Biological Function And Clinical Characteristics Of PRC1 In High-grade Serous Ovarian Cancer

Epithelial ovarian cancer is the most lethal gynecological malignant tumor.In 2018,there were 295,414 new cases and 184,799 deaths worldwide with an increasing trend.Ovarian cancer starts insidiously and has no obvious clinical symptoms in its early stage.Only about 30%of patients can be diagnosed in the early stage and treated in a timely manner due to the inaccurate screening protocols.Although surgical procedures and chemotherapy regimens for ovarian cancer have improved in recent years,more than 70 percent of patients will relapse after standard treatment.Relapsed patients will face the thorny problem of multiple drug resistance,with a 5-year survival rate of only about 50%.High-grade serous ovarian cancer(HGSOC)is one of the types with high degree of malignancy and poor prognosis.There is still no clear consensus on the pathogenesis,molecular regulation mechanism and drug resistance mechanism,and how to effectively prevent,detect and treat the disease in early stage is also an important issue that needs to be solved urgently.Microtubule targeting agents,also known as anti-mitotic drugs,such as paclitaxel,docetaxel,have been widely used in the clinical treatment of various malignant tumors and have significant therapeutic effects.Microtubule targeting drugs can change microtubule dynamics and participate in cell cycle monitoring mechanism,block the cell cycle process,inhibit invasive growth of cells,and initiate programmed cell death of tumor cells,so as to achieve the purpose of anti-tumor.In the process of anti-mitosis,cancer cells would eventually die of cell apoptosis.However,due to the mechanism of cell slippage and death escape and secondary drug resistance,anti-microtubule targeting drugs face certain difficulties in the treatment of tumors.Moreover,anti-microtubule drugs themselves also have many limitations,such as hypersensitivity reaction,cardiovascular toxicity and neurotoxicity,which have certain limitations in clinical use.New generation of anti-microtubule targeting drugs,such as Aurora kinases,SAC proteins,and CENP-E,have not achieved significant results in traditional drugs such as paclitaxel in gynecological tumors due to their toxicity,efficacy and other problems.Therefore,the exploration of new anti-mitotic targets is also one of the hot spots of current research.PRC1(protein regulating cytokinesis 1)is located on human chromosome 15(15Q26.1),which is a microtubule related protein.It is a necessary protein molecule formed in the central region of spindle structure during the transition of cells from middle to late mitosis,and is involved in the important process of cytokinesis.Abnormal expression of PRC 1 will lead to abnormal cell behavior and lead to abnormal cytokinesis,thus further promoting the occurrence and development of tumors.At present,there has been no relevant research report on PRC1 in ovarian cancer.This study will explore the effect of PRC 1 on the proliferation,metastasis and multi-drugs resistance of ovarian cancer cells through in vitro experiments,and further explore the relevant mechanisms.At the same time,clinical tissue samples were used to analyze the expression status of PRC 1 in high-grade serous ovarian cancer through immunohistochemistry,so as to determine its correlation with the occurrence,development and prognosis of tumors.This study will help to reveal the pathogenesis of PRC 1 in high-grade serous ovarian cancer and provide a new molecular target for molecular typing and individualized treatment of this disease.This study will be elaborated from the following two aspects:PART Ⅰ:Biological function and mechanism of PRC 1 in high-grade serous ovarian cancer;PART Ⅱ:Clinical characterization of PRC 1 in high-grade serous ovarian cancer patients.PART Ⅰ:Biological function and mechanism of PRC1 in high-grade serous ovarian cancerBackground:The ability of cells to enter the growth and division cycle accurately is a prerequisite for maintaining normal cell proliferation and genomic stability Cytokinesis is the physical separation of two daughter cells during cell division and is the final stage of the cell cycle.After chromosome separation in the late stage of cell cycle,the cell forms contraction ring to drive the contraction of plasma membrane,thus producing two daughter cells connected by cytoplasmic bridge.However,the instability of tetraploid and chromosome caused by the failure of cell division can promote the occurrence and development of tumor.PRC1 protein molecule was discovered by Wei Jiang et al.,in 1998,when they were searching for the functional substrate of cyclin dependent kinases(CDKS)related to cell cycle regulation.PRC1 protein molecule is an essential protein molecule formed in the central region of the spindle during the transition from metaphase to anaphase of cells,and is involved in the important process of cytokinesis.PRC1 gene is located on human chromosome 15(15Q26.1)and encodes 620 amino acids with a molecular weight of about 72kDa.The microtubules in the cells that normally express PRC1 form normal spindle structure,while the cells that overexpress PRC1 lead to abnormal microtubules in the spindle in the premetaphase and metaphase of the cell cycle,and affect the formation of central structure of the spindle in the anaphase.Abnormal expression of PRC 1 can lead to abnormal cell behavior and lead to abnormal cytokinesis,thus further promoting the occurrence of tumors.PRC1 is negatively regulated by P53 and overexpressed in p5 3-deficient cells,indicating that this gene is tightly regulated in a cancer-specific manner.Therefore,PRC1 may play a corresponding role in a variety of tumors.Preliminary research results have been obtained on PRC1 in liver cancer,gastric cancer,lung adenocarcinoma and other tumors.Its overexpression can promote the proliferation,invasion,drug resistance and other malignant biological behaviors of tumor cells,and lead to poor prognosis of tumor patients.However,no relevant studies on PRC1 have been reported in ovarian cancer,so we conducted this study to explore the malignant biological behavior of PRC1 in ovarian cancer cells and explore the relevant mechanisms.Materials and methods:TCGA,Oncomine and other databases were used to analyze the differential expression of PRC1 in ovarian cancer tissues and normal ovarian epithelial and peritoneal tissues,as well as the differential expression of PRC 1 in different degrees of invasive ovarian tumors.The differential expression of PRC 1 was detected by RT-qPCR and Western blot between high-grade serous ovarian cancer tissue and normal tubal epithelial tissue.Lentiviral vectors with PRC1 overexpression and low expression were constructed to establish corresponding stable ovarian cancer cell lines(A2780,HO8910,SKOV3).MTT proliferation curve,colony formation assay,transwell and scratch test were used to detect the effects of PRC 1 on the proliferation,drug sensitivity,invasion and migration of ovarian cancer cells.Flow cytometry was used to detect the changes of cell cycle,and Western blot was used to detect the effects of PRC 1 on cyclin-related proteins,EMT-related proteins and DNA damage repair proteins.The correlation between PRC1 and FOXM1 was explored by bioinformatics analysis.The A2780 and SKOV3 cell lines were treated by Si-RNA of FOXM1,and the changes of PRC1 were detected by RT-qPCR and Western blot.Correlation between FOXM1 and PRC1 was verified by immunohistochemistry.Ensembl was used to query the promoter sequence of PRC 1,and the potential regulation site of FOXM1 on PRC1 was predicted by Gene regulation and related literature.Dual luciferase Reporter assay was used to verify the regulation site and effect of FOXM1,and the regulation effect of FOXM1 on PRC1 was further verified by rescue assays.Results:1.PRC1 was overexpression in high-grade serous ovarian cancerAnalysis on TCGA and Oncomine databases found that PRC1 expression in serous ovarian cancer was higher than that in normal ovarian tissue and peritoneal tissue,and it was positively correlated with the malignant degree of tumor.The expression of PRC 1 in high-grade serous ovarian cancer was higher than that in normal tubal epithelial tissue by RT-qPCR and Western blot.Immunohistochemistry showed that PRC1 was mainly located in the cytoplasm and the expression of PRC1 in HGSOC tissues was higher than that in normal tubal fimbria tissues.2.PRC1 could promote the proliferation ability of ovarian cancer cells MTT assay showed that the growth rate of ovarian cancer cells was significantly slowed after PRC1 knockdown,and the colony formation assay confirmed that its clonal formation ability was significantly inhibited.Knockdown of PRC 1 resulted in G2 phase arrest of the cells.Western blot analysis of cell proliferation and cycle-related proteins showed that knockdown of PRC 1 significantly inhibited the expression of CCNB1,CCND1,AURKB and CENPE,and up-regulated the expression of P21.The above results were all contrary when PRC1 was overexpression.3.PRC1 could promote the invasion and metastasis ability of ovarian cancer cellsTranswell assay confirmed that PRC1 could significantly enhance the invasion and metastasis ability of ovarian cancer cells and enhance the healing ability when PRC1 was overexpression.Knockdown of PRC 1 significantly reduced the ability of ovarian cancer cells to invade and metastasize.Western blot analysis of EMT-related proteins showed that PRC 1 knockdown could significantly down-regulate the expression of N-cadherin,MMP9,and Slug proteins,and up-regulate the expression of E-cadherin protein.4.PRC1 could mediate multiple drug resistance of ovarian cancer cellsImmunohistochemistry confirmed that PRC1 was overexpressed in platinum-resistant HGSOC tissues.MTT confirmed that overexpression of PRC 1 can reduce the sensitivity of paclitaxel,cisplatin and doxorubicin liposomes chemotherapy.Western blot showed that after PRC1 was knockdown,the expression of C-MYC gene could be significantly down-regulated,and the expressions of DNA damage repair related proteins BRCA1,PARP1 and RAD51 could be significantly down-regulated.5.FOXM1 could transcribe and activate PRC1 promoter and control its expressionImmunohistochemical studies of HGSOC patients and TCGA database studies showed that FOXM1 was correlated with PRC1 expression.The mRNA and protein expression levels of PRC 1 were significantly decreased in ovarian cancer cell lines treated with FOXM1 si-RNA.Dual-luciferase reporter assay showed that FOXM1 regulated the expression of PRC1 by binding directly to-1440～-1445bp of PRC1 promoterSi-RNA of PRC1 was transient in FOXM1 overexpressed cell lines,and transwell assay showed that PRC1 interference could partially eliminate the enhanced invasion and metastasis ability of ovarian cancer cells caused by FOMX1 overexpression.Meanwhile,when si-RNA of FOXM1 was transiently transferred in ovarian cancer cell lines overexpressed by PRC1,the enhanced invasion and metastasis ability caused by PRC1 overexpression could also be significantly inhibited.Conclusion:1.The expression of PRC 1 in high-grade serous carcinoma was higher than that in normal tubal umbrella tissue;2.PRC1 can enhance the proliferation,invasion,migration and drugs resistance of。ovarian cancer cells;3.PRC1 is regulated by the direct transcription of FOXM1.Part Ⅱ:Clinical characteristics of PRC1 in high-grade serous ovarian cancerBackground:Tumor heterogeneity is a key factor leading to tumor invasiveness and drug resistance.High tumor heterogeneity can lead to faster tumor progression and increased drug resistance to chemotherapy,while most of the heterogeneity in tumors is caused by chromosome instability.Another significant feature of tumor cell is the lack of regulation of cell cycle,resulting in uncontrolled proliferation,secondary metastasis,drug resistance and other biological behaviors.With further explore the mechanisms of the human to tumor formation,many involved in human cancer pathophysiology mechanisms have been used as therapeutic targets,including microtubule targeted drugs,such as taxol and vincristine can interfere with the cell cycle in the spindle assembly and the separation of chromosomes,so as to achieve anti-tumor effect,is widely used in clinic.With repeated recurrence of the tumor,drug resistance to these drugs will gradually develop,and anti-microtubules themselves have many limitations,such as hypersensitivity,cardiovascular toxicity,neurotoxicity,and so on.Therefore,it is of great significance to explore new anti-microtubule targeting drugs.PRC 1,as one of the cycle-related proteins,is involved in the key steps of cytokinesis.PRC1 overexpression can lead to abnormal cell division and polyploid formation,thus exacerbating chromosome instability and further leading to tumor heterogeneity,thereby leading to tumor proliferation,invasion and drug resistance.Therefore,targeting PRC1 for anti-tumor therapy may have potential application value,In high-grade serous carcinoma,the expression of PRC1 and its correlation with clinical features have not been reported.To this end,we conducted this study to explore the relationship between the expression of PRC1 and the clinical characteristics,chemotherapy sensitivity,prognosis and other aspects of HGSOC patients,and analyze the correlation between PRC1 and BRCA1/2 mutations,so as to provide a theoretical basis for targeting PRC1 therapy in HGSOC.Materials and methods:TCGA,GSE9891 and other databases were used to analyze the effect of PRC1 on survival in ovarian cancer patients.Immunohistochemical staining analysis was performed on HGSOC tissues constructed in the laboratory,and relevant clinical information was summarized,such as age of onset,FIGO stage,ascites,residual lesions,recurrence interval,sensitivity of platinum chemotherapy,etc.,to study the correlation between PRC1 expression and the above clinical characteristicsThe correlation between the expression of PRC1 and the prognosis of HGSOC patients was analyzed,and the related factors affecting the prognosis were further analyzed.The NGS sequencing platform was used to detect BRCA1/2 mutation,to explore the correlation between PRC1 expression and BRCA1/2 mutation,and to further analyze the clinical characteristics.Results:1.Correlation between PRC1 expression and clinical characteristics.In this study,PRC1 expression and clinical characteristics of 216 HGSOC patients were summarized,and the analysis found that the expression of PRC 1 was not significantly correlated with patients’ age,FIGO stage,CA-125 level,ascites,residual lesions,or family history of malignant tumor.2.Overexpression of PRC 1 could reduce the sensitivity of first-and second-line platinum-based chemotherapy in HGSOC patients.Among 94 HGSOC patients with FIGO Ⅲ-Ⅳ stage who received satisfactory cytoreductive surgery,87 patients(92.6%)achieved partial or complete remission,among which 47 patients(54.0%)were high expression of PRC 1.Seven patients(7.4%)presented primary platinum-resistance and all showed high expression of PRC1,with statistically significant differences between the two groups(p=0.019).In addition,the second-line platinum-based chemotherapy information of 81 patients with platinum-sensitive recurrence was obtained.It was found that 29 patients(29/60,48.3%)had high PRC1 expression in the patients with effective platinum treatment,while 18 patients(18/25,72.0%)had high PRC1 expression in the patients with failed platinum treatment,with statistical difference(p=0.028).3.Overexpression of PRC 1 could shorten the interval of the second recurrence in HGSOC patientsAmong the 88 patients who achieved partial or complete response at first-line treatment,31 patients(31/47,66.0%)with high expression of PRC 1 had sensitive recurrence,with an average recurrence interval of 11.8 months.Sensitivity recurrence was found in 33 patients with low expression(33/41,80.1%),with an average recurrence interval of 14.7 months.The proportion of sensitivity recurrence in patients with low expression was slightly higher than that in patients with high expression,but there was no statistical difference(p=0.127).In the analysis of 31 patients with partial or complete remission after second-line treatment,3 patients(3/14,21.4%)with high expression of PRC 1 had secondary sensitive recurrence,with an average recurrence interval of 4.9 months.There were 12 cases of secondary sensitive recurrence with low expression(12/17,70.6%),and the mean recurrence interval was 7.3 months.High expression of PRC 1 could significantly shorten the second recurrence interval(p=0.011).4.Overexpression of PRC 1 indicated a poor prognosis in HGSOC patientsAnalysis of All databases,GSE17260,GSE9891 and GSE17260 databases revealed that overexpression of PRC 1 could significantly shorten the patients’progression-free survival and overall survival.Analysis of HGSOC patients included in the study showed that the 3-year and 5-year survival rates of patients with high PRC1 expression were lower than those with low PRC1 expression(50.0%vs 73.3%,p=0.006:36.8%vs 62.7%,p=0.003).Overexpression of PRC1 could significantly shorten the overall survival of patients(Median OS,61.0 vs 36.0 months,p=0.012),Multivariate Cox regression analysis revealed that a high expression of PRC 1 protein was a unique independent prognostic factor for HGSOC patients(HR=1.970,95%Cl 1.147-13.384,p=0.014).5.PRC1 was overexpressed in BRCA1/2 wild-type patients and contributed to a poor prognosis.Retrospective analysis of the germline BRCA1/2 sequencing results of 42 HGSOC patients showed that in the BRCA1/2 wild-type patients,the proportion of patients with high PRC1 expression was significantly higher than that of the mutant patients(60.0%vs 23.5%,P=0.019),and high PRC 1 expression could reduce the overall survival of HGSOC patients with BRCA1/2 wild-type(Media OS,69.0 vs 45.0 months,P=0.030).Conclusion:1.PRC1 overexpression could reduce the sensitivity of Platinum-based chemotherapy in HGSOC patients;2.PRC1 overexpression could contribute to early recurrence in HGSOC patients;3.PRC1 overexpression indicated a poor prognosis in HGSOC patients and was an independent prognostic risk factor;4.PRC1 was abnormally overexpressed in HGSOC patients with BRCA1/2 wild-type,which could lead to a poor prognosis.