| Colorectal carcinoma (CRC) is one of the most common malignancies which threaten the health of human. The research of molecular mechanism of CRC begins one of the earliest among all the malignancies. Since the "multigene multistep model of Colorectal carcinogenesis" stated by Vogelstein, a series of genes related to the occurrence and development of CRC have been found. With the changes of life style and diet structure of Chinese people in recent 20 years, the incidence and mortality of CRC continue to increase. However, the early diagnosis and the treatment still lack. Searching for the genes related to CRC and exploring their exact roles are of great significance in finding proper target molecules in diagnosis, treatment, and prognosis of CRC.Our laboratory focused on the research in molecular pathology of CRC. In 1999, we constructed three cDNA libraries using SSH (suppression subtractive hybridization): adenocarcinoma VS adenoma (T-A), adenocarcinoma VS normal mucosa (T-N), adenoma VS normal mucosa (A-N). We have selected a series of differentially expressed genes and continued to explore their functions.The gene of insulin-like growth factor binding protein 7 (IGFBP7) was selected from T-N libraries, which was overexpressed in colorectal adenocarcinoma tissue. By reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining, we confirmed the overexpression of IGFBP7 in CRC tissues, compared to the paired normal mucosa. On the contrary, we found the low expression of IGFBP7 in CRC cell lines. Among the ten CRC cell lines: SW480, SW620, HT29, RKO, SW1116, HCT8, COLO205, Caco2, CW2 and Hce8693, theexpression of IGFBP7 was only detected in SW480 and Caco2 cells. The paradoxical phenomenon of expression of IGFBP7 in vivo and in vitro makes the exact role of 1GFBP7 in CRC puzzled. We want to explore the function of IGFBP7 in CRC cell lines, which is very important for us to better understand the role of IGFBP7 in CRC.We selected two CRC cell lines without endogenous 1GFBP7 expression and with different inherited characteristics, RKO and SW620, as our cell models. We constructed PcDNA3.1(IGFBP7) expression vectors and performed the transfection experiment. The expression of 1GFBP7 mRNA and protein were detected in the transfectants by RT-PCR and western blot. We then began the cell function assays.Growth proliferation assay using cell counting kit-8(CCK-8) showed that IGFBP7 could suppress the growth of CRC cells. A significant decrease in growth of IGFBP7-RKO (p=0.0066, VS control) and IGFBP7-SW620 transfectants (p<0.0001, VS control) was observed. The proliferation of SW480 cells, which normally expressed IGFBP7 and derived from the same patient as SW620, was significantly slower than SW620 (p=0.0108).The soft agar assay showed that colony formation was markedly decreased in IGFBP7 transfectants compared to control vector transfectants. After three weeks of incubation, colony number and size were analyzed. Colony number of IGFBP7 transfectants was significantly less than the control (mean 12/well for RKO transfectants, mean 59/well for control, p =0.0004; mean 18/well for SW620 transfectants, mean 55/well for control, p = 0.0026).The clone size of IGFBP7 transfectants was smaller than the control, and the phenomenon was more significant in RKO cells than in SW620 cells. The discrepancy may be due to the different inherited characteristics of the two cell lines, and the exact mechanism needs further research.A sub-G1 peak 48h after transfection of IGFBP7 was detected by flow cytometric analysis of the DNA content using propidium iodide (PI) staining. More percentages of apoptotic cells (Annexin V positive) in IGFBP7 transfectants were detected by annexin V/PI double staining. Chromatin condensation and margination were observed after transfection of IGFBP7 into RKO cells and SW620 cells by transmission electron microscopy, indicating that IGFBP7 could induce apoptosis in CRC cells. Using western blot, we identified upregulated level of caspase-3 protein in 1GFBP7 transfected RKO cells, indicating that the caspase-3 related pathway may play key roles in IGFBP7's apoptosis induction function in CRC cells.Our results that IGFBP7 could inhibit CRC cell growth were consistent with other laboratories' finding on lGFBP7's function toward prostate cancer cells, breast cancer cells, lung cancer cells, cervical cancer cells and osteosarcoma cells. Recently, studies performed in the our laboratory found that overexpression of IGFBP7 in CRC tissue was correlated with favourable prognosis (p=0.012). Based on these results, we concluded that IGFBP7 was a protective factor against CRC. IGFBP7 played a potential tumor suppressor role against colorectal carcinogenesis.The above research of IGFBP7 solved our puzzle about IGFBP7's exact role in CRC cells. The previous studies in our lab also found that the expression level of IGFBP7 in CRC tissue correlated with the fasting glucose level in patients, indicating 1GFBP7 a probable type 2 diabetes mellitus (DM2) associated gene. Other laboratories also found that IGFBP7 was a potential tumor suppressor gene in prostate cancer, breast cancer and lung cancer. Recently, a possible insulin resistance associated role of IGFBP7 in DM2 was uncovered. Our results together with others indicated that IGFBP7 played an important role in malignancies and DM2. However, the exact signaling pathways and molecular mechanisms of IGFBP7 have not been elucidated yet.To gain further insight into the signaling pathway of IGFBP7, we applied Affymetrix HG133 plus 2.0 chip platform and two-dimensional electrophoresis (2-D) to identify the differentially expressed genes induced by IGFBP7 both at mRNA level and protein level in RKO cells. We performed our study on three single-cell clones (control vector clones as control). The six clones were divided into 3 groups. Cells in the same group were cultured and harvested at the same time to minimize the changes of gene expression due to different cell culture conditions.Collectively, seventy-eight differentially expressed genes reproducible were detected by Affymetrix chip (part of them has been validated by Real time PCR). We did the Gene Ontology analysis of the genes and found some important information. We found that MAPK (Mitogen-Activated Protein Kinase) pathway, insulin/IGF1 (insulin growth factor 1) pathway and TGF- 3 (transforming growth factor- β ) pathway were significantly enriched (as below).MAPK pathway (including ERK1/2, JNK and p38 MAP Kinase pathway): GADD45B(growth arrest and DNA-damage-inducible, beta). FOS (v-fos FBJ murine osteosarcoma viraloncogene homolog), RASAl[RASp21 protein activator (GTPase activating protein) 1] , FLNB[filamin B, beta (actin binding protein 278)]. NR4A1 (nuclear receptor subfamily 4, group A,member 1) .Insulin/IGF1pathway: IRS1 (insulin receptor substrate 1), RASA1[RAS p21 protein activator (GTPase activating protein) 1], FOS (v-fos FBJ murine osteosarcoma viral oncogene homolog). RASA1 and FOS belong to MAPK pathway.TGF- β pathway: CDKN2B [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], ID1 (inhibitor of DNA binding 1, dominant negative helix-loop-helix protein), ID3 (inhibitor of DNA binding 3, dominant negative helix-loop-helix protein), SMAD3(SMAD family member 3 ).The above pathways played important roles in growth and metabolism, which may be responsible for the growth inhibition and metabolism related role of IGFBP7. Further exploring on the exact pathways is needed.Directed Acyclic Graph (DAG) showed that cytoskeleton associated, actin binding genes were significantly enriched, indicating that IGFBP7 possibly influenced the cell cytoskeleton associated function. Remodeling of cytoskeleton may possibly be one of the important mechanisms responsible for IGFBP7's biological behaviour. We observed that IGFBP7-transfected RKO cells exhibited a pronounced anterior-posterior morphology, longer and more narrow than the control. Perhaps these changed cytoskeleton associated genes may lead to, at least in part, the morphology change of RKO cells by IGFBP7.Totally, 16 genes were reproducible in 3 clones, including IGFBP7, TAGLN (Transgelin) , SP140 (SP140 nuclear body protein) , FRMD4A (FERM domain containing 4A) , CALD (caldesmon 1) , NAV3 (neuron navigator 3) , SOX9 [SRY (sex determining region Y)box 9(campomelic dysplasia, autosomal sex-reversal)], STC1(stanniocalcin 1), AREG[amphiregulin(schwannoma-derived growth factor)], ID1 (inhibitor of DNA binding 1, dominant negative helix-loop-helix protein) , IRS1, TACSTD (tumor-associated calcium signal transducer 1) , IER5L (immediate early response 5-like) , CDKN2B[cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], SYNl(synapsin I), LAMB1(laminin, beta l).These data were verified by realtime RT-PCR.In order to clarify whether IGFBP7 could induce these differentially expressed genes in other CRC cell lines, we analyzed the expression of these genes in IGFBP7-SW620 transfectants versus empty vector control. We found that IGFBP7 could downregulate AREG, P15, ID1, IER5L, IRS1, SOX9, STC1, SYN1, and upregulate TAGLN in both in RKO cells and SW620 cells. These genes may be the target signal molecules of IGFBP7, which provide important clues for our futureresearch.The results of 2-D and the mass spectrometry(MS) indicated that IGFBP7 could downregulate ALB(albumin), HSP60 (60 kDa heat shock protein, mitochondrial precursor) , Actin cytoplasmic 1 or 2 , PKM2 (pyruvate kinase, muscle). FARSB (phenylalanyl-tRNA synthetase, beta subunit). Interestingly, we found that IGFBP7 could influence the expression of actin at protein level. Together with the Affymetrix chip results, we concluded IGFBP7 possibly an actin associated, cytoskeleton associated gene.From the above researches of both IGFBP7's biological function and the differentially expressed molecules induced by IGFBP7 in CRC cells, we concluded that:1. IGFBP7 could inhibit the growth, decrease the soft agar colony formation ability, and induce apoptosis in CRC cells. IGFBP7 played a potential tumor suppressor role in colorectal carcinogenesis.2. Both of the Affymetrix data and the proteomics data found that IGFBP7 probably was an actin associated, cytoskeleton related gene. Remodeling the cytoskeleton may be one of the important mechanisms responsible for IGFBP7's biological function.3. The probable signaling pathways of IGFBP7 were MAPK, Insulin/IGF1, TGF- β pathway, which may be responsible for its growth inhibitory and metabolism related biological behaviour.4. In RKO and SW620 cells, IGFBP7 downregulate AREG, P15, ID1, IER5L, IRS1, SOX9, STC1, SYN1, upregulate TAGLN. These genes might be the target genes of IGFBP7 in CRC.5. A combined application of gene transfection, gene expression microarray and 2-D proteomics study is of great significance in searching for gene specific target molecules.
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