter and miRWalk online databases predicted that the actin-binding protein TAGLN2 might be the direct downstream target gene of miR-613.The results of dual-luciferase reporter assay showed that the Rluc/Luc luciferase ratio of WT + mimics group(1.768 ± 0.085)was significantly lower than that of WT + NC group(2.324 ± 0.043,p < 0.05).At the same time,there was no significant difference between the Rluc/Luc luciferase ratio of MUT + mimics group(2.819 ± 0.102)and MUT + NC group(2.761 ± 0.172,p > 0.05).In PTC tissues,the expression levels of TAGLN2 mRNA and protein in PTC were 1.831 ± 0.197 and 1.237 ± 0.145,respectively,which were significantly higher than those in adjacent normal tissues(0.973 ± 0.080,p < 0.001 and 0.836 ± 0.171,p < 0.01).At the same time,overexpression of miR-613 in PTC cells significantly reduced the expression of TAGLN2 mRNA and protein.6.Compared with PTC cells transfected with miR-613,the proliferation,migration and invasion ability of cells co-transfected with miR-613 and TAGLN2 are enhanced;at the same time,the expression level of EMT-related marker proteins in the cells has also changed: the expression level of E-cadherin was down-regulated,while the expression levels of N-cadherin,vimentin,and metal matrix proteinase 2 were up-regulated.7.After up-regulating the expression of TAGLN2,the expression levels of p-PI3 K and p-AKT were increased in TAGLN2 up-regulated PTC cell lines while they were decreased in TAGLN2 downregulated PTC cell lines.While the expression of PI3 K and AKT did not change significantly.Conclusion: 1.Mi R-613 is significantly downregulated in PTC tissues and its downregulation negatively correlated with lymph node metastasis.2.Mi R-613 can significantly inhibit the proliferation,migration and invasion of PTC cells,as well as the epithelial-mesenchymal transition.Overexpression of miR-613 plays an important role in improving the malignant phenotype of PTC.3.Mi R-613 is involved in regulating the expression of actin-binding protein TAGLN2,and overexpression of TAGLN2 offsets the inhibition effects of miR-613 on PTC cells,which may be the biological mechanism of miR-613 in anti-tumor effect in PTC.4.TAGLN2 could upregulated the expression of p-PI3 K and p-AKT,suggesting that TAGLN2 may promote the progress of EMT by activating PI3K/AKT.These results may provide a new therapy target for the PTC. |
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