The Role And Mechanism Of Id1 Involved In The Carcinogenesis And Progression Of Gastric Cancer

Id (Inhibitor of DNA binding/differentiation), 13–20 kDa small proteins, belong to the bHLH transcriptional factor superfamily, which have a HLH domain and a Basic domain. While Id proteins lack a basic domain and form transcriptionally inert heterodimers with other bHLH transcription factors, thereby inhibiting transcription of differentiation associated genes. Basic helix–loop–helix (bHLH) transcription factors have their roles in development and differentiation of cells, thus Id serve to inhibit differentiation by heterodimerization with bHLH proteins. Now four Id members have been cloned.The role of differentiation inhibitor of Id makes them suspected players in tumorigenesis. Four Ids lost their expressions in mature tissues and cells. However, to date, several reports revealed deregulated expression of Id proteins in various tumor types and tumor derived cell lines. Id1 and Id3 are overexpressed even more extensively than the other two family members. There is evidence that Id may contribute to oncogenesis and are involved in a variety of biological processes, including development, proliferation, and angiogenesis, although the carcinogenetic mechanisms of Id proteins have not been fully established. Id1 also seems to be an important factor for G1/S cell cycle transition in certain cell types. Id1 and Id3 are also important regulators of angiogenesis, and participate in recruiting the precursor endothelial cells to the local site of the tumor. Hence there is increasing evidence that deregulated Id1 expression may contribute to various properties of tumor cells, including induction of aberrant cell proliferation, inhibition of differentiation, stimulation of angiogenesis, and induction of genomic instability. In digestive system, Id1 has its deregulated expression in pancreatic cancer, esophageal cancer and colorectal cancer.Gastric cancer is one of the most common malignancies throughout the world, particularly in Eastern Asian countries. The molecular mechanisms of gastric carcinogenesis, however, remain unclear, and in particular, Id1 expression in human gastric cancers has not been examined. To explore whether or not Id1 expression is related to the gastric carcinogenesis, we investigated the expression and role of Id1 in human gastric cancer. Some possible mechanisms of Id1 involved are also our interests.【Objectives】(1) To investigate the expression of Id1 in the gastric cancers and the relationship of expression intensity with the clinicopathologic characteristics of gastric cancer;(2) To examine the effect and possible mechanism of Id1-siRNA on the malignant phenotype of gastric cancer cells and to screen the downstream effectors of Id1; (3) To study the regulation of HIF-1 on Id1 under hypoxia condition in gastric cancer cell and the mechanisms involved.【Methods】(1) The expression of Id1 in gastric cancer was determined by immuno- histochemistry assay. (2) Semiquantitative RT-PCR and Western blot were applied to detect the Id1, Id2 and Id3 mRNA and Id1 protein level in gastric cancer cells. (3) Full-length vector and siRNA vector of Id1 were constructed. (4) MTT assay, colony formation assay on plate and agarose,and nude mice tumor formation assay were used to examine the effect of forced ectopic expression of Id1 on GES-1. (5) MTT assay, FCM, Transwell assays were also used to detect the proliferation, apoptosis, migration effect of Id1-siRNA on the gastric cancer cells. (6) The in vitro proliferation and tube formation assay and in vivo MVD were taken to test the angiogenesis effect by Id1 inhibition in the gastric cancer cells. (7) The concentration of VEGF in the supernatant of Id1-siRNA transfectants were determined by ELISA. (8) The downstream effectors were screened by microarray. The expression of some of the downstream molecules, such as p21, p16 and TIMP4, revealed by silicon analysis were determined by Western blot. (9) The expression of Id1 in gastric cancer cells under hypoxia condition (1%O2) was tested. (10) Id1 promoter activity was evaluated by dual luciferase reporter assay when co-transfected with HIF-1 or under hypoxia condition. (11) The binding of HIF-1 on the promoter of Id1 were verified by in vitro EMSA and in vivo ChIP.【Results】1. Id1 is over-expressed in gastric cancer tissues and cell lines. Western blot analysis performed on gastric tissues found that Id1 was expressed at higher levels in 11(11/15) cancer tissues compared to adjacent tissues. Immunohistochemistry carried out on 99 paraffin-embedded gastric cancer sections showed that Id1 was mainly expressed in the cytoplasm of the epithelial cells, and only occasionally in the nuclei. Id1 expression was at the lower limit of detection in most normal epithelial cells. In contrast approximately 10–30% of examined epithelial cells stained positive in intestinal metaplasia and dysplasia specimens, with an average staining score of 0.33±0.09. A significant difference in staining intensities was also found between these two sets of specimens (p<0.001). Compared to gastric cancer, Id1 expression was significantly weaker in intestinal metaplasia specimens (p<0.001). Further analysis of the clinicopathological characteristics of the 99 gastric cancer specimens analyzed revealed a positive association of Id1 staining intensities with the degree tumor differentiation. In well-differentiated tumor cells, the staining index was 0.66±0.12, while Id1 was detected in most epithelial cells with higher expression (1.4±0.45)in poorly differentiated tumor cells(p<0.05). With respect to the TNM stage, the index of Id1 staining was much higher in patients ofⅢandⅣ(1.31±0.87) than that ofⅠa ndⅡ(0.66±0.52, p<0.001) stage. Western blot analysis on gastric cancer cell lines, SGC7901, AGS, MGC803, MKN45, MKN28, KATOⅢand immortalized gastric mucosa epithelial cell line GES-1 revealed that Id1 was upregulated more prominently in the poorly differentiated cell lines than in the well-differentiated ones and the immortalized gastric mucosal cell line GES-1. Semiquantitative RT-PCR performed in the gastric cancer cell lines and GES-1 showed that most gastric carcinoma cell lines and GES-1 showed high level expression of Id1 mRNA, compared to normal gastric mucosa and similar expression patterns of Id2 and Id3 mRNA were also found. These results further support our model that Id1 might play a role in the tumorigenesis of gastric cancer.2. Forced ectopic expression of Id1 can increase the proliferation and malignancy of GES-1, while Id1-siRNA can reverse the malignant potential of gastric cancer cells.Stable Id1 transfectant of the nontumorigenic gastric cell line GES-1 showed increased proliferation by MTT assay and acquisition of anchorage independent growth in soft agar. The number of plate colonies also increased in contrast to the control cell. However, ectopic expression of Id1 could not induce the tumorigenecity of the immortalized cell GES-1 in the nude mice.To downregulate the expression of Id1 in gastric cancer cells, Id1-specific siRNA vectors were constructed. After cell transfection and antibiotic screening for about 8w, the expression of Id1 in stable transfected cells was determined by Western blot. Id1-siRNA could downregulate the expression of Id1 in gastric cancer cell SGC7901 effectively. Clone 1(IdRNAi1) and Clone 2(IdRNAi2) were picked out and Id1 in these two clones was inhibited by 60% and 90%, respectively. Downregulation of Id1 in SGC7901 decreased their proliferation and abrogates anchorage-dependent and -independent colony formation in gastric cancer cells. Cell cycle progression observed in SGC7901 cells could be arrested by Id1 inhibition at G1 phase. Instant transfection of Id1-siRNA could induce apoptosis in the gastric cancer cells with the increased active form of Casepase3. By in vivo tumor formation assay, transfectants showed a decrease in tumor size and weight when injected into nude mice and the in situ TUNEL and Hochest33258 staining showed that the apoptosis rate of Id1-siRNA was higher than the parental and mock transfection cells. Id1 suppression could also abrogate the abilities to migrate through matrigel on Boyden chamber assay. The concentration of VEGF in the supernatant of Id1-siRNA transfectants were diminished markedly compared to the parental cells. The HUVEC proliferation and tube formation were inhibited when grew in the Id1-siRNA conditioned medium and it was proved by the decreased MVD(micro vessel density)in Id1-siRNA cells in vivo assay. Both in vitro and in vivo nude mice assays suggested that Id1-siRNA had an effect to reverse the malignant potential of gastric cancer. All these above changes were proportional to the level of Id1 expression in these SGC7901 derivative cell lines.The total RNA of SGC7901/Id1iRNA and SGC7901-psilencer cells were extracted. The quality of extracted RNA was evaluated by agarose electrophoresis and analysis of Lab-on-chip. After microarray hybridization and data normalization 1551 genes were up-regulated while 70 genes were down-regulated by Id1RNAi. Among these genes, we tested the expression level of TIMP4 protein level, which was increased in SGC7901/Id1iRNA compared to mock or empty vector transfected cells. P16 and p21 were also showed increased expression by Id1 suppression, which may explain the cell cycle arrest observed in Id1-siRNA transfectants.3. Id1 expression in gastric cancer can be increased under hypoxia and HIF-1 binds to the promoter of Id1 at -924 away from the initial of transcriptional start.Id1 mRNA and protein level in SGC7901 cell were both increased under hypoxia condition and this induction could be reversed by HIF-1 block by specific siRNA. This phenomenon clewed that Id1 could be the downstream effector of HIF-1. By bioinformatics, eight putative HIF-1 binding sites were found in the promoter region(-3000~+100) of Id1 and two important auxiliary sequence CACAG were also found near to the No.6 and 7 HBS. Two different length promoters of Id1 were cloned into PGL-3 basic vector (named PM and PS). PM contains the No.6, No.7 and No.8 HBSs and PS only contains No.8 HBS. The dual luciferase reporter assay demonstrated that transfection of HIF-1 full length or under hypoxia led to an increase of PM compared to empty vector transfection, indicating that HIF-1 caused transactivation of Id1 promoter and then the upregulation of Id1 mRNA and protein. To study the functional sites of HIF-1 binds to the Id1 promoter, EMSA (Electrophoresis mobility shift assay) and ChIP(chromosome immunoprecipitation) were applied and the No.6 HBS(-924 site: 5'-GACGTC-3')away from the transcriptional start were proved to be the functional HRE. The mutations of the three bases (CGT) thus make the binding invalid.【Conclusions】 In conclusion, we provide evidence that Id1 is highly expressed in gastric cancer tissues and strong Id1 expression is associated with poorer differentiation and more aggressive behavior of tumor cells. Knocking down the Id1 expression by specific siRNA can reverse the malignant potential of tumor cells to some degrees. Id1 expression in the gastric cancer cell can be increased by hypoxia and this is due to the HIF-1 binding to the Id1 promoter. Id1 is proved to be a new direct target of HIF-1. The present work give the proof of carcinogenesis function of Id1 on gastric cancer and to our knowledge, for the first time, found the HIF-1 regulation on Id1 in this context.