外源性PTEN基因对人肺癌细胞株SPC-A-1增殖、凋亡及侵袭的影响实验
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摘要
目的观察外源性第10号染色体同源缺失磷酸酶和张力蛋白(PTEN)基因对人肺癌细胞株SPC-A-1增殖、凋亡及侵袭的影响,并初步探讨其调控机制。方法取对数期SPC-A-1细胞转染重组腺病毒Ad-PTENGFP、Ad-GFP,分别设为PTEN组、空载组,并检测转染效率,另取不做处理为对照组。MTT法检测并对比各组细胞增殖情况; Hoechst 33342荧光染色法检测细胞凋亡情况; Transwell试验检测细胞侵袭能力;分别采用实时荧光定量PCR法、蛋白免疫印迹法检测并对比各组细胞中PTEN mRNA和蛋白、粘着斑激酶(FAK)、蛋白激酶B(AKT) mRNA表达情况及p FAK/FAK、p AKT/AKT。结果 FACS法检测重组腺病毒Ad-PTEN-GFP、Ad-GFP对SPC-A-1细胞转染效率分别为(82. 41±5. 46)%、(83. 02±6. 20)%。PENT组MTT试验不同时刻OD值显著低于对照组和空载组(P <0. 05),3组OD值均随时间延长呈显著升高趋势(P <0. 05); Hoechst 33342荧光染色发现,对照组和空载组细胞形态正常,呈弥散均匀荧光,PTEN组部分细胞核浓染,呈月牙形聚集、颗粒状荧光碎片,PENT组细胞凋亡率显著高于对照组和空载组(P <0. 05); PENT组细胞侵袭数目显著少于对照组和空载组(P <0. 05);对照组和空载组OD值、凋亡率及细胞侵袭数目比较差异均无显著性(P> 0. 05);与对照组和空载组比较,PENT组PENT mRNA和蛋白相对表达量显著较高(P <0. 05),p AKT/AKT、p FAK/FAK显著较低(P <0. 05);对照组和空载组PENT mRNA和蛋白相对表达量、p AKT/AKT、p FAK/FAK、3组AKT、FAK mRNA相对表达量比较差异均无显著性(P> 0. 05)。结论外源性PTEN基因可显著抑制SPC-A-1细胞增殖及侵袭,促进其凋亡,推测与抑制AKT/FAK信号通路有关。
Objective To observe the effect of lack of chromosome 10 and tension homologous protein phosphatase( PTEN) gene on proliferation,apoptosis and invasion of human lung cancer cell line SPC-A-1,and to explore its regulatory mechanism. Methods Ad-PTEN-GFP,AD-GFP transfected with recombinant adenovirus in log phase SPC-A-1 cells. The recombinant cells were randomly divided into PTEN group and empty group,and the transfection efficiency was detected. The other treatments were used as the control group. MTT assay was used to detect and compare the proliferation of each group. Apoptosis was detected by Hoechst 33342 fluorescence staining. Cell invasion ability was detected by Transwell test. The expressions of PTEN mRNA and protein,focal adhesion kinase( FAK) and protein kinase B( AKT) mRNA in different groups of cells and pFAK/FAK and pAKT/AKT were detected by real-time fluorescent quantitative PCR and Western blotting,respectively. Results The transfection efficiency of recombinant adenovirus Ad-PTEN-GFP and Ad-GFP to SPC-A-1 cells was( 82. 41 ± 5. 46) % and( 83. 02 ± 6. 20) %,respectively. The OD values of the MTT test in the PENT group were significantly lower than those in the control group and the empty group( P < 0. 05),and the OD values of the three groups increased significantly with time( P < 0. 05). Hoechst 33342 fluorescence staining showed that the cells in the control group and the empty group were normal and diffuse and uniform fluorescence,while some of the cells in the PTEN group were densely stained with crescent-shaped aggregates and granular fluorescent fragments. The apoptosis rate of PENT group was significantly higher than that of control group and empty group( P < 0. 05). The number of cell invasion in the PENT group was significantly lower than that in the control group and the empty group( P < 0. 05). There were no significant differences in OD value,apoptotic rate and cell invasion between the control group and the empty group( P > 0. 05). The relative expressions of PENT mRNA and protein in PENT group were significantly higher than those in control group and empty group( P < 0. 05),while the pENTT/AKT and pFAK/FAK in the PENT group were significantly lower than those in the control group and the empty group( P < 0. 05). There were no significant differences in the relative expressions of PENT mRNA and protein,pAKT/AKT,pFAK/FAK,AKT and FAK mRNA in the control group and the empty group( P > 0. 05).Conclusion The exogenous PTEN gene can significantly inhibit the proliferation and invasion of SPC-A-1 cells and promote their apoptosis,which is presumed to be related to the inhibition of AKT/FAK signaling pathway.
Objective To observe the effect of lack of chromosome 10 and tension homologous protein phosphatase( PTEN) gene on proliferation,apoptosis and invasion of human lung cancer cell line SPC-A-1,and to explore its regulatory mechanism. Methods Ad-PTEN-GFP,AD-GFP transfected with recombinant adenovirus in log phase SPC-A-1 cells. The recombinant cells were randomly divided into PTEN group and empty group,and the transfection efficiency was detected. The other treatments were used as the control group. MTT assay was used to detect and compare the proliferation of each group. Apoptosis was detected by Hoechst 33342 fluorescence staining. Cell invasion ability was detected by Transwell test. The expressions of PTEN mRNA and protein,focal adhesion kinase( FAK) and protein kinase B( AKT) mRNA in different groups of cells and pFAK/FAK and pAKT/AKT were detected by real-time fluorescent quantitative PCR and Western blotting,respectively. Results The transfection efficiency of recombinant adenovirus Ad-PTEN-GFP and Ad-GFP to SPC-A-1 cells was( 82. 41 ± 5. 46) % and( 83. 02 ± 6. 20) %,respectively. The OD values of the MTT test in the PENT group were significantly lower than those in the control group and the empty group( P < 0. 05),and the OD values of the three groups increased significantly with time( P < 0. 05). Hoechst 33342 fluorescence staining showed that the cells in the control group and the empty group were normal and diffuse and uniform fluorescence,while some of the cells in the PTEN group were densely stained with crescent-shaped aggregates and granular fluorescent fragments. The apoptosis rate of PENT group was significantly higher than that of control group and empty group( P < 0. 05). The number of cell invasion in the PENT group was significantly lower than that in the control group and the empty group( P < 0. 05). There were no significant differences in OD value,apoptotic rate and cell invasion between the control group and the empty group( P > 0. 05). The relative expressions of PENT mRNA and protein in PENT group were significantly higher than those in control group and empty group( P < 0. 05),while the pENTT/AKT and pFAK/FAK in the PENT group were significantly lower than those in the control group and the empty group( P < 0. 05). There were no significant differences in the relative expressions of PENT mRNA and protein,pAKT/AKT,pFAK/FAK,AKT and FAK mRNA in the control group and the empty group( P > 0. 05).Conclusion The exogenous PTEN gene can significantly inhibit the proliferation and invasion of SPC-A-1 cells and promote their apoptosis,which is presumed to be related to the inhibition of AKT/FAK signaling pathway.
引文
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[14]任荣,崔晓东,王转花. r BTI通过上调PTEN的表达抑制Hep G2细胞增殖[J].中国生物化学与分子生物学报,2016,32(2):170-177.
[15] Zhou GY,Pan CW,Jin LX,et al. Neoalbaconol inhibits cell growth of human cholangiocarcinoma cells by up-regulating PTEN[J]. Am J Transl Res,2016,8(2):496-505.
[16]刘小美,潘志强,方肇勤,等.黄连-大黄-肉桂复方对肝癌细胞增殖及PTEN-PI3K-AKT/SGK1信号通路的影响[J].上海中医药大学学报,2016,30(1):50-54.
[17] Mon NN,Senga T,Ito S. Interleukin-1βactivates focal adhesion kinase and Src to induce matrix metalloproteinase-9 production and invasion of MCF-7 breast cancer cells[J]. Oncol Lett,2017,13(2):955-960.
[18] Egawa H,Jingushi K,Hirono T,et al. The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN[J]. Sci Rep,2016,6:20574.
[2]张瑞,李静,宋玉芝,等.临床分期为ⅢA(N2)期非小细胞肺癌放射治疗的预后分析[J].肿瘤防治研究,2016,43(6):526-530.
[3] Rescigno P,Lorente D,Dolling D,et al. Docetaxel Treatment in PTEN-and ERG-aberrant Metastatic Prostate Cancers[J]. Eur Urol Oncol,2018,1(1):71-77.
[4] Yang Y,Guo JX,Shao ZQ. miR-21 targets and inhibits tumor suppressor gene PTEN to promote prostate cancer cell proliferation and invasion:An experimental study[J]. Asian Pac J Trop Med,2017,10(1):87-91.
[5] Wu MF,Jian ZH,Huang JY,et al. Post-inhaled corticosteroid pulmonary tuberculosis and pneumonia increases lung cancer in patients with COPD[J]. BMC Cancer,2016,16(1):778-783.
[6] Liu Y,Ni R,Zhang H,et al. Identification of feature genes for smoking-related lung adenocarcinoma based on gene expression profile data[J]. Onco Targets Ther,2016,9:7397-7407.
[7] Luo J,Zhu H,Jiang H,et al. The effects of aberrant expression of LncRNA DGCR5/miR-873-5p/TUSC3 in lung cancer cell progression[J]. Cancer Med,2018,16(1):50-58.
[8] Xu L,Leng H,Shi X,et al. MiR-155 promotes cell proliferation and inhibits apoptosis by PTEN signaling pathway in the psoriasis[J]. Biomed Pharmacother,2017,90:524-530.
[9]端木颖,朱喆辰,汤由之,等. Sox2、Pten、Stat3、Vegf在皮肤恶性黑色素瘤中表达的相关性研究[J].南京医科大学学报:自然科学版,2017,22(11):1395-1397.
[10] Blee AM,He Y,Yang Y,et al. TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer[J]. Clin Cancer Res,2018,24(18):4551-4565.
[11] Wang W,Oguz G,Lee PL,et al. KDM4B-regulated unfolded protein response as a therapeutic vulnerability in PTEN-deficient breast cancer[J]. J Exp Med,2018,215(11):2833-2849.
[12]詹晓娟,戴益琛.抑癌基因PTEN在大肠癌中的功能及机制[J].临床和实验医学杂志,2018,17(5):449-452.
[13]晏群,邹明祥,刘文恩,等. PTEN/PI3K突变对肺癌细胞基因表达谱的影响[J].肿瘤,2016,32(6):420-428.
[14]任荣,崔晓东,王转花. r BTI通过上调PTEN的表达抑制Hep G2细胞增殖[J].中国生物化学与分子生物学报,2016,32(2):170-177.
[15] Zhou GY,Pan CW,Jin LX,et al. Neoalbaconol inhibits cell growth of human cholangiocarcinoma cells by up-regulating PTEN[J]. Am J Transl Res,2016,8(2):496-505.
[16]刘小美,潘志强,方肇勤,等.黄连-大黄-肉桂复方对肝癌细胞增殖及PTEN-PI3K-AKT/SGK1信号通路的影响[J].上海中医药大学学报,2016,30(1):50-54.
[17] Mon NN,Senga T,Ito S. Interleukin-1βactivates focal adhesion kinase and Src to induce matrix metalloproteinase-9 production and invasion of MCF-7 breast cancer cells[J]. Oncol Lett,2017,13(2):955-960.
[18] Egawa H,Jingushi K,Hirono T,et al. The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN[J]. Sci Rep,2016,6:20574.