Expression Of BMP-4 And Its Effect On Cell Biological Characteristics In Papillary Thyroid Carcinoma

BackgroundThyroid cancer,a common malignancy that develop from endocrine organs,is increasing in incidence.The most common form is papillary thyroid carcinoma(PTC).Under standard treatment,PTC generally has an excellent prognosis,but some papillary carcinoma may show high invasiveness,leading to tumor progression,metastasis and postoperative recurrence,as well as a decreased survival rate and quality of life.Studies have shown that 20 to 40%of patients with thyroid cancer suffer from tumor recurrence,and 3 to 15%have distant metastasis.If distant metastasis is present when diagnosed,the prognosis of these patients is even worse.The 5 year survival rate is only 50%.Therefore,studying the process of PTC cell growth,invasion and metastasis,investigation for differential expression in PTC,predicting biomarkers for tumor invasion,metastasis and intervention,are of great significance for further improving the cure rate and survival rate.Epithelial-mesenchymal transition(EMT)is the process by which cancer epithelial cells undergo morphological changes,lose their intercellular contact and acquire properties characteristic of stromal cell components,thereby showing a migratory phenotype.The molecular markers include epithelial cell marker E-Cadherin,keratin,as well as interstitial cell markers such as Vimentin and N-Cadherin etc.Through EMT,tumor cells gain the ability of migration and invasion.EMT plays an important role in metastasis.This is an important theory for explaining tumor metastasis and is a research hotspot in recent years.Studies have shown that EMT plays an important role in the migration of thyroid cancer cells.Bone morphogenetic proteins(BMPs)belong to an extracellular signal molecule family,and is a member of the Transforming growth factor-beta(TGF-β)superfamily.BMPs have many functions,including regulating cell proliferation,differentiation,apoptosis and migration,as well as cell fate determination.Besides,it has been sho’wed that BMPs expression is dysregulated in various malignant tumors and they play an important role in tumor growth,invasion and migration.The role of BMPs is mainly through two signaling pathways,Smad-dependent and Smad-independent.Smad-dependent pathway(bone morphogenetic protein signaling pathway)is a classic signaling pathway.Smads protein is the hub of intracellular signal transduction in the pathway.Receptor-associated Smads(R-Smads)include Smadl,Smad2,Smad3,Smad5 and Smad 8.Smad1,Smad 5 and Smad 8 are involved in BMPs signaling pathway.In addition,BMPs can activate other signaling pathways,such as the ERK and p38MAPK pathways.As a member of the BMPs family,BMP-4 pays an important role in regulating the biological behaviors in various types of cells,such as embryogenesis,development of asymmetric organs,and hematopoietic system.Studies have shown that BMP-4 protein is abnormal in multiple types of tumors,and is related to tumor growth,invasion,migration and prognosis.Moreover,many studies have shown that BMP-4 is related to epithelial-mesenchymal transition of tumor cells.To date,no reports have been reported about the role of BMP-4 in PTC.Previous studies found that invasion and metastasis in PTC is related to epithelial-mesenchymal transition,and BMP-4 not only plays an important role in the induction of epithelial-mesenchymal transition but also in the regulation of the biological behaviors of tumor cells,such as tumor growth.Therefore,How does BMP-4 express in PTC?Is it related to the invasion and metastasis of PTC and whether it has a role in the biological characteristics of PTC cells?What is the signal transduction pathway through which BMP-4 plays a role in PTC?The above questions will be discussed in our study.Part I Expression of BMP-4 in papillary thyroid carcinoma and its correlation with tumor invasion and metastasisObjectiveTo study the expression of BMP-4 in papillary thyroid carcinoma and its correlation with tumor invasion and metastasis.Methods1.Surgically resected PTC specimens and corresponding 41 paracancerous tissues from 82 patients with PTC were collected from the thyroid surgery department of Yantai Yuhuangding Hospital affiliated to Qingdao University medical school.All the selected cases were pathologically diagnosed as PTC,and their clinical and pathological data were also collected.2.Expression of BMP-4 in PTC tissues and paracancerous tissues was detected by immunohistochemistry.3.The full clinical records of all patients were collected to analyze the relevance between BMP-4 expression and the clinical pathological features of PTC,including gender,age,tumor size,whether tumor is multifocal,whether there is capsular invasion,lymph node metastasis and TNM staging.Results1.BMP-4 expression was found in the tissue of PTC,and the BMP-4 protein was diffusely distributed,located in the cytoplasm,with its expression level ranging from weak to strong positive.BMP-4 expression was absent in the paracancerous tissues.2.BMP-4 expression in PTC tissues was significantly different from that of paracancerous tissue.3.There was no correlation between BMP-4 expression and the patient’s gender,multicentricity and lymph node metastasis(P>0.05).BMP-4 expression in patients at the age of 45 or above was higher than that in patients at the age of less than 45 years old(P<0.01).BMP-4 expression in patients with tumor size>1cm was higher than that in patients with tumor size ≤1cm(P<0.01).BMP-4 expression in patients with capsular invasion was higher than that in patients without capsular invasion(P<0.01).BMP-4 expression in TNM stage Ⅲ + Ⅳ patients was higher than that in Ⅰ+ Ⅱ patients(P<0.01).Conclusions1.BMP-4 is expressed in PTC tissues.2.BMP-4 may be related to the occurrence and development of PTC.PTC patients with high expression of BMP-4 are more likely to have tumor invasion and metastasis.Part Ⅱ Effect of BMP-4 on the Biological Characteristics of papillary thyroid carcinoma cellObjective1.To detect the expression of BMP-4 in PTC cell lines B-CPAP,TPC-1 and K1.2.To investigate the effect of BMP-4 on the biological characteristics of PTC cell lines B-CPAP and TPC-1.3.To study the effect of BMP-4 on the expression of molecular marker during epithelial-mesenchymal transition in PTC cell lines B-CPAP and TPC-1.4.To explore the signal pathway of BMP-4 involved in PTC cell lines B-CPAP and TPC-1.Methods:1.Total RNA was isolated from PTC cell.2.The expression of BMP-4 in PTC cell lines B-CPAP,TPC-1 and K1 was detected by Quantitative real-time PCR(qPCR).B-CPAP cell line with low expression of BMP-4 and TPC-1 cell line with high expression of BMP-4 were selected for follow-up experiments.3.B-CPAP cell line was used to establish the overexpressed cell line and TPC-1 cell linewas used to establish the interfered cell line.4.Cell infection① B-CPAP cell line was divided into control group(NC:transfected with invalid vector)and overexpressed group(OE:transfected with recombined BMP-4 lentiviral vector).TPC-1 cell line was divided into control group(NC:transfected with invalid vector)and interfered group(KD:transfected with BMP-4 interference lentiviral vector).② The cell lines grew well after infection.About 72 hours after infection,the expression of green fluorescent protein in the cells was observed by fluorescence microscopy and infection rate was evaluated.③ The overexpression efficiency of BMP-4 in B-CPAP cell line and knockdown efficiency of BMP-4 in TPC-1 cell line were detected by qPCR.5.Cell proliferation of B-CPAP and TPC-1 was detected by MTT assay.6.The apoptosis of B-CPAP and TPC-1 was detected by flow cytometry.7.Transwell cell invasion assay was carried out to identify the effect of BMP-4 on invasion of B-CPAP and TPC-1.8.The expression of E-Cadherin,Vimentin and Smadl in B-CPAP and TPC-1 was detectd by qPCR.9.The expression of E-Cadherin,Vimentin and Smadl in B-CPAP and TPC-1 was detectd by Western Blotting.Results1.BMP-4 expression in B-CPAP was 0.03 fold than that in K1 detected by qPCR.BMP-4 expression in TPC-1 was 1.13 fold than that in K1 detected by qPCR.B-CPAP with low expression of BMP-4 and TPC-1 with high expression of BMP-4 were.selected to proceed with experiments.2.Recombined BMP-4 lentiviral vector was transfected into B-CPAP cell and BMP-4 interference lentiviral vector was transfected into TPC-1 cell.The expression of green fluorescent protein was observed by fluorescence microscope at 72 hours.The infection rate were over 70%in both cell lines.3.qPCR detection indicated that overexpressed BMP-4 in B-CPAP cell was 17.11 fold than that in control group,which was statistically significant(P<0.01).qPCR detection indicated that interfered efficiency of BMP-4 in.TPC-1 cell is 94.4%,which was statistically significant(P<0.01).4.The B-CPAP cell with overexpressed BMP-4 was treated with MTT for 4h after 5 days of cultxure.The OD of overexpressed group was higher significantly than that of control group.The cell proliferation fold was also higher than that in control group significantly(P<0.01).The result indicated that BMP-4 can promote cell proliferation.The OD of interfered group was lower significantly than that of control group.The cell proliferation fold was also lower than that in control group significantly(P<0.01).The result indicated that the reduced BMP-4 can inhibit cell proliferation.5.The B-CPAP cell infected with overexpressed BMP-4 lentiviral vector was detectedapoptosis after 5 days of culture.The results indicated that the apoptosis of control and overexpressed group were less than 5%.The apoptosis of TPC-1 cell infected with interfered BMP-4 lentiviral vector was increased significarntly than that in control group(P<0.01).6.The effect of BMP-4 on invasion of B-CPAP and TPC-1 were detected by Transwell.The metastatic cell amounts of the B-CPAP cell infected with overexpressed BMP-4 lentiviral vector was not observed with significant difference compared with control groups(P>0.05).The number of metastatic cells in TPC-1 cell infected with interfered BMP-4 lentiviral vector was lower than that in the control group,and the difference was statistically significant(P<0.05).7.The expression of E-Cadherin,Vimentin and Smadl in B-CPAP and TPC-1 cell line were detected by qPCR and Western Blotting.The results indicated that there was no statistical difference between overexpressed and control group of B-CPAP cell in E-Cadherin,Vimentin and Smadl expression level(P>0.05).There was no statistical difference between interfered and control group of TPC-1 cell in E-Cadherin and Smadl expression level(P>0.05),but the mRNA level and protein level of Vimentin expression in the TPC-1 cell infected with interfered BMP-4 lentiviral vector were lower than that in control group,with statistical significance(P<0.01),indicating that reduced BMP-4 can inhibit Vimentin expression.Conclusions1.BMP-4 expression was found in PTC cell.2.BMP-4 can promote the proliferation of PTC cell,and may be involved in the apoptosis of PTC cell.3.BMP-4 may be associated with EMT of PTC cell and Vimentin can be regarded as a valuable predictive marker for EMT.BMP-4 may be related to the invasion and metastasis of PTC cell.4.BMP-4 can regulate tumor cell biological activities of PTC independent of Smadl.