RASSF1A基因在人乳腺癌细胞株MCF-7中的表达及对细胞增殖凋亡、体内成瘤能力的影响观察

Expression of RASSF1A in human breast cancer cell line MCF-7 and its effect on cell proliferation, apoptosis and tumorigenic ability in vivo

目的观察Ras相关区域家族1异构体A(RASSF1A)基因在人乳腺癌细胞株MCF-7中的表达及对细胞增殖、凋亡、体内成瘤能力的影响,并探讨其作用的可能机制。方法取MCF-7细胞,分别感染RASSF1A过表达慢病毒oe-RASSF1A和空白载体慢病毒vector,记为oe-RASSF1A组和vector组。两组细胞继续培养72 h,采用qRT-PCR法检测两组细胞中的RASSF1A mRNA。两组细胞在培养24、48、72、96 h时,采用MTS法观察两组细胞增殖能力(以OD值表示细胞增殖能力)。两组细胞继续培养72 h,采用流式细胞仪测算各细胞周期比例。两组细胞继续培养72 h,采用流式细胞仪测算细胞凋亡率。两组细胞继续培养72 h,取1×10~7个细胞重悬于100μL PBS中,分别注射入10只4周龄雌性裸鼠右侧腋下,常规饲养4周后处死,解剖瘤体并称重,以瘤体重表示体内成瘤能力。两组细胞继续培养72 h,采用Western Blotting法检测两组细胞中的RASSF1A、磷脂酰肌醇-3-羟激酶(PI3K)、磷酸化PI3K(pPI3K)、蛋白激酶B(AKT)和磷酸化AKT(pAKT)蛋白。结果oe-RASSF1A组细胞中RASSF1A mRNA相对表达量为13.25±3.20,vector组为1.00±0.03,两组相比,P<0.05。oe-RASSF1A组细胞在培养24、48、72、96 h时的OD值分别为0.36±0.02、0.53±0.06、0.74±0.13、0.91±0.07,vector组分别为0.35±0.03、0.62±0.14、0.95±0.08、1.34±0.21,在培养72、96 h时两组的OD值相比,P均<0.05。oe-RASSF1A组细胞处于G0/G1期、S期、G2/M期的比例分别为60.02%±3.21%、18.30%±2.31%、22.11%±1.03%,vector组分别为63.67%±4.16%、21.04%±2.65%、15.32%±2.52%,oe-RASSF1A组细胞处于G2/M期的比例与vector组相比,P<0.05。oe-RASSF1A组细胞凋亡率为24.38%±5.80%,vector组为4.25%±0.92%,两组相比,P<0.05。注射oe-RASSF1A组细胞的裸鼠成瘤的瘤体重为(63.29±14.03)mg,注射vector组细胞的裸鼠成瘤的瘤体重为(104.58±17.52)mg,两组相比,P<0.05。oe-RASSF1A组细胞RASSF1A、PI3K、pPI3K、AKT、pAKT蛋白相对表达量分别为8.67±1.32、0.86±0.21、0.26±0.15、0.91±0.11、0.30±0.07,vector组分别为1.05±0.04、1.00±0.00、1.01±0.01、1.04±0.04、1.00±0.00,两组细胞RASSF1A、pPI3K、pAKT蛋白相对表达量相比,P均<0.05。结论感染RASSF1A过表达慢病毒oe-RASSF1A的MCF-7细胞RASSF1A mRNA升高;过表达RASSF1A可抑制MCF-7细胞增殖、诱导细胞周期阻滞在G2/M期、促进细胞凋亡、抑制细胞体内成瘤能力,其机制可能与RASSF1A抑制PI3K/AKT信号通路有关。

Objective To observe the expression of RAS-association domain family 1 isoform A(RASSF1 A)gene in the human breast cancer cell line MCF-7 and its effect on cell proliferation,apoptosis and tumorigenic ability in vivo,and to explore the possible mechanism.Methods MCF-7 cells were infected with RASSF1 A over-expressed lentivirus oe-RASSF1 A and blank vector lentivirus vector,respectively,which were recorded as the oe-RASSF1 A group and vector group.The cells of the two groups were cultured for 72 and the RASSF1A mRNA was detected by qRT-PCR.At 24,48,72 and 96 h,the proliferation abilities of the two groups were observed by MTS method(OD value was used to express the cell proliferation ability).The cells of the two groups were cultured for 72 h and the cell cycle ratio was measured by flow cytometry.The cells of the two groups were cultured for 72 h and the apoptosis rate was measured by flow cytometry.The cells of the two groups were cultured for 72 h,and 1× 10^7 cells were suspended in 100 μL PBS,and were injected into the right armpit of ten 4-week-old female nude mice.After 4 weeks of conventional feeding,the cells were killed,the tumor bodies were dissected and weighed,and the tumor weight was used to express the tumorigenic ability in vivo.The cells of the two groups were cultured for 72 h and Western blotting was used to detect RASSF1A,phosphatidylinositol-3-hydroxykinase(PI3K),phosphorylated PI3K(pPI3K),protein kinase B(Akt),and phosphorylated Akt(pAkt)proteins.Results The relative expression of RASSF1A mRNA was 13.25±3.20 and 1.00±0.03 in the oe-RASSF1A group and vector group,respectively,with statistically significant difference(P<0.05).The OD values of cells in the oe-RASSF1 A group were 0.36±0.02,0.53±0.06,0.74±0.13,0.91±0.07,0.35±0.03,0.62±0.14,0.95±0.08,and 1.34±0.21,respectively at 24,48,72 and 96 h,with statistically significant difference in the OD values between the two groups at 72 and 96 h(all P<0.05).The proportion of cells in the G0/G1 phase,S phase and Gq/M phase was 60.02%±3.21%,18.30%±2.31%,and 22.11%±1.03%,respectively,in oe-RASSF1A group,versus 63.67%±4.16%,21.04%±2.65%,and 15.32%±2.52%,respectively,in vector group.The apoptosis rate of oe-RASSF1A group was 24.38%±5.80%,and that of vector group was 4.25%±0.92%,with statistically significant difference(P<0.05).The tumor weight of nude mice injected with oe-RASSF1A cells was(63.29±14.03/mg,and that of nude mice injected with vector cells was(104.58±17.52)mg with statistically significant difference(P<0.05).The relative expression levels of RASSF1A,PI3K,pPI3K,AKT,pAKT protein were 8.67±1.32,0.86±0.21,0.26±0.15,0.91±0.11,0.30±0.07 respectively in the oe-RASSF1A group versus 1.05±0.041.00±0.001.01±0.011.04±0.04 and 1.00±0.00 respectively in the vector group and significant differences were found in the relative expression levels of RASSF1A pPI3K and pAkt(all P<0.05).Conclusions The expression of RASSF1A mRNA in MCF-7 cells infected with RASSF1 A over-expressed lentivirus oe-RASSF1A increases;the over-expressed RASSF1 A could inhibit the proliferation of MCF-7 cells,induce cell cycle arrest in G2/M phase,promote apoptosis,and inhibit the tumorigenicity of cells in vivo.The mechanism may be related to the inhibition of PI3K/Akt signaling pathway by RASSF1 A.