δ-catenin Regulates Proliferation And Apoptosis In Renal Cell Carcinoma Via Promoting β-catenin Nuclear Localization And Activating Its Downstream Target Genes

Objective:δ-catenin,which is encoded by CTNND2 gene,is a E-cadherin related protein and a member of the cateninδsubfamily.δ-catenin was initially identified to be concentrated in brain tissues.But like p120ctn,the another member of this family,δ-catenin contains 10 armadillo repeats units.Through these units,δ-catenin binds to the unique JMD(juxtamembrane domain)on E-cadherin to maintain it stability.Researches have heightened thatδ-catenin is implicated in some cancers.It is reported thatδ-catenin promotes the malignant phenotype of non-small-cell lung cancer by binding to the JMD of E-cadherin.It has also been indicated that the ectopic overexpression ofδ-catenin is associated with the progression of prostate cancer.One reportedδ-catenin causes the alteration of cell cycle and survival gene profiles.In addition,δ-catenin can induce E-cadherin processing and activateβ-catenin-mediated oncogenic signaling.However,the precise role ofδ-catenin in the development and progression of renal cancer is remains unclear.The relationship betweenδ-catenin expression and clinical pathological parameters is still poorly understood.All of these need to be explored.Renal cell carcinoma(RCC)accounts for 90%of all kidney cancers.Advances in early detection techniques have improved the survival rate of RCC.Nevertheless,local progression after treatment,distant metastasis and insensitivity to chemoradiotherapy are still main factors for poor prognosis.Therefore,focusing on studying the molecular mechanisms of RCC is helpful for the discovery of new effective anti-cancer drugs and novel diagnostic markers.Besides the HGF/Met,PI3K/Akt/m TOR and VHL/hypoxia cellular signaling pathways,Wnt/β-catenin pathway is also widely involved in the study in RCC.Wnt signaling and its targeted genes are known to actively participate in different biological processes during embryonic development and renal cancer.Studies have shown that targeting this pathway by inhibiting its intracellular signal transduction can reduce cancer cells viability and inhibit their growth.It has been reported that the ectopic expression ofδ-catenin in prostate cancer can promote tumor progression.Furthermore,δ-catenin can affect the subcellular distribution ofβ-catenin and regulate the expression of its downstream target genes.So,we speculate thatδ-catenin may be associated with RCC.In this study,we detected the expression ofδ-catenin in RCC specimens by Western blot analysis and immunohistochemistry staining,and then analyzed the correlation between its expression and clinicopathological factors.After investigated the relationship betweenδ-catenin and the proliferation and apoptosis of RCC cells,we explored the regulation ofδ-catenin onβ-catenin andβ-catenin-mediated oncogenic signals.In addition,we determined the effect ofδ-catenin on tumor growth in the xenograft mouse model.This understanding ofδ-catenin may help to establish a novel targeted therapy and discovery new diagnostic marker for RCC.Methods:1 Immunohistochemistry staining assay.The slides were heated,dewaxed in xylene,and rehydrated by graded ethanol.Sections were blocked with goat serum after antigen retrieval.The sections were then exposed to antibodies againstδ-catenin or Ki67 overnight at 4℃.After washed with PBS,slides were incubated with HRP-labeled goat anti-rabbit antibody for 1 hour.Before added with DAB,the slides were washed and then counterstained with hematoxylin.Finally,after dehydrated by graded ethanol,the sections were sealed by neutral balsam,and visualized at400×magnification with Olympus microscope.2 Western blot analysis.Cells were collected and lysed with RIPA lysis buffe.The protein concentrations were assessed by Enhanced BCA Protein Assay Kit.After separated by electrophoresis on SDS-PAGE,proteins were transferred onto PVDF membranes,and then incubated with primary antibodies at 4℃ overnight.After incubated with goat anti-rabbit or goat anti-mouse IgG at 37℃ the protein bands were visualized by enhanced chemiluminescence(ECL).The optical density value of the target strip was analyzed by Gel-Pro-Analyzer software.3 Cell lines and cell culture.Normal human renal epithelial cells HK-2 and human renal cancer cell lines A498,ACHN and 786-O were obtained from Procell Life Science&Technology in Wuhan,China.Hk-2、A498 and ACHN were cultured in MEM medium supplemented with 10%fetal bovine serum in a humidified incubator with 5%CO2 at 37℃.786-O cells was cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum under the same conditions.4 Nuclear and cytoplasm protein extraction.The cells were lysed in cytoplasmic lysis buffer A supplemented with PMSF on ice,then added with cytoplasmic lysis buffer B.After briefly vortexed,the mixture was centrifuged at 12 000×g for 5 minutes at 4℃.The supernatant was cytoplasmic extract.The pellet was resuspended with nuclei lysis buffer and deposited in ice with several short vortexes during this period.After centrifugated at 12 000×g for 10 minutes at 4℃,the supernatant was the nuclear extract.The protein concentration was evaluated via Enhanced BCA Protein Assay Kit.5 Plasmid construction and siRNA transfection.Theδ-catenin sequence was found in NCBI,and annealing primers were designed.Extraction of plasmid(pRNAH1.1vector)using plasmid mass preparation kit.The annealing primer was inserted in Prnah1.1 vector.A498 and ACHN cells were transfected byδ-catenin siRNA or NC siRNA encapsulated with Lipofectamine 2000 following the protocol.ACHN cells were transfected byδ-catenin sh RNA plasmids for silencingδ-catenin,and NC sh RNA plasmids was used as the negative control.6 RNA extraction and quantitative realtime PCR(qRT-PCR).According to the protocol,total RNA rapid extraction kit was used to isolate RNA from RCC cells.The RNA was reverse transcribed into c DNA by M-MLV.The c DNA was used to perform real-time PCR with Taq HS Perfect Mix and SYBR Green kit.The data were determined by Exicycler~TM96.The data were analyzed by fluorescence quantitative analyzer.7 CCK-8 assay.Cell viability was detected by Cell counting kit-8(CCK-8)assay.RCC cells were seeded into 96-well plates with 4×10~3 per well beforehand.After being adhered,the cells were transfected with NC siRNA andδ-catenin siRNA;and CCK-8 reagent was added into the 96-well plates with 10μl per well and incubated for 1 hour.The optical density(OD)value at 450 nm was measured with a microplate analyzer at 0,24,48,72 and 96 hours,respectively.8 Immunofluorescence.The slides of cells were fixed with 4%paraformaldehyde and permeabilized with 0.1%triton-100.After blocked by goat serum,primary antibody(Ki67 andβ-catenin)was added to the cells and incubated overnight at 4℃.After PBS washing,Cy3-labeled goat anti-rabbit IgG diluted in PBS was incubated in cells at room temperature.The nuclei were counterstained by DAPI and the samples were sealed.Finally,observed the samples at 400×magnification using the OLUMPUS fluorescence microscope.9 Flow cytometry.RCC cells were treated by Cell Cycle Analysis kit and Annexin V-FITC Apoptosis Detection Kit following the manufacturer’s protocol.The cell cycle and the apoptosis of the cells were detected by Novo Cyte flow cytometer.10 TOP/FOP luciferase reporter assay.δcatenin siRNA was transfected in ACHN cells.After incubation,the cells were transfected with firefly luciferase reporter plasmid TOPflash/FOPflash and vector encoding Renilla luciferase.The firefly and Renilla luciferase activities were determined using the dual luciferase reporter assay according to the manufacturer’s protocol.11 Animal studies.This animal study was approved by the Institutional Animal Ethics Committee of Shengjing Hospital of China Medical University.And this study was carried based on the Guideline for the Care and Use of Laboratory Animals.6-week-old male BALB/c nude mice were randomly divided into two groups with 6 mice in each group.ACHN cells transfected withδ-catenin sh RNA plasmids or negative NC sh RNA plasmids were injected subcutaneously into the right flank of the nude mice.The tumor size was measured every four days,and the tumor volume was calculated as follow:V=(width~2×length)/2.27 days after injection,the mice were sacrificed.Tumors were excised and the weight of tumor was recorded.Results:1.δ-catenin was highly expressed in human renal cancer tissues and correlated with p TNM,tumor stage,and lymph node metastasis.2.δ-catenin was highly expressed in human renal cancer cells.3.δ-catenin promoted renal cancer cell proliferation.4.δ-catenin inhibited apoptosis of renal cancer cells.5.δ-catenin affectedβ-catenin nuclear localization and its downstream gene expression in renal cancer cells.6.δ-catenin promoted tumor growth in ACHN xenograft mouse model.Conclusion:1.δ-catenin was highly expressed in human renal cancer tissues and correlated with poor prognosis of RCC patients.2.δ-catenin was highly expressed in human renal cancer cells and promoted renal cancer cell proliferation and inhibited apoptosis of renal cancer cells.3.In vitro and in vivo assays,δ-catenin affectedβ-catenin nuclear localization and its downstream gene expression in renal cancer cells.