PCa组织Mfn2 mRNA表达及转染Mfn2过表达质粒的人PCa细胞株增殖、迁移、侵袭、凋亡观察

Mfn2 mRNA expression in PCa tissues and observation of proliferation,migration,invasion and apoptosis of human PCa cell line transfected with Mfn2 overexpression plasmid

目的观察前列腺癌(PCa)组织Mfn2 mRNA表达及转染Mfn2过表达质粒的人PCa细胞株增殖、迁移、侵袭、凋亡情况。方法体外培养人PCa细胞株(PC3细胞和DU145细胞),分别转染Mfn2过表达质粒、空白载体质粒及不转染质粒,PC3细胞分别记为A、B、C组,DU145细胞分别记为D、E、F组。CCK-8实验检测各组细胞OD值,细胞集落形成实验测算各组细胞集落数目,以OD值和细胞集落数目表示细胞增殖能力。细胞划痕实验测算各组细胞迁移率,以细胞迁移率表示细胞迁移能力。Transwell实验测算各组细胞侵袭数目,以细胞侵袭数目数表示细胞侵袭能力。流式细胞术测算各组细胞凋亡率,Western blot检测凋亡相关蛋白Bax、Bcl-2、Cleaved caspase-3、caspase-3及PI3K/AKT通路相关蛋白(p-PI3K/PI3K、p-AKT/AKT)。结果PCa、癌旁组织Mfn2 mRNA相对表达量分别为0.36±0.32、0.72±0.35,两者比较,P<0.05。与同组24 h比较,A、B、C组48和72 h的OD值增加(P均<0.05);与同组48 h比较,A、B、C组72 h的OD值增加(P均<0.05);与B、C组比较,A组24、48、72 h的OD值和细胞集落数目减少(P均<0.05)。与同组24 h比较,D、E、F组48和72 h的OD值增加(P均<0.05);与同组48 h比较,D、E、F组72 h的OD值增加(P均<0.05);与E、F组比较,D组24、48、72 h的OD值和细胞集落数目减少(P均<0.05)。与B、C组比较,A组细胞迁移率及细胞侵袭数目减少(P均<0.05);与E、F组比较,D组细胞迁移率及细胞侵袭数目减少(P均<0.05)。与B、C组比较,A组细胞凋亡率及Bax/Bcl-2、Cleaved caspase-3/caspase-3表达增加(P均<0.05);与E、F组比较,D组细胞凋亡率及Bax/Bcl-2、Cleaved caspase-3/caspase-3表达增加(P均<0.05);与B、C组比较,A组p-PI3K/PI3K、p-AKT/AKT蛋白表达降低(P均<0.05);与E、F组比较,D组p-PI3K/PI3K、p-AKT/AKT蛋白表达降低(P均<0.05)。结论PCa组织Mfn2 mRNA的表达低于癌旁组织,转染Mfn2过表达质粒可抑制PCa细胞株增殖、迁移和侵袭,并诱导凋亡,其作用机制可能与抑制PI3K/AKT信号通路有关。

Objective To observe Mfn2 mRNA expression in the prostate cancer(PCa)tissues and proliferation,mi⁃gration,invasion and apoptosis of human PCa cell lines transfected with Mfn2 overexpression plasmid.Methods In vitro culture of human PCa cell lines(PC3 cells and DU145 cells),transfected with Mfn2 overexpression plasmid and blank vector plasmid were categorized as groups A and B;in vitro culture of human PCa cell lines without transfection were taken as the group C;DU145 cells were categorized as groups D,E and F.CCK-8 assay measured the OD values of each group and the cell colony formation assay measured the number of cell colonies in each group,and the OD value and the number of cell colonies were used to indicate the proliferation capacity of the cells.The cell migration rate was measured in the Scratch assay for each group.Transwell assay was used to measure the number of invasion cells in each group.Flow cy⁃tometry was used to measure the apoptosis rate in each group.Western blotting was used to detect apoptosis-related pro⁃teins Bax,Bcl-2,Cleaved caspase-3,caspase-3 and PI3K/AKT pathway-related proteins(p-PI3K/PI3K,p-AKT/AKT).Results The relative expression levels of Mfn2 mRNA in the prostate cancer and adjacent tissues were 0.36±0.32 and 0.72±0.35,respectively,with statistically significant difference(P<0.05).Compared with the same group at 24 h,the OD values of groups A,B and C increased at 48 and 72 h(all P<0.05);compared with the same group at 48 h,the OD values of groups A,B and C increased at 72 h(all P<0.05);compared with groups B and C,the OD values and the num⁃ber of cell colonies decreased at 24,48 and 72 h in the group A(all P<0.05).Compared with the same group at 24 h,the OD values at 48 and 72 h increased in groups D,E,and F(all P<0.05);compared with the same group at 48 h,the OD values at 72 h increased in groups D,E,and F(all P<0.05);compared with groups E and F,the OD values at 24,48,and 72 h and the number of cell colonies decreased in the group D(all P<0.05).Compared with groups B and C,the cell migration rate and the number of invasion cells were reduced in the group A(all P<0.05);compared with groups E and F,the cell migration rate and the number of invasion cells were reduced in the group D(all P<0.05).Compared with groups B and C,apoptosis rate and the expression of Bax/Bcl-2 and Cleaved caspase-3/caspase-3 increased in the group A(all P<0.05);compared with the groups E and F,apoptosis rate and the expression of Bax/Bcl-2 and Cleaved caspase-3/caspase-3 increased in the group D(all P<0.05);compared with the groups B and C,p-PI3K/PI3K and p-AKT/AKT protein expression decreased in the group A(all P<0.05);compared with groups E and F,p-PI3K/PI3K and p-AKT/AKT protein expression decreased in the group D(all P<0.05).Conclusions The expression of Mfn2 mRNA in pros⁃tate cancer tissues is lower than that in adjacent tissues.Transfection of Mfn2 overexpression plasmid inhibits the prolifera⁃tion,migration and invasion of prostate cancer cell lines and induces apoptosis,and its mechanism of action may be relat⁃ed to the inhibition of PI3K/AKT signaling pathway.