Study On Expression,Downregulatioll Mechanism And Function Of RASGRF1in Colon Cancer

Part1Expression and methylation of RASGRF1in human colon cancer tissuesObjectiveTo explore expression and methylation of RASGRF1and its clinicopathological significance in human colon cancer tissues.MethodsImmunohistochemistry and methylation-specific PCR (MSP) was used to detect the protein expression and methylation of RASGRF1in colon cancer tissues and its adjacent non-tumorous colon tissues, respectively. Correlation between the expression and methylation of RASGRF1and clinicopathological factors of colon cancer was analyzed.Results1. Positive expression rate of RASGRF1in colon cancer tissues was lower than that in its adjacent non-tumorous colon tissues. The expression of RASGRF1protein was associated with depth of tumor invasion, lymph node metastasis and tumor TNM stage (all P<0.05), but not with gender, age, tumor size and differention degree (all P>0.05).2. Methylation rate of RASGRF1in colon cancer tissues was higher than that in its adjacent non-tumorous colon tissues. The methylation of RASGRF1was associated with depth of tumor invasion, lymph node metastasis and tumor TNM stage (all P<0.05), but not with gender, age, tumor size and differention degree (all P>0.05).3. The expression of RASGRF1was negatively related to the methylation of RASGRF1in human colon cancer tissues (P<0.05).Conclusions1. Inverse correlation between expression and methylation of RASGRF1in colon cancer tissues may suggest the low expression of RASGRF1may be correlated with its gene promoter methylation in colon cancer tissues.2. The expression and methylation of RASGRF1in colon cancer tissues was correlated with depth of tumor invasion, lymph node metastasis and tumor TNM stage. The abnormal expression and methylation may play an important role in the invasion and metastasis of colon cancer. Part2Expression and methylation of RASGRF1in human colon cancer cells and establishment of RASGRF1overexpression cell modelObjectiveTo explore expression and methylation of RASGRF1in human colon cancer cells and find a suitable cell line to establish RASGRF1overexpression cell model in vitro.MethodsRT-PCR、Western Blot and MSP was used to detect mRNA expression, protein expression and methylation of RASGRF1in human colon cancer cell line HT29, HCT116, SW480and SW620to find a suitable cell line as a RASGRF1overexpression cell model of colon cancer in vitro. The colon cancer cell line of low expression and high methylation RASGRF1was treated by a different concentration of5-Aza-CdR, and RT-PCR was used to detect RASGRF1mRNA expression before and after the treatment. Construction of RASGRF1over-expression vector was used for transfection of the suitable colon cancer cell line. The high expression of RASGRF1in the colon cancer cell was detected by Real-time qPCR and Western blot.Results1. Among mRNA expression of RASGRF1in human colon cancer cell line, SW480cell is the highest, and HCT116cell is the lowest.2. Among protein expression of RASGRF1in human colon cancer cell line, SW480cell is still the highest, and RASGRF1protein decreased significantly in HT29、HCT116and SW620.3. Aberrant methylation of RASGRF1was present in all human colon cancer cell lines, and the degree of methylation was highest in HCT116cell.4. RASGRF1expression could be restored after treatment with5-Aza-CdR in HCT116cell.5. HCT116was chosen to construct RASGRF1overexpression cell model (pcDNA3.2/V5/GW/D-TOPO-RASGRF1) in vitro successfully.Conclusions1. RASGRF1mRNA and protein expression decreased significantly in colon cancer HCT116cell, and high RASGRF1methylation existed in HCT116cell; The decreased expression can be restored by5-Aza-CdR.2. HCT116cell is suitable to build the RASGRF1overexpression cell model in vitro; pcDNA3.2/V5/GW/D-TOPO-RASGRF1can be effectively transfected with HCT116cell, and the RASGRF1expression in HCT116cell is high. Part3Effect of RASGRF1overexpression on the biological characteristics of human colon cancer HCT116and its mechanismObjectiveTo investigate the effects of RASGRF1overexpression on the biological characteristics of human colon cancer cell HCT116in vitro and its mechanism.MethodsExperiments were divided into two groups, overexpression group (HCT116cells were transfected with pcDNA3.2/V5/GW/D-TOPO-RASGRF1plasmids) and blank control group (HCT116cells were transfected with pcDNA3.2/V5/GW/D-TOPO NC plasmids). Cell proliferation was examined by MTT assay; Cell apoptosis was assessed by Annexin V-FITC/PI double staining by flow cytometry; Cell invasive capability were evaluated by Transwell cell invasion assay; Cell migration capability were evaluated by wound-healing assay and Transwell cell migration assay. The expression of epithelial marker E-cadherin, mesenchymal markers fibronectin and vimentin was detected by Western blot analysis.Results1. MTT assay indicated that the proliferation level of HCT116cell in the overexpression group was significantly decreased compared to the blank contral group (P<0.05).2. Annexin V-FITC/PI double staining by flow cytometry showed that the early apoptotic fraction of HCT116cell were3.68±0.26%and4.18±0.25%in the blank contral group and the overexpression group, respectively. Simultaneously, the late apoptotic fraction was23.90±0.38%and24.68±0.82%, respectively. The apoptotic rate of HCT116cell was not significantly different between the two groups (P>0.05).3. Transwell cell invasion assay indicated that the numbers of invaded cells in the overexpression group was extremely less than that in the blank control group (32.2±2.3vs.48.9±2.2)(P<0.01).4. Wound-healing assays showed that HCT116cell in the overexpression group had lower motility compared with the blank control groups. Transwell cell migration assay confirmed that the numbers of migrated cells in the overexpression group was extremely less than that in the blank control group (41.1±5.3vs.66.8±5.5)(P<0.01).5. Western blot analysis showed that the expression level of E-cadherin protein in the overexpression group increased compared with that in the blank control group. However, the expression level of vimentin and fibronectin protein decreased (P<0.05).Conclusions1. RASGRF1overexpression could suppress the growth and proliferation of colon cancer cell HCT116, and reduce invasion and migration capacity of colon cancer cell HCT116, but no significant effect on HCT116cells apoptosis was found. RASGRF1may play an important role in the carcinogenesis and progression of colon cancer.2. RASGRF1overexpression enhanced the recruitment of E-cadherin and decreased the expression of vimentin and fibronectin. RASGRF1may prevent the invasion and metastasis of colon cancer partly via the regulation of EMT.