The Expression Of Genes Associated With Stem Cell And MiR-24-1 & MiR-106a In Colon Cancer

Purpose:1. To investigate the expression of stem cell-related molecules markers include CD133,CD166,Oct4,Sox2,C-myc,Klf4 and Bmi-1 in colon cancer and sorted CD133+ SW620 cell line.2. small interference RNA (siRNA) transfected with pSIREN-Bmi-1-2 under the mediation of Lipofectamine to SW620,so as to obtain stable transfected cloning cells and to observe whether the expression of Bmi-1 had changed in mRNA and protein level.3. To investigate the expression of miR-24-1 and miR-106a in both colon cancer and precancerous tissues and analyze the relatinship between the expression of miR-24-1, miR-106a and C-myc gene,and to discuss the mechanism of action of miR-24-1 and miR-106a in colon cancer.Method:1. To extract RNA from 29 colon cancer tissues and their adjacent ones,detect the mRNA expression of CD133,CD166,Oct4,Sox2,C-myc,Klf4 and Bmi-1 by RT-PCR;2. Flow cytometry analyse colon cancer cells SW620,SW480,Lovo who had the highest expression of CD133 antigen,and choosed the highest one to be sorted.3. To sort SW620 by CD133 MiniMACS, and assay the expression of above genes between the sorted CD133+ cells and the CD133- cells.4. small interference RNA (siRNA) transfected with pSIREN-Bmi-1-2 under the mediation of Lipofectamine to SW620,choose the stable transfected cloning cells by means of the puromycin.5. Detect the mRNA expression of Bmi-1 after transfected with pSIREN-bmi-1-2 compared to before transfected by RT-PCR.6. Detect the protein expression of Bmi-1 after transfected with pSIREN-bmi-1-2 compared to before transfected.7. The TaqMan MicroRNA assays were used to analyze the expression of the miR-24-1 and miR-106a,as well as C-myc gene in both colon cancer and precancerous tissues.Result:1. All 29 samples were proved to be poorly/moderately differentiated adenocarcinoma or myxoadenocarcinoma by pathology.The relative quantity expression of the stem cell-related moleculars markers in the cancer tissues and the paracancerous tissues were CD133(189.1±25.4,49.4±9.6),CD166(120.3±21.4,47.0±9.7),Oct4(152.3±18.2,80.6±12.7),Sox2(43.2±8.2,18.0±3.3),C-myc(234.6±31.1,104.7±17.8),Klf4(189.8±26.1,85.8±13.8)and Bmi-1(266.5±23.3,157.8±19.1)respectively. All genes expression of in cancer tissues were higher than their paracancerous partners (P<0.05).2. Flow cytometry analys showed that SW620 expressed CD133 was 15.1%,SW480 was 8.5%,LOVO was 6.8%,so the SW620 would be the ones which were used to be sorted.3.Flow cytometry analys showed that CD133+ cells was 88.5% in sorted positive SW620 and CD133- cells was 99.9% in sorted negative cell,the expression of 7 stem cell associated genes were higher in CD133+ populations than CD133- ones.4. The optimum concentration of obtaining stable transfected cloning cells was 0.8ug/mL. 5. Bmi-1 mRNA expression level of pSIREN-bmi-1-2 group was lower than that of blank control group by fluorescence quantitative PCR method, the rate of suppression on bmi-1 mRNA is 68%(p< 0.05).6. Bmi-1 protein expression level of pSIREN-bmi-1-2 group was lower than that of blank control group by Western Blot method.7. The results showed that the expression of miR-106a in cancer tissues was higher than the precancerous tissuesin the C-myc positive group(p< 0.05),while the expression of miR-106a in cancer tissues was higher than the precancerous tissues in the C-myc negative ones(p< 0.05),the expression of miR-24-1 showed no difference between the cancer and precancerous tissues, or between positive and negative C-myc control.Conclusion:1. The expression of the stem cell-related moleculars markers CD133,CD166,Oct4,Sox2,C-myc,Klf4 and Bmi-1 in colon cancer tissues were higher than paracancerous ones.2. In the colon cancer cells SW620,SW480 and Lovo,SW620 had the highest expression of CD133 antigen,so SW620 would used to be sorted.3. CD133,CD166,Oct4,Sox2,C-myc,Klf4 and Bmi-1 genes expressed higher in SW620 sorted CD133+ cells compared with CD133- sorted cells.4. Recombinant vector pSIREN-bmi-1-2 could inhibit bmi-1 gene expression on the mRNA and the protein level in colon cancer SW620 cell.5. The expression of miR-106a in both C-myc positive group cancer tissue and the C-myc negative ones were higher than precancerous tissues, miR-106a would be a blood screening target of colon cancer;there is no difference of the expression of miR-24-1 in both C-myc positive group ones and the C-myc negative ones.