The Study For The High-expression Of RASGRF1 And Its Significance In Colorectal Cancer

Background:Colorectal cancer with its high mobility is the third leading malignant tumor wordwide. American Cancer Society had published the research data of colorectal cancer in 2014, which predicted that there would be 136930 new cases and 50310 patients would die of this disease. In China, due to the improving living standard and the change of dietary structure and living environment, the incidence of colorectal cancer was estimated to rise year by year. Colorectal cancer was a serious threat to health and which need to pay more attention. Almost 20% of new cases were diagnosed as metastasic colorectal cancer, and half of the others eventually developed metastasis tumor with overall survival of 20 months. There were many molecules and signal pathways correlated with the development and progression of colorectal cancer. RAS and RAS signal pathway were the most important.Generally RAS protein shares a common biochemical mechanism and act as binary molecular switches with other small GTPase. RAS exhibit high-affinity binding for GDP and GTP, and possess low intrinsic GTP hydrolysis and GDP/GTP exchange activities. GDP/GTP binding is controlled by two main classes of regulatory proteins:guanine nucleotide exchange factors (RASGEFs) promote formation of the active, GTP-bound form, whereas GTPase activating proteins (RASGAPs) accelerate the intrinsic GTPase activity to promote formation of the inactive GDP-bound form. RASGAPs could not bind to RAS protein and lost its function since KRAS gene was mutant. Meanwhile RASGEFs could still play its role as activator of RAS. Then RAS protein could stay in active, GTP-bound form and continuously activate its signal pathway, including RAS-RAF-MEK-ERK pathway and RAS-PI3K-Akt pathway. Finally cell proliferation and apoptosis were modulated and may transform to tumor cell.RASGEFs super family mainly had more than 20 family members, included RASGRF1 and 2, Sosl and 2, RASGRP1-4(CalDAG-GEFs), C3G, Epac 1 and 2 (cAMP-GEFs), RalGDS family members, RalGPS, BCAR3, PDZ-GEFs, MR-GEF, Smg GDS, and phospholipase C(epsilon). There were reports detecting RASGEFs as proto-oncogene product playing pivotal role in tumor formation and progression. RASGRF1 was one of the RASGEF family members. Its high expression was detected in rhabdomyosarcoma and skin cancer, and down-regulation of RASGRF1 may inhibited these cancer cell proliferation and metastasis.Our preliminary study indicated the high-expression of RASGRF1, RASGRP1, RASGRP2, SOS1 in colorectal cancer tissue and colon cancer cell after screening the RASGEF expression profile in colon tissue and colon cancer cell. And down-regulation of RASGRF1 inhibited the activation of RAS protein. However, the expression level of RASGRF1 incolorectal cancer, its clinical significance and its role in the progression of colorectal cancer remains to be elucidated.Aim:In this study, we sought to investigate the expression level of RASGRF1 in colorectal cancer tissue, the adjacent normal tissue and colorectal adenoma tissue, and defined its clinical significance through immunohistochemistry staining. Employing RNA interference, detected the effect of down-regulation of RASGRF1 on KRAS mutant colon cancer cell HCT116 biological function. It will provide a theoretical basis for further elucidation of RASGRF1 in colorectal cancer development and progression, and afford a new molecular target for colorectal cancer early diagnosis and treatment.Method:1. Paraffin-embedded specimens of colorectal cancer tissues paraffin section, related adjacent normal tissues paraffin section and colorectal adenoma tissue paraffin section (n= 35) were collected during the period from 2008 to 2011 in Guangdong General Hospital. Paraffin-embedded colon tissue specimens were sectioned (4 μm thick) and slides prepared using standard techniques. All patients had not accepted preoperative chemotherapy, radiotherapy and immune/biological intervention. Valuation of colorectal cancer was based on the colorectal cancer TNM staging system (the seventh edition) draw up jointly by American Joint Committee on Cancer (AJCC) and Union Internationale against Cancer (UICC).2. To detect the location and expression level of RASGRF1 in colorectal cancer tissue, related adjacent normal tissues and colorectal adenoma tissue by En Vision method. The following are the main steps:paraffin section were baked at 65℃ for 3 h, de-paraffinized and rehydrated in xylene and graded alcohols. Antigens were retrieved by heating in antigen retrieval solution Tris-EDTA in pressure cooker for 3 min, cooled the cooker to room temperature by cold running water, washed the slides with double distilled water twice, followed by incubation in 100 ul 3% H2O2 for 10 minutes in dark at room temperature to destroy endogenous peroxidase activity. Washed the slides with PBS for three times, Non-specific antigen was blocked incubating in 10%sheep serum for 10 min. Then cleaned the sheep serum and incubated the tissue sections with 50ul anti-RASGRF1 primary antibodies overnight at 4℃. Took the slides out of the refrigerator and warmed them in room temperature for 30 minutes, the incubated with a EnVision HRP-conjugated goat anti-rabbit antibody secondary antibody for 40 minutes at room temperature. Sections were washed by PBS for three times, stained with 50-1 00μl DAB working solution and hematoxylin, dehydrated in graded alcohols and xylene and then mounted by neutral resins.3. Transfected the KRAS mutant colon cancer cell HCT116 with LipofectamineTM RNAiMAX:Transfection was conducted when the cell growing in logarithmic growth phase and divided into three groups:RASGRF1 siRNA, negative control and blank. According to the percentage of 200 μL Opti-MEM Reduced serum Medium to 5μL MAX every well, fetched appropriate amount of Opti-MEM Reduced serum Medium and MAX, and mixed them gently as Solution A. Figured out the volume of RASGRF1 siRNA and NC siRNA every well according to the optimum transfection concentration in directions. Mixed 200 μL Opti-MEM Reduced serum Medium and the appropriate volume of RASGRF1 siRNA and NC siRNA every well as Solution B. Blended Solution A and Solution B lightly and incubated in room temperature for 20 minutes. Meanwhile discarded the old culture medium and washed the cells with serum-free medium, added 2mL Opti-MEM Reduced serum Medium every well, followed by adding mixture of Solution A and Solution B, and waggled the cell culture plate gently, and cultured at 37℃ under an atmosphere containing 5% CO2. Transfection efficiency was verified by qPCR.4. Cell proliferation assay:HCT116 were growing in good condition and in logarithmic growth phase, and were digested by trypsin enzyme and then counted and adjusted to 104 cells every well, inoculated the cell suspension to 96-well plate. Placed the plate in the culture chamber and cultured for 24 hours, then transfected the cells with lipofectamine carrying RASGRF1 siRNA and NC siRNA as mentioned before. After culturing 48 hours, added 10μL CCK-8 every well gently, avoiding any bubble, the optical density (OD) value of 24h,48h and 72h was determined at 450 nm in the multimode reader.5. Cell apoptosis assay:HCT116 were growing in good condition and in logarithmic growth phase and tranfected by lipofectamine carrying RASGRF1 siRNA and NC siRNA. Collected the old culture medium and transferred them to a 15mL centrifuge tube and placed the tube on ice. Washed the cell with 2mL ice cold PBS gently and discarded the PBS, followed by adding 0.5ml 0.25% trypsin enzyme without EDTA, and collected the cells. Resuspended the cells with previous culture medium and adjusted its concentration to 1×106/mL. Added 1.25 μl Annexin V-FITC to 0.5 ml cell suspension (about 5×105 cell) in a new centrifuge tube, and incubated in dark in room temperature for 15 minutes. Discarded the supernatant after centrifuging the tube at 1000xg for 5 minutes, added 10 μl Propidium Iodide and keep the sample in dark and ice. The apoptotic rate was detected by flow cytometry.6. Tranfection was conducted when the colon cancer cell HCT116 in good condition and in logarithmic growth phase, collected 1×106 tranfected cell and discarded the supernatant after centrifuging. Washed the cells with ice cold PBS twice and fixed by cold 70% ethanol at 4℃ overnight. Washed the cells with 1 mL PBS again before adding 500uL PBS which contained 50ug/mL PI, 100ug/mL RNase A, 0.2% Triton X-100, incubated in dark and at 4℃ for 30 minutes. When conducting the analysis procedure in flow cytometry,20000-30000 cells should be counted, and the final analytic process was carried through the ModFit software.7. Cell migration assay:colon cancer cells HCT116 that in good condition and logarithmic growth phase, were tranfected by lipofectamine carrying RASGRFl siRNA and NC siRNA. Vacuumed the culture medium and washed by PBS twice. Cells were digested by 0.25% trypsin enzyme and then collected in a clean centrifuge tube, counted and adjusted to 105 cells, and resuspended by 100 ul serum-free medium. Inoculated the cell suspension in the upper chamber of transwell cell culture plate, meanwhile added 600ul complete medium in the lower chamber, cultured at 37℃ under an atmosphere containing 5% CO2.48 hours later, took out the plate and wiped off the cells in the upper chamber with a swab. Cells were fixed in 4% paraformaldehyde solution for 20 minutes, washed the plate before and after 10 minutes crystal violet staining, counted and photographed under a microscope.8. Statistical method:All experimental data was analyzed using the SPSS 13.0 statistical software. Continuous variable in normal distribution was described as (x±s), median and inter-quartile range were recruited to describe the continuous variable in abnormal distribution. The differences of RASGRFl expression level between colorectal cancer tissue, related adjacent normal tissues and colorectal adenoma tissue were assessed by K independent sample nonparametric tests (Kruskal-Wallis H test). Two independent sample nonparametric tests (Mann-Whitney U test) and Kruskal-Wallis H test were recruited to analyze relationship between RASGRF1 expression and clinicopathologic parameters of colorectal cancer patients. Difference of RASGRF1 mRNA expression level of RASGRF1 siRNA 50 nm group, RASGRF1 siRNA 100 nm group, RASGRF1 siRNA 200 nm group and NC siRNA group were assessed by One-way ANOVA. The proliferation rate of 24h,48h,72h in RASGRF1 siRNA group, NC siRNA group and HCT116 group were assessed by ANOVA for repeated measurement and One-way ANOVA. Differences between groups were analyzed by LSD test when the homogeneity of variance was meted, and Dunnett T3 test when heterogeneity of variance. Kruskal-Wallis H test and One-way ANOVA were recruited to analyze the difference of Gl stage cells and S stage cells in RASGRF1 siRNA group, NC siRNA group and HCT116 group.The change of colon cancer cell HCT116 migration ability after tranfecting by RASGRF1 siRNA was evaluated by Kruslal-Wallis H test. P values<0.05 were considered to be statistically significant.Result:1. Immunohistochemistry staining of RASGRF1 in colorectal cancer tissue, related adjacent normal tissues and colorectal adenoma tissueBrown and yellow punctuation in cytoplasm could be defined as RASGRF1 positive. RASGRF1 mainly located in cytoplasm rather than in nucleus both in colorectal cancer tissue, related adjacent normal tissues and colorectal adenoma tissue. There were statistical significances of the RASGRF1 expression level between the three kinds of tissues. RASGRF1 expression level was higher in colorectal cancer tissue than in related adjacent normal tissues and colorectal adenoma tissue (P =0.038). There were no differences between the 17 cases of tubular adenoma and 18 cases of villioustublar adenoma (P=0.171).2. Relationship between RASGRF1 expression level and clinicopathologic parameters of colorectal cancer patientsWe further analyzed the relationship between RASGRF1 expression level and colorectal cancer patient’s clinicopathologic parameters, which indicated that RASGRF1 expression was correlated with sex (P=0.021), histological classification (P=0.009), distant metastasis (P=0.019), TNM stage (P=0.001). RASGRF1 level was higher in female, mucinous adenocarcinoma, with distant metastasis, TNM stage Ⅲ、 Ⅳ patients than male, adenocarcinoma, without distant metastasis, TNM stage Ⅰ、 Ⅱ patients. There was no correlation with age, tumor location, tumor size, differentiation (P>0.05).3. Cell proliferation assay by CCK8KRAS mutant colon cancer cell HCT116 was transfected by lipofectamine carrying RASGRF1 siRNA and NC siRNA.48 hours later, the proliferation capacity changed sharply between three groups (P=0.007), the proliferation rate in RASGRF1 siRNA group was slower than HCT116 group (P=0.005).72 hour after transfection, compared to NC siRNA group and HCT116 group, the proliferation capacity of RASGRF1 siRNA group went an apparent decline (P=0.000 and P=0.000). There were no differences between NC siRNA group and HCT116 group.4. Cell apoptosis assayWe evaluated the apoptosis cell after transfection through flow cytometry. Once RASGRF1 was down-regulated, early stage apoptotic cells, late stage apoptotic cells and total apoptotic cells were increased (P=0.027). RASGRF1 siRNA may have influence on colon cancer cell apoptosis.5. Cell cycle assayColon cancer cell HCT116 was transfected by lipofectamine carrying RASGRF1 siRNA and NC siRNA, the change of cell cycle was assessed by flow cytometry, which showed that Gl stage cells (P=0.039) in RASGRF1 siRNA group were increased, while S stage cells (P=0.000) decreased, compared with NC siRNA group and HCT116 group. Cell may be blocked in G1 stage and stopped mitosis when RASGRF1 was down-regulated.6. Cell migration assay48 hours after inoculated in transwell cell culture plate, cells acrossed the upper chamber in RASGRF1 siRNA group were fewer than those in NC siRNA group and HCT116 group (P=0.003), there were no differences between NC siRNA group and HCT116 group.Conclusion:RASGRF1 expression level was higher in colorectal cancer tissue than in related adjacent normal tissues and colorectal adenoma tissue and was correlated with sex, histological classification, distant metastasis, TNM stage. There was no correlation with age, tumor location, tumor size, differentiation. Down-regulation of RASGRF1 in KRAS mutant colon cancer cell may inhibit cell proliferation, cell division, cell migration and accelerate cell apoptosis.