Study On The Mechanism Of Cyclin CDC50A In Maintaining The Characteristics Of Ovarian Cancer Stem Cells And Its Related Mechanism

BackgroundOvarian cancer is the third commonest but the most lethal gynecological cancer among worldwide. Most patients had advanced stage disease when diagnosed. In the past several decades, the 5-year survival time of advanced ovarian cancer patients is only about 30% after receiving optimal cytoreductive surgery, followed with adjuvant chemotherapy. And over 70% of the patients relapsed within 18 months. The chemoresistance and replase of the tumor is considered to be the main causes of the high mortality. The mechanism for this, however, is not clear, which is the biggest obstacle for ovarian cancer therapy.Cancer stem cell(CSC) is the emerging concept in tumor biology. The CSC theory dicates that the progression, recurrence and the chemoresistance of cancers are governed by a small subpopulation of CSCs within a tumor. Bonnet and Dick first isolated the CD34+CD38" cell subpopulation from leukemia patients which were identified as CSCs. After that, CSCs were found in many solid tumors, such as breast cancer, prostate cancer, colon cancer, and so on. The CSCs possess the characteristics of differentiation, self-renewal, tumorigenicity and play a role in resistance to chemotherapy. As the development of the CSCs theory, lots of methods for isolation and identification of CSCs in solid tumors have been established, which plays an important role in new research of cancer treatment.Recently, ovarian caner has been postulated to imitate the CSC model. Many CSCs markers found in other solid tumors are used for identification and isolation of ovarian caner stem cells, such as CD 133、CD117、CD44、ALDH1、EPCAM, while CD44 is known as the CSCs marker for breast cancer and pancreas cancer, and CD133 is the marker for colon cancer. As a result, the main method to isolate ovarian cancer stem cells is still the sorting of side population cells (side population, SP) by now. So it is very important to looking for a specific marker to research ovarian cancer stem cells.In the early stage of our work, we used quantitative proteomic techniques based on the stable isotope labeling and mass spectrometry to compare the membrane protein of SP and non-SP cells, and screened a candidate marker- transmembrane protein 30A(TMEM30A, also called CDC50A). And the CDC50A+ cells were identified to possess the CSCs properties by a series studies in vivo and in vitro. Thus, CDC50A is considered as a marker for ovarian CSCs. In this research, we continued to identified the role of CDC50A in maintain the CSCs properties and the related mechanisms in ovarian cancer.Methods1. Down-regulation of CDC50A+ cells proportion in SKOV3 cells. The available CDC50A shRNA was screened by Western blot after transfection of 293T cells. Packaging shRNA with lentiviral vector, and then infected SKOV3 cells to silence their CDC50A gene. The silence efficiency was detected by flow cytometry. SKOV3 cells with down-regulation of CDC50A+ cells proportion (SKOV3 CDC50A-GFP) and negative control cells (SKOV3 NC-GFP) were sorted by fluorescence activated cell sorting (FACS). The proportion of CDC50A+ cells was analysed by flow cytometry in SKOV3 CDC50A-GFP and SKOV3 NC-GFP cells.2. The identification of the CSCs properties of SKOV3 cells with down-regulation of CDC50A+ cells proportion. The sphere formation and passage capability of SKOV3 CDC50A-GFP and SKOV3 NC-GFP cells was detected after cultured in serum-free suspension medium, and the enrichment of CDC50A in sphere was identified by flow cytometry. The difference of resistance to cisplatin of the two populations was tested by MTT assays. Equal number of SKOV3 CDC50A-GFP and SKOV3 NC-GFP cells were implanted into NSG mice, and then the capability of tumorigenicity of the two population was identified.3. The search for related mechanisms of CDC50A to maintain the CSCs properties in ovarian cancer by RNA sequencing of CDC50A+ and CDC50A- cells. Tumor tissues of 3 ovarian cancer patients were collected and tumor cells were isolated. CDC50A+ and CDC50A- cells were sorted by FACS. RNA was extracted from the two population cells, and then was used to sequence. Then the differentially-expressed genes were identified which were used to look for CDC50A-associated signal pathways.Results1. Down-regulation of CDC50A+ cells proportion in SKOV3 cells successfully. Of the 4 candidate CDC50A shRNA, the sequence of TMEM30A-homo-974 was certified to have the highest silencing efficiency in 293T cells by Western blot. The target sequence is GCCTGTGAACTGGCTTAAACC. The CDC50A shRNA packaged with lentiviral vector was used to infect the 293T cells and proved to down-regulate the CDC50A expression by over 80%. After the lentiviral vector infecting SKOV3 cells, the infection efficiency of CDC50A-GFP and NC-GFP was 87.9% and 81.6% respectively. After sorted by FACS, the CDC50A+ cells in SKOV3 NC-GFP and CDC50A-GFP populations was 0.48% and 0.12%. It showed that the CDC50A+ cells could be down- regulated successfully via lentiviral infection vector with CDC50A shRNA.2. The CSCs properties of SKOV3 cells with down-regulation of CDC50A+ cells proportion declined. Spheres were formed in two populations, and compared with SKOV3 NC-GFP cells, SKOV3 CDC50A-GFP cells were less capable of generating spheres, and couldn’t passage, while SKOV3 NC-GFP cells can passage consecutively. Besides, most of spheres formed by SKOV3 NC-GFP cells could be detected under the fluorescence microscope, while little spheres formed by SKOV3 CDC50A-GFP cells could. The CDC50A+ cells in SKOV3 NC-GFP and CDC50A-GFP spheres was enriched to 19.7% and 6.7% respectively. By MTT assays, the IC50 of cisplatin of SKOV3 CDC50A-GFP and NC-GFP cells was 1.33±0.14ug/ml and 3.05±0.39 ug/ml, with significant difference(P=0.01). The tumor formation experiment showed that implantation of 103 SKOV3 NC-GFP cells could formation a tumor in NSG mice, while the same number SKOV3 CDC50A-GFP cells couldn’t.3. Several related mechanisms of CDC50A to maintain the CSCs properties were found in ovarian cancer by RNA sequencing of CDC50A+ and CDC50A- cells.92,59 and 251 differentially-expressed genes (P<0.01) were found between the CDC50A+ and CDC50A- cells in 3 primary ovarian cancer tissues respectively. By RNA spectrum analysis and Bioinformatics technology, several pathways were verified associated CDC50A in maintaining the CSC properties in ovarian cancer, including ATPase H+ transporting V1 subunit F, MALAT1, MAPK signaling pathway and regulation of the function of ribosome and splicesome.Conclusion1. The CDC50A+ cells proportion of SKOV3 cells can be down-regulated efficiently after infecting by the lentiviral vectors of CDC50A shRNA.2. The CSCs properties of SKOV3 cells with CDC50A+ cells proportion down-regulation declined. They have lower capabilities of sphere formation, self-renewal, differentiation and tumorigenicity, and becomes sensitive to cisplatin.3. CDC50A plays an important role in maintain the ovarian CSCs’ biological properties and functions mainly via the mechanisms of ATPase H+ transporting V1 subunit F.