| Objective: Thyroid cancer is the most common malignant tumor of the endocrine system.In recent years,the incidence of thyroid cancer has been increased significantly worldwide,accounting for about 5%-10% of female malignant tumors.Papillary thyroid cancer(PTC)is the most common pathological subtype of thyroid cancer,constituting85%–90 % of all thyroid malignancies.Although most of PTCs had a good prognosis with a combination of radioiodine treatment and levothyroxine suppression therapy after complete surgical intervention,there was still a significant increase in tumor recurrence and cancer-related mortality in some patients with advanced PTC due to extensive invasion and metastasis.Hence,it is essential to study the molecular mechanism of the occurrence and development of thyroid cancer,which is very important to find effective new methods of diagnosis and treatment.Bcl-2-associated athanogene(BAG)are a family of anti-apoptotic genes which have been found in recent years.There are conserved BAG domains at the C-terminal of BAG protein family,which can interact with heat shock protein HSP70/HCP70 and produce a wide range of cellular biological effects,including apoptosis,tumorigenesis,neurodifferentiation,stress response and cell cycle,so BAG family proteins are also recognized as Co-chaperone.Among all BAG family members,BAG5 is the only protein that contains multiple BAG domains.In recent years,the related research of BAG5 was mainly focused on Parkinson's disease.Although a few reports suggested a potential function of BAG5 in tumorigenesis,its function in thyroid cancer had not been investigated yet.In the previous results of this study,we found that expression of BAG5 was significantly increased in papillary thyroid cancer tissues,but its specific mechanism remained to be clarified.Fibronectin(FN)family is closely related to epithelial-mesenchymal transformation(epithelial-mesenchymal transition,EMT).EMT plays an important role in tumor invasion and metastasis,such as decreased expression of epithelial markers E-cadherin and increased expression of mesenchymal markers N-cadherin and FN,which are both the most important features of EMT.Fibronectin 1(FN1)is an member of the FN family,which is up-regulated in many human cancers,including thyroid cancer.It has been confirmed that FN1 could promote the invasion and metastasis of thyroid cancer.In the previous study using SILAC followed by spectrometry,we screened that fibronectin 1(FN1)was significantly decreased in cells that with downregulating BAG5.Therefore,we speculated that FN1 might be the downstream target molecule of BAG5,and the upregulated expression of BAG5 in PTC promoted invasion and metastasis of PTC by promoting FN1 expression,but the specific mechanism of action needs to be further verified and analyzed.The purpose of this study was to explore the different protein expression of BAG5 in the PTC tissues relative to adjacent non-cancerous tissues and the functions in the PTC cell line.It was demonstrated that BAG5 could play its role in promoting migration and invasion in PTC by regulating FN1.In addition,we want to explain the specific mechanism of BAG5 regulating FN1 at the translation level.Methods: 1.We collected 84 pairs of PTC tissues and adjacent non-cancerous tissues treated by thyroid surgery in the First Affiliated Hospital of China Medical University between March 2018 and May 2018.The protein expression of BAG5 was detected in 84 pairs of PTC tissues and adjacent non-cancerous tissues by Western blot and specific immunohistochemical staining.2.We cultured PTC cell lines TPC1,K1,BCPAP and IHH4.The basic protein expression of BAG5 was detected by Western blot analysis in each cell line.3.K1 and TPC1 cell lines were infected with lentivirus containing empty or BAG5 construct,while IHH4 and BCPAP cell lines were infected with gRNA guided BAG5 using CRISPR/Cas9 system.Both the BAG5 expression was confirmed by Western blot analysis.4.Cell counting assay and colony formation were used to detect the cell proliferation ability of PTC cells with BAG5 overexpression or knockdown.Transwell was used to detect the ability of migration and invasion.5.Using SILAC followed by spectrometry,we selected the differential protein expression in control and BAG5 knockdown IHH4 cells.6.FN1 mRNA expression in BAG5 knockdown or overexpression PTC cells were analyzed using real-time RT-PCR,and the protein expression of FN1 was detected by Western blot analysis.We also detected the protein expression of FN1 in 84 pairs of PTC tissues and adjacent non-cancerous tissues by Western blot and specific immunohistochemical staining.7.Control and BAG5 knockdown PTC cells were transfected with empty and FN1 containing eukaryotic expression vectors.FN1 expression was confirmed using Western blot analysis.Transwell assay confirmed that overexpression of FN1 significantly weakened the suppressive effects of BAG knockdown on migration and invasion of BCPAP and IHH4 cells.8.FN1 expression of control and BAG5 overexpression PTC cells was knocked down using lentivirus containing shRNAs specific against FN1(shFN1).FN1 knockdown efficiency was confirmed using Western blot analysis.Transwell assay confirmed that knockdown of FN1 significantly decreased migration and invasion of TPC1 and K1 cells.9.Co-IP demonstrated that BAG5 did not interact with FN1.10.Control and BAG5 knockdown PTC cells were treated with vehicle,lysosome inhibitor E64 D and pepstatin A,or proteasome inhibitor MG132,FN1 expression was analyzed using Western blot.Control and BAG5 knockdown PTC cells were treated with the protein synthesis inhibitor cycloheximide for the indicated time,FN1 expression was analyzed using Western blot.11.Ribosome Profiling was used to detect the localization of FN1 mRNA in BAG5 knockdown PTC cells.12.Luciferase activity assays was used to detect the luciferase activity of the reporter construct including FN1 3'UTR in control and BAG5 knockdown IHH4 or control and BAG5 overexpression TPC1 cells.13.RNA sequence screened that some miRNAs were significantly upregulated by BAG5 knockdown.Five miRNA databases Targetscan?PITA?miRmap?microT and miRanda were used to predict which most likely combine with 3'UTR of FN1.14.The expression of miR-144-3p in BAG5 knockdown or overexpression PTC cell lines was detected by qRT-PCR.IHH4 cells were cotransfected with miR-144-3p mimic or antagomir and luciferase containing wild-type(WT)or potential miR-144-3p binding site mutant FN13'UTR.Luciferase activity assays was used to detect luciferase activity of reporter containing FN1 3'UTR.15.BAG5 knockdown or BAG5 overexpression PTC cells were transfected with miR-144-3p antagomir or miR-144-3p mimic.FN1 expression was analyzed using Western blot analysis.Transwell was used to detect the the changes of migration and invasion ability.16.The correlation between the expression of BAG5 and FN1 in 84 cases of PTC tissues was statistically analyzed by Pearson's coefficient tests.Results: 1.The expression of BAG5 protein was significantly increased in thyroid papillary cancer,when compared with those in peripheral normal tissues.2.Knockdownor overexpression of BAG5 demonstrated no obvious effects on proliferation of PTC cells,as assessed by viable cell count and colony formation.3.BAG5 knockdown significantly suppressed migration and invasion,while BAG5 overexpression significantly promoted migration and invasion of PTC cells.4.Using SILAC followed by spectrometry,we selected the differential protein expression of FN1.5.The expression of FN1 protein was also significantly increased in thyroid papillary carcinoma,when compared with those in peripheral normal tissues.We found positive correlation of BAG5 and FN1 protein expression was observed in papillary cancer tissues.6.Overexpression or knockdown of FN1 could significantly reverse the effect of BAG5 on migration and invasion of PTC cell lines.7.BAG5 regulated the expression of FN1 at the protein level,but Co-IP confirmed that there was no direct interaction between them.8.BAG5 did not affect the protein degradation and stability of FN1 in PTC cells.9.Ribosome Profiling indicated that BAG5 knockdown had obvious effect on distribution of FN1 mRNA on monosomes or polysomes.10.Luciferase activity assays exhibited that BAG5 knockdown or overexpression significantly could affect the luciferase activity of the reporter construct including FN1 3'UTR in PTC cells.RNA sequence screened miR-144-3p.Five miRNA databases Targetscan?PITA?miRmap?microT and miRanda predicted that 3'UTR of FN1 was potential target for miR-144-3p.11.miR-144-3p antagomir significantly increased the protein expression of FN1 and the invasive capacity of IHH4 and BCPAP cells with BAG5 knockdown,while mi R-144-3p mimic significantly decreased the protein expression of FN1 and the invasive capacity of TPC1 and K1 cells with BAG5 overexpression.Conclusion: BAG5 is highly expressed in PTC tissues,which promotes the migration and invasion of PTC cells.BAG5 plays a important role in promoting migration and invasion in PTC by upregulating FN1 through miR-144-3p at the translation level. |
PDF Full Text Request