| Thyroid cancer is the most common endocrine malignancy,and is one of the most rapidly growing malignancy in many countries over the last several decades.Despite advanced diagnostic and therapeutic approaches that have greatly improved long-term survival from thyroid cancer,a significant proportion of patients exhibit locoregional recurrence or distant metastases,which have great impact on the patients' prognosis.Our research team believed that better understanding of the molecular mechanisms underlying carcinogenesis and progression in papillary thyroid cancer and finding potential novel diagnosis markers and novel therapeutic targets would contribute to improving cure rate,reducing the recurrence rate and ameliorating the patients' prognosis.The pathogenesis of thyroid cancer is very complex,among them gene mutation and abnormal expression plays a vital role in the development of thyroid cancer.Gene expression is regulated by micro RNAs in cells.In thyroid cancer,a lot of micro RNAs have been confirmed to regulate tumor progression and various malignant biological characteristics of tumor cells.MiR-497,a member of the micro RNA-15 family,is considered as a tumor suppressor and negatively regulates the development of many solid malignancies in vivo.However,its role in papillary thyroid cancer has not been systematically studied and reported.Therefore,we wondered that if there is miR-497 abnormal expression in papillary thyroid cancer and the correlation between the abnormal expression of miR-497 and the pathogenesis of papillary thyroid cancer?Then,this research further explored the regulatory effect of miR-497 on the biological characteristics of papillary thyroid cancer cells TPC-1 and its specific mechanisms.Early studies have shown that miR-497 may be involved in theregulation of tumor cells' biological behaviors such as proliferation,invasion and migration.Therefore,we investigated the effect of miR-497 on papillary thyroid cancer cell TPC-1's proliferation,invasion and migration.Micro RNAs always regulate cellular biology function by target genes.It could regulate the translation of target genes at the post-transcriptional level through complete or incomplete binding with the messenger RNA of target gene,and further affect the expression of related proteins,thereby participated in the regulation of cell pathophysiology process,such as cell growth,differentiation,proliferation,cell cycle and cell apoptosis.Therefore,we need to predict the target gene by bioinformatics analysis,and validate it by reading the literature and doing corresponding experiments.In our present study,we focused on the regulatory effect of miR-497 on the development of papillary thyroid cancer and its related mechanisms.Part? observed the expression of miR-497 in papillary thyroid cancer tissue and its association with the clinicopathological features of human papillary thyroid cancer.Part?discussed the regulatory effect of miR-497 on cells' biological behaviors in papillary thyroid cancer cell TPC-1 and its specific mechanism.Part? verified the regulatory effect and its target gene of miR-497 on papillary thyroid cancer in nude mice xenografts.Part? The expression of miR-497 in papillary thyroid cancer and its clinical significanceWe selected Fresh frozen tissue of papillary thyroid cancer patients as our research object,and detected the expression of miR-497 by quantitative real-time PCR(q RT-PCR)assay.Then its association with the clinicopathological features of human papillary thyroid cancer was analyzed by statistical analysis.Results were as follows:(1)The expression of miR-497 decreased in papillary thyroid cancer tissue.Compared with corresponding adjacent normal tissues,the expression abundance of miR-497 in papillary thyroid cancer tissue was 0.31±0.03.Statistical analysis showed that the expression of miR-497 in papillary thyroid cancer tissue was remarkably reduced.(2)The expression of miR-497 was closely related to the TNM staging ofpapillary thyroid cancer.38.7% of the early stage(stage I-?)papillary thyroid cancer tissues showed low miR-497 expression and 88.2% of the advanced stage(stage ?-?)thyroid cancer tissues showed low miR-497 expression.Furthermore,the expression of miR-497 in the advanced stage(stage ?-?)papillary thyroid cancer tissues was significantly lower than that in the early stage(stage I-II).(3)The expression of miR-497 was closely related to the lymph node metastasis of papillary thyroid cancer.The expression of miR-497 in papillary thyroid cancer tissues with lymph node metastasis was significantly lower in the patients with lymph node metastases compared to the patients without lymph node metastases.Statistical analysis also showed that 14(93.3%)out of 15 patients with lymph node metastasis had a low miR-497 expression,while Only 13(39.4%)out of 33 patients without lymph node metastasis had a low miR-497 expression.(4)The expression of miR-497 was not related to patients' gender,age,tumor size and T stage.Part? The effect of miR-497 on cellular biological behavior of papillary thyroid cancer cell TPC-1 and its mechanismIn this part,we transfected the miR-497 mimic into the TPC-1 cell line with low miR-497 expression to mimic the expression of endogenous miR-497,and then assayed the cell proliferation,migration and invasion ability to further explore the effect of miR-497 on the biological function of papillary thyroid cancer cell line TPC-1;and predict its possible target genes by bioinformatics methods.Results were as follows:(1)MiR-497 mimic could mimic the function of endogenous miR-497.After transfected with miR-497 mimics in TPC-1 cells,the expression of miR-497 was significantly increased.(2)Over-expression of miR-497 inhibited TPC-1 cell proliferation.Compared with negative control group(miR-NC group),after transfected with miR-497 mimic for 48 h and 72 h in TPC-1 cells,cell viability was obviously decreased,and the cell colony formation rate decreased to(38.79±4.21)%in miR-497 over-expression group(miR-497 group)(P<0.01 vs.miR-NC).(3)Over-expression of miR-497 inhibited TPC-1 cell invasion and migration.Aftertransfected with miR-497 mimic,the related migration rate of TPC-1cells in miR-NC group and miR-497 group was(1.0±0.05)and(0.46±0.03),respectively(P<0.01 vs.miR-NC);and the invasion cells number was(421.56±12.03)and(237.69±7.54)%,respectively(P<0.01 vs miR-NC).(4)BDNF gene was the direct target gene of miR-497.Bioinformatics retrieve showed that there was a recognition site of miR-497 on the 3'non-coding region(3'UTR)of BDNF m RNA.The dual luciferase reporter assay showed that co-transfected with the wild-type BDNF gene 3'UTR luciferase expression vector(WT-3'UTR)and miR-497 mimic inhibited luciferase activity.(5)Over-expression of miR-497 inhibited BDNF expression and PI3K/AKT activity in TPC-1 cells.After transfected with miR-497 mimic in TPC-1 cells,the m RNA and protein expression level of BDNF were both decreased(P<0.01);and the phosphorylation level of BDNF gene's down-stream regulatory molecules PI3K(Tyr458)and AKT(Ser473)phosphorylation site was remarkably decreased(P<0.01).(6)Effect of si-BDNF transfection on BDNF m RNA expression and protein expression in TPC-1 cells was detected.Si-BDNF inhibited BDNF m RNA and protein expression in TPC-1 cells.Si-BDNF inhibited TPC-1 cell peoliferation,colony formation capacity,migration and invasion after transfection.(7)MiR-497 expression was negatively related to BDNF expression in papillary thyroid cancer tissues.Compared with corresponding adjacent normal tissues,the m RNA expression of BDNF in papillary thyroid cancer tissue increased 4.02±0.09 fold.Spearman's correlation analysis showed miR-497 expression was negatively related to BDNF m RNA expression in papillary thyroid cancer tissues(r=-0.651,p<0.01).(8)The transfection efficacy of miR-497 mimic and BDNF overexpression vector.Compared with miR-497 group,co-transfection with miR-497 mimic and BDNF over-expression vector in TPC-1 cells for 48 h could increase the m RNA and protein expression of BDNF gene.(9)Over-expression of BDNF could partly reverse the inhibitory effect of miR-497 on TPC-1 cell proliferation.Compared with miR-497 group,both cell proliferation and cell colony formation rate in co-transfection group(miR-497+BDNF group)were obviously increased.(10)Over-expression of BDNF could partly reverse the inhibitory effect of miR-497 on TPC-1 cell migration and invasion.Comparedwith miR-497 group,both related cell migration rate and invasion cell number in co-transfection group(miR-497+ BDNF group)were obviously increased.Part? The inhibitory effect of miR-497 on TPC-1 cell nude mice xenografts and its mechanismIn this part,we used nude mice subcutaneously transplanted tumor model to evaluate the inhibitory effect of miR-497 on papillary thyroid cancer in vivo and its mechanism.First nude mice subcutaneous injection of TPC-1 cells which stable expression miR-497 or negtive micro RNA control was used to modeling nude mice xenografts model;then tumor growth was evaluated by measuring tumor size and weight.Then we detected the expression of miR-497,BDNF genes and its downstream PI3K/AKT signal pathway.Results were as follows:(1)miR-497 could inhibit the growth of TPC-1 cell nude mice xenografts.15 days after the injection of cells in the two group nude mice(n=10),all these mice showed transplantation tumor,and it means the model was successfully constructed.While compared with negative control group(TPC-1/miR-NC),transplantation tumor grew slower and the tumor volume was smaller in experimental group(TPC-1/miR-497).Moreover,tumor weight weighing results showed that the mean tumor weight in negative control group(TPC-1/miR-NC)and experimental group(TPC-1/miR-497)were(1.32±0.09)g and(0.56 ±0.04)g;tumor weight in TPC-1/ miR-497 group was obviously lower than that in TPC-1/miR-NC group(P<0.01).(2)The expression of miR-497 and BDNF m RNA in TPC-1 cell nude mice xenografts.Compared with TPC-1/miR-NC group,the expression of miR-497 was extraordinarily escalated in TPC-1/miR-497 group(P<0.01),while the expression of BDNF m RNA was extraordinarily reduced(P<0.01).(3)The protein expression of BDNF and activity of PI3K/AKT signal pathway in TPC-1 cell nude mice xenografts.Compared with TPC-1/miR-NC group,the expression of BDNF m RNA was extraordinarily reduced in TPC-1/miR-497 group(P<0.01);meanwhile the phosphorylation level of BDNF gene's down-stream regulatory molecules PI3K(Tyr458)and AKT(Ser473)phosphorylation site wasremarkably decreased(P<0.01),while the whole protein level of PI3 K and AKT was not obviously changed.Conclusion:1.MiR-497 expression was obviously decreased in papillary thyroid cancer tissues.Down-regulation of miR-497 could be related to the clinical stages and lymph node metastasis.2.MiR-497 mimic could inhibit the proliferation,migration and invasion of human papillary thyroid cancer cell line TPC-1.MiR-497 act as a tumor suppressor by negatively regulating of BDNF gene and inhibiting the activation of its downstream PI3K/AKT signal pathway.3.MiR-497 could inhibit the growth of TPC-1 cell nude mice xenografts.The inhibitory effect of miR-497 on the growth of TPC-1 cell nude mice xenografts could be related to direct inhibition of BDNF gene and the activation of its downstream PI3K/AKT signal pathway. |
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