| BackgroundOvarian cancer is the most common malignancy of the female reproductive system with the highest mortality rate.Although platinum-based chemotherapy combined with ideal tumor cell reduction(CRS)has achieved a high response rate in ovarian cancer(OC)patients,the 5-year survival rate in advanced patients is only about 30%[1-3]Recurrence and resistance to chemotherapy are the main causes[4].The way to improve this condition is to explore the mechanisms of recurrence and drug resistance of ovarian cancer.A subset of tumor cells with self-renewal,differentiation,and tumor initiation capacity is known as tumor stem cell(CSCs)or tumor initiation cell(TlCs)[5].Tumor cells with the characteristics of stem cells(SC)was first raised in 1858,when Virchow suggested that cancer might originate from immature cells.In 1875,Coenheim improved this model by suggesting that embryonic-like cells were retained in adult tissue and developed into cancer after being activated later in life[6-7].In heterogeneous ovarian tu1ors,a very small fraction of the population(usually less than 2%of the cells)has a stronger tumorigenicity and differentiation thal other tumor cells[9-10].There are many reasonable reasons for the concept of OC as a SC disease,firstly,because of the clinical process of OC.This recurrent tumor is a heterogeneous group of chemo-sensitive drug-resistant cells,which suggests that there is a differentiatiol ability in the surviving cell population after chemotherapy[9].More mesenchymal components were expressed in epithelial cells of ovary(OSE)at gene level,and the differentiation degree of it was lower[11].Howto isolate ovarian cancer stem cells has become a hot issue[8]It is of great significance for targeted treatment of OC to search for specific surface marker protein and explore its mechanism.Supported by the National Natural Science Foundation of China,our research group selected candidate protein CDC50A as the OCSCs surface marker.And successful isolation and purification of CDC50A cells from primary tissue cells,in vitro and vivo experiments showed that CDC50A was a marker protein on the surface of OCSCs.The up-regulated expression of CDC50A can significantly enhance the SC characteristics of OC.The P4 family associated with CDC50A was further screened by RT-PCR technique.ATPase family phospholipid transporting is widely distributed.It is a multistage transmembrane transporter with adenosine triphosphate as its energy,which is involved in the transport of a variety of substances and important functional proteins in life-sustaining activities[12].It is believed that the ATPase family can be used in conjunction with the CDC50A to transfer the phospholipid from the extracellular matrix to the cytoplasmic leaflet on the inner side of the cell double-phospholipid layer[13].In tumor studies,ATP8A1 is associated with tumor invasion and metastasis[14],ATP8B1 and ATP11B are associated with chemotherapy resistance[15-16],and ATP11C is associated with apoptosis[17].Hiroyuki Takatsu,et al[18]have found that CD 50 protein(mainly CDC50A)is required for P4-ATP enzymes of Category 5(ATP 10A,ATP 1 OB and ATP 10D)and Class 6(ATP 11 A,ATP 11B and ATP 11C)to withdraw from endoplasmic reticulum(ER)and complete the final subcellular localization.Our research found that in OC cell lines,ATP11A and ATP11B could enhance the characteristics of CDC50A as stem cell marker by inhibiting early apoptosis and enhancing the drug resistance.On this basis,this study will investigate the effect of stable low expression of CDC50A on the characteristics of OCSC through OC cell lines.Sphere formation experiment and NSG mouse tumorigenic model were used to investigate the effect of down-regulated expression of ATP11A and ATP11B on SC function in OC cell lines.The CDC50A+and CDC50A-cells in samples of human OC were sequenced by genome sequencing technology,and the mechanism of CDC50A playing the role of stem cell surface markers was further explored.Methods1、To observe the specificity of CDC50A as a marker of OCSCs by down-regulating the expression of CDC50A in ovarian cancer cell line.The lentivirus vector of shCDC50A was constructed,transfected it into 293FT cells.Virus produced by 293FT cells was used to infect SKOV3 and OVCAR4 cell lines and the stable cell lines(skov3-shCDC50A and ovcar4-shCDC50A)were obtained by flow sorting.The cells treated with certain concentration of cisplatin were tested by apoptosis assay and drug resistance assay(cck-8 assay)to detect the changes in apoptosis rate and cell viability.2、CDC50A+and CDC50A-cells from human ovarian cancer samples were sequenced by high-throughput sequencing technology to explore the stem cell function mechanism of CDC50A.Tumor tissues of 2 patients with OC were collected and OC cells were isolated.CDC50A+and CDC50A-cells were obtained by flow cytometry.Micro DN A and RN A extraction techniques were used to extract DNA and RNA,respectively.3、To explore the interaction mechanism between CDC50A and ATP 11A and ATP 11B by down-regulating the expression of ATP 11A and ATP 11B in ovarian cancer cell lines.1)Down-regulation the expression of ATP11A and ATP11B in ovarian cancer cell lines,the changes in CDC50A expression level were verified by RT-PCR and western-blot.shATP11A,shATP11B plasmid was screened and constructed.After identification of shRNA,lentivirus vector was constructed and infected with SKOV3-CDC50A cell line.The stable co-expression genome(SKOV3-CDC50A-shATP11A,SKOV3-CDC50A-shATP11B)cell line were obtained by flow analysis technique.RT-PCR and western-Blot was used to identify the changes of CDC50A expression.2)Sphere formation assay and tumor formation in NSG mice verify the changes in stem cell function of ovarian cancer cell lines with stable low expression of ATP11A and ATP11B.In vitro experiment:the ability of sphere formation in different groups(SKOV3,SKOV3-CDC50A,SKOV3-CDC50A-shNC,SKOV3-CDC50A-shATP11A,SKOV3-CDC50A-shATP11B)was detected.In vivo experiment:the model of ovarian cancer transplantation in NSG mice was established by using the stable cell lines obtained above.Results1、Down-regulation the expression of CDC50A in OC cell lines,the stem cell characteristic was significantly weakened.Apoptosis and cell viability significantly decreased after cisplatin treatment.After cisplatin treatment(6ug/ml),the percentage of apoptotic cells(early apoptosis and late apoptosis)in SKOV3-shCDC50A group was significantly higher than SKOV3-shNC(47.92±11.43 vs 22.98±10.02,P<0.024).The percentage of apoptotic cells in OVCAR4-shCDC50A was significantly higher than that in OVCAR4-shNC(43.95±0.778 vs 25.71±1.158,P<0.001);Under a certain concentration of cisplatin(9ug/ml),the cell viability of different cell populations was measured at different time(24h,48h,72h).The cell viability of SKOV3-shCDC50A was significantly lower than SKOV3-shNC(24h:the average cell viability was 19.11%vs 44.51%,P=0.0178;48h:9.33%vs 35.69%,P=0.024;72h:0.26%vs 11.50%,P<0.001).The cell viability of OVCAR4-shCDC50A population was significantly lower than OVCAR4-shNC group(24h:22.33%vs 29.33%,P=0.041;48h:2.90%vs 5.93%P=0.021;72h:0.63%vs 3.13%,P<0.001).2、There was no significant difference between CDC50A+and CDC50A-cells at exon level.In total,1617 protein-coding genes were significantly differentially expressed(FDR<0.1).Overrepresentative tests indicated that differentially expressed genes(DEGs)were significantly enriched(q value-0.0229)with respect to the Gene Ontology molecular function(MF)category of phospholipid-translocating ATPase activity.Of 23 total genes with phospholipid-translocating ATPase activity,7 were significantly differentially expressed,including ATP 11 A,ATP 11 B,ATP8B2,ATP8B4,ATP10A,ABCA1 and ABCA7.3、Down-regulation the expression of ATP 11A and ATP 11B in ovarian cancer cell lines,the expression of CDC50A in gene and protein levels significantly decreased,and sphere formation ability and tumor formation ability significantly decreased.Comparing the ability of sphere formation among SKOV3-shNC,SKOV3-CDC50A,SKOV3-CDC50A-shNC,SKO V3-CDC50A-shATP 11 A,SKOV3-CDC50A-shATP11B.The sphere formation ability among SKOV3-CDC50A was significantly higher than SKOV3-shNC(mean number of balls:18.67 vs 7.68,P=O.021).The sphere number of SKOV3-CDC50A-shATP11A and SKOV3-CDC50A-shATP11B was significantly lower than that of SKO V3-CDC50A-shNC(8.67 vs 15,P<0.01;8.67 vs 15,P=0.021).The tumorigenicity of ovarian cancer cell lines with low expression of ATP11A or ATP11B was significantly decreased.5×105 cells could induce tumorigenesis in mice.The mice were killed at the same time(52 days after tumor inoculation),the weight of transplanted tumor in SKOV3-CDC50A-shNC group was significantly higher than that in SKOV3-CDC50A-shATP11A,SKOV3-CDC50A-shATP11B(P=0.003,0.0028,respectively).Further flow analysis showed that the absolute value of CDC50A/Lin-cells in SKOV3-CDC50A-shNC group was significantly higher than that in SKOV3-CDC50A-shATP11A,SKOV3-CDC50A-shATP11B(P=0.037,0.032 respectively).Conclusions1、Down-regulation expression of CDC50A could significantly weaken the stem cell characteristics of ovarian cancer cells,and further proving that CDC50A is a specific marker of OCSCs.2、The sequencing results of complete exons and transcriptome genes of CDC50A+and CDC50A-cells suggested that CDC50A may regulate the function of OCSCs through ATP family at the metabolic level.3、In ovarian cancer cell lines,down-regulation the expression of ATP11A or ATP11B can significantly reduce the expression of CDC50A and weaken the characteristics of OCSCs of CDC50A+cells.Suggesting that CDC50A+ovarian cancer cells play the stem cell role through ATP11A or ATP11B. |
PDF Full Text Request