| Objective:To clarify the effects of FOXP 4 by regulating FBXW 7 on the proliferation,migration and epithelial mesenchymal transformation of papillary thyroid carcinoma(PTC)cells,and further verify the tumor of nude mice in vivo,and explore the potential molecular mechanism of interaction.Methods:1.The expression level of FOXP 4 in papillary thyroid carcinoma,medullary thyroid carcinoma and undifferentiated thyroid carcinoma tissues.The expression level of FOXP 4in PTC tissues and PTC carcinoma(PTC)was also tested,and the expression of FOXP 4in PTC and clinical factors were analyzed.2.Western Blot tested the expression of FOXP 4protein in four thyroid cells(including normal thyroid follicular epithelial cells Nothy-ori3-1 and three thyroid cancer cells B-CPAP,TPC-1,and KTC-1).3 In the in vitro experiments,To investigate the mechanism of the regulatory effect of FOXP 4 on PTC cells,We first performed a screening of stable transfer cell lines,Selected the sh RNA-FOXP 4-6 # with the lowest knockdown efficiency,TPC-1,KTC-1 cells were then transfected with sh RNA-FOXP 4 and sh RNA-NC to establish PTC cell lines with low expression of FOXP 4,TPC-1,KTC-1 cells were also transfected with pc PC-NC and pc DNA-FOXP 4 to establish PTC cell lines overexpressing FOXP 4,After potency testing using WB and RT-q PCR,Cell proliferation,migration were measured by by by vitro formation assay,Flow cytometry experiments to verify the cell cycle changes after cell transfection,Western blot Changes of FOXP 4 and EMT-related markers(E-cadherin,N-cadherin and Vimentin).4 To detect the interaction of FOXP 4 with downstream molecules,we sequenced sh RNA-FOXP 4 and sh RNA-NC by Seq-RNA technology,screened the FOXP 4-downstream binding molecule FBXW 7 for verification,and verified FBXW 7 expression after knockdown of FOXP 4 by WB and RT-q PCR,and verified by response assay.We verified the direct binding of FOXP 4 to FBXW 7 by a Chip assay.5 To reveal the localization of FOXP 4,we isolated the nucleus and cytoplasmic RNA of TPC-1and KTC-1 cells and examined the distribution of FOXP 4 in the nucleus and cytoplasm by RT-q PCR.6 To verify the effect of FOXP 4 on the regulation of FBXW 7 expression,we examined the effect of FOXP 4 silencing or overexpression on FBXW 7 expression levels in TPC-1,KTC-1 cells using RT-q PCR and Western blot-based,respectively.7 To investigate the FOXP 4 promotion through the regulation of FBXW 7.Results:1.FOXP 4 expression was significantly upregulated in different types of thyroid cancer tissues(P <0.05),and FOXP 4 levels were negatively correlated with FBXW 7levels in PTC cells.2.The expression levels of FOXP 4 in the three PTC cells(B-CPAP,TPC-1,KTC-1)were significantly higher than that of normal thyroid follicular epithelial cells Nothy-ori3-1(P <0.05).3 The expression level of FOXP 4 was significantly correlated with apoglandal tumor invasion and lymph node metastasis in PTC patients.There was no significant association with patient sex,clinical stage,age,and BRAF gene mutations.4 In vitro,the potency assays showed that we successfully established the FOXP4 overexpression and silencing PTC cell lines.5 FOXP 4 was significantly increased in PTC cells(P <0.001),FBXW 7(P <0.01),EMT marker E-cadherin(P <0.01),and N-cadherin and Vimentin(P <0.001).In addition,the level of E-cadherin expression was significantly negatively associated with FOXP 4 expression in PTC(P <0.01),while the level of N-cadherin and Vimentin were significantly positively associated with FOXP 4expression(P <0.001).6.In cell cloning assay,CCK-8 and Transwell chamber experiments,cell proliferation and migration ability after FOXP 4 silencing decreased significantly(P <0.01),while FOXP 4 overexpression significantly promoted cell proliferation and migration ability(P <0.001).7 In the study to Western blot the progression of cellular EMT by FOXP 4 overexpression or silencing,the expression levels of N-cadherin and Vimentin were significantly decreased in the sh RNA-FOXP 4 group(P<0.05)group compared with the sh RNA-NC group.Compared with the pc DNA-NC group,N-cadherin and Vimentin expression were significantly higher in the pc DNA-FOXP 4group(P <0.01),and E-cadherin expression was significantly decreased in the pc DNA-FOXP 4 group(P <0.05).9 The results of Ch IP experiments indicate that FOXP 4can directly bind to FBXW 7.10 Cytoplasmic / nuclear isolation experiments indicate that FOXP 4 is mainly expressed in the nucleus,accounting for about 2 / 3 of the total.11 In studying the effect of FOXP 4 gain-of-function and loss on FBXW 7 expression,FBXW 7expression was increased in PTC cells after silenced FOXP 4(P <0.05);while FOXP 4overexpression of FOXP 4 significantly reduced FBXW 7 expression(P <0.05).12 In experiments investigating the effect of FOXP 4 on FBXW 7 on cell clone formation,clone formation capacity of cells co-transfected with sh-FBXW 7 was restored compared with cells cotransfected with sh RNA-FOXP 4 and sh RNA-NC,respectively(P <0.01).13 In experiments investigating the effect of FOXP 4 by regulating the migration of FBXW 7,the cells cotransfected with sh RNA-FOXP 4 co-transfected respectively were significantly increased compared with cells cotransfected with sh RNA-FOXP 4(P <0.001).14 In western blot experiments in which FOXP 4 regulated EMT progression by FBXW 7 on PTC cells,si-FBXW 7 effectively reversed the increase of E-cadherin expression by sh RNA-FOXP 4 knockdown(P <0.001),and si-FBXW 7 effectively reversed the decrease of N-cadherin and Vimentin expression by sh RNA-FOXP 4 knockdown(P<0.001).15 In the experiment of FOXP 4 in nude mice,the tumor volume and tumor weight were significantly reduced after FOXP 4 knockdown compared with the control group(P <0.01).16 The expression level of E-cadherin was significantly increased in FOXP 4 knockdown PTC nude mice(P <0.001),which is consistent with our in vitro cell results.17 FOXP 4 knockdown significantly reduced Ki-67 protein expression in tumor tissues(P <0.05).18 decreased knocklevels of N-cadherin and Vimentin in tumor tissues(P <0.001).Conclusion: FOXP 4 promotes the proliferation,migration,mesenchymal transition of epithelium and growth of PTC by regulating FBXW 7.FOXP 4 may be used as an effective evaluation index for the invasive efficacy of thyroid cancer,as well as as a promising research target for diagnosis and treatment. |
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