Expression Of C3G In Colorectal Cancer And Its Effect On Cell Proliferation And Migration

Bankground and objectives: Colorectal cancer(CRC)is one of the most common cancers with significant morbidity and mortality worldwide,and ranks second only to lung cancer and gastric cancer in overall morbidity in China.With the changes of life-style,environment,diet and other factors,the incidence of CRC increases year by year.Because the early symptoms of CRC are not typical,most patients have entered the advanced stage when diagnosed.The pathogenesis of CRC involves the activation and inactivation of multiple oncogenes,including P53,K-ras,APC and so on.With the advance of medical level,targeted therapy has achieved good effect on CRC.However,gene mutation and human drug resistance make it important to explore new targets for CRC gene therapy.C3G,a guanine nucleotide exchange factor for Ras and Rap1 protein,can receive growth factor,integrin,hormone,cytokine and other stimulation to regulate Ras and Rap1 binding GTP or GDP to complete signal transmission.Previous studies have shown that C3 G is widely expressed in humans but has tissue specificity.For example,the expression of C3 G is up-regulated in lung cancer,liver cancer,ovarian cancer,breast cancer and neuroblastoma,and is involved in cell migration,proliferation,adhesion,differentiation and apoptosis,while the expression of C3 G is down-regulated in cervical cancer and chronic lymphoblastic leukemia.In addition,C3 G plays a different role in different tumors,for example up-regulated C3 G promotes the migration and invasion in ovarian cancer cells,but inhibits the movement of breast cancer cells.In cervical cancer the C3 G is down-regulated,but it promotes the invasion of cancer cells.Due to the unknown mechanism of C3 G in CRC,we will explore the relationship between C3 G expression level,clinicopathological characteristics,and prognosis in CRC.Besides,we will construct a C3 Gi cell line to investigate the effects of changes in C3 G on the proliferation and migration of colorectal cancer cells.Methods:1.We collected 69 pairs of CRC and its adjacent normal tissue,these specimens obtained from patients who underwent surgery at the affiliated hospital of southwest medical university between July 2013 and June 2014.In addition,we collected 31 pairs of fresh CRC and normal tissue from May to August 2019.For all patients,detailed data including basic information,pathological diagnosis,and tumor grade were obtained from the hospital records,and C3 G protein and mRNA was detected by immunohistochemistry and real-time quantitative PCR respectively.We followed up to analyze the relationship between C3 G protein expression and clinicopathological features and prognosis,and the deadline was September 2,2019.2.The level of C3 G in FHC,HCT-116 and HCT-8 cells was detect by Western Blot and RT-PCR.We were used siRNA transfection into colorectal cancer cell line to interference with C3 G expression,and fluorescence transfection and real-time quantitative PCR to detect gene silencing effect.Finally,Western Blot was used to verify the result of the interference of C3 G protein.3.CCK-8 and Transwell assays were used to detect the effects of C3 G on the proliferation and migration of colorectal cancer cells.Results:1.Expression of C3 G in CRC and adjacent normal tissues: C3 G protein was localized in the cytoplasm and cell membrane,and the positive expression was yellow or brown granules.In CRC and adjacent tissues,the positive rate of C3 G staining was 60.87%(42/69)and 34.78%(24/69)(P<0.05).At the same time,C3 G mRNA expression in CRC was(5.86±2.03)times higher than in normal tissues(P<0.05).2.The relationship between C3 G expression and clinicopathological features,prognosis: Chi square test showed that the high expression of C3 G in CRC is associated with TNM stage,tumor invasion depth,lymphatic metastasis,and CEA level,but not to age and gender.According to Kaplan Meier survival curve analysis,the 5-year survival rates of patients with high and low expression of C3 G protein were 32.43%(12/37)and 60.0%(15/25)(P< 0.05).3.Differential expression of C3 G in colorectal cancer cells and Intestinal mucosal cell: Western Blot was used to detect the expression of C3 G in HCT-116,HCT-8 and FHC,and the relative expression levels were 1.043 ± 0.116,0.899 ± 0.149 and 0.551 ± 0.155.Compared with FHC,the expression of C3 G in colorectal cancer cell line was higher(P< 0.05).The relative expression levels of C3 G mRNA in HCT-116 and HCT-8 were 3.582±0.561 and 2.645±0.612(P < 0.05).4.C3 Gi colorectal cancer cell line Construction: After using si-C3G-1,si-C3G-2,si-C3G-3,si-C3G-NC and si-C3G-PC sequences to transfect HCT-116 cells,the relative expression of C3 G mRNA were 0.296 ± 0.064,0.305 ± 0.986,0.465 ± 0.146 and 0.411 ± 0.109(P<0.05).We selected high interference efficiency sequence si-C3G-1 to transfect HCT-8 and HCT-116 cell lines,and then detected luorescence transfection effect.Finally,we used Western Blot detect the C3 Gi cell protein expression,the results showed that:(HCT-116)vs.(si-HCT-116)=(0.967 ± 0.145)vs.(0.278 ± 0.168)(P<0.05),(HCT-8)vs.(si-HCT-8)=(0.704 ± 0.230)vs.(0.196 ± 0.033)(P<0.05).5.C3 G silencing can inhibit the proliferation and migration of colorectalcancer cells: after si-RNA transfection with colorectal cancer cells,we wereused CCK-8 and Transwell to detect the cell number and motility.The resultsshowed that when C3 G of colorectal cancer cells(HCT-8,HCT-116)wassilenced,cell proliferation and migration were inhibited.Conclusion:1.C3 G was localized in cytoplasm and membrane,and the expression of C3 G in colorectal cancer cells and tissues was higher than normal cells and tissues.2.The up-regulated C3 G may be related to the occurrence and development of colorectal cancer,and it is one of the risk factors for poor prognosis of colorectal cancer patients.3.Silencing C3 G in colorectal cancer cell can inhibit the proliferation and migration.The successful construction of C3 Gi cell line in this experiment laid a foundation for the follow-up study to explore molecular mechanisms.