The Effect Of CYC1 On Biological Behavior Of Serous Ovarian Cancer And Its Mechanism

Research objective and backgroundOvarian cancer is one of the most common malignant tumors in female reproductive organs.Serous ovarian cancer(SOC)accounts for the majority of ovarian epithelial carcinoma,including high-grade serous carcinoma with high malignancy and poor prognosis.Ovarian cancer patients after treatment,there are often chemotherapy recurrence and multiple drug resistance occurred.Therefore,the study on the pathogenesis of ovarian cancer and the discovery of early monitoring indicators are particularly important,which can lay a foundation for further guiding clinical treatment.CYC 1(Cytochrome c-1)is an important sub-unit of mitochondrial complex ?,which can rapidly promote the organization of cell oxidation and reduction process with a rapid enzymatic function in the process of biological oxidation.CYC1 is highly expressed in many malignant tumors,such as ductal carcinoma in situ,nasopharyngeal carcinoma,neuroblastoma,and osteosarcoma.CYC1 is involved in mitochondrial apoptosis pathway,inhibits AMPK activation and promotes tumor proliferation and metastasis.In this study,gene expression profiling sequencing and Protein Mass Spectrometry technology were used to find that CYC1 expression in serous ovarian cancer was higher than that in fimbriae tubae.A series of functional experiments have verified the function of CYC1,which plays a role in promoting tumor invasion and metastasis,proliferation,regulating cell cycle and chemotherapy resistance in SOC.Meanwhile,the downstream molecular mechanism of CYC1 was explored through high-throughout sequencing technology,and the potential binding gene and related mechanisms were discussed.Research methods:1.Collect serous ovarian cancer tissue(SOC)and normal fimbriae tubae tissue(FT)from patients with benign diseases.The expression of CYC1 in tissues was detected by Western Blot and RT-PCR.Immunohistochemistry was used to compare the expression of CYC1 with the prognosis of HGSOC and FT tissue microarrays(3 slides).2.Stable cell lines with overexpression and interference of CYC1 were constructed.Cell growth activity was detected by MTT assay,invasion and migration ability by Transwell assay,cell cycle progression by flow cytology,cell cycle and EMT-related proteins detection by Western Blot.3.The cells were stimulated with cisplatin and olaparib at a certain concentration gradient,the expression of CYC1 protein was detected,the growth activity of cells was measured by MTT method,and the long-term growth activity of cells was verified by plate cloning.Flow cytology combined with TUNEL assay was used to detect the apoptosis of CYC1.4.The RNA-seq method was used to search for the downstream molecular mechanism of CYC1,and the genes with high abundance were verified by Western Blot and RT-PCR.Research results:1.The mRNA and protein expression of CYC1 in serous ovarian cancer was significantly higher than that of oviduct umbrella.The TCGA data set analysis of ovarian cancer showed that CYC1 had abnormal copy number amplification in the SOC,and the copy number amplification was related to the protein and mRNA expression of CYC 1.2.Immunohistochemical analysis of the expression of CYC1 in tissue microarrays showed that patients with high expression of CYC1 had a short overall survival,and high expression of CYC1 was associated with poor prognosis of SOC.3.CYC1 enhanced the proliferation ability of serous ovarian cancer cells.MTT growth curve and plate cloning experiments showed that both overexpression of CYC1 could increase the growth rate of cells,while cell growth slowed down significantly after CYC1 interference.Downregulated expression of CYC1 cell cycle arrest in G1/S phase,CYC1 overexpression will cause the transformation of G0/G1 phase to S phase.After interference with CYC1,the expression of cyclin-related proteins CCNB1,CCNE1 and CDKs decreased,and the expression of dependent kinase inhibitor proteins p21 and p27 increased.4.CYC1 promotes the invasion and migration ability of serous ovarian cancer cells.After CYC1 expression is reduced,the invasion and migration ability of A2780 cells and HO8910 cells is weakened,while overexpression promotes the invasion and migration ability of cells.After CYC1 interference,the expression of epithelial protein markers increased,the expression of interstitial marker decreased,and the corresponding EMT transcription factors MMP9,Snail and slug decreased.5.The high expression of CYC1 can promote the growth of subcutaneous transplantation tumor of human ovarian cancer.The average weight of the experimental group was significantly greater than that of the control group,and the marker Ki67 of immunohistochemical staining cell proliferation was increased.6.CYC1 promotes chemotherapy resistance of SOC,and the expression of CYC1 protein increases in turn with the increase of cisplatin concentration.Drug-resistant MTT assay shows that overexpression of CYC1 promotes the growth of A2780,SKOV3 and HO8910 cells.Plate cloning experiments showed that interfering CYC1 reduced the long-term growth activity of cells.After CYC1 expression was reduced,apoptosis-related protein expression was increased,and apoptotic cells in early and late stages were increased,indicating that CYC1 exerts drug resistance by inhibiting apoptosis.7.Silencing CYC1 sensitized olaparib on BRCA wild-type ovarian cancer cells.With the increase of olaparib concentration,the activity of A2780 and SKOV3 cells decreased in the silencing group compared with the control group.8.CYC1 regulates downstream molecules in a series of important pathways,and according to RNA-seq sequencing,it down-regulates the expression richness of tumor pathways significantly.The mRNA levels of downstream molecules were verified.After the interference of A2780 cells with CYC1,the expression levels of CCNE2 and BIRC3 decreased significantly,and the expression levels of FGF18,EGFR,WNT5B,NFKBIA and FZD4 all decreased.For CCNE2 and BIRC3 with high abundance,the expression of CCNE2 and BIRC3 decreased after CYC1 expression was reduced.Research conclusion:1.CYC1 expression in serous ovarian cancer is significantly higher than that of fimbriae tubae,which is related to poor prognosis of SOC.2.As an adverse prognostic factor for ovarian cancer,CYC1 promotes the malignant biological behavior of serous ovarian cancer and can be used as a therapeutic target for ovarian cancer.3.CYC1 promotes the chemotherapy-resistance of SOC,and knockdown of CYC1 can increase the sensitivity of chemotherapy-resistance.4.CYC1 plays a role in promoting cancer by regulating various important molecules such as CCNE2 and BIRC3.