The Studies Of MicroRNA-21-3p In Colorectal Cancer

Background and purposeColorectal cancer(CRC) is one of the most commonly digestive tract malignant tumors. In most cities of China, with the improvement of living standards and the changes of eating habits, the incidence and mortality of CRC is increasing and patients are tending to become younger. Its incidence increases with the age starting from 40, arriving at its peak among the age 60 to 75. And younger, familial aggregation and genetic susceptibility are the characteristics of the disease in our country. Because of the tumors’ growth speed is slow, it needs a long time to produce symptoms and signs. Therefore, when the patients with some symptoms gp to the hospital to see doctor, the colorectal cancer cells metastasis has happended which is the main cause of death in CRC and the direct reason of recurrence after operation. Now the mian treatment for colorectal cancer is surgery, sometimes with radiation and chemotherapy, but both of them have poor effect. So it is important to explore the potential mechanism of those diseases.MiR-21-3p is one of numerous mature micrornas which is from pre-mi R21 after processed by nucleic acid enzyme Dicer in cytoplasm. Its matching mi RNA mi R-21-5p has high abundance and its function and mechanism in tumor has been researched completely. All the results show that mi R-21-5p has higher abundance in the tumor tissues, and plays same role in different tissue tumors. So whether mi R-21-3p does the same work like oncogene mi R-21-5p?Many esearch have found that mi R-21-3p Many research have found that there is a up-regulated expression of mi R-21-3p in breast cancer, laryngeal cancer, kidney cancer, ovarian cancer, endometrial cancer and other tumors, suggesting that mi R-21-3p involves in the regulation of tumors, but the mechamism is not clear. In addition, there is no report about mi R-21-3p in colorectal cancer which needs to be further studied.Methods1. Mature mi R-21-3p expression in colorectal cancer tissues and cells was detected using Real-time RT-PCRMature mi R-21-3p expression in tissues and cells was detected using Real-time RT-PCR. These specimens include samples of colorectal cancer tissue and matched normal colonic mucosa of 27 patients. While colorectal cancer eleven human colonic carcinoma cell lines include DLD-1、HCT-8、Caco-2、HCE-8693、RKO、SW480、SW620、HT29、Lo Vo、HCT116、LS174T.2. Effect of mi R-21-3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined(1)A lentiviral expression vector LV3-mi R-21-3p was ordered. HCT116 and SW480 cells were transduced with puromycin and subsequently FACS-sorted for green fluorescent protein(GFP). Confirmation of stable transfection of the plasmids was obtained using the mi R q RT-PCR assay. The colorectal cancer cell lines were called HCT116/SW480-LV3-mi R-21-3p. The cell proliferation, invasion and migration ability was evaluated using CCK8 method, wound scratch, transwell migration, invasion assay;(2)The mi R-21-3p mimics was transiently transfected into colorectal cancer cell lines HCT116 and SW480. The change of EMT(epithelial-Mesenchymal Transition) index was evaluated using using Western Blot and cell immunofluorescence experiment;(3)Using nude mice as the whole visualization animal model, the mice subcutaneous tumor experiment and the immunohistochemical experiment detected the effect of tumorigenicity and the change of EMT index; Pulmonary metastatic carcinoma in mice model was constructed to observe the metastasis in the lung tissue of mice.3. Statistical analysisSPSS 17.0 software was used for statistical analysis. Data were presented as Mean±SD of at least 3 independent experiments. The clinical samples’ differences were analyzed using the Paired-sample T test. The results of RT-PCR in cells were analyzed through Independent-Samples T test. Wound scratch, transwell migration, invasion assay were analyzed through Independent-Samples T test. P values of <0.05 were considered statistically significant.Result1. mi R-21-3p expression was detected in colorectal cancer tissues and cellsIn 27 fresh colorectal cancer paired tissues, fluorescence quantitative PCR test results show that the expression of mi R-21-3p in colorectal cancer tissues was significantly higher than normal mucosa(t=-5.365,p=0.000). The expression of mi R-21-3p in RKO cell is much higher than other 10 kinds of cells.2. Effect of mi R-21-3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined(1)The established stable cell line HCT116 and SW480 expressing of mi R-21-3p was named HCT116/SW480-LV3- mi R-21-3p. The expression of mi R-21-3p in HCT116/SW480-LV3- mi R-21-3p was much higher than that of HCT116/SW480 group LV3-NC. The difference was statistically significant(t=-15.019,p=0.000;t=-83.302,p=0.000) showed significant moderating effect.(2)Compared with LV3-NC, proliferation properties of HCT116 and SW480 transfected with the LV3-mi R-21-3p were also detected by CCK8 assay(p=0.075;p=0.058). The difference was statistically insignificant showed insignificant moderating effect.(3)The number of migratory HCT116 and SW480 cells transfected with the LV3-mi R-21-3p was more than mi R mimics control, the difference was statistically significant(t=-32.986,p=0.000;t=-11.620,p=0.000). The number of invasive HCT116 and SW480 cells transfected with the mi R-21-3p mimics was more than mi R mimics control, the difference wasstatistically significant(t=-9.257, p=0.000; t=-8.552,p=0.000).(4)RT-PCR results indicated that compared with mi R mimics control, increased mi R-21-3p expression levels were measured in HCT116 and SW480 cells after transfected with mi R-21-3p mimics. The difference was statistically significant(t=-26.508, p=0.000; t=-60.562, p=0.000) showed significant moderating effect; Western Blot and cell immunofluorescence results showed that with increased mi R-21-3p expression, HCT116 and SW480 enhanced EMT ability.(5) nude mice subcutaneous tumor immunohistochemical experiment showed that compared with LV3-NC, HCT116 and SW480 cells transfected with the LV3-mi R-21-3p enhanced EMT ability. Nude mice lung metastatic carcinoma showed the invasive ablity that HCT116 and SW480 cells transfected with the LV3-mi R-21-3p was stronger than mi R mimics control.Conclusion1. The expression of mi R-21-3p in the colorectal cancer tissues is much higher than in the normal tissue;2. MiR-21-3p can promote invasion and magration of colorectal cancer cell lines, its target genes will be researched in the subsequent experiments;...