RAC1对人舌鳞癌CAL27细胞迁移、侵袭的影响
|
摘要
目的 :构建RAC1基因小干扰RNA(small interfering RNA,si RNA),探讨RAC1对人舌鳞癌CAL27细胞侵袭、迁移的影响及其分子机制。方法:针对人RAC1基因设计并合成3条si RNA,通过脂质体介导转染进3组人舌鳞癌细胞CAL27中,以抑制RAC1的表达;同时设置阴性对照组(转染无关核苷酸序列)和空白对照组(未转染),采用实时荧光定量PCR检测细胞中RAC1 m RNA的表达,Western免疫印迹实验检测RAC1、PAK1、LIMK1蛋白的表达,通过划痕实验及Transwell细胞侵袭实验检测转染后细胞迁移和侵袭能力的变化。采用SPSS 18.0软件包对数据进行统计学分析。结果:细胞转染48 h后,与阴性对照组和空白对照组相比,RAC1干扰组细胞RAC1 m RNA和蛋白表达均显著下降(P<0.05),PAK1、LIMK1蛋白表达显著下降(P<0.05),细胞迁移和侵袭能力显著降低(P<0.05)。结论:沉默RAC1基因可抑制舌鳞癌CAL27细胞的侵袭和转移,其机制可能与细胞内PAK1、LIMK1的表达下调有关。
PURPOSE: To investigate the effect of RAC1 on invasion and migration of human squamous cell carcinoma of tongue cells of CAL27, as well as the possible molecular mechanism. METHODS: The specific small interfering RNAs(si RNAs) was designed and constructed on the basis of RAC1 gene sequence. Through liposome mediation, these three RAC1 siRNAs were transfected into CAL27 cells to inhibit RAC1 expression. Meanwhile, negative control group and blank control group were transfected with random sequence NC-si RNA and liposome, respectively. The m RNA of RAC1 were detected by real-time fluorescence quantitative polymerase chain reaction and the protein expression of RAC1,PAK1, LIMK1 were examined by Western blotting. The migration and invasion ability were analyzed by wound healing assay and Transwell chamber test. One-Way ANOVA and student's t test were used for data analysis with SPSS 18.0 software package. RESULTS: The protein expression of RAC1 was decreased significantly 48 h after transfection( P<0.05),the expression of PAK1 and LIMK1 were suppressed(P<0.05), the migration and invasion ability of CAL27 cells was also significantly inhibited(P <0.05). CONCLUSIONS: Silencing RAC1 gene can inhibit migration and invasion ability of human squamous cell carcinoma of tongue CAL27 cells, and the mechanism may be related to down-regulation of PAK1 and LIMK1 expression in tongue cancer cells.
PURPOSE: To investigate the effect of RAC1 on invasion and migration of human squamous cell carcinoma of tongue cells of CAL27, as well as the possible molecular mechanism. METHODS: The specific small interfering RNAs(si RNAs) was designed and constructed on the basis of RAC1 gene sequence. Through liposome mediation, these three RAC1 siRNAs were transfected into CAL27 cells to inhibit RAC1 expression. Meanwhile, negative control group and blank control group were transfected with random sequence NC-si RNA and liposome, respectively. The m RNA of RAC1 were detected by real-time fluorescence quantitative polymerase chain reaction and the protein expression of RAC1,PAK1, LIMK1 were examined by Western blotting. The migration and invasion ability were analyzed by wound healing assay and Transwell chamber test. One-Way ANOVA and student's t test were used for data analysis with SPSS 18.0 software package. RESULTS: The protein expression of RAC1 was decreased significantly 48 h after transfection( P<0.05),the expression of PAK1 and LIMK1 were suppressed(P<0.05), the migration and invasion ability of CAL27 cells was also significantly inhibited(P <0.05). CONCLUSIONS: Silencing RAC1 gene can inhibit migration and invasion ability of human squamous cell carcinoma of tongue CAL27 cells, and the mechanism may be related to down-regulation of PAK1 and LIMK1 expression in tongue cancer cells.
引文
[1]Lee DY,Kang SH,Kim JH,et al.Survival and recurrence of resectable tongue cancer:Resection margin cutoff value by T classification[J].Head Neck,2018,40(2):283-291.
[2]Lam L,Logan RM,Luke C,et al.Retrospective study of survival and treatment pattern in a cohort of patients with oral and oropharyngeal tongue cancers from 1987 to 2004[J].Oral Oncol,2007,43(2):150-158.
[3]Al-Rajhi N,Khafaga Y,El-Husseiny J,et al.Early stage carcinoma of oral tongue:prognostic factors for local control and survival[J].Oral Oncol,2000,36(4):508-514.
[4]章硕,姜浩,苏琦.Rac1与肿瘤的研究进展[J].国际病理科学与临床杂志,2011,31(5):410-414.
[5]Tan W,Palmby TR,Gavard J,et al.An essential role for Rac1 in endothelial cell function and vascular development[J].FASEBJ,2008,22(6):1829-1838.
[6]Malla RR,Gopinath S,Gondi CS,et al.Cathepsin B and u PAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma[J].Cancer Gene Ther,2011,18(6):419-434.
[7]Du J,Liu J,Smith BJ,et al.Role of Rac1-dependent NADPH oxidase in the growth of pancreatic cancer[J].Cancer Gene Ther,2011,18(2):135-143.
[8]Bosco EE,Nakai Y,Hennigan RF,et al.NF2-deficient cells depend on the Rac1-canonical Wnt signaling pathway to promote the loss of contact inhibition of proliferation[J].Oncogene,2010,29(17):2540-2549.
[9]Zhao SY,Sun Y,Lai ZS,et al.Inhibition of migration and invasion of colorectal cancer cells via deletion of Rac1 with RNA interference[J].Mol Cell Biochem,2009,322(1-2):179-184.
[10]Pan Y,Bi F,Liu N,et al.Expression of seven main Rho family members in gastric carcinoma[J].Biochem Biophys Res Commun,2004,315(3):686-691.
[11]Roberts AW,Kim C,Zhen L,et al.Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense[J].Immunity,1999,10(2):183-196.
[12]Giannone G,Dubin-Thaler BJ,Rosier O,et al.Lamellipodial actin mechanically links myosin activity with adhesion site formation[J].Cell,2007,128(3):561-575.
[13]Ridley AJ.Rho GTPases and cell migration[J].J Cell Sci,2001,114(6):2713-2722.
[14]Etienne-Manneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629-635.
[15]Bokoch GM.Biology of the p21-activated kinases[J].Annu Rev Biochem,2003,72:743-781.
[16]Benner GE,Dennis PB,Masaracchia RA.Activation of an S6/H4kinase(PAK 65)from 72 human placentae by intramolecular and intermolecular autophosphorylation[J].J Biol Chem,1995,270(36):21121-21128.
[17]Roig J,Traugh JA.Cytostatic p21 G protein-activated protein kinase gamma-PAK[J].Vitam Horm,2001,62:167-198.
[18]Rudel T,Bokoch GM.Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2[J].Science,1997,276(5318):1571-1574.
[19]Connolly JO,Simpson N,Hewlett L,et al.Rac regulates endothelial morphogenesis and capillary assembly[J].Mol Biol Cell,2002,13(7):2474-2485.
[20]Balasenthil S,Sahin AA,Barnes CJ,et al.p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells[J].J Biol Chem,2004,279(2):1422-1428.
[21]Holm C,Rayala S,Jirstrom K,et al.Association between Pak1expression and subcellular localization and tamoxifen resistance in breast cancer patients[J].J Natl Cancer Inst,2006,98(10):671-680.
[22]Carter JH,Douglass LE,Deddens JA,et al.Pak-1 expression increases with progression of colorectal carcinomas to metastasis[J].Clin Cancer Res,2004,10(10):3448-3456.
[23]罗婧,张琍.LIM激酶与肿瘤迁移、侵袭的研究进展[J].中国现代医药杂志,2014,16(10):102-105.
[24]Andrianantoandro E,Pollard TD.Mechanism of action filament turn over by severing and nucleation at different concentrations of ADF/cofilin[J].Mol Cell,2006,24(1):13-23.
[25]Delorme V,Machacek M,Der Mardirossian C,et al.Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks[J].Dev Cell,2007,13(5):646-662.
[26]Aznar S,Lacal JC.Rho signals to cell growth and apoptosis[J].Cancer Lett,2001,165(1):1-10.
[27]Garrett TA,Van Buul JD,Burridge K.VEGF-induced Rac1activation in endothelial cells is regulated by the guanine nucleotide ex-change factor Vav2[J].Exp Cell Res,2007,313(15):3285-3297.
[2]Lam L,Logan RM,Luke C,et al.Retrospective study of survival and treatment pattern in a cohort of patients with oral and oropharyngeal tongue cancers from 1987 to 2004[J].Oral Oncol,2007,43(2):150-158.
[3]Al-Rajhi N,Khafaga Y,El-Husseiny J,et al.Early stage carcinoma of oral tongue:prognostic factors for local control and survival[J].Oral Oncol,2000,36(4):508-514.
[4]章硕,姜浩,苏琦.Rac1与肿瘤的研究进展[J].国际病理科学与临床杂志,2011,31(5):410-414.
[5]Tan W,Palmby TR,Gavard J,et al.An essential role for Rac1 in endothelial cell function and vascular development[J].FASEBJ,2008,22(6):1829-1838.
[6]Malla RR,Gopinath S,Gondi CS,et al.Cathepsin B and u PAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma[J].Cancer Gene Ther,2011,18(6):419-434.
[7]Du J,Liu J,Smith BJ,et al.Role of Rac1-dependent NADPH oxidase in the growth of pancreatic cancer[J].Cancer Gene Ther,2011,18(2):135-143.
[8]Bosco EE,Nakai Y,Hennigan RF,et al.NF2-deficient cells depend on the Rac1-canonical Wnt signaling pathway to promote the loss of contact inhibition of proliferation[J].Oncogene,2010,29(17):2540-2549.
[9]Zhao SY,Sun Y,Lai ZS,et al.Inhibition of migration and invasion of colorectal cancer cells via deletion of Rac1 with RNA interference[J].Mol Cell Biochem,2009,322(1-2):179-184.
[10]Pan Y,Bi F,Liu N,et al.Expression of seven main Rho family members in gastric carcinoma[J].Biochem Biophys Res Commun,2004,315(3):686-691.
[11]Roberts AW,Kim C,Zhen L,et al.Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense[J].Immunity,1999,10(2):183-196.
[12]Giannone G,Dubin-Thaler BJ,Rosier O,et al.Lamellipodial actin mechanically links myosin activity with adhesion site formation[J].Cell,2007,128(3):561-575.
[13]Ridley AJ.Rho GTPases and cell migration[J].J Cell Sci,2001,114(6):2713-2722.
[14]Etienne-Manneville S,Hall A.Rho GTPases in cell biology[J].Nature,2002,420(6916):629-635.
[15]Bokoch GM.Biology of the p21-activated kinases[J].Annu Rev Biochem,2003,72:743-781.
[16]Benner GE,Dennis PB,Masaracchia RA.Activation of an S6/H4kinase(PAK 65)from 72 human placentae by intramolecular and intermolecular autophosphorylation[J].J Biol Chem,1995,270(36):21121-21128.
[17]Roig J,Traugh JA.Cytostatic p21 G protein-activated protein kinase gamma-PAK[J].Vitam Horm,2001,62:167-198.
[18]Rudel T,Bokoch GM.Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2[J].Science,1997,276(5318):1571-1574.
[19]Connolly JO,Simpson N,Hewlett L,et al.Rac regulates endothelial morphogenesis and capillary assembly[J].Mol Biol Cell,2002,13(7):2474-2485.
[20]Balasenthil S,Sahin AA,Barnes CJ,et al.p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells[J].J Biol Chem,2004,279(2):1422-1428.
[21]Holm C,Rayala S,Jirstrom K,et al.Association between Pak1expression and subcellular localization and tamoxifen resistance in breast cancer patients[J].J Natl Cancer Inst,2006,98(10):671-680.
[22]Carter JH,Douglass LE,Deddens JA,et al.Pak-1 expression increases with progression of colorectal carcinomas to metastasis[J].Clin Cancer Res,2004,10(10):3448-3456.
[23]罗婧,张琍.LIM激酶与肿瘤迁移、侵袭的研究进展[J].中国现代医药杂志,2014,16(10):102-105.
[24]Andrianantoandro E,Pollard TD.Mechanism of action filament turn over by severing and nucleation at different concentrations of ADF/cofilin[J].Mol Cell,2006,24(1):13-23.
[25]Delorme V,Machacek M,Der Mardirossian C,et al.Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks[J].Dev Cell,2007,13(5):646-662.
[26]Aznar S,Lacal JC.Rho signals to cell growth and apoptosis[J].Cancer Lett,2001,165(1):1-10.
[27]Garrett TA,Van Buul JD,Burridge K.VEGF-induced Rac1activation in endothelial cells is regulated by the guanine nucleotide ex-change factor Vav2[J].Exp Cell Res,2007,313(15):3285-3297.