Mechanism Of Circ-PGAM1 Affecting The Biological Behaviors Of Epithelial Ovarian Cancer By Regulating The MiR-542-3p/CDC5L/PEAK1 Pathway

Objective: Epithelial ovarian cancer(EOC)is the most common type of malignant ovarian tumor,and it is mostly of late stages at the time of diagnosis.Despite continuous improvement of treatment approaches in recent years,the overall survival of epithelial ovarian cancer is still poor,mainly due to lack of effective preventive measures,difficulty of early diagnosis,and relapses caused by chemotherapy resistance.Epithelial ovarian cancer,especially type II epithelial ovarian cancer,mostly contains inheritable mutations,including mutations of TP53 gene,chromosomal instability,somatic mutations,and defects of homologous recombination,resulting in activation of a series of proto-oncogenes,inactivation of tumor suppressor genes,and abnormal signal pathways that lead to the development of epithelial ovarian cancer.Therefore,finding out the key genes and regulatory pathways in the occurrence and development of epithelial ovarian cancer can promote early diagnosis of ovarian cancer and improve the survival of ovarian cancer patients.Circular RNA(circ RNA)is an endogenous noncoding RNA with complex functions,including mi RNA sponge function as ce RNA,regulation of gene transcription and translation,and interaction with proteins.Some circ RNAs can even encode proteins.In recent years,the role of mi RNA sponges is getting to be more widely studied as in the field of malignant tumors.Circ-PGAM1 belongs to circ RNA and is transcribed from its parent gene PGAM1(Pseudopodium enriched atypical kinase 1).It had been reported that PGAM1 could promote tumor growth and metastasis in small cell lung cancer,pancreatic ductal adenocarcinoma,and oral squamous cell carcinoma.Mi R-542-3p belongs to micro RNA.It was reported that mi R-542-3p could promote the expression of P53 by activating the targets of P53,and could bind to tumor-promoting genes such as Survivin,thereby inhibiting cell proliferation,migration and invasion,and promoting the cell apoptosis,and therefore played a role in tumor suppression in various cancers.By the analysis of bioinformatic database,it was predicted that there was a binding site between circ-PGAM1 and mi R-542-3p.CDC5L(Cell division cycle 5-like)is widely expressed in mammals and participates in the regulation of pre-m RNA splicing.It can also act as a transcription factor to activate downstream target genes.It had been found that the expression of CDC5L was higher in liver cancer,colon cancer,and prostate cancer than that in normal tissues and that CDC5 L played a tumor-promoting role.By bioinformatic analysis,it was predicted that there was a binding site between mi R-542-3p and the 3’UTR of CDC5 L m RNA.PEAK1(Pseudopodium enriched atypical kinase 1)is a non-receptor tyrosine kinase with catalytic activity,which is involved in regulating the cytoskeleton and cell migration.It had been proven to play a tumor-promoting role in a variety of tumors.Through bioinformatic analysis,it was predicted that CDC5 L might regulate the expression of PEAK1 by binding to the promoter of PEAK1.Based on the above facts,this study aimed to investigate the expression of circ-PGAM1,mi R-542-3p,CDC5 L and PEAK1 in EOC and normal ovarian tissues in surgical specimens,and to study the effects of circ-PGAM1,mi R-542-3p,CDC5 L,and PEAK1 on biological behaviors of ovarian cancer cells,and further to study the underlying mechanisms of their interactions.This study focused on the expression and function of circ-PGAM1 and its related genes in EOC tissues and cells,and further aimed to reveal the molecular mechanisms of EOC occurrence and development and to search for more specific biomarks related to EOC,in order to achieve early diagnosis and treatment of EOC,and to improve the treatment effect and survival of EOC patients.Methods: 1.Epithelial ovarian cancer tissues and normal ovarian tissues were collected from patients undergoing surgeries at the Department of Obstetrics and Gynecology,Shengjing Hospital of China Medical University.Human ovarian cancer cell lines including SKOV3,CAOV3,OVCAR3,ES-2,and human embryonic kidney cells 293T(HEK-293T)were cultured.2.Real-time PCR was used to detect the expression of circPGAM1 and mi R-542-3p in ovarian cancer tissues and cells.Fluorescence in situ hybridization(FISH)experiment was used to detect the location of circ-PGAM1 and mi R-542-3p in ovarian cancer cells(CAOV3 and OVCAR3).Immunohistochemistry was used to detect the location of CDC5 L and PEAK1 proteins in ovarian cancer tissue.Western blot was used to detect the expression of CDC5 L and PEAK1 protein in ovarian cancer tissues and cells.3.Cell lines(CAOV3 and OVCAR3)with circPGAM1 silencing,mi R-542-3p overexpression and silencing,CDC5 L overexpression and silencing,and PEAK1 overexpression and silencing were constructed.CCK-8 assay was used to detect the proliferation of CAOV3 and OVCAR3.Transwell assay was used to detect the migration and invasion of CAOV3 and OVCAR3.Flow cytometry was used to detect the apoptosis of CAOV3 and OVCAR3.Western blot was used to detect the expression of CDC5 L,PEAK1,ERK1/2,p-ERK1/2,JAK1,p-JAK1,JAK2,and p-JAK2.4.Dual-luciferase reporter assay was used to detect the interactions and binding sites between circ-PGAM1 and mi R-542-3p,and between mi R-542-3p and CDC5 L.5.The binding effect of circ-PGAM1 and mi R-542-3p was detected by RNA binding protein immunoprecipitation assay(RIP),and the binding effect of CDC5 L and the PEAK1 promoter was detected by chromatin immunoprecipitation(Ch IP)experiment.6.The effect of circ-PGAM1 and mi R-542-3p on tumor growth was measured by tumor xenograft experiment in nude mice,and the expressions of CDC5 L,PEAK1,ERK1/2,p-ERK1/2,JAK2 and p-JAK2 in tumor tissues were detected.Results: 1.The expression of Circ-PGAM1 in epithelial ovarian cancer tissue was upregulated,while the expression of linear PGAM1 in epithelial ovarian cancer tissue was unchanged compared with normal ovarian tissue.The expression of circ-PGAM1 in CAOV3 and OVCAR3 was higher than the other two cell lines among ovarian cancer cell lines SKOV3,CAOV3,OVCAR3,and ES-2.Therefore,CAOV3 and OVCAR3 were selected as the cell lines to be used in this study.Circ-PGAM1 was mainly expressed in the cytoplasm.Silencing circ-PGAM1 inhibited the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,while promoted cell apoptosis.2.The expression of mi R-542-3p was down-regulated in epithelial ovarian cancer tissues.It was detected by FISH that mi R-542-3p was also mainly expressed in the cytoplasm.Overexpression of mi R-542-3p inhibited the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,while promoted cell apoptosis.3.Dualluciferase reporter assay confirmed that circ-PGAM1 and mi R-542-3p have a specific binding effect,and RIP assay confirmed that the binding of circ-PGAM1 and mi R-542-3p involved the RNA-induced silencing complex(RISC).Silencing of circ-PGAM1 promoted the expression of mi R-542-3p,while overexpression of mi R-542-3p downregulated the expression of circ-PGAM1.4.Silencing of circ-PGAM1 plus overexpression of mi R-542-3p inhibited the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,and promoted cell apoptosis.While silencing of mi R-542-3p offset the tumor suppressive effect of simply silencing circ-PGAM1.5.The CDC5L protein was mainly expressed in the cytoplasm and was also slightly expressed in the nucleus.The expression of CDC5 L in epithelial ovarian cancer tissue was significantly higher than that in normal ovarian tissue.Dual-luciferase reporter assay confirmed that CDC5 L and mi R-542-3p had a targeted binding effect.Silencing of circ-PGAM1 inhibited the expression of CDC5 L m RNA and protein;overexpression of mi R-542-3p also inhibited the expression of CDC5 L m RNA and protein.Silencing of mi R-542-3p promoted the expression of CDC5 L m RNA and protein.Silencing of circ-PGAM1 plus overexpression of mi R-542-3p significantly inhibited the expression of CDC5 L m RNA and protein,but there was no significant change in the expression of CDC5 L m RNA and protein after silencing circ-PGAM1 plus silencing mi R-542-3p.6.Overexpression of CDC5 L promoted the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,and inhitbited cell apoptosis.Silencing of CDC5 L inhibited the malignant biological behavior of CAOV3 and OVCAR3 cells.In addition,CDC5 L could directly bind to the promoter of PEAK1.Overexpression of CDC5 L promoted the expression of PEAK1 protein,and silencing of CDC5 L inhibited the expression of PEAK1 protein.7.The expression of PEAK1 protein in epithelial ovarian cancer tissue was significantly higher than that in normal ovarian tissue,and PEAK1 was mainly expressed in cytoplasm.Overexpression of PEAK1 promoted the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,inhibited cell apoptosis.Silencing o PEAK1 inhibited the proliferation,migration and invasion of CAOV3 and OVCAR3 cells,and promoted cell apoptosis.After overexpressing PEAK1,the expression levels of p-ERK1/2 and p-JAK2 increased significantly,but the expression of p-JAK1 did not change significantly,confirming that PEAK1 could regulate the activity of ERK1/2 and JAK2 signaling pathways but had no effect on JAK1 signaling pathways.8.Co-transfection of mi R-542-3p overexpression plasmid and CDC5 L overexpression or CDC5 L non-3’UTR overexpression plasmid confirmed that the proliferation,migration and invasion levels of cells co-transfected with mi R-542-3p overexpression and CDC5 L non-3’UTR overexpression plasmids were higher than those of mi R-542-3p plus CDC5 L overexpression group,and the apoptosis level was accordingly reduced.In addition,western blot assays revealed that the expression levels of PEAK1,p-ERK1/2 and p-JAK2 in the mi R-542-3p overexpression plus CDC5 L non-3’UTR overexpression group were significantly higher than those in the mi R-542-3p overexpression plus CDC5 L overexpression group.This demonstrated that mi R-542-3p inhibited the expression of CDC5 L,and then reduced the expression of PEAK1,thereby regulating the activity of ERK1/2 and JAK2 signaling pathways.9.Tumor xenograft experiment in nude mice demonstrated that silencing of circ-PGAM1 or overexpression of mi R-542-3p alone could inhibit tumor growth,while silencing of circ-PGAM1 and simultaneous over-expression of mi R-542-3p could inhibit tumor growth to the greatest extent.In addition,the expression levels of CDC5 L,PEAK1,pERK1/2,and p-JAK2 in circ-PGAM1 silencing or mi R-542-3p overexpression group were reduced,but the group of silencing circ-PGAM1 plus overexpressing mi R-542-3p had the lowest expression levels of CDC5 L,PEAK1,p-ERK1/2 and p-JAK2.Conclusions: 1.Circ-PGAM1,mi R-542-3p,CDC5 L and PEAK1 were all endogenously expressed in epithelial ovarian cancer tissues and cells.The expression of circ-PGAM1,CDC5 L and PEAK1 was up-regulated in epithelial ovarian cancer tissues while the expression of mi R-542-3p down-regulated in epithelial ovarian cancer tissues.2.Circ-PGAM1,CDC5 L and PEAK1 promoted the malignant biological behaviors of ovarian cancer cells,while mi R-542-3p inhibited the malignant biological behaviors of ovarian cancer cells.3.Circ-PGAM1 and mi R-542-3p could target each other at the predicted binding site and negatively regulate the expression of each other.Silencing of circ-PGAM1 inhibited the malignant biological behaviors of ovarian cancer cells.4.Mi R-542-3p could target the 3’UTR of CDC5 L and inhibited the expression of CDC5 L,and then played an inhibitive role in malignant biological behaviors of ovarian cancer cells.5.CDC5 L could directly bind to the promoter region of PEAK1 and promote its transcription,and promoted the malignant biological behaviors of ovarian cancer cells.6.Overexpression of PEAK1 activated ERK1/2 and JAK2 signaling pathways,and promoted the malignant biological behaviors of ovarian cancer cells.7.Silencing circ-PGAM1 and over-expressing mi R-542-3p in vivo inhibited tumor growth to the greatest extent,inhibited the expression of CDC5 L and PEAK1 proteins,and inhibited the ERK1/2 and JAK2 signaling pathways,which further demonstrated that circ-PGAM1 promoted the malignant progression of epithelial ovarian cancer by regulating the mi R-542-3p/CDC5/PEAK1 pathway.