Expression Of COL5A2 In Ovarian Tumor Microenvironment And Its Mechanism Of Promoting Ovarian Cancer

BackgroundOvarian cancer is the main cause of the death of gynecological tumors in the world.Due to the lack of more effective early diagnosis methods,high recurrence rate and chemoresistance,the prognosis of most patients with ovarian cancer is still poor.Therefore,it is urgent to explore the mechanism of the occurrence and development of ovarian cancer and find more effective ovarian cancer targets.Recent studies have shown that the research of tumor cells alone cannot explain many phenomena in tumors,so the concept of tumor microenvironment has attracted more and more attention in tumor research.In addition to tumor cells,tumor microenvironment mainly includes extracellular matrix,cancer associated fibroblasts(CAFs),extensive vascular network and immune cells.Studies have found that tumor cells need to interact with other cells,especially cancer associated fibroblasts to promote tumor progression.Exosomes can stably carry mi RNA,lnc RNA,protein molecules and other substances which are not easy to be degraded in the tumor microenvironment,so exosomes play a key role in the interaction between tumor cells and other cells.COL5A2 belongs to collagen family and is an important part of extracellular matrix(ECM)in tumor microenvironment.Its high expression was found in proteomics and database of a variety of tumors and is closely related to the poor prognosis of tumors.Therefore,COL5A2 may be a potential target for tumor therapy.However,at present,the research on COL5A2 mainly focuses on the detection of clinical samples,and its specific mechanism in tumor is not clear.In the tumor microenvironment,collagen is closely related to cancer associated fibroblasts.Therefore,taking COL5A2 as the core to clarify the specific mechanism of the interaction between ovarian cancer cells and ovarian cancer associated fibroblasts in the ovarian tumor microenvironment can provide a theoretical basis for the development of new treatment strategies for ovarian cancer.ObjectiveThis study aims to explore the expression and main cell sources of COL5A2 in ovarian tumor microenvironment and take exosomes as the beginning point to explore the specific regulatory mechanism of COL5A2 expression in ovarian tumor microenvironment.At the same time,this study clarifies the effect and specific mechanism of COL5A2 expression changes on the progression of ovarian cancer in the ovarian tumor microenvironment.Methods1.Study on the mechanism of COL5A2 expression in ovarian tumor microenvironment(1)Normal ovarian tissues and ovarian cancer tissues were collected for immunohistochemical staining of COL5A2 to detect the expression of COL5A2 in ovarian cancer.And the correlation between COL5A2 and the prognosis of ovarian cancer was analyzed by Kaplan-Meier plotter database.(2)The main cell sources of COL5A2 were analyzed by Human Protein Atls database.Ovarian cancer and normal ovarian tissues were collected for Masson staining and Immunohistochemical staining of ?-SMA to detect the relationship between collagen deposition and cancer associated fibroblasts in ovarian cancer.Primary human ovarian cancer associated fibroblasts were extracted by collagenase digestion,and the extracted primary human ovarian cancer associated fibroblasts were identified by Western blot,cellular immunofluorescence assay and optical microscope.The expression of COL5A2 protein,m RNA and secretion of COL5A2 protein in primary human ovarian cancer associated fibroblasts and ovarian cancer cell lines ES-2 and SKOV3 were detected by Western blot,q RT-PCR and ELISA to study the main cell sources of COL5A2 in the ovarian tumor microenvironment.(3)The supernatant of ovarian cancer cell lines ES-2 and SKOV3 and the supernatant of ovarian cancer cell lines ES-2 and SKOV3 treated with exosome inhibitor GW4869 were co-incubated with CAFs.The expression of COL5A2 protein and m RNA and the secretion of COL5A2 protein were detected by Western blot,q RTPCR and ELISA.(4)The exosomes of ovarian cancer cells were extracted by ultracentrifugation,and identified by electron microscope,Western blot and dynamic light scattering.The extracted ovarian cancer cell exosomes were co-incubated with CAFs,and the effect of ovarian cancer cell exosomes on the activation function of CAFs and the expression,secretion of COL5A2 protein were studied by Western blot,q RT-PCR,ELISA,and cell contraction assay.(5)ITGB1 regulating COL5A2 was screened by bioinformatics analysis.The stable cell line with knockdown of ITGB1 was constructed by lentivirus transfection.Ovarian cancer cell exosomes and ovarian cancer cell exosomes after knockdown of ITGB1 were extracted and incubated with CAFs.The effects of ITGB1 carried by ovarian cancer cell exosomes on the activation function of CAFs and the expression,secretion of COL5A2 protein were studied by Western blot,q RT-PCR,ELISA and cell contraction assay.2.Study on the mechanism of COL5A2 on the proliferation,migration and invasion of ovarian cancer(1)The effect of COL5A2 on ovarian cancer in nude mice was studied by establishing subcutaneous tumor model and intraperitoneal implantation and metastasis model,detecting tumor size and quality,drawing growth curve,calculating Ki67 positive rate and counting the number of metastatic tumors in liver and intestine.The effect of COL5A2 on the proliferation of ovarian cancer was detected by CCK8 experiment and Ed U experiment,and the effect of COL5A2 on the migration and X invasion of ovarian cancer was detected by cell scratch experiment and Transwell experiment.(2)The possible receptors of COL5A2 were screened by bioinformatics analysis.Silencing the receptor with small interfering RNA or blocking the receptor binding site with blocking antibody,and then adding COL5A2.The effect of COL5A2 on the proliferation of ovarian cancer after silencing or blocking the receptor was detected by CCK8 experiment and Ed U experiment.The effect of col5a2 on the migration and invasion of ovarian cancer after silencing or blocking the receptor was detected by cell scratch experiment and Transwell experiment.Then the colocalization of COL5A2 and the receptor was detected by cellular immunofluorescence assay.(3)Identify the downstream signal pathway of COL5A2.COL5A2 was coincubated with ovarian cancer cells,and the expressions of p-FAK,p-PI3 K and p-AKT in ovarian cancer cells were detected by Western blot.Then,after silencing or blocking the receptor,COL5A2 was added to detect the expression of p-FAK,p-PI3 K and pAKT in ovarian cancer cells by Western blot.Finally,FAK/PI3K/Akt signal pathway inhibitor was added.The correlation between the activation of FAK/PI3K/Akt signal pathway and the effects of col5a2 on the proliferation,migration and invasion of ovarian cancer was detected by Western blot,CCK8 experiment,Ed U experiment,cell scratch experiment and Transwell experiment.Results1.Study on the mechanism of COL5A2 expression in ovarian tumor microenvironment(1)COL5A2 is highly expressed in ovarian cancer tissues compared with normal ovarian tissues.And the prognosis of ovarian cancer was worse when COL5A2 was highly expressed by database analysis.(2)Database analysis showed that COL5A2 mainly came from fibroblasts.Collagen deposition in ovarian cancer is closely related to cancer associated fibroblasts.Subsequently,primary human ovarian cancer associated fibroblasts were successfully extracted.Through verification in the extracted primary human ovarian cancer associated fibroblasts and ovarian cancer cell lines ES-2 and SKOV3,it was found that COL5A2 mainly came from cancer associated fibroblasts,not ovarian cancer cells.(3)The ability of ovarian cancer cell supernatant treated with exosome inhibitor GW4869 to stimulate the expression of CAFs and secrete COL5A2 decreased significantly,indicating that the exosomes secreted by ovarian cancer cells play a key role in ovarian cancer cell supernatant.(4)The exosomes secreted by ovarian cancer cells can activate the function of CAFs and promote the expression and secretion of COL5A2.(5)ITGB1 is the major protein carried by ovarian cancer cell exosomes and affects the function of CAFs.ITGB1 knockdown ovarian cancer cell exosomes inhibited their ability to activate the function of CAFs and promote the expression and secretion of COL5A2.It shows that ovarian cancer cells activate the function of CAFs and promote the expression and secretion of COL5A2 by secreting exosomes carrying ITGB1.2.Study on the mechanism of the effect of COL5A2 on the proliferation,migration and invasion of ovarian cancer(1)COL5A2 can promote the proliferation,migration and invasion of ovarian cancer in vivo and in vitro.(2)After COL5A2 was co-incubated with ovarian cancer cells,the proliferation,migration and invasion of ovarian cancer cells were significantly enhanced.However,when COL5A2 was added after silencing or blocking ITGAV,the proliferation,migration and invasion of ovarian cancer cells were significantly reduced,indicating that COL5A2 can combine with ITGAV on the surface of ovarian cancer cells and promote the proliferation,migration and invasion of ovarian cancer cells.(3)After COL5A2 was co-incubated with ovarian cancer cells,the expression of p-FAK,p-PI3 K and p-AKT in ovarian cancer cells increased significantly.However,when COL5A2 was added after silencing or blocking ITGAV,the expression of p-FAK,p-PI3 K and p-AKT in ovarian cancer cells decreased significantly.Moreover,FAK/PI3K/AKT signaling pathway inhibitor can inhibit the ability of COL5A2 to promote the proliferation,migration and invasion of ovarian cancer.It shows that COL5A2 can activate the FAK/PI3K/AKT signal pathway of ovarian cancer cells by combining with ITGAV on the surface of ovarian cancer cells,so as to promote the proliferation,migration and invasion of ovarian cancer.ConclusionOvarian cancer cells activate ovarian cancer associated fibroblasts and promote their expression and secretion of COL5A2 by secreting exosomes carrying ITGB1.COL5A2,which is widely expressed and secreted,can act as the signal molecule feedback on ovarian cancer cells,bind to ITGAV receptor on the surface of ovarian cancer cells,and then activate FAK/PI3K/AKT signal pathway of ovarian cancer cells to promote the proliferation,migration and invasion of ovarian cancer.In the ovarian tumor microenvironment,there is a positive feedback mechanism between ovarian cancer cells and ovarian cancer associated fibroblasts.The interaction between them promotes the progression of ovarian cancer.Innovation and significanceThis study clarified the specific mechanism of the interaction between cancer cells and cancer associated fibroblasts in the ovarian tumor microenvironment.To clarify the role of cancer associated fibroblasts in the tumor microenvironment provides a theoretical basis for targeting cancer associated fibroblasts to treat ovarian cancer and provides a new idea for the treatment of ovarian cancer.In addition,COL5A2 is a key molecule in the interaction between ovarian cancer cells and ovarian cancer associated fibroblasts,suggesting that COL5A2 may become a new target for the treatment of ovarian cancer.