| Objective:To detect the expression levels of estrogen receptor-related receptor α(ERRα)and peroxisome proliferator-activated receptor γ(PPARγ)genes in immortalized ovarian epithelial cells IOSE80,ovarian cancer cell HO8910 and its highly metastatic sub-strain HO8910 PM cell,to investigate the effect of regulating ERRα or PPARγ on the expression of each other,and analyze the effect and potential mechanism of regulating ERRα or PPARγ on the invasion and migration ability of ovarian cancer cell.Methods: The human immortalized ovarian epithelial cell IOSE80 and ovarian cancer cell lines HO8910 and HO8910 PM were established in vitro.The m RNA and protein expression levels of ERRα and PPARγ in the three cells were detected by real-time q PCR and Western blot techniques.Lentiviral eukaryotic cell over-expression vectors containing full-length ERRα and PPARγ c DNAs were constructed.HO8910 cells were selected to transfect ERRα over-expression vector to construct over-expressing strain OE-ERRα;HO8910PM cells were selected to treat with ERRα specific inhibitor XCT790,and the effect of regulating ERRα on PPARγ expression was observed.HO8910 cells were selected to establish the PPARγ over-expressing strain OE-PPARγ in the same way,HO8910 PM cells were selected to treat with PPARγ specific inhibitor GW9662,and the effect of regulating PPARγ on ERRα was observed.The cell scratch test and the trans-membrane cell experiment were used to observe the differences in the invasion and migration ability of ovarian epithelial cell and ovarian cancer cells and the changes in invasion and migration ability after regulating the expression levels of ERRα and PPARγ genes.Western blot technique was used to detect the expression of epithelial marker E-cadherin and interstitial marker Vimentin in cells after different treatments.Bioinformatics was used to analyze the correlation between ERRα and PPARγ proteins,the proteins interacting with them,and their association with epithelial‐mesenchymal transformation markersResults: 1.Compared with the ovarian epithelial cell IOSE80,the expression levels of ERRα gene m RNA in ovarian cancer cell HO8910 and high-metastasis sub-strain HO8910 PM cells were higher,and the relative expression levels were 1.24±0.10 and 3.09±0.35 times of normal epithelial cells(P all <0.05).Similarly,compared with the ovarian epithelial cell IOSE80,the ovarian cancer cells HO8910,HO8910 PM PPARγ gene m RNA expression levels are also higher,the expression levels were 2.14±0.66 and 72.12±7.47 times of normal epithelial cells(P all <0.05).Western blot analysis showed that the expression of PPARγ protein in IOSE80,HO8910 and HO8910 PM coincided with the m RNA level,showing a gradual upward trend(0.04±0.001,0.85±0.05,0.99±0.06,P<0.05).The expression of ERRα protein in HO8910 and HO8910 PM is consistent with the m RNA expression trend(0.55±0.14,0.64±0.02,0.88±0.11,P HO8910 vs HO8910PM<0.05,P IOSE80 vs HO8910PM<0.05),but the expression level of ERRα in HO8910 is higher than that of IOSE80,and the difference is not significant(P IOSE80 vs HO8910>0.05).2.The ERRα over-expression lentiviral vector was transfected into ovarian cancer cell HO8910 to establish ERRα high expression cell group OE-ERRα.Compared with HO8910,the expression levels of ERRα and PPARγ m RNA in OE-ERRα cell increased significantly(11.01±2.16 times,63.07±18.90 times,P all<0.01),the protein expression of ERRα and PPARγ also increased significantly(0.64±0.02 vs 1.71±0.04,0.83±0.08 vs 1.37±0.08,P all<0.01);compared with non-drug-treated ovarian cancer cell HO8910 PM,the expression of ERRα and PPARγ m RNA in the XCT790-treated group was significantly reduced(0.16±0.03 times,0.008±0.001 times,P all<0.01),The expression levels of ERRα and PPARγ proteins were significantly reduced(0.87±0.11 vs 0.38±0.03,0.97±0.02 vs 0.64±0.08,P all<0.01).3.Transfect the PPARγ over-expression lentiviral vector into ovarian cancer cell HO8910 to establish the PPARγ high expression cell group OE-PPARγ.Compared with HO8910,the expression levels of ERRα and PPARγ m RNA of OE-PPARγ cell increased significantly(5.06±0.59 times,8.31±2.52 times,P all<0.01),the protein expression of ERRα and PPARγ also increased significantly(0.64±0.02 vs 1.32±0.03,0.83±0.08 vs 1.86±0.07,P all<0.01);compared with non-drug-treated ovarian cancer cell HO8910 PM,the expression of ERRα and PPARγ m RNA in the GW9662-treated group was significantly reduced(0.31±0.13 times,0.05±0.02 times,P all<0.01),the expression levels of ERRα and PPAR proteins were reduced(0.87±0.11 vs 0.53±0.03,0.97±0.02 vs 0.47±0.04,P all<0.01).4.The cell closure rates of the cells IOSE80,HO8910 and HO8910 PM were(16.41 ± 3.03)%,(24.33 ± 2.43)% and(36.26 ± 2.43)%,respectively.The migration ability of cells gradually increased(PIOSE80 vs HO8910<0.05,PIOSE80 vs HO8910PM<0.01,PHO8910 vs HO8910PM<0.01).After over-expressing genes ERRα and PPARγ,the OE-ERRα cell scratch area closure rate was(35.31 ± 0.56)%(P <0.01),and the OE-PPARγ cell scratch area closure rate was(30.43 ± 1.07)%(P <0.05).After inhibiting the ERRα and PPARγ of HO8910 PM cells with specific inhibitors,the closed area of the scratched area of the cells in the XCT790-treated group was(14.88 ± 1.05)%,and the closed area of the scratched area of the cell in the GW9662-treated group was 24.01 ± 1.64)%,compared with HO8910 PM,the closure rate of the scratch area of the drug-treated groups were reduced,and the differences were statistically significant(P <0.01).Pearson correlation analysis showed that ERRα m RNA expression level was positively correlated with cell scratch area closure rate(r=0.67,P <0.01),and PPARγ m RNA expression level was also positively correlated with cell scratch area closure rate(r=0.77,P< 0.01).5.The cell numbers of the cell lines IOSE80,HO8910 and HO8910 PM were(11.00 ± 3.27),(112.33 ± 12.28),(265.67 ± 30.66),and the comparison of the two cells showed that the invasion ability of the three cells gradually Increase(PIOSE80 vs HO8910<0.01,PIOSE80 vs HO8910PM<0.01,PHO8910 vs HO8910PM<0.01).After over-expression of HO8910 cell lines ERRα and PPARγ,the measured number of OE-ERRα cells and OE-PPARγ cells increased significantly(482.33 ± 24.50,PHO8910 vs OE-ERRα<0.01;607.33 ± 21.70,PHO8910 vs OE-PPARγ< 0.01).After treating HO8910 PM cells with specific inhibitors to down-regulate ERRα and PPARγ,the number of cell perforations in the XCT790-treated group was(107.67 ± 10.34),and the cell penetration in the GW9662-treated group was(131.67 ± 19.15).Compared with the drug-treated group,the number of cell penetrating membranes decreased,and the differences were statistically significant(P<0.01).Pearson correlation analysis showed that the expression level of ERRα m RNA was positively correlated with the number of trans-membrane cells(r=0.78,P<0.01),and the expression level of PPARγ m RNA was also positively correlated with the number of trans-membrane cells(r=0.58,P <0.01).6.Compared with ovarian epithelial cell IOSE80,the expression level of epithelial marker E-cadherin in cancer cell HO8910 and its high-metastasis sub-strain HO8910 PM cells is lower(0.51±0.01,0.33±0.04,0.23±0.03,P all<0.05),and the expression of interstitial marker Vimentin was higher(0.58±0.02,0.76±0.02,0.83±0.03,P all<0.05).Compared with ovarian cancer cell HO8910,the expression of E-cadherin in the OE-ERRα cell with high expression of ERRα and the OE-PPARγ cell with high expression of PPARγ both decreased(0.40±0.06,0.25±0.07,0.28±0.04,P all<0.05),while the expression of interstitial marker Vimentin increased(0.72±0.03,0.80±0.02,0.86±0.02,P all<0.05).Compared with ovarian cancer cell HO8910 PM,the expression level of epithelial marker E-cadherin in HO8910 PM cells that specifically inhibited ERRα or PPARγ was increased(0.26±0.02,0.36±0.04,0.49±0.04,P all<0.05),while the expression of interstitial marker Vimentin decreased(0.79±0.10,0.60±0.06,0.44±0.02,P all<0.05).7.ERRα and PPARγ proteins interact with NCOA1,EP300,CREBBP,LEP,ADIPOQ,CEBPB,FABP4,etc.,and these proteins are involved in gene transcription activation,transcription modification,and cellular energy metabolism.The interaction between ERRα,PPARγ proteins and epithelial-mesenchymal transition markers E-Cadherin and Vimentin was analyzed.The results showed that ERRα,PPARγ and E-Cadherin,Vimentin all have a co-expression relationship.Conclusion:1.The expression level of ERRα and PPARγ in ovarian cancer cells is higher than that of ovarian epithelial cells,and it is positively correlated with the invasion and metastasis ability of ovarian cancer cells.Cells with higher expression levels of ERRα and PPARγ have stronger invasion and metastasis ability.2.In ovarian cancer cells,up-regulating the expression of ERRα can increase the expression level of PPARγ,inhibiting the expression of ERRα and causing the expression level of PPARγ to decrease;studies on the regulation of PPARγ have shown that up-regulating the expression level of PPARγ gene leads to an increase in ERRα expression.Inhibiting the expression level of PPARγ also caused the down-regulation of ERRα expression,suggesting that there should be a synergistic effect between the two genes.3.Both up‐regulation and down‐regulation of ERRα and PPARγ can cause the metastasis and invasive ability of ovarian cancer cells to increase or decrease,and the two genes have the same synergistic effect.4.The expression level of epithelial marker E‐cadherin is lower in ovarian cancer cells with stronger invasion and metastatic ability,while the expression level of interstitial marker Vimentin is higher.The invasion and migration ability of ovarian cancer cells may be related to the epithelial‐mesenchymal transition mechanism.5.In cells with higher expression levels of ERRα or PPARγ,the expression level of E-cadherin is lower,while that of Vimentin is higher;in cells with lower expression levels of ERRα or PPARγ,the expression level of E-cadherin is higher while the expression level of Vimentin is lower.6.There are related proteins between ERRα and PPARγ,and they have a co-expression relationship with E-cadherin and Vimentin,respectively.The mechanism of ERRα and PPARγ regulating the invasion and migration ability of ovarian cancer cells may be involved in the epithelial-mesenchymal transition of malignant tumor cells. |
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