| BackgroundOvarian cancer is one of the three major gynecological malignancies,of which 85-90%are epithelial ovarian cancer(EOC).As the ovary is located deep in the pelvic cavity,the early symptoms are not obvious,and most of them are in the late stage when they are diagnosed.Surgery combined with chemotherapy is the main treatment,but more than 70%of patients with advanced ovarian cancer still die of tumor recurrence,metastasis and chemotherapy resistance.The 5-year survival rate is much lower than other cancers,which is 20-30%.It seriously threatens women’s lives and health.Therefore,exploring the mechanism of the occurrence and development of ovarian cancer is of great significance to improve the prognosis of patients with ovarian cancer.Exonucleoside triphosphate diphosphate hydrolase 5(ENTPD5)mediates the catabolism of glycolic acid in the cell core and the N-glycosylation of proteins in the endoplasmic reticulum.Many studies have shown that ENTPD5 plays an important role in a variety of malignant tumors.The expression of ENTPD5 can promote the proliferation of pancreatic cancer cells,while inhibiting the expression of ENTPD5 can reduce the ability of breast cancer cells to invade and metastasize to the lung.In prostate cancer,the higher the expression of ENTPD5,the higher the degree of prostate cancer.Overexpression of ENTPD5 can regulate Bcl-2 homeostasis through protein kinase Ca,and increase the resistance of prostate cancer to cisplatin.ENTPD5 plays a key role in cancer proliferation,migration and drug resistance.It may become a target molecule for early diagnosis or monitoring curative effect of cancer.Is ENTPD5 involved in the occurrence,development and drug resistance of ovarian cancer?In the previous study,we found that the expression of ENTPD5 was negatively correlated with the survival rate of patients with ovarian cancer,and the preliminary experimental study showed that the expression of ENTPD5 in EOC was significantly higher than that in adjacent tissues.Based on the previous study,this study will further explore the correlation between ENTPD5 and EOC proliferation,migration and drug resistance and its preliminary molecular mechanism,which has important clinical significance for whether ENTPD5 and its target molecules can become a new target for the diagnosis and treatment of platinum secondary drug-resistant ovarian cancer.ObjectiveTo study the role of ENTPD5 in the proliferation,migration and cisplatin resistance of epithelial ovarian cancer,and explore the potential mechanism of ENTPD5 regulating the function of epithelial ovarian cancer cells.Method1.82 cases of EOC tissue samples and 6 cases of normal FT tissue samples were collected from January 2018 to June 2021.RT-qPCR,WB and immunohistochemical were performed to detect the expression level of ENTPD5 in EOC tissue and normal FT tissue.The correlation between ENTPD5 and clinicopathological characteristics was statistically analyzed by the chi-square test.2.ENTPD5 was knockdown or overexpressed in EOC cells OVCAR8 and SKOV3.Plate cloning experiment,3D cell culture,scratch healing experiment and flow cytometry were used to study the effects of ENTPD5 on proliferation,migration,cycle and apoptosis of EOC cells.RT-qPCR and WB were used to detect the effects of ENTPD5 knockdown on the expression of GRP78、p-eIF-2α and CHOP.3.Immunohistochemistry was performed to analyze the expression level of ENTPD5 in platinum sensitive EOC tissues and platinum resistant EOC tissues.The Cisplatin concentration gradient method was used to establish cisplatin resistant ovarian cancer cells SKOV3/DDP.RT-qPCR and WB were performed to detect the effect of cisplatin on endoplasmic reticulum stress of SKOV3 and SKOV3/DDP cells,the expression level of ENTPD5 and the effect of cisplatin on ENTPD5 expression.CCK8 and flow cytometry were used to detect the effect of ENTPD5 knockdown on cisplatin resistance of ovarian cancer SKOV3/DDP cells.The expression of GRP78 was detected by WB after ENTPD5 knockdown.ResultLENTPD5 is highly expressed in epithelial ovarian cancer tissues1.1 The mRNA expression level of ENTPD5 in epithelial ovarian cancer tissue(2.18±1.34)was significantly higher than that in normal fallopian tube tissue(0.50±0.230)(P<0.05).The protein expression level of ENTPD5 in epithelial ovarian cancer tissue(1.37±0.53)was significantly higher than that in normal fallopian tube tissue(0.532±0.24)(P<0.05).The immunohistochemical score in epithelial ovarian cancer tissue(7.21±3.89)was significantly higher than that in normal fallopian tube tissue(0.50±0.55)(P<0.001).1.2 High expression of ENTPD5 was correlated with FIGO stage and metastasis in patients with epithelial ovarian cancer(P<0.05).2.ENTPD5 is involved in the regulation of proliferation and migration of epithelial ovarian cancer cells.2.1 Compared with the control group,,the clone formation rate of SKOV3 and OVCAR8 in epithelial ovarian cancer cells was significantly decreased(OVCAR8:46.32±1.74%VS37.45±3.24%,SKOV3:40.72±2.12%VS28.42±1.04%,P<0.01),the scratch healing ability was significantly inhibited(OVCAR8:32.34±3.54%VS46.43±4.42%,SKOV3:45.41±3.76%VS62.63±5.87%,P<0.05),and the proportion of apoptosis was significantly increased(OVCAR8:8.67±0.30VS18.64±0.31,SKOV3:3.57±0.34VS6.67±0.35,P<0.01),cell cycle arrest in G0/G1 phase(OVCAR8:35.20±1.20VS42.70±2.53,SKOV3:41.26±3.07 vs 59.28±3.54,P<0.05)after ENTPD5 knockdown.ENTPD5 Overexpression promoted the proliferation and migration of ovarian cancer cells in vitro(P<0.05).ENTPD5 knockdown significantly inhibited the growth of EOC cells in 3D cell model(24.9±3.18 vs 16.9±2.82,P<0.01).2.2 Compared with the control group,the mRNA and protein expression levels of GRP78,p-eiF-2α and CHOP in OVCAR8 and SKOV3 cells were decreased significantly after ENTPD5 knockdown(P<0.01).3.ENTPD5 is involved in cisplatin regulation in epithelial ovarian cancer3.1 The immunohistochemical score of ENTPD5 In platinum treated resistant ovarian cancer tissues(8.2±2.39)was significantly higher than that in platinum treated sensitive ovarian cancer tissues(4.5±2.18)(P<0.01),.3.2 Ovarian cancer cisplatin resistant cell line SKOV3/DDP was successfully constructed,and its resistance to cisplatin(IC50:4.69±0.20)μg/ml)was significantly higher than SKOV3(IC50:0.82±0.30)μg/mL)(P<0.01).3.3 Cisplatin induced a significant increase in the mRNA expression levels of endoplasmic reticulum stress genes GRP78,CHOP and PERK in SKOV3 cells(P<0.01).the expression of GRP78 protein and apoptotic protein PARP-1 increased with the increase time of cisplatin treatment,but there was no significant change in drug-resistant cells.The expression level of ENTPD5 in cisplatin resistant cells SKOV3/DDP(8.02±1.87)was significantly higher than that in cisplatin sensitive cells SKOV3(1.00±0.00)(P<0.001).The protein expression level of ENTPD5 in cisplatin resistant cells SKOV3/DDP(2.31±0.26)was significantly higher than that in cisplatin sensitive cells SKOV3(1.00±0.00)(P<0.01).The expression of ENTPD5 in SKOV3 cells decreased significantly with the increase of cisplatin concentration(P<0.01),but increased significantly in SKOV3/DDP cells(P<0.01).3.4 ENTPD5 knockdown significantly enhanced the sensitivity of SKOV3/DDP cells to cisplatin(IC50:4.76±0.30vs3.54±0.529μg/mL,P<0.01).Cisplatin induced apoptosis increased significantly after ENTPD5 knockdown(apoptosis rate:23.43±3.41 vs 8.84±1.57%,P<0.01).3.5 ENTPD5 knockdown significantly inhibited the expression of GRP78 protein in SKOV3/DDP cells(P<0.01).ConclusionENTPD5 participates in the pathogenesis of ovarian cancer by regulating the proliferation,migration and cisplatin-resistance of epithelial ovarian cancer cells,GRP78/p-eif-2α/Chop pathway may be the main molecular mechanism. |
PDF Full Text Request