| Background and ObjectiveThyroid cancer(TC),derived from follicular epithelial,is the common tumor in head and neck.The growing incidence of TC,accounting for 94% of endocrine malignancies,have rocketed to 3 folds in the past three decades.And about 80–85%histotype of TC is papillary thyroid carcinoma(PTC).The five-year survival rate of most PTCs,effectively treated by conventional managements including thyroidectomy,radioiodine ablation and adjuvant long-term thyrotropin suppression therapy,is over the 95%.However,traditional therapeutic strategies are not excellent for individuals who are diagnosed with metastasis and extra-thyroidal invasion and stagnate the ten-year survival rate under 10%.MicroRNAs(miRNAs)are a class of highly conserved,small non-coding RNAs participating in the development and progression of various tumors by base-pairing with the 3?-untranslated regions(UTRs)of target mRNAs at post-transcriptional level.Recent investigations have indicated that PTC and most carcinomas are characterized by aberrant expression of miRNAs,which presenting its value for diagnosis,treatment and prognosis.Based on our previous miRNA microarray analysis,mi R-146b-5p is distinctly up-regulated in PTC tissues compared with the adjacent non-cancerous thyroid tissues.Youben Fan et al.demonstrated that miR-146b-5p promotes TPC-1 cells metastasis by targeting ZNRF3.And ET Kimura et al.confirmed that miR-146b-5p enhances TPC-1 cells proliferation via targeting SMAD4.Nevertheless,the underlying molecular mechanism by which mi R-146b-5p aggravates the malignant phenotypes of PTC cells is far from being fully elucidated.Coiled-coil domain-containing 6(CCDC6)is originally recognized in chimeric genes caused by chromosomal translocation involving the RET proto-oncogene in some PTCs.Angela Celetti et al.showed that CCDC6 acting as an substrate of ATM(ataxia telangiectasia-mutated gene)mediates cellular response to DNA damage and its deficiency is associated with thyroid tumorigenesis,and they also found loss of CCDC6 was strongly correlated with the lymph node metastasis in Non-small cell lung cancer(NSCLC).Alfredo Fusco et al.verified the hypothesis that downregulation of CCDC6 contributed to thyrocyte pathological proliferation by enhancing transcriptional activity of CREB1(cAMP-response element binding protein1),and then took part in carcinogenesis of PTC,and they also indicated that up-regulation of miR-130b-3p increases CREB1 activity in thyroid adenomas by targeting CCDC6.According to these results,it is reasonable to hypothesis that CCDC6 wild type(wt)might serve as a new tumor suppressor involving in multiple biological processes and to assume that miR-146b-5p contributes to the malignant phenotype of PTC by down-regulating CCDC6.In the present study,the correlations between clinical parameters of PTC and miR-146b-5p were verified in PTC tissues and cell lines.Gain-and loss-of-function studies were performed to investigate the the effects of miR-146b-5p,CCDC6,and their interaction on the biological behaviors of papillary thyroid carcinoma cell lines in vitro.The volume and weight of the tumors were applied to determined the functions of miR-146b-5p,CCDC6,and their interaction on the proliferation of TPC-1 cells in vivo.Dual-Luciferase Reporter assays were conducted to confirm that CCDC6 was a direct target of miR-146b-5p.And the the interaction of CCDC6 and clinical characteristics of PTC were detected in paraffin-embedded PTC tissues and its normal correlations.Hereby,we have discussed the interaction between miR-146b-5p and CCDC6 on the development and progression of PTC for thetargeted therapy.Materials and Methods1.87 PTC tissues and the corresponding non-cancerous tissues were collected from thyroidectomy in the The First Affiliated Hospital of Zhengzhou University during March,2015 to November,2015.All patients had not received radiotherapy and chemotherapy before operation and written informed consent has been obtained.All samples were immediately snap-frozen in liquid nitrogen after tumor resection and reserved at-80°C refrigerator.All specimens were diagnosed by two expert endocrine pathologists and 19 PTC tissues with extra-thyroidal invasion,39 PTC tissues with cervical lymph node metastasis,and 47,13,4,23 PTC tissues belonging to TNM I,TNM II,TNM III,and TNM IV respectively,included.qRT-PCR analyses were performed to detect the expression levels of miR-146b-5p in 87 PTC tissues and the adjacent non-cancerous tissues,and their correlations with clinical characterizations.And qRT-PCR analyses were conducted to detect the different endogenous expression levels of miR-146b-5p in TPC-1,BCPAP,and Nthy-ori 3-1cell lines;2.CCK-8,wound-healing and matrigel transwell,flow cytometry analyses for cell cycle and apoptosis assay were performed to evaluate the effects of miR-146b-5p down-regulation on viability,migration,metastasis,apoptosis,and cell cycle progression of TPC-1 and BCPAP cells in vitro,respectively;3.Bioinformatics analysis and Dual-Luciferase Reporter assays were performed to demonstrate that CCDC6 is a direct target of miR-146b-5p;4.CCK-8,wound-healing and matrigel transwell assay were performed to estimate whether CCDC6 down-regulation could attenuate the viability,migration,and metastasis respectively,which were induced by miR-146b-5p down-regulation;5.Transplantation tumor experiment and IHC were conducted to evaluate the effects of miR-146b-5p inhibition or CCDC6 over-expression on the proliferation of TPC-1 cells in vivo;6.IHC were performed to detect the expression levels of CCDC6 in 185paraffin-embedded PTC tissues and its normal correlations with clinical characteristics;7.Statistical analyses were performed using SPSS 17.0 and Prism v6.0 software.One-way analysis of variance or two-tailed Student's t-test were used for quantitative variables.Chi-squared tests were used for qualitative variables.Data were shown as mean ± s.e.m.P < 0.05 was considered significant.Results1.MiR-146b-5p expressions in PTC tissues were 2.20 times as high as in the corresponding non-cancerous tissues,and in female patients were 1.28 times as high as in male patients(p<0.05).And the expressions had a positive correlation with extra-thyroidal invasion and TNM stages(p<0.05);2.(1).In miR-146b-5p inhibitor group of TPC-1 cells,the absorbance at 72 h was decreased to 1.18±0.05,the width of wound-healing within 24 h was subtle changed and the average amount of cells penetrating Matrigel membrane was decreased to 26,the S phase was decreased to(16.02%),compared with negative control(p<0.05).The apoptosis rate was(1.73%±0.09),compared with negative control(p>0.05);(2).In miR-146b-5p inhibitor group of BCPAP cells,the absorbance at 72 h was decreased to 1.12±0.04,the width of wound-healing within 24 h was subtle changed and the average amount of cells penetrating Matrigel membrane was decreased to 28,the S phase was decreased to(13.63%),compared with negative control(p<0.05).The apoptosis rate was(2.53%±0.18),compared with negative control(p>0.05);3.Conserved sequence 1742-1749 and poorly conserved sequence 1920-1927 of CCDC6 3'UTR is a direct target of miR-146b-5p;4.(1).In miR-146b-5p group of TPC-1 cells,the absorbance at 72 h was increased to 2.14±0.05,the width of wound-healing within 24 h was distinctly shortened and the average amount of cells penetrating Matrigel membrane was increased to 109,the S phase was increased to(24.58%),compared with negative control(p<0.05).In miR-146b-5p+Lv-CCDC6 group of TPC-1 cells,the absorbance at 72 h was decreased to 1.91±0.02,the width of wound-healing within 24 h was largeand the average amount of cells penetrating Matrigel membrane was decreased to 63,the S phase was decreased to(20.21%),compared with miR-146b-5p group(p<0.05);(2).In miR-146b-5p group of BCPAP cells,the absorbance at 72 h was increased to 3.04±0.07,the width of wound-healing within 24 h was distinctly shortened and the average amount of cells penetrating Matrigel membrane was increased to 112,the S phase was increased to(25.33%),compared with negative control(p<0.05).In miR-146b-5p+Lv-CCDC6 group of TPC-1 cells,the absorbance at 72 h was decreased to 2.07±0.04,the width of wound-healing within 24 h was large and the average amount of cells penetrating Matrigel membrane was decreased to 57,the S phase was decreased to(20.68%),compared with miR-146b-5p group(p<0.05);5.In miR-146b-5p inhibitor group,the volume and weight of tumors was 366.5±37.5mm3,408.5±10.71 mg after 28 days implantation respectively,compared with negative control(p<0.05).In CCDC6 over-expression group,the volume and weight of tumors was326.5 ± 19.5mm3,363.1 ± 31.97 mg after 28 days implantation respectively,compared with negative control(p<0.05);6.In CCDC6 over-expression group,tumor sections IHC staining intensities of CCDC6 were stronger than negative control group,and the tumor sections,derived from miR-146b-5p inhibitor group,staining intensities of CCDC6 were stronger than negative control group(p<0.05).7.The CCDC6 expression levels in paraffin-embedded PTC tissues had a negative correlation with extra-thyroidal invasion,lymph node metastasis,and TNM stages in PTC tissues(p<0.05).(Table 4.1)Conclusions1.Consistent with the previous microarray results,miR-146b-5p expression was higher in PTC tissues than the corresponding non-cancerous tissues and had a positive correlation with miR-146b-5p expression and TNM clinical stages of PTC.Down-regulation of miR-146b-5p inhibited the malignant phenotypes of TPC-1 and BCPAP in vitro and in vivo,which demonstrated miR-146b-5p served as oncogene in the development and progression of PTC;2.Dual-Luciferase Reporter assays confirmed that CCDC6 was a direct target of miR-146b-5p.Down-regulation of CCDC6 promoted the malignant phenotypes of TPC-1 and BCPAP,which inhibited by miR-146b-5p down-regulation in vitro and in vivo;3.CCDC6 expressions were absent or reduced in paraffin-embedded PTC tissues and had a negative correlation with the malignant clinicopathologies of PTC,which demonstrated CCDC6 were serving as anti-tumor gene in the development and progression of PTC. |
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