Objective:To investigate the effect of nuclear receptor subfamily 2 group F member 2(NR2F2)on the biological behaviors of human ovarian cancer SKOV3 cells,and to clarify its molecular mechauism and provide the new idea for treatment of ovarian cancer.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)Database analyse the expression level of NR2F2 gene in ovarian tissue,and analyse its correlation with clinical prognosis of ovarian cancer patients.The human ovarian cancer SKOV3 cells were divided into control group and NR2F2 over-expression(NR2F2 OE)group,which were transfected with mCherry control virus and NR2F2 OE over-expression virus,respectively,when the cell deusity reached 70％,and the stable transfection SKOV3 cell lines were screened with puromycin(puro)48h lafter.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the transfection efficiencies of the cells;RT-qPCR method was used to detect the expression levels of NR2F2 and sex-determining region Y-box 2(SOX2)mRNA in the cells in two groups;Western blotting method was used to detect the expression levels of NR2F2,ATP-binding cassette superfamily G member 2(ABCG2),and programmed cell death 1-ligand 1(PD-L1)protcins in the cells in two groups.CCK-8 assay was used to detect the proliferation activities of the cells in two groups;Wound assay was used to detect the migration rates of the cells in two groups;Transwell chamber assay was used to detect the number of transmembrane cells;Spheroidization assay was used to detect the numbers of spheroids in the cells;peripheral blood mononuclear cells(PBMCs)-mediated tumor cell killing assay was used to detect the relative densities of surviving tumor cells;CCK-8 assay was used to detect the half maximal inhibitory concentration(IC50)of paclitaxel(PTX)and carboplatin(CBP).Results:Compared with normal ovarian tissue,the expression level of NR2F2 gene in ovarian tumor tissue was decreased(P＜0.05),and decreased with the improvement of clinical pathological grading of ovarian tumor.The patients with higher expression level of NR2F2 gene had better clincal prognosis.The SKOV3 cells with NR2F2 over-expresson were successfully constructed,and the expression levels of NR2F2 mRNA and protein in the cells in NR2F2 OE group were increased compared with control group(P＜0.001).The CCK-8 assay results showed that compared with control group,the proliferation activities of the cells in NR2F2 OE group were decreased at different time points(1,2,3,and 4 d)(P＜0.05 or P＜0.01).The cell wound assay results showed that compared with control group,the migration rate of the cells in NR2F2 OE group was decreased(P＜0.001).The Transwell assay results showed that compared with control group,the number of transmembrane cells in NR2F2 OE group was decreased(P＜0.01).Compared with control group,the number of the spheroids in NR2F2 OE group was decreased(P＜0.05),and the expression levels of SOX2 mRNA(P＜0.01)and protein(P＜0.001)were increased.Compared with control group,the relative density of surviving tumor cells in NR2F2 OE group was decreased,but the difference was not significant(P＜0.05),and the expression level of PD-L1 protein was decreased(P＜0.05).Compared with control group,the proliferation activities of cells in NR2F2 OE group were decreased(P＜0.05),and the drug sensitivities of the cells to PTX and CBP were enhanced(P＜0.05);the IC50 of PTX was significantly reduced,while the IC50of CBP could not be calculated due to excessively high drug concentration;the expression level of ABCG2 protein was decreased(P＜0.05).Conclusion:The over-expression of NR2F2 may inhibit the proliferation,migration,and invasion of the human ovarian cancer SKOV3 cells,decrease the expression levels of SOX2,PD-L1 and ABCG2 proteins,suppress the stemness and immune evasion ability of the SKOV3 cells,and enhance the sensitivities of the SKOV3 cells to PTX and CBP.

Objective:To discuss the effect of long non-coding RNA(lncRNA)LINC00973 on the migration,invasion,and distant metastasis of epithelial ovarian cancer,and to clarify its molecular mechanism.Methods:The human ovarian cancer SKOV3 and OVCAR3 cells were divided into EF1a-FH empty vector control group(control group),LINC00973 overexpression group(LINC00973 OE group),U6-shRNA empty vector control group(SHV group),and LINC00973 knockdown group(LINC00973 KD group),and were transfected with lentivirus containing nonsense sequence(pLent-EF1a-FH-CMV-copGFP-P2A-Puro),LINC00973 overexpression,nonsense sequence(pLent-U6-shRNA-CMV-copGFP-P2A-Puro)and LINC00973 shRNA,respectively,followed by puromycin screening to obtain stably transfected SKOV3 and OVCAR3 cells.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of target genes in the cells in various groups;wound healing assay was used to detect the migration rate of the cells in various groups;Transwell chamber assay was used to detect the number of transmembrane cells in various groups;The mice were divided into control group(WT group),LINC00973 OE group,and LINC00973 KD group,with 4 mice in each group.SKOV3 wild-type cells,LINC00973 OE cells,and LINC00973 KD cells were intraperitoneally injected into the mice in various groups,respectively,to establish the epithelial ovarian cancer intraperitoneal implantation and metastasis model;HE staining was used to observe the morphology of the colon and liver tissues of the mice in various groups;RNA-secquencing(RNA-seq)was used to analyze the differentially expressed genes between SHV and LINC00973 KD groups in the SKOV3 cell line;RT-qPCR method was used to detect the mRNA expression levels of LINC00973 in the normal ovarian epithelial cells IOSE-80 and epithelial ovarian cancer cells SKOV3,A2780 and OVCAR3,the mRNA expression levels of LINC00973,Vimentin,Snail family transcriptional repressor 1(Snail),Twist family basic helix-loop-helix transcription factor 1(Twist),zinc finger E-box binding homeobox 1(ZEB1),zinc finger E-box binding homeobox 2(ZEB2),CXCL8 and matrix metalloproteinase(MMP)16 in the cells in various groups,and the mRNA expression levels of LINC00973,Vimentin and Twist in liver and colon tissues of the mice in various groups.Results:Compared with normal ovarian epithelial cells IOSE-80,the expression level of LINC00973 mRNA in the epithelial ovarian cancer cells SKOV3,OVCAR3 and A2780 was significantly increased(P<0.01),with the highest expression level of LINC00973 in SKOV3 and OVCAR3 cells,which were therefore selected for subsequent experiments.In SKOV3 and OVCAR3 cells,compared with control group,the expression level of LINC00973 mRNA in the cells in LINC00973 OE group was increased(P<0.01);compared with SHV group,the expression level of LINC00973 mRNA in the cells in LINC00973 KD group was decreased(P<0.05 or P<0.01),indicating successful construction of LINC00973 overexpression and knockdown cell lines.In SKOV3 cells,compared with control group,the mRNA expression levels of Vimentin and Twist in LINC00973 OE group were increased(P<0.05 or P<0.01),while no significant difference was observed in Snail mRNA expression level(P>0.05);compared with SHV group,the mRNA expression levels of Vimentin,Snail and Twist in LINC00973 KD group were decreased(P<0.01).In OVCAR3 cells,compared with control group,the mRNA expression levels of Vimentin,Snail and Twist in LINC00973 OE group were increased(P<0.01);compared with SHV group,the expression levels of Vimentin,Snail,and mRNA Twist in LINC00973 KD group were decreased(P<0.01).The wound healing assay results showed that compared with control group,the wound healing rates of the SKOV3 and OVCAR3 cells in LINC00973 OE group were significantly increased(P<0.01);compared with SHV group,the wound healing rates of the cells in LINC00973 KD group were significantly decreased(P<0.01).The Transwell chamber assay results showed that compared with control group,the numbers of transmembrane cells of the SKOV3 and OVCAR3 cells in LINC00973 OE group were significantly increased(P<0.01);compared with SHV group,the numbers of transmembrane cells in LINC00973 KD group were significantly decreased(P<0.01).Compared with WT group,the number of peritoneal nodules in LINC00973 OE group was increased,with rough liver surface and multiple nodules formed on mesentery and colon surface,and the expression levels of LINC00973,Vimentin,and Twist mRNA in colon tissue were increased(P<0.01);compared with WT group,no nodules were formed in the peritoneal cavity of LINC00973 KD group,with smooth liver surface,no nodules in liver tissue,and decreased expression levels of LINC00973,Vimentin,and Twist mRNA,and no nodules were observed on mesentery and colon surface.The HE staining results showed that compared with WT group,the multiple lesions were observed in liver and colon tissues in LINC00973 OE group,manifested as uneven cell size,irregular shape,unclear cell boundaries,increased nuclear division,and uneven red staining in cytoplasm,while in LINC00973 KD group,the cells in liver and colon tissues were arranged neatly with regular shape,and uniform distribution of nuclei and cytoplasm.The RNA-seq results showed that compared with SHV group,no key signaling pathways related to tumor metastasis were enriched in LINC00973 KD group,and the transcription levels of metastasis-related genes CXCL8,MMP16,ZEB1,and ZEB2 were decreased.The RT-qPCR results showed that compared with control group,the expression levels of ZEB1,ZEB2,CXCL8,and MMP16 mRNA in the cells in LINC00973 OE group were significantly increased(P<0.01);compared with SHV group,the expression levels of ZEB1,ZEB2,CXCL8,and MMP16 mRNA in the cells in LINC00973 KD group were significantly decreased(P<0.01).Conclusion:LINC00973 can up-regulate the expression of metastasis-related factors Vimentin,Snail,Twist,ZEB1,ZEB2,CXCL8,and MMP16,and promote the migration,invasion,and distant metastasis of epithelial ovarian cancer.

Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.