Effect Of YKL-40 Gene Silencing On Angiogenesis And Anti-apoptotic Of Endometrial Cancer

Endometrial cancer(EC)is the third most common malignant tumor of female in the world,the most common gynecological malignant tumor in developed countries and the second most common gynecological malignant tumor in developing countries.In the United States,there are more than 60,000 new diagnoses and more than 10,000 deaths a year.The incidence of EC is increasing year by year,and tends to be younger,threatens the females life and influences patients quality of life seriously.Although the survival rate of patients with EC can be improved by surgery in the early stage of cancer,it is difficult to achieve a satisfactory therapeutic effect for patients with metastasis or recurrence.Therefore,the search for effective therapeutic targets is of great significance for the clinical treatment of EC.A large number of studies have confirmed that Chitinase-3-like Protein-1(YKL-40)is highly expressed in various malignant tumor tissues and serum,and is closely related to the survival time and prognosis of patients,which has potential value in early diagnosis,treatment and prognosis of tumors.In order to explore the key target of EC,previous studies from our research team showed that YKL-40 was highly expressed in the tissues and serum of patients with EC,and serum YKL-40 had the clinical value of predicting the prognosis of EC.In addition,YKL-40 was highly expressed in a variety of EC cell lines(HEC-1A ? HEC-1B ? ishikawa ? RL-952),among which the relative expression of YKL-40 was the highest in HEC-1A cell line,so HEC-1A cell line was selected as the cell model for our subsequent study.We had showed that small interfering RNA(si RNA)silencing YKL-40 gene can inhibit the proliferation,migration,invasion and anti-apoptosis of HEC-1A cells.Based on the previous study,the present study investigates the mechanism of si RNA silencing YKL-40 gene on angiogenesis and anti-apoptosis in EC both in vitro and in vivo,and aim to provide a theoretical foundation for the clinical treatment targeting YKL-40.PART ? EFFECTS OF YKL-40 GENE SILENCING ON VEGF/VEGFR-2,ERK1/2 AND PI3K/AKT SIGNALING PATHWAYS IN HEC-1A CELLSObjective Based on the previous study,we established the HEC-1A cell line of human endometrial cancer with YKL-40 gene silencing by si RNA,and investigated the expression of YKL-40 gene silencing on VEGF/VEGFR-2,ERK1/2 and PI3K/AKT signaling pathways-related proteins in HEC-1A cells.Methods 1.The lentivirus vector si RNA-YKL-40 and the negative control empty vector si RNA-NC were transfected into HEC-1A cells,respectively.2.HEC-1A cells were divided into three groups: experimental group(si YKL-40)was transfected with si RNA-YKL-40,negative control group(si NC)was transfected with si RNA-NC,and blank control group(Ctrl)was untransfected.3.Quantitative Real-time PCR(q PCR)and western blotting were used to verify the gene-silencing effect of YKL-40 in HEC-1A cells.4.Tube Formation Assay of human umbilical vein endothelial cells(HUVECs)was used to detect the effect of YKL-40 gene silencing on angiogenic ability in vitro.5.Western blotting was performed to detect the expression levels of VEGF,VEGFR-2,p VEGFR-2(Tyr1175),ERK1/2 and PERK1/2(Thr202/Tyr204)protein.6.HEC-1A cells were treated with different concentrations of cisplatin(DDP)(0,3.125,6.25,12.5,25,50 or 100 mmol/L)for 48 h,the cell viability of HEC-1A cells was measured by methyl thiazolyl tetrazolium(MTT)assays.And then calculated the 50% inhibitory concentraton(IC50)of cisplatin acting on HEC-1A cells.Each group of HEC-1A cells was treated with IC50 of cisplatin and were grouped as follows: Ctrl: HEC-1A cells not treated with cisplatin;DDP+Ctrl: Ctrl+IC50 cisplatin;DDP+si NC: si NC + IC50 cisplatin;and DDP+ si YKL-40: si YKL-40 + IC50 cisplatin.Western blotting was used to detect the expression of PI3K?p PI3K(Tyr458)?AKT and p AKT(Ser473)protein.Results 1.After transfection for 96 h,the transfected HEC-1A cells expressed green fluorescent protein(GFP),and the transfection efficiency exceeded 85%.After puromycin selection,GFP was stably expressed in HEC-1A cells,and the transfection efficiency did not change significantly after subculture.2.Detection of YKL-40 silencing gene after transfection: the q PCR results indicated that the expression of YKL-40 m RNA in si YKL-40 was significantly decreased,the relative level(0.26±0.06)was lower than that of si NC(1.01±0.20)and Ctrl(1.12±0.44)(P<0.05).Western blotting analysis showed that the relative expression of YKL-40 in si YKL-40(1.52±0.16)was downregulated compared with the si NC and Ctrl(6.21±0.10,5.65±0.19,respectively,P<0.05).3.The tube numbers of si YKL-40 was 6.67±3.51,which was significantly less than that of si NC and Ctrl(33.67±10.96,33±4,respectively),the difference was statistically significant(F=14.96,P= 0.005).4.Western blotting showed that the expression level of VEGF,p VEGFR2(Tyr1175)and p ERK1/2(Thr202/Tyr204)were notably reduced in si YKL-40(P<0.05).5.Treatment of HEC-1A cells with cisplatin resulted in the decline of cell viability in a dose-dependent manner,the cell viability of HEC-1A cells demonstrated a significant decrease as cisplatin concentrations increased,and the activity of cells in si YKL-40 decreased more significantly compared with the si NC and Ctrl(P<0.05).The IC50 of cisplatin is 30mmol/L.We used the cisplatin IC50(30 ?mol/L)to treat HEC-1A cells for 48 hours,western blotting showed that the expression of AKT,p AKT(Ser473)and p PI3K(Tyr458)in DDP+si YKL-40 were lower compared to that in Ctrl ? DDP+Ctrl and DDP+si NC(P<0.05).Conclusion 1.Successful establishment of HEC-1A cell line stably silencing YKL-40 gene.2.Silencing of YKL-40 gene by si RNA significantly could inhibit angiogenic ability in vitro.3.Silencing of YKL-40 gene could reduce the expression of VEGF,p VEGFR2 and p ERK1/2.4.Silencing of YKL-40 gene could down-regulated the protein expression of p PI3K(Tyr458)?AKT and p AKT(Ser473)to enhance the sensitivity of cisplatin-induced apoptosis of HEC-1A cells.PART ? EFFECT AND MECHANISM OF YKL-40 GENE SILENCING ON THE GROWTH OF ENDOMETRIAL CANCER XENOGRAFT IN NUDE MICEObjective To investigate the effect and mechanism of silencing of YKL-40 gene by si RNA on the growth of human endometrial cancer xenografts in nude mice.Methods 1.21 female BALB/c nude mice were randomly divided into three groups(n=7 per group): si YKL-40,si NC and Ctrl.The three groups of cells(1×107/0.2ml/mouse)were inoculated subcutaneously to the nude mice.After tumor formation,the long diameter(L)and short diameter(S)of the subcutaneous tumor in nude mice were measured every 3 days,and the tumor growth curve was drawn.In the 5th week,the nude mice were sacrificed by cervical dislocation,and then the volume and weight of isolated tumor tissues were measured.2.Hematoxylin-eosin staining(HE)was used to observe the histopatholog-ical changes of tumor tissues in nude mice.3.Immunohistochemistry and western blotting were performed to detect the expression of YKL-40 protein of tumor tissues in nude mice.4.Immunohistochemistry and western blotting were used to examine the expression of VEGF?EGFR protein of tumor tissues in nude mice.Results 1.The tumor formation time of si YKL-40 was(9.29±1.50)d,which was significantly longer than Ctrl and si NC [(4.29±0.76)?(4.14±0.90)d,respectively].The volume and weight of si YKL-40 transplanted tumor tissues were significantly lower than those of Ctrl and si NC,and the difference was statistically significant(P<0.01).Compared with Ctrl and si NC,the tumor inhibition rates of si YKL-40 were 78.67% and 79.87%,respectively.2.HE staining showed that the tumor cells were found to have a large amount of necrosis and some nuclear pyknosis in si YKL-40.However,in si NC and Ctrl,the tumor cells had cytologic atypia and nuclear fission,the nuclei were deeply stained,and the nuclear pyknosis and necrosis were rare.3.Image Pro Plus6.0 software(IPP)was used to analyzed the pictures of immunohistochemistry,results showed that the expression of YKL-40 was reduced in si YKL-40 compared with si NC and Ctrl(P<0.01).Western blotting revealed that the protein expression of YKL-40 was significantly decreased in si YKL-40 compared with the other two groups,(P<0.01).4.IPP analyzed the pictures of immunohistochemistry,which showed that VEGF and EGFR were weakly expressed in si YKL-40 but positively expressed in si NC and Ctrl.The expression levels of VEGF and EGFR in si YKL-40 were significantly decreased compared with si NC and Ctrl(P<0.01).Western blotting analysis demonstrated that the protein expression of VEGF and EGFR in si YKL-40 were downregulated compared with si NC and Ctrl(P<0.01).Conclusion 1.Silencing of YKL-40 gene by si RNA could inhibit the growth of human endometrial cancer xenografts in nude mice.2.Silencing of YKL-40 gene by si RNA may inhibit the growth of xenografts in nude mice by down-regulating the expression of VEGF and EGFR proteins.