Neuralized1a Regulates The Asymmetric Cell Dividing Of Mouse Lewis Lung Carcinoma Stem Cells

ObjectiveTo establish an asymmetric dividing cell line(LLC-ASD cells)derived from mouse Lewis lung carcinoma cancer cells(LLC-Parental cells),and to investigate its stemness features.Then,figure out the role of asymmetric dividing in the self-renew of cancer stem cells according to the study of LLC-ASD cells.Methods(1)In order to obtain asymmetrically dividing LLC cells(LLC-ASD cells)derived from LLC-Parental cells,8 times of consecutive culture,enrichment and collection of floating spheriod forming cells followed by 5 times of consecutive single cell cloning were conducted.(2)Immunofluorescence assay was used to visualize and quantify the rate of asymmetric division in LLC-ASD cells labeled by Brd U.(3)For comparing the stem characteristics of LLC-Parental and LLC-ASD,RT-q PCR,clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay were conducted.(4)In vivo,LLC-Parental cells and LLC-ASD cells were subcutaneously transplanted in nude mice to determine the effect of the difference in stem cell like properties on tumorigeneicy.(4)The same lung transplantation into tumor experiment in mice were used to compare the differences in cancer biology characteristics.(5)Screen out the target gene from the candidate genes regulating asymmetric dividing(6)Design and purchase siRNAs of the target gene,and transfect the LLC-ASD cells with the siRNAs,then verify the inference effect of the si RNAs by RT-q PCR and WB respectively.(7)Immunofluorescence assay was used to visualize and quantify the rate of asymmetric division in transfected LLC-ASD cells labeled by Brd U.(8)For comparing the stem characteristics of transfected LLC-ASD cells,clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay were conducted.(9)In vivo,transfected LLC-ASD cells were subcutaneously transplanted in nude mice to determine the effect of the siRNA inference in stem cell like properties on tumorigeneicy.Results(1)Asysmmetric dividing cells were found in LLC-ASD cell culture through the BrdU immunofluorescence assay and the rate of asymmetric division in the anaphase cells was as high as 50%.(2)According to the clonogenic assay in 6-well plate(P<0.001),single cell spheroid formation assay with agar in 6-well plate(P<0.05)and 96-well-plate single cell cloning assay(P<0.01),the results in LLC-ASD cells showed that they were more prominent than those in the LLC-Parental cells.(3)In vivo,the tumor metastatic ability of LLC-ASD was enhanced than that of LLC-parental when transplanted to the C57 mice.(5)Further,the tumorigenic ability of LLC-ASD cells was also increased compared to that of LLC-parental cells when subcutaneously transplanted to the nude mice.(6)The candidate asymmetric dividing regulator gene of somatic cells Neuralized1 a were selected.(7)The expression of Neuralized1 a were surpressed in the experimental group.(8)According to the clonogenic assay in 6-well plate(P<0.001),single cell spheroid formation assay with agar in 6-well plate(P<0.001)and 96-well-plate single cell cloning assay(P<0.01),the results in the experimental group showed that they were less prominent than those in the control group.(9)At last,the tumorigenic ability of the experimental group was also decreased compared to that of the control group when subcutaneously transplanted to the nude mice(P<0.05).ConclusionThe asymmetric dividing cell line derived from mouse Lewis lung carcinoma cancer cell line(LLC-ASD cells)is established which exhibits stemness properties.Besides,Neuralized1 a regulates the asymmetric cell dividing in the LLC-ASD cells.