Effect And Mechanism Of TFF3 On Epithelial-mesenchymal Transition Of Papillary Thyroid Carcinoma TPC-1 Cell

Thyroid cancer has become one of the most common malignant endocrine system tumors,and its incidence is increasing year after year.Papillary thyroid carcinoma is the most common pathological type in thyroid carcinoma,accounting for about 85%.Nowsdays the therapeutic options for thyroid tumor mainly through operation,but there are still some patients with cancer cells distant metastases to lymph node tissue or normal thyroid tissue.And this is proven to be the main cause of death of thyroid cancer patients.Recent studies indicate that epithelial-mesenchymal transition(EMT)is closely related to metastasis in thyroid cancer.EMT is the process that the epithelial cells acquire mesenchymal characters,losing epithelial phenotype,and further enhance their mobility.Therefore,it is particularly important to find the markers involved in the regulation of EMT in thyroid cancer cells.Trefoil factor three(TFF3)is a number of trefoil factor family peptides.In recent years,some scholars have pointed out that TFF3 may be a potential oncogene.However,there are few studies reported about whether TFF3 can regulate the process of EMT in thyroid papillary carcinoma.In the previous experiment,we found that TFF3 was highly expressed in PTC;Then sh RNA-TFF3-TPC-1 cell line and sh RNAC-TPC-1 cell line were successful established by lentivirus transfection.The aim of this study is to investigate the mechanism of TFF3 on the process of EMT in thyroid papillary carcinoma cell lines.First,we used immunohistochemistry to detect the expression of TFF3 and Snail expression in thyroid carcinoma tissue microarrays.The correlation between snail expression and clinical data such as pathological staging,tumor size and lymph node metastasis was analyzed.And the correlation between TFF3 expression and Snail expression was analyzed.Then,wound scratch assay was used to detect the migration of papillary thyroid carcinoma cell line sh RNA-TFF3-TPC-1(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line after silencing TFF3;Transwell assay was used to detect the invasion of papillary thyroid carcinoma cell line sh RNA-TFF3-TPC-1(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line after silencing TFF3;Colony forming assay was used to detect the change of colony forming ability of sh RNA-TFF3-TPC-1 cell line(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line after TFF3 silencing.Finally,Real time quantitative PCR was used to detect the changes of EMT markers(E-cadherin,N-cadherin,Snail,Slug)m RNA in sh RNA-TFF3-TPC-1 cell line(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line;the changes of EMT markers(E-cadherin,N-cadherin,Snail,Slug)and MAPK pathway related protein ERK1/2 in sh RNA-TFF3-TPC-1 cell line(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line were detected by immunocytochemical staining;The changes of EMT markers(E-cadherin,N-cadherin,Snail,Slug)and MAPK pathway related proteins(ERK1/2,p-ERK1/2)in sh RNA-TFF3-TPC-1 cell line(sh RNA-TFF3),sh RNAC-TPC-1 cell line(sh RNAC)and TPC-1 cell line were detected by Western blot.Our results showed that TFF3 was significantly increased in PTC as compared to para-carcinoma tissue;Snail was significantly increased in PTC as compared to para-carcinoma tissue.IA value of Snail is 0.5331 + 0.0190 vs 0.0976 + 0.0026(P<0.001),and IA value of TFF3 is 0.4614 + 0.1031 vs 0.0812 + 0.006(P<0.001).Snail expression was positively correlated with pathology grade(P=0.007,r=0.6978),tumor size(P=0.002,r=0.738),lymph node metastasis(P=0.037,r=0.651)and TFF3 expression(P=0.001,r=0.8006).Wound scratch assay results showed that sh RNA-TFF3 cells had significantly slower migration rate compared to sh RNAC cells and TPC-1 cells.There was no significant migration rate difference between TPC-1 group and sh RNAC group;TFF3 inhibited invasive capacities of TPC-1 cell lines in vitro.OD value of sh RNA-TFF3-TPC-1 group was obviously lowerer than that in TPC-1 group and sh RNAC-TPC-1 group.There was no significant difference between OD value of TPC-1 group and sh RNAC group;TFF3 inhibited cloning ability capacities of TPC-1 cell lines in vitro.The colony results showed that the number of colonies in sh RNA-TFF3-TPC-1 group(106±20)was fewer than that in TPC-1 group(478±35)and sh RNAC-TPC-1 group(493±32).There was no significant difference between TPC-1 group and sh RNAC group.Immunocytochemical staining results showed that the proteins of Snail、Slug、N-cadherin、ERK1/2 were down regulated in sh RNA-TFF3 group,while E-cadherin positively expressed;The proteins of Snail、Slug、N-cadherin、ERK1/2 were positively expressed in TPC-1 group,while E-cadherin down regulated;The proteins of Snail、Slug、N-cadherin、ERK1/2 were positively expressed in sh RNAC group,while E-cadherin down regulated expressed.q PCR results showed that m RNA levels of Snail,Slug,N-cadherin was decreased and E-cadherin was increased in sh RNA-TFF3-TPC-1 group compared with TPC-1 group and sh RNAC-TPC-1 group;There was no significant m RNA expression difference between TPC-1 group and sh RNAC-TPC-1 group;Western boltting results showed protein expression levels of Snail,Slug,N-cadherin,ERK,p-ERK was decreased and E-cadherin was increased in sh RNA-TFF3-TPC-1 group compared with TPC-1 group and sh RNAC-TPC-1 group;There was no significant protein expression difference between TPC-1 group and sh RNAC-TPC-1 group.Our results show that TFF3 is associated with the invasion and metastasis of papillary thyroid carcinoma.Knockdown TFF3 can reduce the migration ability,invasion ability and clone formation ability of TPC-1 cell lines.TFF3 may modulate EMT process via MAPK signaling pathway in TPC-1 cells.