PPARγ基因表达对子宫内膜癌细胞迁移、侵袭及增殖能力的影响

Effects of PPARγ gene expression on cell migration, invasion, and proliferation in endometrial cancer cells

目的 探讨过氧化物酶增殖体激活受体γ（PPARγ）基因表达对子宫内膜癌细胞迁移、侵袭及增殖能力的影响.方法 选取子宫内膜癌细胞系ECC-1（ER阳性）及KLE（ER阴性）细胞,分别转染PPARγ基因表达载体（PPARγ载体组）及PPARγ小分子干扰RNA（siRNA）质粒（PPARγsiRNA组）,以转染无义序列siRNA（siRNA无义序列组）、空载体（空载体组）者为阴性对照,以只加入脂质体者（空白对照组）为空白对照.转染48 h后,实时逆转录（RT）-PCR技术及蛋白印迹法（western blot）检测细胞中PPARγmRNA及蛋白表达水平的变化;western blot检测细胞中Wnt信号传导通路中关键蛋白——β连接素（β-catenin）及髓细胞增生原癌基因编码蛋白（C-myc）蛋白表达水平的变化;体外迁移、侵袭实验检测细胞迁移、侵袭能力的变化,活细胞计数（CCK-8）法检测细胞增殖能力[以吸光度（A）值表示].结果 转染48 h后,实时RT-PCR技术和western blot检测显示,PPARγ载体组ECC-1及KLE细胞中PPARγ mRNA（分别为5.18 ±0.99、4.54±0.89）和蛋白（分别为1.45±0.12、1.30±0.13）的表达水平升高,β-eatenin（分别为0.44 ±0.06、0.46±0.04）、C-myc（分别为0.42±0.08、0.30±0.11）蛋白表达水平降低,PPAR γ siRNA组ECC-1及KLE细胞中PPAR γ mRNA（分别为0.48 ±0.08、0.53±0.11）和蛋白（分别为0.41±0.04、0.49±0.05）的表达水平降低,β-catenin（分别为1.18±0.12、0.89±0.07）、C-myc（分别为0.91 ±0.08、0.77 ±0.12）蛋白的表达水平升高,分别与siRNA无义序列组、空载体组、空白对照组比较,差异均有统计学意义（P＜0.05）.体外迁移、侵袭实验检测显示,PPAR γ载体组ECC-1、KLE细胞的迁移细胞数[分别为（129±9）、（119±9）个]及侵袭细胞数[分别为（63±12）、（68±16）个]减少,PPARγ siRNA组ECC-1、KLE细胞的迁移细胞数[分别为（201±14）、（170±11）个]及侵袭细胞数[分别为（142±9）、（138±7）个]增多,分别与siRNA无义序列组、空载体组、空白对照组比较,差异均有统计学意义（P＜0.05）.CCK-8法检测显示,PPARγ载体组ECC-1、KLE细胞的A值（分别为0.66±0.14、0.78 ±0.06）均低于siRNA无义序列组、空载体组、空白对照组,PPARγ siRNA组ECC-1、KLE细胞的A值（分别为1.42±0.16、1.23±0.04）均高于siRNA无义序列组、空载体组、空白对照组,分别比较,差异均有统计学意义（P＜0.05）.结论 上调PPARγ基因表达可抑制子宫内膜癌细胞的迁移、侵袭及增殖,而下调PPARγ基因表达可促进子宫内膜癌细胞的迁移、侵袭及增殖.

Objective To observe the effects of differentially expressed peroxisome proliferatoractivated receptor γ （PPAR γ） on cell migration,invasion and proliferation of endometrial cancer cells.Methods Two endometrial cancer cell lines ECC-1 （ER positive） and KLE （ER negative） cells were used in this study.To up or down regulate PPARγ expression,the transient transfection by using PPARγ expression vector （PPARγ expression vector group） and PPARγ small interference RNA （PPARγ siRNA group） were done.The negative control groups were cells transfected by nonsence sequence siRNA （siRNA non sence sequence group） or empty vector （empty vector group）.At the same time,cells only added with liposome were used as blank control group.Then,quantitative real time （RT）-PCR and western blot were used to detect PPARγexpression both in mRNA and protein levels.To assess the expression levels of Wnt signaling pathway,western blot was performed to analysis protein levels of β-catenin and C-myc.The effects on cell migration,invasion and proliferation using in vitro transwell migration,invasion assays and cell counting kit-8 （CCK-8） assay were further be examined.Results After transfection for 48 hours,quantitative RT-PCR and western blot showed that PPARγmRNA （5.18 ± 0.99,4.54 ± 0.89） and protein （1.45 ± 0.12,1.30 ± 0.13） expression levels significantly increased and the protein levels of β-catenin （0.44 ± 0.06,0.46 ± 0.04） and C-myc （0.42 ± 0.08,0.30 ± 0.11） decreased in PPAR γ expression vector group,while in PPARγ siRNA group,PPARγ mRNA （0.48 ± 0.08,0.53 ± 0.11） and protein （0.41 ±0.04,0.49 ±0.05） expression levels decreased and the protein levels of 3-catenin （1.18 ±0.12,0.89 ±0.07） and C-myc（0.91 ±0.08,0.77 ±0.12） increased significantly compared with control groups （all P 〈 0.05）.In vitro migration and invasion assay indicated that the migratory and invasive cell numbers of PPARγ expression vector group （ECC-1：129 ± 9,63 ± 12 ; KLE：119 ± 9,68 ± 16） were significantly decreased,while the migratory and invasive cell numbers of were PPARγ siRNA group （ECC-1：201 ± 14,142 ±9 ; KLE：170 ± 11,138 ± 7） increased significantly compared with those in control groups（all P 〈 0.05）.CCK-8 assay showed that A values （0.66 ±0.14,0.78 ±0.06） in PPARγexpression vector group were lower than those in control groups,and in PPARγ siRNA group,A values （1.42 ± 0.16,1.23 ± 0.04） were higher than those in control groups,and there were statistically significant difference among them （all P 〈 0.05）.Conclusion Up-regulated PPARγ gene expression could inhibit endometrial cancer cell migration,invasion and proliferation abilities,and down-regulated PPARγ gene expression could promote endometrial cancer cell migration,invasion and proliferation abilities.