Study On The Mechanism Of OCT4B1 Induced Colorectal Cancer Cell Epithelial Mesenchymal Transition To Obtain Stem Cell Characteristics

Objective:Previous research has confirmed that OCT4B1 gene induces the occurrence of EMT in colorectal cancer cells to obtain the stemness characteristics,this study explore the regulatory mechanism.Methords:Based on the previous research,The level of OCT4B1 m RNA in human colorectal cancer stem cells(SW480 CSCs)by suspension culture was significantly higher than that of its parental cells(SW480).OCT4B1 gene was silenced by lentivirus mediated in SW480 CSCs(SW480 CSCs-RNAi).Compared with negative control group(SW480CSCs-NC),the relative expression of OCT4B1 m RNA decreased obviously.The above four groups of cells were tested as follows:(1)The level of m RNA and protein expression of PLK1,which regulates the process of EMT,was detected by RT-q PCR and Western blot respectively.To observe whether the results showed the same variation tendency with that in OCT4B1;(2)Screening mi RNAs with OCT4B1 and PLK1 changes in the opposite trend,which have binding sites for PLK1 by using mi RNA chip detection technology;(3)verifying the results consistency by RT-q PCR between the expression of mi RNA and chip testing results in four groups of cells;(4)Dual luciferase assay was used to detect the direct regulation of mi RNA and PLK1.Results:(1)The relative expression of PLK1 m RNA in parental cells(SW480)and SW480 CSCs were(2.30±0.15)vs.(7.40±0.10);in SW480 CSCs-NC 与 SW480CSCs-RNAi,the results were(4.57±0.17)vs.(2.13±0.09),all the difference was statistically significant(P<0.01);the protein expression of PLK1 in parental cells(SW480)and SW480 CSCs were(0.68±0.05)vs.(1.16±0.08),in SW480 CSCs-NCand SW480 CSCs-RNAi,the results were(1.50±0.11)vs.(0.27±0.06),all the difference was statistically significant(P<0.01).It was suggested that PLK1 and OCT4B1 showed the same trendency.(2)compared with OCT4B1 and PLK1,the changes of mi R-8064 was in the opposite direction by mi RNA chip detection technology.Bioinformatics software(Target Scan human)found mi R-8064 had a binding site with PLK1;(3)The relative expression of mi R-8064 m RNA in parental cells(SW480)and SW480 CSCs were(1.00±0.12)vs.(0.40±0.06);in SW480 CSCs-NC and SW480 CSCs-RNAi,the results were(0.12±0.03)vs.(0.93±0.02),all the difference was statistically significant(P<0.05).The results indicated mi R-8064 m RNA was in accordance with chip test results.(4)Dual luciferase assay confirmed that Plk1 is a direct target gene of mir-8064.Conclusion:OCT4B1 induces EMT to obtain stem cell characteristics,the mechanism of which is related to the inhibition of mi RNA-8064 expression by OCT4B1 and the expression of target gene PLK1 in colorectal cancer SW480 cells.