The Role And Mechanism Of TWIST In Gastric Cancer And Its Angiogenesis

【Background】: TWIST was originally identified in Drosophila as a protein involved in the regulation of diverse developmental processes, particularly in the formation of mesoderm. The human TWIST gene is located at chromosome 7p21.2.The TWIST protein consists of 201 amino acids and its molecular weight is about 28kd. TWIST protein is a transcription factor that belongs to the family of basic helix-loop- helix (bHLH) protein. The mechanism of bHLH molecules require that they either homo- or heterodimerize with other bHLH molecules, form a bipartite DNA-binding domain and bind to a conserved E-box region (CANNTG) on the promoter of genes which activate or inhibit transcription.Recent studies have shown that TWIST is a potential oncogene and be involved in the protection of apoptosis via both p53-dependent and independent pathways. To date several studies revealed that TWIST played crucial roles in tumorigenesis, invasion and metastasis, angiogenesis and drug resistance, but its accurate molecular mechanisms remain unclear. However, there have been little available data on the role of TWIST in the development and progession of gastric cancer; the molecule involved in the regulation of TWIST and its mechanism was still unclear. In this study, we have constructed several gastric cancer cell lines with different expression of TWIST. We observed the effects of TWIST on the biological behavior of gastric cancer cells and explored its roles in the development, progression and angiogenesis of gastric cancer. We researched for the molecule involved in the regulation of TWIST and its mechanisms.【Objectives】: (1) To investigate the expression of TWIST in gastric cancer tissue and cells and analyze the relationship of expression intensity with the clinicopathologic characteristics of gastric cancer. (2) To explore the effects of TWIST protein on malicious biological behaviours and tumor angiogenesis of gastric cancer. (3) To study the relationship of hypoxia with TWIST and the molecular mechanism.【Methods】: (1) The expression and sucellualr location of TWIST protein in gastric cancer tissues was investigated by immunohistochemistry and the relationship between the TWIST expression and the clinicopathologic characteristics was statistically analyzed. (2) The expression of TWIST mRNA and protein in gastric cancer cells and immortalized gastric epithelial cells were measured by semiquantitative RT-PCR and Western blot, respectively. The expression of TWIST protein in fresh gastric cancer tissues and normal gastric tissues was detected by Western blot. (3) The pcDNA3-TW vector which contained the full length of TWIST cDNA and the TWIST siRNA vector were transfected into gastric cancer cells SGC7901 respectively, followed by screening and verifying. (4) MTT assay, Flow cytometry, wound-healing assay, cell invasion assay and nude mice tumor formation assay were used to detect the proliferation, apoptosis, cycle, migration and invasion, and tumorigenesis effects of TWIST on the gastric cancer cells. The apoptosis related molecules were dectede by Western blot. (5) The concentration of VEGF in the supernatant of transfectants was determined by ELISA. (6) The expression of factorⅧrelated antigen in xenograft tumor was detected by immunohistochemistry and the MVD was compared among different xenograft tumors. (7) The hypoxia model in vitro was established and the expression of TWIST mRNA and protein was dected respectively under hypoxia condition. The changes of TWIST in HIF-1αsiRNA transfected cells under hypoxia and the regulation relationship of HIF-1 and TWIST were observed. (8) The TWIST reporter gene vector was constructed; The TWIST promoter activity was evaluated by dual luciferase reporter assay when co-transfected with HIF-1 or under hypoxia condition. (9) The possible binding sites of HIF-1 on the promoter of TWIST were selected by Chromatin Immunoprecipitation Assay (ChIP). (10) The functional binding site of HIF-1αon the promoter of TWIST was determined by Electrophoretic Mobility shift DNA-binding Assay (EMSA).【Results】: (1) Immunohistochemistry carried out on 76 paraffin-embedded gastric cancer sections showed that TWIST was mainly expressed in the cytoplasm of the epithelial cells, and only occasionally in the nuclei. TWIST expression was very low and at the lower limit of detection in most normal epithelial cells. TWIST expression was significantly increased in 35 (46.1%) of the 76 cancers. The positive rate of TWIST was higher in moderately and poorly differentiated gastric cancers compared to that in well-differentiated (p<0.05) and in the tumors with lymph-node metastasis compared to that without metastasis (node positive rate 60.4%; node negative rate 21.4%; P<0.05). (2) The expression of TWIST protein was higher levels in fresh cancer tissues compared to that in adjacent normal tissues. (3) The expression of mRNA and protein of TWIST in gastric cancer cell lines were up-regulated compared to that in GES-1 detected by semiquantitative RT-PCR and Western blot, respectively. (4) The TWIST specific siRNA plasmid pSilencer-TW1 and pSilencer-TW2 were constructed successfully. The plasmid pSilencer-TW1, pSilencer-TW2, and pcDNA3-TW were transfected into SGC7901 cell lines respectively. The cell lines with forced ectopic or knocked out expression of TWIST were established and identified, and named as SGC7901/pcDNA3-TW and SGC7901/pSi-TW1, respectively. (5) The growth of the sense transfectants was accelerated while the RNAi transfectants was slowed down, showed by MTT assay. (6) The FCM indicated that the proliferation index of SGC7901/pcDNA3-TW was higher compared to control group (45.4 vs 40) while the SGC7901/pSi-TW1 was lower than that of control group (32.8 vs 39.7).(7) The FCM also showed that the apoptosis rate was lower in sense transfectants while higher in RNAi transfectants when compared to vector transfectants. (8) Western blot revealed that the Bcl-2 and the Bcl-2/bax ratio increased in sense transfectants, while the Bcl-2 and the Bcl-2/bax ratio decreased in RNAi transfectants. (9) The wound-healing assay showed that the migration ability were elevated in sense transfectants while cut down in RNAi transfectants. (10) The Transwell assay indicated that the number of the invasive cells through the membrane were much more in sense transfectants than in RNAi transfectants (p<0.05). (11) The weights of xenograft tumor from sense transfectants were higer than that from RNAi transfectants (p<0.05); the volumes of xenograft tumor from sense transfectants were bigger than that from RNAi transfectants (p<0.05). (12) The concentration of VEGF in the supernatant of sense transfectants were increased while decreased in RNAi transfectants when compared to the control cells. (13) The stain of factorⅧ-related antigen was positive in the microvessel endothelial of nude mice xenograft tumors. The microvessel density was higher in the sense transfectants while lower in the RNAi transfectants when compared to control groups (p<0.05). (14) RT-PCR and Western blot results indicated that the expression of HIF-1αmRNA was unchanged, yet the HIF-1αprotein was increased under hypoxia condition. Both the expression of TWIST mRNA and protein of SGC7901 cells were upregulated under hypoxia condition while the HIF-1αspecific RNAi transfected SGC7901 cells were not induced. (15) By biological information analysis, six potential binding sites of HRE were located in the upstream region of the initial of transcription (-1442～+152). A length of 1.6 kb promoter of TWIST was cloned into PGL3-enhancer vector and identified right by sequencing and enzyme digestive. (16) The dual luciferase reporter assay demonstrated that the transfection of HIF-1αsense plasmids or under hypoxia could lead to a 3.0～4.5-fold increase of enzyme activety compared to empty vector transfection. (17) ChIP assay indicated that a length of 200bp DNA was amplified which located in -1014～-799bp of the TWIST promoter(containing three potential HRE binding sites). (18) To determine the functional sites of HIF-1αbinding to the TWIST promoter, EMSA and Super shift assay were applied and -1003～-997 site away from the transcriptional start was proved to be the functional binding site of HIF-1α.【Conclusion】: (1) Our results suggest that up-regulation of TWIST plays a role in the neoplastic transformation to gastric cancer and subsequent development of lymph nodes metastasis. TWIST may serve as a useful prognostic marker for predicting the development of metastasis and a target for therapy in gastric cancer. (2) The roles of TWIST in the development and progression of gastric cancer may be associated with cell cycle, apoptosis, invasion, and angiogenesis mechanisms. (3) TWIST expression in the gastric cancer cell can be induced by hypoxia and this is due to the HIF-1αbinding to the HRE site in the TWIST promoter region.