The Roles And Mechanism Of Notch3 In Renal Cancer Development

Objective:After bladder cancer,renal cancer is the second most common genitourinary tumor worldwide.About two-thirds of renal cancers are renal clear cell cancer(RCCC)which arise from the urinary tubule epithelial cells of renal parenchyma.Moreover,Biallelic gene Von Hippel Lindau(VHL)in60-80%of renal clear cell cancer is inactivate,which leads to the accumulation of hypoxia induced factor(HIF)in cells.Despite advances in surgical and chemoradiotherapy technique,most of the patients are in the middle and late stage at the time of initial diagnosis and the prognosis is poor.Therefore,optimizing individual therapy according to biological and pathlogical character of renal cancer and exploring potential new molecular targets would be of great importance.Notch3,a transmembrane protein receptor,binding with a Notch ligand results in three proteolytic cleavages of the receptor,which leads to the release of the Notch intracellular domain(NICD).Subsequently,NICD translocates into the nucleus,where it interacts with the DNA?binding factors[CBF1/RBP?J,Su(H),Rbpsuh and Lag?1,CSL]to regulate the expression of downstream genes.To date,the expression pattern of Notch3 and the relationship of Notch3 and HIF in human renal cancer has not been explored and its biological roles remain unclear.In the present study,the protein expressions of Notch3 and HIF were examined in renal cancer tissue specimens using immunohistochemistry and their correlation with clinicopathological factors were analyzed.The effects of NICD inhibition and knocked down Notch3 on cell proliferation and cell cycle of renal cancer cell lines were respectively futhur studyed in order to explore its mechanism in renal cancer.Methods:1.Patients and specimens:57 cases of RCCC(47 males and 10females)underwent surgical resection and their corresponding paracancer tissues were collected from Department of Urinary Surgery of General Hospital of Northern Theater Command between January 2012 and October2019,the median age was 58 years.The pathological diagnosis and tumor grade were evaluated for sections stained with hematoxylin and eosin.Tumors were classified according to the American Joint Committee on Cancer(AJCC)classification guidelines(2010).2.Immunohistochemistry:paraffin sections of tissue specimens were made and successively deparaffinized in xylene and hydrated in gradient ethanol.Then,the antigen of sections were retrieved in citrate buffer at 95?for 15min and hydrogen peroxide was applied to block endogenous peroxide activity.The non-specific binding were blocked by incubating with normal goat serum for.The sections were respectively incubated with Notch3 and HIF-1?rabbit polyclonal antibodies at 1:200 and 1:400 dilution at 4?overnight.Rabbit immunoglobulin was used as a negative control.After washing with PBS,the sections were incubated in biotinylated goat antirabbit or antimouse serum Ig G at room temperature for 15 min.Immunoreactivity was visualized with streptavidin–biotin conjugated with horseradish peroxidase,and 3,3?-diaminobenzidine tetrahydrochloride(DAB)under microscope.After that,secion was counterstained with hematoxylin-eosin,then gradient alcohol and xylene dehydrated and mounted.Ten views were randomly selected per slide,from which hundred cells were randomly selected by two independent blinded pathologists.The cells,immunostaining of which was higher than that of the background cell,were considered to be positive.The protein expressions of Notch3 and HIF-1?were semiquantitativly scored according to both the number and intensity of staining cell.The staining intensity was categorized as follows:0,negative;1,light yellow;2,light brown;3,brown.The percentage of positive tumor cells over hundred tumor cells in the denominator was scored as 0,0-5%;1,5-25%;2,26-50%;3,51-75%,and4,76-100%.The tumor samples with a final score?3 were regarded as low expression,tumor samples with a final score?3 were finally determined as high expression.3.Cell culture and RO4929097 treatment:786-O and ACHN cell lines were respectively cultured in RPMI1640 and MEM containing 10%fetal calf serum.The cells were respectively treated with 0?M,5?M,10?M,20?M RO4929097 for 48 h and Western blotting was used to check NICD3levels.4.Hypoxic culture:The cells were cultured under 2%O₂conditions or with 200?M cobalt chloride(Co Cl₂)treatment for 48h to mimic hypoxia effect.5.Transfection:The coding sequence of NICD3 was cloned into a p3x FLAG-CMV-14 vector.Plasmid DNAs were amplified in competent E.coli DH5a cells.Then the vector was transfected into cells of the 786-O and ACHN cells according to the manufacture of Lipofectamine3000.6.Establishment of stable Notch3 knock-down cells:Notch3 short hairpin(sh)RNA plasmids were transfected into 786-O cells using Polybrene.After 48h,2?/?l puromycin was added into the medium to kill the cells without Notch3sh RNA plasmid.The medium was replaced every 2-3 days with the fresh medium containing puromycin.After 2 weeks,the cell clones were picked up and amplified,and were identified by the western blot analysis.7.Cell Counting Kit 8(CCK 8)analysis:Cells were plated in 96-well plates.Culturing in normoxia or hypoxia for 48 h,CCK8 working solution was added into cells and the optical density of absorbance was measured by spectrophotometer at 490nm.8.5-Ethynyl-2'-deoxyuridine(Ed U)assay:Ed U incorporation assay was performed using Cell-Light?Ed U Apollo?488according to the following protocol.After culturing in normoxia and hypoxia for 48h,cells were cultured in the medium with 10?M Ed U for 2 h,and washed with cold PBS,resuspended in 500?l of 1×Apollo reaction buffer,and incubated at room temperature for 30 min.Then cells were washed with PBS containing 0.5%Triton X-100,stained with 1×Hoechst33342 reaction buffer for 5 min at room temperature.Cells were observed using invert immunofluorescent microscope at low magnification or checked by flow cytometer.9.6-Well colony formation assay:Cells were trypsinized and plated in 6-well dishes at 500 cells/well densities.Every 48 h,the media was replaced with fresh media,and the plates were incubated under normoxic and hypoxic conditions,respectively.10 days later,the cells were fixed and stained with 10%methylene blue in 70%ethanol and wells were observed and taken pictures under a light microscope at low magnification.The integrated optical density(IOD)of each well was analyzed by Image-Pro Plus6.0.10.Cell cycle analysis:After culturing in normoxia and hypoxia for 48h,cells were collected,fixed by incubating in 75%alcohol,and stained in PBS containing propidium iodide.The flow cytometry and Mod Fit software was used to analyze the percentage of cell cycle phase.11.Mitochondrial membrane potential analysis:After culturing in normoxia and hypoxia for48h,cells were collected and stained by JC-1 working solution.Then,the flow cytometry was used to analyze the percentage of JC-1 green cell in total cells.12.Western bloting:Total proteins from cells were extracted using RIPA lysis buffer and quantified using the Bradford method.Samples of 50?g of protein were separated by 10%SDS-PAGE gel and transferred to PVDF membrance.Then the membrances were respectively incubated overnight at4°C with antibody against Notch3,HIF-2?,cyclin D1,CDK4,PCNA,m TOR,pm TOR,p70S6K and pp70S6K,followed by incubation with secondary HRP-conjugated goat anti-rabbit or anti-mouse Ig G.The bounded proteins were visualized using enhance chemiluminescent(ECL)reagent.13.Statistical analysis:A?² test or Fisher exact test was used to examine possible correlations between Notch3 and HIF-1?expression and between their expression and clinicopathologic factors through SPSS version 11.5.Data are presented as the mean±standard deviation.One-way ANOVA was utilized for multiple comparisons followed by Tukey's post hoc test.P<0.05 was considered to indicate statistically significant differences.Results:1.The expressions of Notch3 and HIF-1?in renal cancer were significantly associated with the pathological grades and the clinical stages,but not with the gender,age and lymph node metastasis.(1)Notch3 protein was mainly localized in cell nuclei.The positive staining was found in 46 out of 57(80.70%)renal cancer tissues and 15 out of 57(26.31%)their corresponding paracancer tissues.The difference between the two samples was statistically significant.(?²=33.886,P=0.000).(2)HIF-1?protein was mainly localized in cell cytoplasm and membrance.The positive staining was found in 32 out of 57(56.14%)renal cancer tissues and 8 out of 57(14.04%)their corresponding paracancer tissues.The difference between the two samples was statistically significant.(?²=22.184,P=0.000).(3)The high expressions of Notch3 in well differentiated and medium differentiated carcinomas were 90.63%and 68.00%,respectively.The difference between the two pathological grades wasn't statistically significant.(?²=4.613,P=0.045).The high expressions of Notch3 in the clinical stage?,?,?and?were 61.90%,91.67%,87.50%and 100.00%,respectively.The difference between the four clinical stages was statistically significant.(?²=6.455,P=0.065).(4)The high expressions of HIF-1?in well differentiated and medium differentiated carcinomas were 68.75%and 40.00%,respectively.The difference between the two pathological grades was statistically significant.(?²=4.711,P=0.030).The high expressions of HIF-1?in the clinical stage?,?,?and?were 28.58%,70.83%,75.00%and 75.00%,respectively.The difference between the four clinical stages was statistically significant(?²=9.965,P=0.012).(5)The chi-square test results showed that there was a strong positive correlation between Notch3 and HIF-1?expressions in these cancer tissues(?=0.264,P=0.005).2.The roles and underlying mechanism of Notch3 in the proliferation of RCC cell lines in normoxia and hypoxia.(1)RO4929097 dose-dependently inhibited the expression of NICD3.(2)Hypoxia induced the proliferation of RCCC,while RO4929097 treatment decreased cc RCC cell proliferation in normoxia and hypoxia.CCK-8 assay showed that a significant increase in proliferation was induced by hypoxia in the control cells of 786-O(P<0.01),but not ACHN,and the proliferation of 786-O and ACHN cells was significantly inhibited by RO4929097 in normoxia and hypoxia(P<0.05).Subsequently,in Ed U incorporation assay,it was observed that the numbers of the Ed U-positive cells were significantly increased in response to hypoxia in both the 786-O and ACHN cells(P<0.01),whereas they were significantly reduced by RO4929097 treatment.Moreover,as compared with that in normoxia,the inhibitive effect of RO4929097 treatment in hypoxia was more obvious(P<0.05).The colony formation assay results demonstrated that,in normoxia,the sum of the IODs was significantly reduced with RO4929097 treatment in786-O,but not in ACHN cells.Hypoxic culture for 10 days induced a significant increase in the sum of the IODs in 786-O and ACHN cells,which was decreased by RO4929097 treatment(P<0.05).(3)RO4929097 regulated the cell cycle progression of RCC cells.Hypoxia decreased the percentage of G₁ phase cells compared with that in normoxia(P<0.05),and RO4929097treatment significantly increased the percentage of G₁ phase cells in both normoxia(P<0.05)and hypoxia(P<0.01).In addition,the increase range of the percentage of G₁ phase cells induced by RO4929097 in hypoxia was higher compared with that in normoxia.Consistently with the results of cell cycle analysis,hypoxia upregulated the expression of cyclin D1 and CDK4(P<0.01),while RO4929097 downregulated their expression(P<0.01).3.There was a link between Notch3 and HIF-2?.(1)The efficiency of transfecting the reconstructed plasmid containing NICD3 in 786-O and ACHN cells were 47.0%and 168.1%,respectively.(2)Hypoxia didn't affect the expressions of the full length of Notch3(Notch3FL),NICD3 with transmembrane domain(TD+NICD3)and NICD3.(3)Notch3 regulated the expression of HIF-2?induced by 2%O₂.In 786-O cells,it was 2%O₂,but not Co Cl₂,increased the expression of HIF-2?,and the expression of HIF-2?in ACNH cells was increased by Co Cl₂ and 2%O₂.Under conditions of 2%O₂ for 48 h,the expression of HIF-2?was significantly decreased by RO4929097,which was reversed by transfecting the plasmid containing NICD3.Moreover,transfecting the plasmid containing NICD3 also reversed the expressions of CDK4,cyclin D1 and PCNA(P<0.01).4.Establishment of stable Notch3 knock-down 786-O cells:(1)After transfecting the sh RNA plasmid targeting Notch3,the cell clones were selected by puromycin and identified by western blotting.Notch3 knock-down led to the decrease of NICD3 by 70%and Ed U incorporation assay showed that Notch3 knock-down inhibited the proliferation of 786-O cells.(2)Notch3 knock-down inhibited the expression of main moleculars of m TOR signaling and induced the cell apoptosis.(1)Hypoxia increased the expressions of m TOR,p70S6K and pp70S6K proteins(P<0.05),but not p m TOR.Under normoxic condition,Notch3 knock-down significantly reduced the expressions of m TOR,pm TOR,p70S6K and pp70S6K proteins(P<0.05),while under hypoxic condition,Notch3 knock-down significantly reduced the expressions of m TOR and p70S6K proteins(P<0.01).Futhurmore,the role of inhibition under hypoxic condition was greater than that under normoxic condition.(3)Notch3 knock-down induced the cell apoptosis of 786-O in both normoxia and hypoxia.Conclusion:1.Notch3 and HIF-1?overexpressed in renal cancer tissues and were signidicantly related to the pathological grade and the clinical stage.There was a positive correation between the expressions of Notch3 and HIF-1?.2.Decreasing production of NICD3 inhibited the cell proliferation of786-O and ACHN cells in normoxia and hypoxia through its regulation to cell cycle.3.Notch3 affected the expression of HIF-2?in786-O and ACHN cells.4.Establishment of stable Notch3 knock-down 786-O cells would provide a base for the further research.5.Notch3 knock-down induced the cell apoptosis via its regulation to m TOR signaling.