PTEN对苦参碱抑制LoVo细胞的影响

Influence of PTEN on matrine inhibiting LoVo cells

目的探讨人第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome 10,PTEN)在苦参碱诱导结肠癌LoVo细胞凋亡及抑制增殖、迁移、侵袭中的作用。方法结肠癌LoVo细胞,分为空白对照组(不转染不加药)、转染组(转染PTEN-siRNA)、转染加药组(转染PTEN-siRNA并加入苦参碱1mg/mL)、阴性对照组(转染NC-siRNA)、阴性转染加药组(转染NC-siRNA并加入苦参碱1mg/mL)、加药组(不转染但加入苦参碱1mg/mL)。空白对照组和转染组采用MTT法检测细胞残余活性,各组采用细胞划痕实验检测细胞迁移距离,采用Transwell法检测细胞穿膜数量,采用Western blot法检测细胞PTEN、Akt、p-Akt、bax和bcl-2蛋白表达情况。结果培养48h后,转染组不同苦参碱浓度作用下细胞残余活性均高于空白对照组(P<0.05),且呈浓度和时间依赖性;24、48h时空白对照组细胞迁移距离[(21.60±0.14)、(11.65±0.39)mm]明显长于转染组[(11.45±0.11)、(3.40±0.28)mm]和转染加药组[(13.15±0.25)、(6.01±0.28)mm],短于阴性转染加药组[(23.10±0.81)、(14.85±0.32)mm]和加药组[(23.65±0.03)、(15.40±0.27)mm](P<0.05);与空白对照组比较,转染组和转染加药组细胞穿膜数量均增加,阴性转染加药组和加药组细胞穿膜数量均减少;转染组PTEN(0.37±0.04)、bax(0.84±0.11)蛋白表达量明显低于空白对照组(0.57±0.06、1.03±0.02)、加药组(1.21±0.04、1.51±0.03)和阴性转染加药组(1.31±0.02、1.49±0.19)(P<0.05),Bcl-2蛋白表达量(1.19±0.04)明显高于空白对照组(0.67±0.11)、加药组(0.44±0.03)和阴性转染加药组(0.48±0.03)(P<0.05);空白对照组PTEN、Akt(0.98±0.02)及bax蛋白表达量低于加药组(Akt为1.22±0.14)和阴性转染加药组(Akt为1.12±0.07),高于转染组(Akt为0.97±0.05)和转染加药组(Akt为0.96±0.07)(P<0.05)。结论苦参碱可上调PTEN基因表达,明显抑制LoVo细胞增殖、迁移、侵袭及诱导细胞凋亡与PI3K-Akt信号通路有关。

Objective To explore the influence of phosphatase and tensin homolog deleted on chromosome 10 （PTEN） on matrine induced apoptosis and inhibiting proliferation, migration and invasion of colon cancer LoVo cells. Methods Colon cancer LoVo cells were divided into blank control group （with no transfection and no matrine）, transfection group （transfecting PTEN-siRNA）, transfection-matrine group （transfecting PTEN-siRNA and adding 1 mg/mL matrine）, negative control group （transfecting NC-siRNA）, negative transfection-matrine group （transfecting NC siRNA and adding 1 mg/mL matrine）, and matrine group （without transfection but adding 1 mg/mL matrine）. Blank control group and transfection group were detected cell proliferation ability by MTT assay. Cell migration distance was examined by scratch assay. Cell invasion count was examined by transwell assay. The expression levels of PTEN, Akt, p-Akt, Bax and Bcl-2 were detected by Western blot method. Results The proliferation ability of LoVo cells after being incubated with varying concentrations of matrine was significantly higher in tranfestion group than that in blank control group after being cultured for 48 hours （P〈0.05）, showing concentration and time dependence. Within 24 and 48 hours, the migration distances in blank control group （（21.60±0.14）, （11.65±0.39） mm） were significantly longer than those in transfection group （（11.45±0.11）, （3.40±0.28） mm） and transfection-matrine group （（13.15±0.25）, （6.01±0.28） mm）, and significantly shorter than those in negative transfection-matrine group （（23.65±0.03）, （15.40±0.27） mm） （P〈0.05） and matrine group （（23. 65±0. 03）, （15. 40±0. 27） mm） （P〈0.05）. The counts of cell invasion in transfection group and transfection-matrine group increased significantly, and the counts of cell invasion in matrine group and negative transfection-matrine group decreased in comparison with blank control group. The expressions of PTEN （0.37±0.04） and bax （0.84±0.11） in transfection group were significantly lower than those in control group （0.57± 0.06, 1.03±0.02）, matrine group （1.21±0.04, 1.51±0.03） and negative transfection-matrine group （1.31±0.02,1.49±0.19） （P〈0.05）. The expression of bel 2 protein in transfection group （1.19±0.04） was significantly higher than that in blank control group （0.67±0.11）, matrine group （0.44±0.03）, and negative matrine group （0.48±0.03） （P〈0. 05）. The expression levels of PTEN, Akt （0.98±0.02） and bax in blank control group were significantly lower than those in matrine group （Akt： 1. 22±0. 14） and negative matrine group （Akt： 1. 12±0. 07） （P〈0.05）, and signficantly higher than those in transfection group （Akt： 0.97 ± 0.05） and transfection-matrine group （Akt： 0. 96 ± 0. 07） （P〈 0. 05）. Conclusion Matrine can upregulate proliferation, migration and invasion, and induce apoptosis, the expression of PTEN, significantly inhibit LoVo cell which is correlated with PI3K-Akt signaling pathways.