RhoC-siRNA与Rapamycin联合作用对肝癌细胞生物学行为影响的实验研究
|
摘要
本实验为了证实沉默RhoC基因表达与靶向抑制mTOR- Raptor复合物功能对肝癌细胞的侵袭力及增殖具有协同抑制作用,并探讨其作用机制;选取人肝癌细胞系Bel-7402为研究对象,根据实验设计分为对照组、溶媒组、Rapamycin组、阴性质粒组、阴性质粒联合Rapamycin组、RhoC-siRNA组、RhoC-siRNA联合Rapamycin组。首先通过RT-PCR及Western Blot方法确认了RhoC-siRNA质粒对RhoC基因的沉默效应,并选取了对人肝癌细胞系Bel-7402增殖抑制有效的Rapamycin浓度。然后,通过MTT法检测各组Bel-7402细胞的增殖情况;通过RT-PCR法检测、比较各组MMP-2、VEGF、CDK4、Cyclin-D1的基因表达水平。
结果显示,RhoC-siRNA质粒转染及Rapamycin均可降低肿瘤细胞的MMP-2、VEGF、CDK4、Cyclin D1基因表达水平,并抑制其增殖,具有统计学意义(P<0.05)。而RhoC-siRNA联合Rapamycin组的MMP-2、VEGF、CDK4、Cyclin D1基因表达水平显著低于其他各组,具有统计学意义(P<0.05)。
由此我们得出结论,沉默RhoC基因与抑制mTOR- Raptor复合物功能均可降低与人肝癌细胞Bel-7402侵袭及转移密切相关的MMP-2、VEGF等基因,以及细胞周期相关的CDK4、Cyclin-D1基因表达水平;而在沉默RhoC基因基础上联合抑制mTOR- Raptor复合物功能,进一步加强了上述效应,将可能进一步抑制侵袭、转移、增殖等肝癌发展的各关键环节。
The radical resection rate of Hepatocellular carcinoma (HCC) increases greatly than before while the high invasion and metastasis rates after surgical treatment become the bottleneck for HCC. The processes of invasion and metastasis include degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis and so on.
RhoC protein, one of the homologous of Rho family, is an important member of Rho GTPases. It plays a diverse role in cell biology, including cell growth, differentiation and metastasis. mTOR (mammalian target of rapamycin) is the hub of multiple cellular signaling tranduction pathways by enchancing protein transcription through the activation of target molecule p70S6 kinase (S6K 1) and eukaryotic translation initiation factor eIF4E.
The activation of effective molecules MMPs, VEGF, CDK, Cyclin, Actin plays an important role of HCC development process. Relative researches show that the overexpression of RhoC or the activation of mTOR enhances the expression of MMP-2、VEGF、CDK、CyclinD1.
The function of RhoC and mTOR is different while the respective activation induces the similar cell biological changes. The activation of RhoC could lead to tumor cellular proliferation, differentiation, invasion, metastasis through MAPK and PI3K/Akt signal transduction pathway. mTOR is also the downstream target molecule of PI3K/Akt signal transduction pathway. So we suppose that the expression of MMP-2、VEGF、CDK4、CyclinD1 could be regulated by RhoC and mTOR at two different levels and the combination of RhoC and mTOR suppression may downregulate the expression of MMP-2、VEGF、CDK4、CyclinD1 much deeply in order to increase the suppressive effect of HCC invasion, metastasis and proliferation.
The methods are showed as followings:
(1) RNA interfering vector targetting RhoC and siRNA-scramble were successfully constructed and transfected into liver cell Bel-7402 with significant inhibiting effeciency confirmed by RT-PCR and Western- blot.
(2) The inhibiting effeciency of Bel-7402 by rapamycin at differernt concentration using MTT is conducted in order to choose an effective inhibiting concertration. The inhibiting effeciency in transfected Bel-7402 cells as high as 34.78% while the concentration of rapamycin was 9.14mg/L,and we took the concentration.
(3) The cellular proliferation and growth characteristics were conducted using MTT in negative control group, Solvent group, rapamycin group, siRNA-scramble group, siRNA-scramble and rapamycin group, RhoC-SiRNA group, RhoC-SiRNA and rapamycin group. The expression of MMP-2、VEGF、CDK4、Cyclin-D1 in different groups were measured by RT-PCR.
The results are showed as followings:
The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2、VEGF、CDK4、Cyclin-D1. The suppressive effect was significantly higher than that in negative control group, Solvent group, siRNA-scramble group; The suppressive effects of MMP-2、VEGF、CDK4、Cyclin-D1 expression in RhoC-SiRNA and rapamycin group were notably lower than that in RhoC-SiRNA group or rapamycin group respectively. The expressions of MMP-2、VEGF、CDK4、Cyclin-D1 were no remarkable differences among the negative control group, Solvent group, siRNA-scramble group.
The RhoC gene silencing or suppression of mTOR could suppress the tumor cell proliferation. The Cell Proliferation inhibiting rate was significantly higher than that in negative control group, solvent group, siRNA-scramble group in a time-dependent manner, suggesting that the tumor cell proliferation inhibiting effect be achieved by down-regulating CDK4、Cyclin-D1. The cell proliferation inhibiting rate in RhoC-SiRNA and rapamycin group was significantly higher than that in RhoC-SiRNA group or rapamycin group respectively. The cell proliferation inhibiting rates were no remarkable differences among the negative control group, solvent group, siRNA-scramble group.
Conclusion:The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2、VEGF、CDK4、Cyclin-D1 related to HCC invasion, proliferation and angiogenesis. MTT confirmed that the RhoC gene silencing or suppression of mTOR inhibited the proliferation of tumor cells through down-regulating the expression of CDK4、Cyclin-D1. The combination of RhoC gene silencing and suppression of mTOR could down-regulate the expression of MMP-2、VEGF to attenuate the tumor cell invasion, metastasis and proliferation. The RhoC gene silencing could also reduce the tumor cell migration and adhesion.
So the combination of the RhoC gene silencing and suppression of mTOR could notably suppress the HCC invasion and metastasis by degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis in order to cure the tumor.
This research lays a foundation to select RhoC as target molecule in tumor gene therapy both theoretically and experimentally. The combination of the RhoC gene silencing and suppression of mTOR could improve the target treatment effect of HCC, as well as the relationship between RhoC and mTOR.
结果显示,RhoC-siRNA质粒转染及Rapamycin均可降低肿瘤细胞的MMP-2、VEGF、CDK4、Cyclin D1基因表达水平,并抑制其增殖,具有统计学意义(P<0.05)。而RhoC-siRNA联合Rapamycin组的MMP-2、VEGF、CDK4、Cyclin D1基因表达水平显著低于其他各组,具有统计学意义(P<0.05)。
由此我们得出结论,沉默RhoC基因与抑制mTOR- Raptor复合物功能均可降低与人肝癌细胞Bel-7402侵袭及转移密切相关的MMP-2、VEGF等基因,以及细胞周期相关的CDK4、Cyclin-D1基因表达水平;而在沉默RhoC基因基础上联合抑制mTOR- Raptor复合物功能,进一步加强了上述效应,将可能进一步抑制侵袭、转移、增殖等肝癌发展的各关键环节。
The radical resection rate of Hepatocellular carcinoma (HCC) increases greatly than before while the high invasion and metastasis rates after surgical treatment become the bottleneck for HCC. The processes of invasion and metastasis include degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis and so on.
RhoC protein, one of the homologous of Rho family, is an important member of Rho GTPases. It plays a diverse role in cell biology, including cell growth, differentiation and metastasis. mTOR (mammalian target of rapamycin) is the hub of multiple cellular signaling tranduction pathways by enchancing protein transcription through the activation of target molecule p70S6 kinase (S6K 1) and eukaryotic translation initiation factor eIF4E.
The activation of effective molecules MMPs, VEGF, CDK, Cyclin, Actin plays an important role of HCC development process. Relative researches show that the overexpression of RhoC or the activation of mTOR enhances the expression of MMP-2、VEGF、CDK、CyclinD1.
The function of RhoC and mTOR is different while the respective activation induces the similar cell biological changes. The activation of RhoC could lead to tumor cellular proliferation, differentiation, invasion, metastasis through MAPK and PI3K/Akt signal transduction pathway. mTOR is also the downstream target molecule of PI3K/Akt signal transduction pathway. So we suppose that the expression of MMP-2、VEGF、CDK4、CyclinD1 could be regulated by RhoC and mTOR at two different levels and the combination of RhoC and mTOR suppression may downregulate the expression of MMP-2、VEGF、CDK4、CyclinD1 much deeply in order to increase the suppressive effect of HCC invasion, metastasis and proliferation.
The methods are showed as followings:
(1) RNA interfering vector targetting RhoC and siRNA-scramble were successfully constructed and transfected into liver cell Bel-7402 with significant inhibiting effeciency confirmed by RT-PCR and Western- blot.
(2) The inhibiting effeciency of Bel-7402 by rapamycin at differernt concentration using MTT is conducted in order to choose an effective inhibiting concertration. The inhibiting effeciency in transfected Bel-7402 cells as high as 34.78% while the concentration of rapamycin was 9.14mg/L,and we took the concentration.
(3) The cellular proliferation and growth characteristics were conducted using MTT in negative control group, Solvent group, rapamycin group, siRNA-scramble group, siRNA-scramble and rapamycin group, RhoC-SiRNA group, RhoC-SiRNA and rapamycin group. The expression of MMP-2、VEGF、CDK4、Cyclin-D1 in different groups were measured by RT-PCR.
The results are showed as followings:
The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2、VEGF、CDK4、Cyclin-D1. The suppressive effect was significantly higher than that in negative control group, Solvent group, siRNA-scramble group; The suppressive effects of MMP-2、VEGF、CDK4、Cyclin-D1 expression in RhoC-SiRNA and rapamycin group were notably lower than that in RhoC-SiRNA group or rapamycin group respectively. The expressions of MMP-2、VEGF、CDK4、Cyclin-D1 were no remarkable differences among the negative control group, Solvent group, siRNA-scramble group.
The RhoC gene silencing or suppression of mTOR could suppress the tumor cell proliferation. The Cell Proliferation inhibiting rate was significantly higher than that in negative control group, solvent group, siRNA-scramble group in a time-dependent manner, suggesting that the tumor cell proliferation inhibiting effect be achieved by down-regulating CDK4、Cyclin-D1. The cell proliferation inhibiting rate in RhoC-SiRNA and rapamycin group was significantly higher than that in RhoC-SiRNA group or rapamycin group respectively. The cell proliferation inhibiting rates were no remarkable differences among the negative control group, solvent group, siRNA-scramble group.
Conclusion:The RhoC gene silencing or suppression of mTOR could down-regulate the expression of MMP-2、VEGF、CDK4、Cyclin-D1 related to HCC invasion, proliferation and angiogenesis. MTT confirmed that the RhoC gene silencing or suppression of mTOR inhibited the proliferation of tumor cells through down-regulating the expression of CDK4、Cyclin-D1. The combination of RhoC gene silencing and suppression of mTOR could down-regulate the expression of MMP-2、VEGF to attenuate the tumor cell invasion, metastasis and proliferation. The RhoC gene silencing could also reduce the tumor cell migration and adhesion.
So the combination of the RhoC gene silencing and suppression of mTOR could notably suppress the HCC invasion and metastasis by degradation of extracellular matrix, cellular migration, adhesion, proliferation, angiogenesis in order to cure the tumor.
This research lays a foundation to select RhoC as target molecule in tumor gene therapy both theoretically and experimentally. The combination of the RhoC gene silencing and suppression of mTOR could improve the target treatment effect of HCC, as well as the relationship between RhoC and mTOR.
引文
[1] Zalcman G, Closson V, Linares-Cruz G, Lerebours F, Honore N, Tavitian A. and Olofsson, B. Regulation of Ras-related RhoB protein expression during the cell cycle [J]. Oncogene. 1995 18; 10(10): 1935-1945.
[2] Rattan R,et al. Rho/ROCK pathway as a target of tumor therapy [J]. J Neurosci Res, 2006, 83(2): 243-255.
[3] Kleer CG, et al. RhoC GTPase expression as a potential marker of lymph node metastasis in squamous cell carcinomas of the head and neck [J]. Clin Cancer Res, 2006, 12(15): 4485-4490.
[4] van Golen K. L, et al. RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells[J]. Neoplasia, 2000, 2(5): 418-425.
[5] Ikomal T, Takahashi T, Nagano S, et al. A definitive role of RhoC in metastasis of orthotopic lung cancer in mice [J]. Clin Cancer Res, 2004, 10(3): 1192-1200.
[6] Wang W, yang LY, Huang GW, et al. Gwnomic analysis reveals RhoC as a potential marker in hepatocellular carcinoma with poor prognosis [J]. Br J Cancer, 2004, 90(12): 2349-2355.
[7] Pille J Y, Denoyelle C, Varet J, et a1.Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo[J]. Mol Ther, 2005, 11(2): 267-274.
[8] Park CM, Park MJ, Kwak HJ, et a1. Ionizing radiation enhances matrixmetalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidyli- nositol 3-kinase/Akt signaling pathways [J]. Cancer Res, 2006, 66(17): 8511-8519.
[9] Ip YC, Cheung ST, Leung KL, et al. Mechanism of metastasis by membrane type 1-matrix metalloproteinase in hepatocellular carcinoma [J]. World J Gastroenterol, 2006, 11(40): 6269-6276.
[10] Heimberger AB, et al. Mechanisms of action of rapamycin in gliomas [J]. Neuro-oncol. 2005, 7(1): 1-11.
[11] Villalonga P, Ridley AJ. Rho GTPases and cell cycle control[J]. Growth Factors, 2006, 24(3): 159-164.
[12]谢淑丽,孙梅,朱明光,等. RNAi沉默RhoC基因对肝癌细胞生长和转移潜力的影响[J].中国实验诊断学. 2008, 12(10): 1212-1215.
[13]石铮,王家兴,何庆良,等. RhoC反义基因转染对人胆管癌QBC939细胞生长的抑制作用[J].福建医科大学学报, 2005, 39(1): 16-19.
[14]郑文建,吕新生. RhoC基因对肝癌细胞促血管生长因子表达的影响[J].中国普通外科杂志, 2007, 16(12): 1162-1165.
[15] Tominaga Y, Tamguney T, Kolesnichenko M, Bilanges B, Stokoe D. Translational deregulation in PDK-1-/-embryonic stem cells[J]. Mol Cell Biol, 2005, 25(19): 8465-8475.
[16] Tsurutani J, West KA, Sayyah J, Gills JJ, Dennis PA. Inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathwaybut not the MEK/ERK pathway attenuates laminin-mediated small cell lung cancer cellular survival and resistance to imatinib mesylate or chemotherapy[J]. Cancer Res, 2005, 65(19): 8423-8432.
[17]邱兴峰,王效民,殷平,等.肝癌组织Rho蛋白表达与微血管密度的关系[J].世界华人消化杂志, 2006, 14(7): 671-675.
[18]王渝,熊晶等.西罗莫司对肝癌HepG2细胞的增殖及mTOR, HIF-1α的抑制作用[J].世界华人消化杂志, 2009, 17(31): 3241-3243.
[19] Young SK, Hyun SH, Jae CK, et al. Inhibitory effect of rapamycin on corneal neovascularization in vitro and in vivo[J]. IOVS, 2005, 46(2): 454-460.
[20] Semela D et al. Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma [J]. Journal of Hepatology, 2007, 46(5), 840-848.
[21] Hartwell LH, kastan MB. Cell cycle control and cancer [J]. Science, 1994, 266(5192): 1821-1828.
[22] Oshiro N, Yoshino K, Hidayat S, et al.Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function [J]. Genes Cell, 2004, 9(4): 359-366.
[23] Sarbassov DD, A1i SM, Kim DH, et al.Rictor a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-inde-pendent pathway that regulates the cytoskeleton [J]. Curr Biol, 2004, 14(14): 1296-1302.
[24] Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulationof Akt/PKB by the rictor-mTOR complex [J]. Science, 2005, 307(5712): 1098-1101.
[25] TANG ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma [J]. J Cancer Res Clin Oncol, 2004, 130(4): 187-196.
[26] Kusama T, Mukai M, Tatsuta M, et al. Inhibition of transendothelial migration and invasion of human breast cancer cells by preventing geranylgeranylation of Rho [J]. Int J Oncol, 2006,29(1):217-223.
[27] Kandpal RP. Rho GTPase activating proteins in cancer phenotypes [J]. Curr Protein pept Sci, 2006, 7(4): 355-365.
[28] Lang JY, Chen H, Zhou J, et al. Antimetastatic effect of salvicine on human breast cancer MDA-MB-435 orthotopic xenograft is closely related to Rho-dependent Pathway[J]. Clinical Cancer Research, 2005, 11(9): 3455-3464.
[29] Schumacher G, Oidtmann M, Rueggeberg A, et al. Sirolimus inhibits growth of human hepatoma cells alone or combined with tacrolimus, while tacrolimus promotes cell growth[J]. World J Gastroenterol. 2005, 11: 1420-1425.
[30] Clark EA, Golub TR, Lander ES, et al .Genomic analysis of metastasis reveals an essential role for RhoC [J]. Nature, 2000, 406(6795): 532-535.
[31] Okabe H, Satoh S, Kato T, et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray; Identification of genes involved in viral carcinogenesis and tumor progression [J]. CancerRes, 2001, 61(5): 2129-2137
[32] Shi, J. and Wei L, Rho kinase in the regulation of cell death and survival [J]. Arch Immunol Ther Exp (Warsz), 2007, 55(2): 61-75.
[33] Gervaise L, Patrice G, Pierre P. Rho kinases in cardiovascular Physiology and pathophysiology [J]. Circulation Research, 2006, 98(3): 322-334.
[34] Oude Weernink PA, Schulte P, Guo Y, et al. Stimulation of phosphatidy- linositol-4-phosphate 5-kinase by Rho-kinase. [J]. J Biol Chem, 2000, 275(14): 10168-10174.
[35] Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, Yang W, Smith TL, Shi D, Yu D. Activation of the Akt/mammalian target of Rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers [J]. Clin Cancer Res, 2004, 10(20): 6779-6788.
[36] Sahin F, Kannangai R, Adegbola O, et al. mTOR and P70 S6 Kinase expression in primary liver neoplasms [J]. Clin Cancer Res, 2004, 10(24): 8421-8425.
[37] Theret N, Musso O, Turlin B, et al. Increased extracellular matrix remodeling is associated with tumor progression in human hepatocellular carcinomas [J]. Hepatology, 2001, 34(1): 82-88
[38]彭利,王顺祥,张风瑞,等.基质金属蛋白酶-9和血管内皮生长因子与原发性肝癌侵袭转移关系研究[J].中华肝胆外科杂志, 2003, 9(1): 42-44.
[39]张明满,严律南,苟兴华等.腺病毒介导反义基质金属蛋白酶-2抑制肝癌生长和血管生成的研究[J].中华实验外科杂志, 2005, 22(11):1325-1327.
[40]郑文建,梁平,赵弘智,韩克强. RhoC-siRNA表达载体的构建及其对肝癌细胞体外侵袭能力的影响[J].中华消化外科杂志, 2007, 6(1): 35-38.
[41] Sherr CJ. Cancer cell cycle [J]. Science, 1996, 274(5293): 1672-1677.
[42] Michalides RJ. Cell cycle regulators: mechanisms and their role in aetiology, prognosis, and treatment of cancer. [J]. J Clin pathol 1999, 52(8): 555-568.
[43] Alao JP, et al.The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention[J]. Mol Cancer, 2007, 6: 24.
[44] Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm [J].Nat Rev Cancer, 2009, 9(3): p.153-166.
[45]郑文建,梁平,赵弘智.RhoC-siRNA表达载体对肝癌细胞生长速度及侵袭力的影响[J].第三军医大学学报, 2007, 29(18): 1779-1782.
[46] Hopfner M, Schuppan D, Scherubl H. Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer [J]. World J Gastroenterol, 2008, 14(1): 1-14.
[47] Poon RT, Lau C, Pang R, et al. High serum vascular endothelial growth factor levels predict poor prognosis after radiofrequency ablation of hepatocellular carcinoma: importance of tumor biomarker in ablative therapies [J]. Ann Surg Oncol, 2007, 14(6): 1835-1845.
[48] Lang SA, Gaumann A, Koehl GE, et al. Mammalian target of rapamycin isactivated in human gastric cancer and serves as a target for therapy in an experimental model [J].Int J Cancer, 2007, 120(8): 1803-1810.
[49] Matei D, Kelich S, Cao L, et a1. PDGF BB induces VEGF secretion in ovarian cancer [J]. Cancer Biol Ther, 2007, 6(12): 1951-1959.
[50] Wang W, Xu GL, Jia WD, Wang ZH, Li JS, Ma JL, Ge YS, Xie SX, Yu JH. Expression and correlation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor and microvessel density in experimental rat hepatocarcinogenesis [J].J Int Med Res 2009, 37(2): 417-425
[51] Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour [J].Nat Rev Cancer 2008, 8(9): 705-713.
[52] Hannon, GJ. RNA interference[J]. Nature, 2002, 418(6894): 244-251.
[53] Megumi Iiizumi et al.RhoC Promotes Metastasis via Activation of the Pyk2 Pathway in Prostate Cancer. [J]. Cancer Research 2008, 68(18): 7613-7620.
[54] Simon C, Hicks MJ, Nemechek AJ, et al. PD098059, an inhibitor of ERK1 activation, attenuates the in vivo invasiveness of head and neck squamous cell carcinoma [J]. Br J Cancer, 1999, 80(9): 1412-1419.
[55] Okajima E, Thorgeirsson UP. Different regulation of vascular endothelial growth factor expression by the ERK and p38 kinase pathways in V-ras, v-raf, and v-myc transformed cells [J]. Biochem Biophys Res Commun, 2000, 270(1): 108-111.
[56] Harris VK, Coticchia CM, Kagan BL, et al. Induction of the angiogenic modulator fibroblast growth factor-binding protein by epidermal growthfactor is mediated through both MEK/ERK and p38 signal transduction pathways [J]. J Biol Chem, 2000, 275(15): 10802-10811.
[57] Remacle-Bonnet MM, Garrouste FL, Heller S, et al. Insulin-like growth factor-Ⅰprotect colon cancer cells from death factor-induced apoptosis by potentiating tumor necrosis factor alpha-induced mitogen-activated protein kinase and nuclear factor kappaB signaling pathways[J]. Cancer Res 2000, 60(7): 2007-2017.
[58] Baek JH, Jang JE, Kang CM, et al. Hypoxia|induced VEGF enhances tumor survivability via suppression of serum deprivation| induced apoptosis [J]. Oncogene, 2000, 19(40): 4621-4631.
[2] Rattan R,et al. Rho/ROCK pathway as a target of tumor therapy [J]. J Neurosci Res, 2006, 83(2): 243-255.
[3] Kleer CG, et al. RhoC GTPase expression as a potential marker of lymph node metastasis in squamous cell carcinomas of the head and neck [J]. Clin Cancer Res, 2006, 12(15): 4485-4490.
[4] van Golen K. L, et al. RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells[J]. Neoplasia, 2000, 2(5): 418-425.
[5] Ikomal T, Takahashi T, Nagano S, et al. A definitive role of RhoC in metastasis of orthotopic lung cancer in mice [J]. Clin Cancer Res, 2004, 10(3): 1192-1200.
[6] Wang W, yang LY, Huang GW, et al. Gwnomic analysis reveals RhoC as a potential marker in hepatocellular carcinoma with poor prognosis [J]. Br J Cancer, 2004, 90(12): 2349-2355.
[7] Pille J Y, Denoyelle C, Varet J, et a1.Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo[J]. Mol Ther, 2005, 11(2): 267-274.
[8] Park CM, Park MJ, Kwak HJ, et a1. Ionizing radiation enhances matrixmetalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidyli- nositol 3-kinase/Akt signaling pathways [J]. Cancer Res, 2006, 66(17): 8511-8519.
[9] Ip YC, Cheung ST, Leung KL, et al. Mechanism of metastasis by membrane type 1-matrix metalloproteinase in hepatocellular carcinoma [J]. World J Gastroenterol, 2006, 11(40): 6269-6276.
[10] Heimberger AB, et al. Mechanisms of action of rapamycin in gliomas [J]. Neuro-oncol. 2005, 7(1): 1-11.
[11] Villalonga P, Ridley AJ. Rho GTPases and cell cycle control[J]. Growth Factors, 2006, 24(3): 159-164.
[12]谢淑丽,孙梅,朱明光,等. RNAi沉默RhoC基因对肝癌细胞生长和转移潜力的影响[J].中国实验诊断学. 2008, 12(10): 1212-1215.
[13]石铮,王家兴,何庆良,等. RhoC反义基因转染对人胆管癌QBC939细胞生长的抑制作用[J].福建医科大学学报, 2005, 39(1): 16-19.
[14]郑文建,吕新生. RhoC基因对肝癌细胞促血管生长因子表达的影响[J].中国普通外科杂志, 2007, 16(12): 1162-1165.
[15] Tominaga Y, Tamguney T, Kolesnichenko M, Bilanges B, Stokoe D. Translational deregulation in PDK-1-/-embryonic stem cells[J]. Mol Cell Biol, 2005, 25(19): 8465-8475.
[16] Tsurutani J, West KA, Sayyah J, Gills JJ, Dennis PA. Inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathwaybut not the MEK/ERK pathway attenuates laminin-mediated small cell lung cancer cellular survival and resistance to imatinib mesylate or chemotherapy[J]. Cancer Res, 2005, 65(19): 8423-8432.
[17]邱兴峰,王效民,殷平,等.肝癌组织Rho蛋白表达与微血管密度的关系[J].世界华人消化杂志, 2006, 14(7): 671-675.
[18]王渝,熊晶等.西罗莫司对肝癌HepG2细胞的增殖及mTOR, HIF-1α的抑制作用[J].世界华人消化杂志, 2009, 17(31): 3241-3243.
[19] Young SK, Hyun SH, Jae CK, et al. Inhibitory effect of rapamycin on corneal neovascularization in vitro and in vivo[J]. IOVS, 2005, 46(2): 454-460.
[20] Semela D et al. Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma [J]. Journal of Hepatology, 2007, 46(5), 840-848.
[21] Hartwell LH, kastan MB. Cell cycle control and cancer [J]. Science, 1994, 266(5192): 1821-1828.
[22] Oshiro N, Yoshino K, Hidayat S, et al.Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function [J]. Genes Cell, 2004, 9(4): 359-366.
[23] Sarbassov DD, A1i SM, Kim DH, et al.Rictor a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-inde-pendent pathway that regulates the cytoskeleton [J]. Curr Biol, 2004, 14(14): 1296-1302.
[24] Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulationof Akt/PKB by the rictor-mTOR complex [J]. Science, 2005, 307(5712): 1098-1101.
[25] TANG ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma [J]. J Cancer Res Clin Oncol, 2004, 130(4): 187-196.
[26] Kusama T, Mukai M, Tatsuta M, et al. Inhibition of transendothelial migration and invasion of human breast cancer cells by preventing geranylgeranylation of Rho [J]. Int J Oncol, 2006,29(1):217-223.
[27] Kandpal RP. Rho GTPase activating proteins in cancer phenotypes [J]. Curr Protein pept Sci, 2006, 7(4): 355-365.
[28] Lang JY, Chen H, Zhou J, et al. Antimetastatic effect of salvicine on human breast cancer MDA-MB-435 orthotopic xenograft is closely related to Rho-dependent Pathway[J]. Clinical Cancer Research, 2005, 11(9): 3455-3464.
[29] Schumacher G, Oidtmann M, Rueggeberg A, et al. Sirolimus inhibits growth of human hepatoma cells alone or combined with tacrolimus, while tacrolimus promotes cell growth[J]. World J Gastroenterol. 2005, 11: 1420-1425.
[30] Clark EA, Golub TR, Lander ES, et al .Genomic analysis of metastasis reveals an essential role for RhoC [J]. Nature, 2000, 406(6795): 532-535.
[31] Okabe H, Satoh S, Kato T, et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray; Identification of genes involved in viral carcinogenesis and tumor progression [J]. CancerRes, 2001, 61(5): 2129-2137
[32] Shi, J. and Wei L, Rho kinase in the regulation of cell death and survival [J]. Arch Immunol Ther Exp (Warsz), 2007, 55(2): 61-75.
[33] Gervaise L, Patrice G, Pierre P. Rho kinases in cardiovascular Physiology and pathophysiology [J]. Circulation Research, 2006, 98(3): 322-334.
[34] Oude Weernink PA, Schulte P, Guo Y, et al. Stimulation of phosphatidy- linositol-4-phosphate 5-kinase by Rho-kinase. [J]. J Biol Chem, 2000, 275(14): 10168-10174.
[35] Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, Yang W, Smith TL, Shi D, Yu D. Activation of the Akt/mammalian target of Rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers [J]. Clin Cancer Res, 2004, 10(20): 6779-6788.
[36] Sahin F, Kannangai R, Adegbola O, et al. mTOR and P70 S6 Kinase expression in primary liver neoplasms [J]. Clin Cancer Res, 2004, 10(24): 8421-8425.
[37] Theret N, Musso O, Turlin B, et al. Increased extracellular matrix remodeling is associated with tumor progression in human hepatocellular carcinomas [J]. Hepatology, 2001, 34(1): 82-88
[38]彭利,王顺祥,张风瑞,等.基质金属蛋白酶-9和血管内皮生长因子与原发性肝癌侵袭转移关系研究[J].中华肝胆外科杂志, 2003, 9(1): 42-44.
[39]张明满,严律南,苟兴华等.腺病毒介导反义基质金属蛋白酶-2抑制肝癌生长和血管生成的研究[J].中华实验外科杂志, 2005, 22(11):1325-1327.
[40]郑文建,梁平,赵弘智,韩克强. RhoC-siRNA表达载体的构建及其对肝癌细胞体外侵袭能力的影响[J].中华消化外科杂志, 2007, 6(1): 35-38.
[41] Sherr CJ. Cancer cell cycle [J]. Science, 1996, 274(5293): 1672-1677.
[42] Michalides RJ. Cell cycle regulators: mechanisms and their role in aetiology, prognosis, and treatment of cancer. [J]. J Clin pathol 1999, 52(8): 555-568.
[43] Alao JP, et al.The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention[J]. Mol Cancer, 2007, 6: 24.
[44] Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm [J].Nat Rev Cancer, 2009, 9(3): p.153-166.
[45]郑文建,梁平,赵弘智.RhoC-siRNA表达载体对肝癌细胞生长速度及侵袭力的影响[J].第三军医大学学报, 2007, 29(18): 1779-1782.
[46] Hopfner M, Schuppan D, Scherubl H. Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer [J]. World J Gastroenterol, 2008, 14(1): 1-14.
[47] Poon RT, Lau C, Pang R, et al. High serum vascular endothelial growth factor levels predict poor prognosis after radiofrequency ablation of hepatocellular carcinoma: importance of tumor biomarker in ablative therapies [J]. Ann Surg Oncol, 2007, 14(6): 1835-1845.
[48] Lang SA, Gaumann A, Koehl GE, et al. Mammalian target of rapamycin isactivated in human gastric cancer and serves as a target for therapy in an experimental model [J].Int J Cancer, 2007, 120(8): 1803-1810.
[49] Matei D, Kelich S, Cao L, et a1. PDGF BB induces VEGF secretion in ovarian cancer [J]. Cancer Biol Ther, 2007, 6(12): 1951-1959.
[50] Wang W, Xu GL, Jia WD, Wang ZH, Li JS, Ma JL, Ge YS, Xie SX, Yu JH. Expression and correlation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor and microvessel density in experimental rat hepatocarcinogenesis [J].J Int Med Res 2009, 37(2): 417-425
[51] Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour [J].Nat Rev Cancer 2008, 8(9): 705-713.
[52] Hannon, GJ. RNA interference[J]. Nature, 2002, 418(6894): 244-251.
[53] Megumi Iiizumi et al.RhoC Promotes Metastasis via Activation of the Pyk2 Pathway in Prostate Cancer. [J]. Cancer Research 2008, 68(18): 7613-7620.
[54] Simon C, Hicks MJ, Nemechek AJ, et al. PD098059, an inhibitor of ERK1 activation, attenuates the in vivo invasiveness of head and neck squamous cell carcinoma [J]. Br J Cancer, 1999, 80(9): 1412-1419.
[55] Okajima E, Thorgeirsson UP. Different regulation of vascular endothelial growth factor expression by the ERK and p38 kinase pathways in V-ras, v-raf, and v-myc transformed cells [J]. Biochem Biophys Res Commun, 2000, 270(1): 108-111.
[56] Harris VK, Coticchia CM, Kagan BL, et al. Induction of the angiogenic modulator fibroblast growth factor-binding protein by epidermal growthfactor is mediated through both MEK/ERK and p38 signal transduction pathways [J]. J Biol Chem, 2000, 275(15): 10802-10811.
[57] Remacle-Bonnet MM, Garrouste FL, Heller S, et al. Insulin-like growth factor-Ⅰprotect colon cancer cells from death factor-induced apoptosis by potentiating tumor necrosis factor alpha-induced mitogen-activated protein kinase and nuclear factor kappaB signaling pathways[J]. Cancer Res 2000, 60(7): 2007-2017.
[58] Baek JH, Jang JE, Kang CM, et al. Hypoxia|induced VEGF enhances tumor survivability via suppression of serum deprivation| induced apoptosis [J]. Oncogene, 2000, 19(40): 4621-4631.