Expression And Significance Of LRRC15 In Microenvironment Of Epithelial Ovarian Cancer

Objective:To investigate the expression and prognostic significance of leucine rich repeat sequence(LRRC)protein superfamily members in the tumor parenchyma and interstitial microenvironment of epithelial ovarian cancer.To explore the relationship between LRRC15 expression level and immune invasion of epithelial ovarian cancer as a new target for tumor diagnosis and treatment.On this basis,further analysis of its impact on primary platinum resistance.Methods:1.The clinical data and transcriptional profiles of OC patients in TCGA(N=427),GTEx(N=88)and GEO data sets(GSE40266 and GSE40595)were analyzed by R software,the differentially expressed genes of LRRC superfamily in high-grade serous ovarian cancer(HGSC)were obtained.Using GEPIA2,Kaplan-Meier Plotter,Gene MANIA,Linked Omics,TIMER2.0,UCSC Xena and other public online databases to further analyze the relationship between differential expression genes(DEGs)and clinical prognosis,immune infiltration,to construct protein interaction(PPI)network.2.The Nomogram prognosis model was constructed.Through univariate and multivariate Cox regression analysis,clinicopathologic factors such as age,stage and LRRC15 expression level were included in the prognosis nomogram.3.Through real-time quantitative reverse transcriptase polymerase chain reaction(q RT-PCR),the m RNA expression level of LRRC15 in MRC-5 cell line was detected before and after 48 hours of TGF-β1 induction.4.The tumor tissues of EOC patients who received surgical treatment in the affiliated hospital of Qingdao University from January 2014 to December 2021 were collected and followed up.20 cases of primary platinum resistance have been identified,and 30 cases of platinum sensitivity have been randomly matched from the patient database,a total of 50 tumor tissue samples.All patients obtained informed consent.Immunohistochemistry(IHC)was used to detect the expression level of LRRC15,PDPN and CD8 in the above samples and analyze their relationship with primary platinum resistance.The diagnostic value of LRRC15 in EOC was evaluated by drawing receiver operating characteristic curve(ROC).Results:1.Comprehensive analysis of public databases found that LRRC15 and LRRC32 were differentially expressed and significantly enriched in TGF-βprotein family-related pathways in HGSC Stroma and cancer associated fibroblast(CAFs),it is suggested that the LRRC superfamily may be involved in the tumor-promoting effect of TGF-βin the progression of HGSC.Only LRRC15 remained highly expressed in intact tumor tissues,including the stroma and parenchyma,and LRRC32 showed an overall downward trend in EOC tumor tissues.High expression of LRRC15 is associated with worse clinical outcomes.In the aspect of immune infiltration,the high expression of LRRC15 was positively correlated with the infiltration of CAFs,and negatively correlated with the infiltration of CD8+T cells and B cells(p<0.05).2.Using TCGA-OV data,Nomogram prognosis model was established.The 1-year,3-year and 5-year overall survival(OS)calibration curve in the validation group is close to the ideal curve,indicating that the Nomogram model can accurately predict the prognosis of OC patients.3.The q RT-PCR results found that LRRC15 was consistent with PDPN(podoplanin),both showing higher expression in MRC-5 cell lines 48 h after TGFΒ1induction(P<0.05).It is suggested that LRRC15 is indeed highly expressed in CAFS and may be involved in the regulation of TGF-βpathway together with PDPN.4.IHC staining confirmed that LRRC15 was always expressed at the same location as PDPN,and LRRC15 was more widely expressed in matrix components.The infiltration of CD8+T cells decreased with the increase of LRRC15 expression in tissues of patients with advanced OC.Spearman correlation analysis showed that LRRC15 was positively correlated with PDPN expression(P<0.05),negatively correlated with CD8 expression(P<0.05),and PDPN was negatively correlated with CD8 expression(P<0.05).The expression of LRRC15 contributes to the proliferation of PDPN~+CAF and promotes the formation of mesenchymal ovarian cancer.To some extent,it inhibits the infiltration of CD8+T cells and promotes the formation of immunosuppressive microenvironment.5.Further statistical analysis showed that the expression of LRRC15 and PDPN in platinum-resistant group was significantly higher than that in platinum-sensitive group,while the expression of CD8 was the opposite(P<0.001).The diagnostic ROC curve observed that the predictive ability of LRRC15 was relatively accurate in predicting drug resistance and sensitivity results(AUC=0.818,CI=0.699-0.938).This suggests that LRRC15 may be a potential therapeutic target for reversing the primary platinum resistance of OC.Conclusions:1.LRRC15 and LRRC32 in the LRRC superfamily may be involved in the tumor-promoting role of TGFΒpathway in the progression of HGSC.2.LRRC15,as a biomarker with high expression in stroma,suggests that EOC has a poor prognosis and promotes the formation of immunosuppressive microenvironment.3.LRRC15 may be a potential therapeutic target for reversing primary platinum resistance in OC.