| Objective:Ovarian cancer,one of the female reproductive system diseases,is the most common malignancies of the female reproductive system.Most patients find it at an advanced stage and because of the lack of effective early diagnosis methods.In 2020,there were 313,959 new cases of ovarian cancer and 207,252 deaths,making it the eighth largest cancer in the world for female.Now,the treatment of ovarian cancer is still based on tumor cell reduction combined with chemotherapy.The improvement of long-term survival rate is still unsatisfactoryalthough the application of multiple targeted drugs has opened up a new era in the treatment of ovarian cancer in recent years.Therefore,it is very important for the diagnosis,disease monitoring and treatment of ovarian cancer to reveal the latent mechanism of how the ovarian cancer occur and develop at the molecular level and to search for effective tumor markers and therapeutic targets.NDUFC1 is an accessory subunit of human NADH-ubiquinone oxidoreductase(Complex I)with 76 amino acids.Located in the mitochondrial inner membrane,NDUFC1 is involved in the assembly and structural stability of complex I..Recent studies have confirmed that the expression of NDUFC1 is up-regulated inliver cancerand in gastric cancer,and its abnormal expression can lead to the dysfunction of complex I and thus lead to the occurrence and development of cancer.However,there are no relevant studies on its effect on the occurrence and development of ovarian cancer.In previous studies,we found that the ovarian cancer tissuesexpress NDUFC1 higher than the one in normal ovarian tissues through bioinformatics analysis.This study will profoundly verify the expression of NDUFC1 in ovarian cancer tissues and analyze the association between its expression and pathological data and prognosis of patients with ovarian cancer.A series of in vivo and in vitro experiments were conducted to investigate the effect of NDUFC1 on malignant biological behavior of ovarian cancer and its related mechanisms,providing a novel theoretical basis for the diagnosis and gene targeted therapy of ovarian cancer.Methods:Part I:1、Bioinformatics analysis was applied to analyze the difference in expression of NDUFC1 between ovarian cancer and normal ovarian tissue;2、Collected79 cases of patients with primary ovarian cancer undergoing surgery in Shengjing Hospital Of China Medical University and 23 cases of normal ovarian tissues undergoing ovariectomy due to other diseases.Immunohistochemical methods were used to detect the expression of NDUFC1 in ovarian cancer tissues and normal ovarian tissues;3、Immunohistochemical method was applied to detect ovarian cancer tissue chip to study the correlation between NDUFC1 expression level and clinicopathological information of patients,and Kaplan-Meier was used to analyze the association between NDUFC1 expression level and prognosis of patients with ovarian cancer.Part II:1、Discrepancies in m RNA expression of NDUFC1 in ovarian cancer cell lines and normal ovarian cell lines detected by q RT-PCR;2、Ovarian cancer cell lines A2780 and HO-8910 were infected with lentivirus to construct stable cell lines with low expression of NDUFC1.The knockdown rate was checked on by q RT-PCR and Western Blot;3、The effect of knocking down NDUFC1 on the proliferation of ovarian cancer cells was digged by Celigo cell counting,the effect of knocking down NDUFC1 on the apoptosis of ovarian cancer cells was probed by Annexin V-APC single staining method,the effect of knocking down NDUFC1 on the cell cycle was explored by flow cytometry,the cell scratch test and Transwell test were used to inquiry the effect on the migration and invasion ability of ovarian cancer cells;4、The tumor model of ovarian cancer cells with NDUFC1 knocked down was created in nude mice,and the NDUFC1’s effect on the proliferation of ovarian cancer cells was further verified in vivo;5、Western Blot was used to verify the effect of differential expression of NDUFC1 on the expression level of related proteins in NF-κB signaling pathway;6、In addition,the overexpressed NDUFC1 cells were treated with NF-κB signaling pathway inhibitor NF-κB-in-1,and the expression of NF-κB signaling pathway related proteins was detected by Western Blot.The effect on proliferation and apoptosis of NF-κB signaling pathway was detected by CCK8 assay and Annexin V-APC single stain method.Part III:1、Potential molecular targets regulated by NDUFC1 were screened using TMT proteomics,and PIR,CLU and TLE1 with significant differential expression were selected for follow-up screening;2、The expression of PIR,CLU and TLE1 after NDUFC1 knockdown was verified by rt-q PCR and Western Blot;3、The interaction between NDUFC1 and PIR protein was further confirmed by Co-IP assay;4、To further verify that NDUFC1 is mediated by PIR-mediated NF-κB signaling pathway,ovarian cancer cell lines A2780 was infected with lentivirus to institutesteady cell lines with high expression of NDUFC1,low expression of PIR,and high expression of NDUFC1/low expression of PIR.CCK-8 assay,flow cytometry double staining apoptosis assay and Western Blot were used to probe the effect of NDUFC1 on proliferation and apoptosis of ovarian cancer cells and downstream NF-κB signaling pathway.Results:Part I:1、According to the data analysis of GSE66957 in the TCGA,GTEx and GEO databases,the expression level of NDUFC1 m RNA in ovarian cancer tissues was remarkably up-regulated compared with that in normal ovarian tissues;2 、Immunohistochemical results demonstrated that the expression of NDUFC1 in ovarian cancer was strikingly higher than that in normal ovarian tissue,and the expression of NDUFC1 gene was positively correlated with pathological data such as pathological grade,T value,clinical stage and recurrence;3、Kaplan-Meier survival analysis proved that the expression level of NDUFC1 was notabaly correlated with overall survival and disease-free survival of ovarian cancer patients.Part II:1、Two ovarian cancer cell lines A2780 and HO-8910 with NDUFC1 knockdown were constructed successfully,andtheir phenotypes were further verified.Celigo cell counting assay showed that NDUFC1 knockdown inhibited the proliferation of ovarian cancer cells.The results of apoptosis experiment displayed that knockdown of NDUFC1 increased the apoptosis rate of ovarian cancer cells.Cell cycle assay results showed that NDUFC1 knockdown affects the cell cycle of ovarian cancer cells and arrest the cell cycle in G2 phase.Cell scratch assay and Transwell assay demonstrated that knockdown of NDUFC1 restrained the migration and invasion ability of ovarian cancer cells;2、NDUFC1 knockdown inhibited tumor growth in nude mice;3、Western Blot results showed that the expressions of p-P50,p-P65(S536),p-IKBα related to NF-κB pathway were down-regulated after knocking down NDUFC1.After overexpression of NDUFC1,the expressions of p-P50,p-P65 and p-IKBα related to NF-κB pathway were up-regulated;4、The effect of overexpression of NDUFC1 on proliferation and apoptosis of ovarian cancer cells can be reversed by adding NF-KB pathway inhibitor NF-κB-IN-1 to overexpression of NDUFC1 cells.Part III:1、The results of TMT proteomics showed a total of 570 differentially expressed proteins after knocking down NDUFC1,of which 331 proteins were up-regulated and239 proteins were down-regulated.PIR,CLU and TLE1 with significant differentially expressed proteins were selected for follow-up screening;2、The consequences of q RT-PCR exhibited that PIR expression was down-regulated and the expression of CLU and TLE1 were up-regulated after NDUFC1 silencing;3、The results of Western blot showed that the expression of PIR、CLU and TLE1 were down-regulated after NDUFC1silencing;4、The Co-IP experiment verified the combination of NDUFC1 and PIR;5、PIR knockdown /NDUFC1 overexpression plasmid was constructed to infect A2780 cells.CCK-8 assaydemonstrated that overexpression of NDUFC1 promoted cell proliferation,silencing of PIR inhibited cell proliferation,and silencing of PIR could reverse the effect of overexpression of NDUFC1 on the proliferation of ovarian cancer cells;6、Apoptosis assay demonstrated that overexpression of NDUFC1 inhibited apoptosis,silencing PIR promoted apoptosis,and silencing PIR could reverse the effect of overexpression of NDUFC1 on apoptosis of ovarian cancer cells;7、Western blot analysis showed that NF-κB pathway related proteins p-P50,p-P65,p-IKBα was down-regulated after knocking down of PIR.PIR silencing could reverse the effect of overexpression of NDUFC1 on NF-κB pathway of ovarian cancer cells.Conclusions:NDUFC1 is involved in the development of ovarian cancer,and high expression of NDUFC1 is associated with poor prognosis.2.The expression level of NDUFC1 influences the proliferation,apoptosis,migration,invasion and tumor growth of ovarian cancer cells,as well as the ovarian cancer cell cycle.3.NDUFC1 affects the proliferation and apoptosis of ovarian cancer by activating the NF-κB pathway through PIR. |
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