| Objective: To investigate the effect of pLL3.7-CMV-LMO3-IRES-puro recombinant lentivirus on the biological function of glioma SHG44 cells and to provide a theoretical basis for the prevention and treatment of malignant glioma.Methods: 1.The LMO3 gene was constructed into the pLL3.7-CMV-IRES-puro vector,and the lentivirus was packaged,and the lentivirus was used to infect the glioma SHG44 cell line.Puromycin screening LMO3 dual-label over-expression stably transfected strain and control stabilizing strain;2.The cells were divided into control group,Vector group and LMO3 group using CCK8,EdU kit and flow cytometry to detect the proliferation activity and cell cycle distribution of glioma SHG44 cells;3.Through the cell migration experiment under the microscope to observe and record the 24 h,48h wound healing of each group of cells to determine the migration of glioma cells;4.The nude mice were subcutaneously inoculated with LMO3 group and vector group glioma SHG44 cells respectively.The tumorigenicity and tumor mass of nude mice were observed to evaluate the effect of LMO3 expression on the tumorigenicity of nude mice.5.The glioma SHG44 cells were stimulated with hEGF and inhibitors U0126 and PD98059,respectively.Some of them were used for EdU to detect the proliferation of SHG44 cells after treatment with hEGF and U0126.The other cells were collected and Western blot was used to detect the expression of LMO3 after MEK1 and p-MEK1,ERK,p-ERK,CyclinD1 protein expression,to explore the effect of LMO3 protein expression on the proliferation inhibition of glioma SHG44 cells.Results: 1.The successful screening of glioma SHG44 cell line with LMO3 double-stranded overexpression and stable metastasis,Western Blot detection of Flag-LMO3 expression in LMO3 group was positive,Vector group no Flag-LMO3 expression was statistically significant(P<0.05);2.Compared with LMO3 group,the cell viability of the control group at 24 h,48h,72 h,and 96 h was significantly higher than that of the LMO3 group(P<0.05),but there was no statistical difference between the Control group and the Vector group(P>0.05);3.The DNA replication activity was detected by specific reaction of EdU and Apollo fluorescent dye.Compared with LMO3 group,the positive rate of Control group was significantly higher than that of LMO3 group(P<0.05).However,there was no statistical difference between Control group and Vector group(P>0.05);4.Cell cycle results showed that overexpression of LMO3 resulted in an increase in the proportion of G1 phase cells in glioma cells,a decrease in the proportion of cells in S phase,and apparently arrested cells in G1 phase,but not in S phase;5.There was no significant difference in the number of migrating cells between 24 h and 48 h in Control and Vector groups(P>0.05),while the number of cells migrating in LMO3 group was significantly lower than that in Control group(P<0.05);6.In vivo test results: Tumor formation rate in nude mice was 100%;subcutaneous tumorigenesis in nude mice confirmed that LMO3 overexpression significantly inhibited the in vivo tumorigenicity of glioma SHG44 cells;tumor tissue weights obtained after ligation showed that tumor mass in the LMO3 group was significantly lower than The vector group was statistically different(P<0.05);7.The activity of SHG44 cells was detected by EdU 0.5 h after hEGF stimulation.Compared with LMO3 group,the positive rate of cells in Control group was higher than that of LMO3 group(P<0.05);8.U0126 treatment of SHG44 cells,EdU test results suggest: Control,Vector,LMO3 three groups of cells positive rate was no statistically significant difference(P>0.05);9.hEGF stimulated glioma SHG44 cells.The results of Western Blot showed that the expression of MEK1,p-MEK1,ERK and p-ERK in LMO3 group was up-regulated when compared with the Vector group(P<0.05).At 0.5 h,the expression of MEK1,p-MEK1,ERK protein in LMO3 group was still increased compared with Vector group(P<0.05),but the expression of p-ERK decreased significantly(P<0.05);when stimulated for 1 h,it was associated with Vector group.In contrast,the expression of MEK1,p-MEK1,ERK and p-ERK was down-regulated in LMO3 group(P<0.05).Compared with Vector group,the expression of CyclinD1 was up-regulated in LMO3 group at three time points and stimulated by hEGF.The time of the two groups increased the expression of CyclinD1 protein(P<0.05);10.U0126 and PD98059 treatment of SHG44 cells Western Blot results: U0126 treatment,compared with Vector group,LMO3 group MEK1 protein expression was no significant difference(P>0.05);while p-MEK1 and p-ERK protein expression was down-regulated,The expression of ERK and CyclinD1 increased with statistical significance(P<0.05);PD98059 treatment results showed that the expression of MEK1,p-MEK1,p-ERK and CyclinD1 in LMO3 group was significantly higher than that in Vector group(P<0.05);10.U0126 and PD98059 treatment of SHG44 cells Western Blot results: U0126 treatment,compared with the Vector group,LMO3 group MEK1 protein expression was no significant difference(P>0.05);while p-MEK1 and p-ERK protein expression was downregulated,ERK,The increase of CyclinD1 protein expression was statistically significant(P<0.05).PD98059 treatment results showed that compared with Vector group,the expression of MEK1,p-MEK1,p-ERK,and CyclinD1 protein in LMO3 group was significantly increased(P<0.05).).Conclusions: 1.LMO3 up-regulation inhibits the proliferation of SHG44 cells,and arrests the cell cycle in G1 phase while inhibiting the migration of SHG44 cells;2.In vivo experiments LMO3 group nude mice slowed down the growth of the tumor further confirmed the overexpression of LMO3 inhibition of glioma cell proliferation;3.LMO3 regulates the progression of the cell cycle and may not be related to changes in CyclinD1 protein.The exact mechanism is not clear.LMO3 overexpression through the MEK-ERK signaling pathway regulates the biological characteristics of gliomas.It is speculated that the upregulation of LMO3 may regulate the biological activity of glioma by decreasing the expression of ERK phosphorylation protein. |
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