| [Objective]The research of oncomolecularbiology and molecular genetics manifest the mechanism of activation of many proto-oncogene and deactivation of anti-oncogenes. The development of Colorectal cancer involved the changes of a sets of genes in differentiation stees. There are a lot of kinds of proto-oncogene being actived and anti-oncogene being inactivated. Four and a half LIM protein 2 (FHL2) is the only protein with the LIM domain, belongs to the member of LIM protein family. LIM domains are present in many proteins that have diverse cellular roles as regulators of gene expression, cyto-architecture, cell adhesion, cell motility and signal transduction. The human four-and-a-half-LIM-only protein family consists of the members of FHL1, FHL2, FHL3, FHL4 and ACT. They are expressed in a cell- and tissue-specific manner and participate in various cellular processes, including regulation of cell survival, transcription and signal transduction. Here, we review the current knowledge of the best-studied member of this family, FHL2.It localized mainly in nucleus and can shuttle between cytoplasm and nucleus. FHL2 is a proteinum product of oncogene codogenic.FHL2 is particularly intriguing because it can function as either a repressor or activator of target proteins in a cell type-dependent fashion, and interacts with other proteins. FHL2 protein expression was nearly undetectable in normal tissue samples. However, many cancerous tissues expressed higher levels of FHL2 than normal tissues, suggesting this gene might be a novel oncogene, In this study, we investigated the role of FHL2 in growth and differentiation in colon cancer.[Methods]1,The expression of FHL2 protein in clinical specimens: Normal or colon cancer tissues with different stage of differentiation were obtained in the Nanfang Hospital (Guangzhou, China)from June to December 2005: (1) FHL2 expression was detected by immunohistochemistry. (2) FHL2 expression in colon cancer tissues and matched normal tissues as detected by Western blotting.2,In vitro expression: (1) Protein expression of FHL2 in colon cancer cell lines SW620, SW1116, SW480, DLD1, HCT15, HT29, LoVo and Colo205 was detected by Western blotting. (2) FHL2 expression in stable transfectants of LoVo cells expressing vector or antisense FHL2 was detected by Western blotting. (3) Morphology of stable transfectants of LoVo/Vector and LoVo/FHL2-AS1 was observed under phase-contrast microscope. (4) Stable transfectants of LoVo cells was stained withα-tubulin to show the cytoplasm and Hochest 22358 to show the nuclei, the visualized under by fluorescent microscopy. (5) Expression of CEA and E-cadherin in LoVo/Vector and LoVo/FHL2-AS stable transfectants was detected by RT-PCR. (6) Stable transfectants of LoVo/Vector and LoVo/FHL2-AS1 stained with rhodamine-phallotoxin with F-actin filaments being visualized were under fluorescent microscopy. (7) The expression of cox-2, survivin, c-jun and hTERT in stable transfectants of LoVo cells was detected by RT-PCR. (8) LoVo/Vector and LoVo/FHL2-AS stable transfectants were seeded into a 96 well plate in triplicate at a density of 10,000 cells/well for 12 h. The medium was replaced with RPMI 1640 containing 0.1%, 0.4%, 2% or 10% FBS for additional 48 h, and then cell growth was assessed by MTT assay. The data obtained were statistically analyzed by SPSS 12.0. The results with different treatments were compared using a two-tailed Student's t test and considered significant if pvalue was less than 0.05.[Results]1,Detection of FHL2 protein in clinical specimen: (1) FHL2 protein was mainly localized in nucleus. FHL2 protein was detectable only in few normal tissues. However, all colon cancer tissues expressed higher levels of FHL2 normal tissues and the expression pattern is negatively correlated with cell differentiation. The lower differentiation cancer tissues expression higher levels of FHL2; (2) Western blotting assay showed that colon cancer tissues expressed higher level of FHL2 than matched normal tissues.2,Suppression of FHL2 expression induced differentiation in colon cancer cells: (1) Protein expression of FHL2 in colon cancer cell lines SW620, SW1116, SW480, DLD1, HCT15, LoVo and Colo205 was relatively high. However, it is nearly undetectable in semi-differentiated HT29 cells. (2) We established stable LoVo transfectant expression vector control or FHL2-AS gene. Western blotting assay showed that LoVo/FHL2-AS expressed lower levels of FHL2 protein compared with LoVo/Vector. (3) The stable transfectants of LoVo/Vector displayed a round or flat morphology with a short cytoplasmic process. However, LoVo/FHL2-AS transfectant exhibited lengthened or shuttle-shape morphology. Long or dendritic-like cytoplasmic processes were visible under phase-contrast microscope. (4) To distinguish cytoplasm and nucleus, we stained the stable transfectants withα-tubulin antibody by immunofluorescence to display the cytoplasm and Hochest22358 to display the nuclei. Similar morphological changes with that observed under phase-contrast microscope were found. Furthermore, LoVo/FHL2-AS stable transfectants exhibited a decreased nuclear:cytoplasmic ratio by mainly increasing the cytoplasm plot and slightly decreasing the size of nuclei. (5) The pretreatment with ATRA induced similar morphological changes to that of LoVo/FHL2-AS transfectant. (6) RT-PCR showed that suppression of FHL2 increased expression of the differentiation markers of colon cancer, CEA and E-cadherin. (7) F-actin staining showed weak and aggregated F-actin filaments exhibited in LoVo/Vector transfectants. Suppression of FHL2 displayed uniform and highly structured array of thick F-actin filaments at cell-adherent regions and throughout the cytoplasm. Importantly, typical microvilli-like actin filaments that protruded out of the cytoplasm plot were observed only in LoVo/FHL2-AS transfectants. (8) The mRNA expression of survivin, cox-2, hTERT, and c-jun were downregulated in stable transfectants of FHL2 antisense. (9) The cell proliferation by MTT assay of LoVo/Vector were 113.8±5%, 126.4±6% and 162.5±5% when cultured in the presence of 0.4%, 2% and 10% FBS, respectively, while those of LoVo/FHL2-AS1 and LoVo/FHL2-AS2 were 103.3±3%, 110.3±5% and 115.2±9%, and 107.8±6%, 113.5±3% and 135.5±4%.[Conclusions]1,Protein expression of FHL2 localized mainly in nucleus, FHL2 expression levels was negatively correlated with the differentiation degree of colon cancer.2,Suppression of FHL2 gene expression in the colon cancer cell. induced morphological changes and the decreased nuclear:cytoplasmic ratio, suggesting that FHL2 might involved in the de-differentiation of colon cancer cells.3,Antisense FHL2 inhibited serum-dependent cancer cell growth and suppressed expression of other oncogenes. It suggested that FHL2 might play important role in carcinogenesis of colon cancer.
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