The Association Between NORAD And The Transformation Of Ovarian Carcinoma Associated Fibroblasts

Objective:The purpose is to investigate the expression level and clinical significance of long non-coding RNA(lnc RNA)non-coding RNA activated by DNA damage(NORAD)in epithelial ovarian carcinoma(EOC)tissues and plasma samples.We also analyze the diagnostic value of NORAD in EOC and the prognostic value of NORAD in ovarian carcinoma(OC).Based on this foundation,the expression level of NORAD in the tumor microenvironment of OC and its correlation with the transformation of ovarian cancer-associated fibroblasts(OCAFs)were investigat ed.Methods:1.With high-throughput transcriptome sequencing of lnc RNA,the serum exosomes from patients with ovarian high-grade serous carcinoma and healthy volunteers was performed on the Illumina sequencing platform by RNA-sequencing(RNA-seq)technology.Then,we screened out NORAD with the most significant differential expression levels.2.The preoperative plasma samples and EOC tissues of 50 patients with EOC who underwent surgery in the Affiliated Hospital of Qingdao University from November 2019 to December 2020 were collected.And the preoperative plasma samples,normal ovarian tissues and normal oviduct tissues of 50 patients who underwent hysterectomy and bilateral adnexectomy due to uterine myoma in the same period were collected.The expression level of NORAD in the above samples was detected by real-time quantitative reverse transcription polymerase chain reactions(q RT-PCR).The clinical data of the above patients were collected to analyze the relationship between the expression level of NORAD a nd the clinicopathological parameters(age,FIGO stage,differentiation,histotype,TP53 status,ascites and lymph node metastasis)of EOC patients.The diagnostic value of NORAD in EOC was evaluated by drawing receiver operating characteristic curve(ROC),and the survival analysis was carried out by using Kaplan Meier plotter database.3.The datasets of GSE40595 and GSE40266 were analyzed by R2 database platform.The expression level of NORAD in the stromal tissue of ovarian carcinoma was analyzed.And the expression level of NORAD was analyzed in human telomerase-immortalized ovarian fibroblast line NOF151 treated with 5ng/m L of TGF-β1 for 48 hours.After stimulated by TGF-β1 or indirectly co-cultured with SKOV3,human embryonic lung fibroblasts MRC5 was detected by q RT-PCR to test the expression of α-smooth muscle actin(α-SMA)and fibroblast activation protein(FAP).And we also detected the expression level of NORAD after the transformation of MRC5 into OCAFs.We use si RNA to interfere with the expression of NORAD in OCAFs.QRT-PCR and cellular immunofluorescence were used to detect the changes of related gene expression levels,which confirmed that NORAD was related to the transformation of OCAFs.Results:1.On the illumina sequencing platform,high-throughput transcriptome sequencing of lnc RNA found that NORAD was overexpressed in serum exosomes from patients with ovarian high-grade serous carcinoma(P<0.05).2.The expression level of NORAD in EOC tissues was higher than that in normal ovarian tissues and normal oviduct tissues.The expression level of NORAD was correlated with FIGO stage,histotype,ascites and lymph node metastasis(all P<0.05).The expression level of NORAD in the plasma of EOC patients was higher than that of the control group,and was correlated with FIGO stage and ascites(all P<0.05).The expression level of NORAD in EOC plasma has certain diagnostic value(AUC=0.772,95%CI=0.676～0.869).The high expression of NORAD is closely related to the 5-year progression-free survival(PFS)of OC patients(P<0.05).The expression level of NORAD in OC tissues has prognostic value in FIGO stage,differentiation,TP53 status and chemotherapy treatment(all P<0.05).3.By analyzing datasets of GSE40595 and GSE40266 in R2 database,we confirmed the overexpression of NORAD in the stromal tissue of ovarian carcinoma(P<0.05).And we also found that NORAD was overexpressed in human telomerase-immortalized ovarian fibroblasts treated with 5ng/m L of TGF-β1 for 48hours(P<0.05).Accordingly,we speculate that NORAD is overexpressed in the ovarian carcinoma microenvironment and can affect the transformatio n of OCAFs.Currently,the identification of cancer associated fibroblasts(CAFs)is mainly by detecting the expression levels of specific expression proteins such as α-SMA and FAP.By experiments,we confirmed that the expression levels of α-SMA and FAP in MRC5 were increased significantly after the stimulation of 5 ng /ml TGF-β1 or indirectly co-cultured with SKOV3 for 24 hours(all P < 0.05).That is,after indirect co-culture,normal fibroblasts(NFS)were transformed into OCAFs.In addition,the expression level of NORAD in OCAFs was increased compared to the NFs(P <0.05).We further found that the expression of NORAD in fibroblasts was regulated by TGF-β1 and showed the dependence of time and concentration(all P<0.05).By interfering with the expression of NORAD in OCAFs by si RNA,the expression of NORAD was significantly decreased and the transformation of OCAFs was inhibited(all P<0.05).Conclusions:1.NORAD was found to be overexpressed in serum exosomes from patients with ovarian high-grade serous carcinoma by high-throughput sequencing.2.NORAD is highly expressed in EOC tissue and plasma,and provides a theoretical basis of the diagnosis and prognosis of OC.3.NORAD is related to the transformation of OCAFs,suggesting that NORAD may become a new target for OC treatment.