Molecular Biological Mechanisms Of RASSF1 Gene Associated With High Glucose Environment In Chondrocytes (ATDC5)

Part1 Effect of different glucose concentrations on ATDC5 cell and their RASSF1 gene expressionObjective:To investigate the effect of different glucose concentrations on the proliferation of ATDC5 cells and their RASSF1 gene expression.Methods:(1)ATDC5 cells were treated by configuring different concentrations of glucose(5.5 mmol/L,25 mmol/L,50 mmol/L,100mmol/L)medium and cultured for 24 h,48 h,respectively.The value-added ability of ATDC5 cells in different treatment groups was detected by CCK8 assay.The time point with a difference in CCK8 was selected as the culture time for subsequent experiments.(2)The expression of RASSF1 protein in ATDC5 cells cultured for 48 hours under different glucose concentration treatments was detected by protein immunoblotting(Western blot).Results:The results showed no significant difference in cell proliferation between the groups at 24 hours of culture(P >0.05).The cells in the 25 mmol/L glucose-treated group proliferated significantly(P <0.05)compared to the 5.5 mmol/L glucose-treated group at48 hours of incubation,while the cells in the 50 mmol/L glucose-treated group did not proliferate significantly(P <0.05)compared to the 5.5 mmol/L glucose treated group,the cells in the 100 mmol/L glucose-treated group did not proliferate significantly(P <0.05)compared to the 5.5 mmol/L glucose treated group.After 48 h culture,the expression level of RASSF1 gene protein in 25mmol/L glucose treated ATDC5 cells was significantly decreased compared with 5.5 mmol/L glucose treated ATDC5 cells(P<0.05).The expression level of RASSF1 gene protein in ATDC5 cells treated with 50mmol/L glucose was significantly decreased(P<0.05).Conclusions:High glucose(50mmol/L、100mmol/L)treatment of ATDC5 cells inhibited their proliferation,and high glucose(25mmol/L 、 50mmol/L)treatment inhibited the expression of the RASSF1 protein.Part2 Effect of knockdown of RASSF1 gene on ATDC5 in chondrocyteObjective:The RASSF1 gene of ATDC5 was knocked down in vitro to explore the effects of this gene on chondrocyte proliferation,migration and on AKT/m TOR and RA signaling pathways.Methods:(1)ATDC5 cells were infected with lentivirus containing empty vector using lentiviral infection technique,and ATDC5 cells were cultured for one week using a complete medium containing 2 μg/m L of puromycin to obtain stably infected ATDC5 cell lines carrying empty vector for subsequent experiments.ATDC5 cells were infected with lentivirus containing knockdown RASSF1 gene,and ATDC5 cells were cultured with a complete medium containing 2 μg/m L of puromycin for one week to obtain stably infected RASSF1 knockdown ATDC5 cell lines for subsequent experiments.(2)The 96-well plates were incubated with empty ATDC5 cells and ATDC5 cells infected with the knockdown RASSF1 gene for 24 h,48 h,and 72 h.CCK8 measured cell proliferation at different time points.(3)The effect of the knockdown of RASSF1 on the migration of ATDC5 cells was examined in the scratch assay by incubating two groups of cells for 24 h and 48 h.(4)The expression of RALDH2,CYP26A1,AKT in both groups of cells was detected by q RT-PCR.(5)Western Blot was used to detect the expression of AKT,m TOR in both groups of cells.Results:RASSF1 knockdown stable strain was successfully constructed.Compared with NC group,cell proliferation was significantly inhibited in RASSF1 group at 24 h(P<0.05).Knockdown of RASSF1 inhibited the migration ability of ATDC5 cells.q RT-PCR showed that knockdown of RASSF1 gene inhibited the expression of RALDH2,CYP26A1,AKT(P <0.05).Western blot showed that knockdown of RASSF1 inhibited AKT,m TOR phosphorylation(P <0.05).Conclusions:Reducing RASSF1 inhibited cell proliferation,cell migration,cellular RA signaling pathway,cellular AKT/m TOR signaling pathway.