KLF16对胃癌细胞增殖、克隆能力的影响及机制
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摘要
目的探讨Krüppel样转录因子16(KLF16)对胃癌细胞增殖、克隆能力的影响及机制。方法取处于对数生长期的BGC-823和SGC-7901细胞,每种细胞分为3组,sh-KLF16 1#、2#、3#组采用脂质体Lipofectamine3000,分别将sh-KLF16 1#、sh-KLF16 2#、sh-KLF16 3#转染至细胞中,sh-NC组将shRNA无关序列转染至细胞中,以不转染shRNA的细胞为空白对照组。Real-time PCR法检测KLF16 mRNA相对表达量。CCK-8法检测胃癌细胞增殖活性,细胞克隆形成试验检测胃癌细胞克隆能力。Western blotting法检测细胞中细胞周期相关蛋白p21和细胞周期蛋白依赖性激酶4(CDK4)的表达。结果 BGC-823细胞中,空白对照组,sh-NC组,sh-KLF16 1#、2#、3#组KLF16 mRNA相对表达量分别为1.13±0.10、1.06±0.15、0.57±0.11、0.72±0.14、0.39±0.13;SGC-7901细胞中,空白对照组,sh-NC组,sh-KLF16 1#、2#、3#组KLF16 mRNA相对表达量分别为1.09±0.12、1.02±0.14、0.59±0.15、0.53±0.11、0.31±0.15;两种细胞中,sh-KLF16#3组的KLF16 mRNA相对表达量低于sh-KLF16#1组和shKLF16#2组(P均<0.05)。在BGC-823和SGC-7901细胞中,干扰KLF16基因表达后,细胞增殖受抑制。BGC-823细胞中,空白对照组、sh-NC组、sh-KLF16 3#组细胞克隆数目分别为(180.45±19.32)、(189.83±22.43)、(109.32±24.31)个,SGC-7901细胞中,空白对照组、sh-NC组、sh-KLF16 3#组细胞克隆数目分别为(148.41±21.43)、(152.95±24.75)、(87.82±18.43)个,sh-KLF16 3#组细胞克隆数目低于空白对照组、sh-NC组(P均<0.05)。在BGC-823和SGC-7901细胞中,干扰KLF16基因表达后,p21蛋白表达增加(P均<0.05),而CDK4蛋白表达下降(P均<0.05)。结论干扰KLF16基因表达,可抑制细胞增殖和克隆形成能力,其机制可能与上调p21蛋白和降低CDK4蛋白表达有关。
Objective To detect the effects and mechanism of Krüeppel-like factor 16(KLF16) on the proliferation and clone ability of gastric cancer cells. Methods The BGC-823 and SGC-7901 cells in the logarithmic growth phase were taken,each kind of cells was divided into 3 groups. The Sh-KLF16 1#,2#,and 3# were transfected into cells by liposome Lipofectamine3000,respectively,which were taken as the sh-KLF16 1 #,2 #,and 3 # groups. The shRNA independent sequence was transfected into sh-NC group,and the cells without transfection of shRNA was taken as the blank control group. The relative expression of KLF16 mRNA was detected by real-time PCR. The proliferation of gastric cancer cells was detected by CCK-8,and gastric cancer cell clone ability was detected by cell clone formation assay. Western blotting was used to detect the expression of cell cycle-related proteins p21 and CDK4. Results In BGC-823 cells,the relative expression of KLF16 mRNA in the blank control group,sh-NC group,sh-KLF16 1#,2#,and 3# groups was 1. 13 ±0. 10,1. 06 ± 0. 15,0. 57 ± 0. 11,0. 72 ± 0. 14,and 0. 39 ± 0. 13,respectively. In SGC-7901 cells,the relative expression of KLF16 mRNA in the blank control group,sh-NC group,sh-KLF16 1#,2#,and 3# groups was 1. 09 ± 0. 12,1. 02± 0. 14,0. 59 ± 0. 15,0. 53 ± 0. 11,and 0. 31 ± 0. 15,respectively. The relative expression of KLF16 mRNA in the shKLF16 3# group was lower than that in the sh-KLF16 1# group and sh-KLF16 2# group(P < 0. 05). CCK-8 results showed that in the BGC-823 and SGC-7901 cells,the cell proliferation was inhibited after interfering KLF16 gene expression. In the BGC-823 cells,the number of cell clones in the blank control group,sh-NC group,and sh-KLF16 3 # group was180. 45 ± 19. 32,189. 83 ± 22. 43,and 109. 32 ± 24. 31,respectively; in the SGC-7901 cells,the number of cell clones in the blank control group,sh-NC group,and sh-KLF16 3 # group was 148. 41 ± 21. 43,152. 95 ± 24. 75,and 87. 82 ±18. 43,respectively; the number of cell clones in the sh-KLF16 3# group was lower than that in the blank control group and sh-NC group(P < 0. 05). Western blotting showed that in the BGC-823 and SGC-7901 cells,after interfering KLF16 gene expression,the expression of p21 protein increased,and the expression of CDK4 protein decreased(both P < 0. 05). Conclusion Interfering KLF16 gene expression can inhibit the cell proliferation and colony forming ability,and its mechanism may be related to the regulation of the expression of p21 and CDK4.
Objective To detect the effects and mechanism of Krüeppel-like factor 16(KLF16) on the proliferation and clone ability of gastric cancer cells. Methods The BGC-823 and SGC-7901 cells in the logarithmic growth phase were taken,each kind of cells was divided into 3 groups. The Sh-KLF16 1#,2#,and 3# were transfected into cells by liposome Lipofectamine3000,respectively,which were taken as the sh-KLF16 1 #,2 #,and 3 # groups. The shRNA independent sequence was transfected into sh-NC group,and the cells without transfection of shRNA was taken as the blank control group. The relative expression of KLF16 mRNA was detected by real-time PCR. The proliferation of gastric cancer cells was detected by CCK-8,and gastric cancer cell clone ability was detected by cell clone formation assay. Western blotting was used to detect the expression of cell cycle-related proteins p21 and CDK4. Results In BGC-823 cells,the relative expression of KLF16 mRNA in the blank control group,sh-NC group,sh-KLF16 1#,2#,and 3# groups was 1. 13 ±0. 10,1. 06 ± 0. 15,0. 57 ± 0. 11,0. 72 ± 0. 14,and 0. 39 ± 0. 13,respectively. In SGC-7901 cells,the relative expression of KLF16 mRNA in the blank control group,sh-NC group,sh-KLF16 1#,2#,and 3# groups was 1. 09 ± 0. 12,1. 02± 0. 14,0. 59 ± 0. 15,0. 53 ± 0. 11,and 0. 31 ± 0. 15,respectively. The relative expression of KLF16 mRNA in the shKLF16 3# group was lower than that in the sh-KLF16 1# group and sh-KLF16 2# group(P < 0. 05). CCK-8 results showed that in the BGC-823 and SGC-7901 cells,the cell proliferation was inhibited after interfering KLF16 gene expression. In the BGC-823 cells,the number of cell clones in the blank control group,sh-NC group,and sh-KLF16 3 # group was180. 45 ± 19. 32,189. 83 ± 22. 43,and 109. 32 ± 24. 31,respectively; in the SGC-7901 cells,the number of cell clones in the blank control group,sh-NC group,and sh-KLF16 3 # group was 148. 41 ± 21. 43,152. 95 ± 24. 75,and 87. 82 ±18. 43,respectively; the number of cell clones in the sh-KLF16 3# group was lower than that in the blank control group and sh-NC group(P < 0. 05). Western blotting showed that in the BGC-823 and SGC-7901 cells,after interfering KLF16 gene expression,the expression of p21 protein increased,and the expression of CDK4 protein decreased(both P < 0. 05). Conclusion Interfering KLF16 gene expression can inhibit the cell proliferation and colony forming ability,and its mechanism may be related to the regulation of the expression of p21 and CDK4.
引文
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[11]Wei D,Kanai M,Jia Z,et al.Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells[J].Cancer Res,2008,68(12):4631-4639.
[12]Zhang N,Zhang J,Shuai L,et al.Krüppel-like factor 4 negatively regulatesβ-catenin expression and inhibits the proliferation,invasion and metastasis of gastric cancer[J].Int J Oncol,2012,40(6):2038-2048.
[13]Patel NV,Ghaleb AM,Nandan MO,et al.Expression of the tumor suppressor Krüppel-like factor 4 as a prognostic predictor for colon cancer[J].Cancer Epidemiol Biomarkers Prev,2010,19(10):2631-2638.
[14]Yoda T,Mc Namara KM,Miki Y,et al.KLF15 in breast cancer:a novel tumor suppressor[J].Cell Oncol(Dordr),2015,38(3):227-235.
[15]Daftary GS,Lomberk GA,Buttar NS,et al.Detailed structuralfunctional analysis of the Krüppel-like factor 16(KLF16)transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology[J].J Biol Chem,2012,287(10):7010-7025.
[16]Wang J,Galvao J,Beach KM,et al.Novel roles and mechanism for Krüppel-like factor 16(KLF16)regulation of neurite outgrowth and ephrin receptor A5(Eph A5)expression in retinal ganglion cells[J].J Biol Chem,2016,291(41):21422.
[17]Jang MK,Lee S,Jung MH.RNA-Seq analysis reveals a negative role of KLF16 in adipogenesis[J].PLo S One,2016,11(9):e0162238.
[18]Fernandez-Zapico ME,Lomberk GA,Tsuji S,et al.A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth[J].Biochem J,2011,435(2):529-537.
[2]Wang D,Li C,Xu Y,et al.Clinicopathological characteristics and prognosis of alpha-fetoprotein positive gastric cancer in Chinese patients[J].Int J Clin Exp Pathol,2015,8(6):6345-6355.
[3]Jiang W,Cui J,Xie D,et al.Sp/KLF family and tumor angiogenesis in pancreatic cancer[J].Curr Pharm Des,2012,18(17):2420-2431.
[4]Marrero-Rodríguez D,Taniguchi-Ponciano K,Jimenez-Vega F,et al.Krüppel-like factor 5 as potential molecular marker in cervical cancer and the KLF family profile expression[J].Tumour Biol,2014,35(11):11399-11407.
[5]Ray A,Alalem M,Ray BK.Loss of epigenetic Kruppel-like factor4 histone deacetylase(KLF-4-HDAC)-mediated transcriptional suppression is crucial in increasing vascular endothelial growth factor(VEGF)expression in breast cancer[J].J Biol Chem,2013,288(38):27232-27242.
[6]Yin L,Wang JP,Xu TP,et al.Downregulation of Kruppel-like factor 2 is associated with poor prognosis for nonsmall-cell lung cancer[J].Tumour Biol,2015,36(4):3075-3084.
[7]Kaczynski JA,Conley AA,Fernandez ZM,et al.Functional analysis of basic transcription element(BTE)-binding protein(BTEB)3and BTEB4,a novel Sp1-like protein,reveals a subfamily of transcriptional repressors for the BTE site of the cytochrome P4501A1gene promoter[J].Biochem J,2002,366(Pt 3):873-882.
[8]Torre LA,Bray F,Siegel RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[9]Satolli MA,Buffoni L,Spadi R,et al.Gastric cancer:the times they are a-changin'[J].World J Gastrointest Oncol,2015,7(11):303-316.
[10]Cravo M,Fidalgo C,Garrido R,et al.Towards curative therapy in gastric cancer:faraway,so close[J].World J Gastroenterol,2015,21(41):11609-11620.
[11]Wei D,Kanai M,Jia Z,et al.Kruppel-like factor 4 induces p27Kip1 expression in and suppresses the growth and metastasis of human pancreatic cancer cells[J].Cancer Res,2008,68(12):4631-4639.
[12]Zhang N,Zhang J,Shuai L,et al.Krüppel-like factor 4 negatively regulatesβ-catenin expression and inhibits the proliferation,invasion and metastasis of gastric cancer[J].Int J Oncol,2012,40(6):2038-2048.
[13]Patel NV,Ghaleb AM,Nandan MO,et al.Expression of the tumor suppressor Krüppel-like factor 4 as a prognostic predictor for colon cancer[J].Cancer Epidemiol Biomarkers Prev,2010,19(10):2631-2638.
[14]Yoda T,Mc Namara KM,Miki Y,et al.KLF15 in breast cancer:a novel tumor suppressor[J].Cell Oncol(Dordr),2015,38(3):227-235.
[15]Daftary GS,Lomberk GA,Buttar NS,et al.Detailed structuralfunctional analysis of the Krüppel-like factor 16(KLF16)transcription factor reveals novel mechanisms for silencing Sp/KLF sites involved in metabolism and endocrinology[J].J Biol Chem,2012,287(10):7010-7025.
[16]Wang J,Galvao J,Beach KM,et al.Novel roles and mechanism for Krüppel-like factor 16(KLF16)regulation of neurite outgrowth and ephrin receptor A5(Eph A5)expression in retinal ganglion cells[J].J Biol Chem,2016,291(41):21422.
[17]Jang MK,Lee S,Jung MH.RNA-Seq analysis reveals a negative role of KLF16 in adipogenesis[J].PLo S One,2016,11(9):e0162238.
[18]Fernandez-Zapico ME,Lomberk GA,Tsuji S,et al.A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth[J].Biochem J,2011,435(2):529-537.