摘要
第一部分
目的:探讨肝细胞粘附分子(hepaCAM)对肾癌增殖的影响及机制。
方法:将携带hepaCAM基因的重组质粒转染786-0细胞,RT-PCR、western blot鉴定hepaCAM基因在786-0中的表达及其对c-Myc表达的影响;细胞计数观测细胞增殖活力变化;流式细胞术检测细胞周期的变化。
结果:上调hepaCAM基因表达可抑制肾癌786-0增殖,阻止细胞周期于G0/G1期,并伴随着c-Myc蛋白表达降低。
结论:上调hepaCAM基因可抑制肾癌786-0增殖,并下调c-Myc蛋白表达而对c-Myc基因水平无影响,说明hepaCAM抑制肾癌增殖可能是通过调节c-Myc翻译后水平实现的。
第二部分
目的:探讨c-Myc特异性小分子抑制剂10058-F4对肾癌增殖的影响,进一步研究hepaCAM对c-Myc的具体调节作用及抑制肾癌增殖的作用机制。
方法:MTT检测10058-F4对肾癌增殖作用的时间依赖性和剂量依赖性;FCM检测细胞周期;Western blot检测hepaCAM对c-Myc稳定性、磷酸化及c-Myc下游分子P21和cyclin D1表达的影响。
结果:10058-F4可抑制肾癌增殖并阻止细胞周期于G0/G1期,上调hepaCAM表达后,c-Myc的稳定性降低; c-Myc磷酸化增加; c-Myc下游分子cyclin D1表达下调、p21表达上调。
结论:C-Myc特异性小分子抑制剂10058-F4可抑制肾癌增殖,其作用与hepaCAM抑制肾癌增殖作用相当,hepaCAM可同时下调c-Myc蛋白水平表达,说明hepaCAM可能通过抑制c-Myc蛋白水平表达从而抑制肾癌增殖,与以上相符的是,hepaCAM可使c-Myc蛋白稳定性降低,促进其泛素化降解,而T58位c-Myc磷酸化是其泛素化降解的前题,上调hepaCAM表达后T58位c-Myc磷酸化增加,说明hepaCAM是通过增加T58位c-Myc磷酸化增加促进其降解,进而调节其相应下游分子cyclin D1、p21等表达抑制肾癌增殖。
PART ONE
Objective: To investigate the effect of hepatocyte cell adhesion molecule (hepaCAM) on proliferation of renal cell carcinoma and its mechanisms.
Methods: The vector containing hepaCAM gene was transfeceted into 786-0 cells, the expression of hepaCAM and c-Myc in 786-0 cells were measured by RT-PCR and western blot; Cell proliferation was detected by cell counting; the cell cycle was analyzed by FCM.
Results: Up-regulated the expression of hepaCAM gene in 786-0 cells can inhibit cell proliferation, which accompany with down-regulation of c-Myc.
Conclusion: we demonstrated that re-expression of hepaCAM caused an accumulation in G0/G1 phase in 786-0 cells. This reaction was accompanied by a substantial reduction of c-Myc expression through using an ectopic hepaCAM expression system. Nevertheless, re-expression of hepaCAM can result in apparent reduction of c-Myc protein while no corresponding reduction of c-Myc mRNA. This suggests that this reaction might take place at a post-transcriptional level rather than transcriptional one.
PART TWO
Objective: To investigate the effect of the small molecule c-Myc inhibitor, 10058-F4, on proliferation of renal cell carcinoma and the mechanisms of hepaCAM inhibits renal cell carcinoma proliferation through regulating c-Myc.
Methods: Cell proliferation was detected by MTT assay; The proliferation of 786-0 cells was analyzed by FCM; Western blot was used to detected the effects of hepaCAM on the stability and phosphorylation of c-Myc and the targets of c-Myc, P21 and cyclin D1.
Results: we showed a comparable decrease in proliferation and G0/G1 accumulation of 786-0 and RC-2 cells after treatment with a small molecule c-Myc inhibitor, 10058-F4. re-expression of hepaCAM decreased c-Myc stability by increasing the proportion of c-Myc phosphorylation on T58 and the expression of P21 was increased and the expression of cyclin D1 was decreased.
Conclusion: we showed a comparable decrease in proliferation and G0/G1 accumulation of 786-0 and RC-2 cells after treatment with a small molecule c-Myc inhibitor, 10058-F4. It demonstrated that down regulation of c-Myc was an essential process in controlling growth inhibitory actions of hepaCAM. Nevertheless, re-expression of hepaCAM can result in apparent reduction of c-Myc protein while no corresponding reduction of c-Myc mRNA. This suggests that this reaction might take place at a post-transcriptional level rather than transcriptional one. Consistent with these findings, hepaCAM decreased c-Myc stability by increasing the proportion of c-Myc phosphorylation on T58 which can be abrogated by a proteasomal inhibitor (MG132). Thus, our results imply that the decrease in c-Myc protein expression, resulting from ectopic expression of hepaCAM, may contribute to inhibition of proliferation in these cells.
引文
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[1] Mei Chung Moh, Lay Hoon Lee, Xiaodong Yang, Shali Shen, HEPN1, a novel gene that is frequently down-regulated in hepatocellular carcinoma, suppresses cell growth and induces apoptosis in HepG2 cells[J]. Journal of Hepatology 39 (2003) 580–586.
[2] LINDA FAVRE-KONTULA, ALEXANDRE ROLLAND, LILIA BERNASCON, GlialCAM, an Immunoglobulin-Like Cell Adhesion Molecule is Expressed in Glial Cells of the Centra Nervous System[J]. GLIA 56(2008)633–645.
[3] Moh MC, Zhang C, Luo C, Lee LH, Shen S, Structural and Functional Analyses of a Novel Ig-like Cell Adhesion Molecule, hepaCAM, in the Human Breast Carcinoma MCF7 Cells[J], J Biol Chem. 280 (2005) 27366–27374.
[4] Chung Moh M, Hoon Lee L, Shen S, Cloning and characterization of hepaCAM, a novel Ig-like cell adhesion molecule suppressed in human hepatocellular carcinoma, J Hepatol. 42 (2005) 833–841.
[5] Chung Moh M, Hoon Lee L, Shen S, Cloning and characterization of hepaCAM, a novel Ig-like cell adhesion molecule suppressed in human hepatocellular carcinoma[J], J Hepatol. 42 (2005) 833–841.
[6] Xun C, Luo C, Wu X, Zhang Q, Yan L, Shen S, Expression of hepacam and its effect on proliferation of tumor cells in renal cell carcinoma[J], Urology. 75 (2010) 828-34.
[7] Pan, Cuicui, Wu, Xiaohou, Luo, Chunli, Yang, Shuzhe, Pu, Jun, Wang, Chunyuan, Shen, Shali, Exon 2 Methylation Inhibits hepaCAM Expression in Transitional Cell Carcinoma of the Bladder[J], Urologia internationalis . 85 (2010) 347-54.
[8] Moh, M C, Tian, Q, Zhang, T, Lee, L H, Shen, S, The immunoglobulin-like cell adhesion molecule hepaCAM modulate cell adhesion and motility through direct interaction with the actin cytoskeleton[J], J Cell Physiol. 219 (2009) 382-91.
[9] Lee LH, Moh MC, Zhang T, Shen S, The immunoglobulin-like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373-MG cells[J], J cell Biochem. 107 (2009) 1129-1138.
[10] Shuzhe Yang, Xiaohou Wu, Chunli Luo, Cuicui Pan, Jun Pu, Expression and clinical significance of hepaCAM and VEGF in urothelial carcinoma[J], World JUrol. 28 (2010) 473–478.
[11] He Y, Wu X, Luo C, Wang L, Lin J, Functional significance of the hepaCAM gene in bladder cancer[J], BMC Cancer. 10 (2010).
