The Role And Mechanism Of HOXD10 In Invasion And Metastasis Of Clear Cell Renal Cell Carcinoma

Objective: Clear Cell Renal Cell Carcinoma(cc Rcc)is one of the most common malignant tumors of urinary system.In recent years,its morbidity and mortality have been rising,and morbidity crowd displays a younger trend in average age.Patients with early stages renal cell cancer can be treated with surgery,and the cure rate is high.In patients with advanced renal cancer,the5-year survival rate is less than 20% due to the presence of distant metastasis.Metastasis and recurrence are the main causes of death in patients with advanced renal cell carcinoma.Therefore,it is of great significance for the treatment of renal cell carcinoma to actively explore the molecular mechanism of invasion and migration of clear cell renal cell carcinoma and to find new therapeutic targets.HOXD10 is a member of the HOX gene family,and its expression product is an important transcription factor that plays an important role in the regulation of cell proliferation,apoptosis,differentiation,angiogenesis,invasion,and metastasis of tumor cells.It is known that the genes regulated by HOXD10 include adhesion molecules,growth factors,and extra-cellular proteins.Currently,several studies have shown that HOXD10 is closely related to the development of various tumors such as breast cancer and colon cancer.However,the function and mechanism of HOXD10 in renal cell carcinoma are still unclear.Epithelial-mesenchymal transition(EMT)is closely related to the invasion and metastasis of cancer,in the process of EMT,the molecular phenotypes necessary for migration and invasion are significantly altered:up-regulation of mesenchymal markers and the loss of epithelial markers et al.The E-cadherin as the main regulatory factor of epithelial phenotype,it is essential to maintain adhesion between epithelial cells and to maintain homeostasis,deletion of E-cadherin expression is relatively common in cancers developing from the epithelial cell.This study aims to detect the expression level of HOXD10 in clear cell renal cell carcinoma tissues and carcinoma cell line,and observe the ability of cell proliferation,colony formation,invasion and migration by knocking down and over-expressing HOXD10 in renal carcinoma cell lines.To further study the molecular mechanism of HOXD10 in regulating the invasion and migration of renal carcinoma cells and the changes in phenotypes of EMT-related molecules,and to provide an experimental basis for the selection of new therapeutic targets for clear cell renal cell carcinoma.Besides,the efficacy and safety of everolimus as a second-line treatment after the first-line treatment were evaluated by following up on 21 patients with metastatic renal cell carcinoma.Methods:1.Detection of HOXD10 expression levels in renal clear cell carcinoma tissues and cell lines.The expression levels of HOXD10 in 72 cases of renal carcinoma tissues and cell lines 786-O and ACHN were detected by quantitative real-time PCR,Western-blot,and immunohistochemistry,the correlation between the level of HOXD10 and clinicopathological parameters of patients was analyzed.2.Functional analysis of HOXD10 on the malignant biological behavior of renal carcinoma in vitro.The over-expression and knockdown efficiency of HOXD10 in ACHN and 786-O cell lines were detected by quantitative real-time PCR and Western-blot;the effect of over-expression and knockdown of HOXD10 on the invasion and migration ability of ACHN and 786-O cell lines was detected by Transwell and wound-healing assays.MTS was used to observe the proliferation ability of HOXD10 on ACHN and 786-O cell lines.The effect of HOXD10 on the colony formation ability of ACHN and 786-O cells was observed by plate cloning assay.3.The molecular mechanism of HOXD10 on invasion and migration of renal carcinoma cells and changes of EMT molecular phenotype.Promo bioinformatics software predicted E-cadherin may be a target gene of HOXD10;the expression levels of E-cadherin in 72 cases of renal carcinoma tissues by quantitative real-time PCR,the correlation between the level of E-cadherin and clinicopathological parameters of patients were analyzed.Chromatin immunoprecipitation(Ch IP)and dual luciferase reporter assay confirmed that HOXD10 binds to the 3'UTR region of E-cadherin.The effects of over-expression and knockdown of HOXD10 on the expression of E-cadherin and EMT-related molecules vimentin and ?-catenin were further verified by quantitative real-time PCR and Western-blot.4.Safety and efficacy of everolimus in second-line treatment for advanced metastatic renal cell carcinoma21 patients with metastatic renal cell carcinoma were observed and followed up,and the efficacy and adverse events of everolimus as second-line treatment after the progress of first-line targeted therapy was analyzed.Results:1.The expression of HOXD10 in 72 cases of clear cell renal cell carcinoma tissues was significantly lower than that in normal renal tissues(P<0.01),In addition,the expression of HOXD10 was closely related to the clinical stage of renal cell carcinoma(P<0.05),but not related to histological grade or lymph node metastasis(P>0.05);Immunohistochemical results showed that HOXD10 was weak positive in clear cell renal cell carcinoma tissues,by contrast,strongly expressed in normal renal tissues.The positive particles were brown-yellow and mainly located in the nucleus.Quantitative real-time PCR and Western-blot results showed that the expression level of HOXD10 in 786-O and ACHN cell lines was significantly lower than that in human embryonic kidney cell 293T(P <0.01);2.The results of Transwell and wound-healing assays showed that over-expression of HOXD10 could significantly inhibit the invasion and migration of ACHN cell lines(P <0.01),by contrast,knock down of HOXD10 could improve the invasion and migration ability of ACHN cells(P <0.01);MTS results showed that over-expression of HOXD10 could inhibit the proliferation of renal carcinoma cell lines(P<0.05);Colony formation assays demonstrated that over-expression of HOXD10 could significantly reduce the colony formation ability of renal carcinoma cells(P<0.01).3.The PROMO databases jointly screened E-cadherin as a downstream target gene of HOXD10,Ch IP and Dual-luciferase reporter assay further verified that HOXD10 binds to the 3'UTR region of E-cadherin;The expression of E-cadherin in 72 cases of renal clear cell carcinoma was detected by quantitative real-time PCR,compared with normal renal tissues,E-cadherin expression in cancer tissues was significantly lower(P <0.01),In addition,the expression of E-cadherin was closely related to the clinical stage of renal cell carcinoma(P <0.05),further analysis showed that HOXD10 and E-cadherin were positively correlated in the same batch of cancer tissues(Spearman Rs=0.454,P <0.01);Quantitative real-time PCR and Western-blot further verified that over-expression of HOXD10 could promote the expression of E-cadherin and reduce the expression of Vimentin,?-catenin,and other EMT-related molecules.However,knocking down HOXD10 expression could produce the opposite result.These results indicated that HOXD10 inhibited Epithelial-mesenchymal transition in renal carcinoma cells.4.The median PFS was 6.3 months,which was slightly higher than previous reports(median PFS 5.1 or 5.4 months).There were 3 patients who achieved PR,12 achieved SD,and 6 experienced PD,and the disease control rate(DCR)was 15/21(71.4%);the incidence of AEs(any grade)was 90%(19/21)with everolimus.Conclusions:1.HOXD10 was down-regulated in CCRCC tissues and renal carcinoma cell lines.2.HOXD10 inhibited proliferation,colony formation,invasion and migration ability of renal carcinoma cells.3.HOXD10 inhibited the invasion and migration ability of renal carcinoma cells by targeting E-cadherin to affect epithelial-mesenchymal transition(EMT).4.Everolimus was still a safe and effective second-line treatment for metastatic renal cell carcinoma when first-line treatment failed.