The Mechanism And Influence Of MTOR Overexpression On Proliferation Of NIH3T3 Fibroblasts

Objective:To investigate the proliferation and secreton of fibronectin(FN)from NIH3T3 fibroblasts after tansfection with mammalian target of rapamycin(mTOR)gene,and to explore the probable mechanism of mTOR gene on proliferation and secretion of FN of NIH3T3 fibroblasts after treatment these cells with rapamycin,so that the expressions of FN gene and protein,the expressions of Akt,mTOR, p70S6K genes and proteins were checked.Methods:The plasmids of recombinant pcDNA3/mTOR was transformed into competent cells(DH-αEscherichia coli(E.coli)) prepared with the conventional CaCl₂ method.Then it was extracted with the alkaline lysis.The recombinant vector pcDNA3/mTOR was determined by restriction enzyme digestion and sequencing analysis. Then the pcDNA3/mTOR plasmid was transfected into NIH3T3 fibroblasts with electroporation method.The positive cell clones were selected with G418.Then the transfected stable cell clones were obtained, which were treated with rapamycin.The mRNA and protein expressions of Akt,mTOR,p70S6K and FN were determined by RT-PCR and Western blotting analysis respectively.MTT assay was employed to check the proliferation of NIH3T3 fibroblasts.The SPSS11.0 software was used for the statistical analysis.The t test and F test or x² test was employed to analysis these date respectively in this research.The P＜0.05 was considered to be statistically significant.Results:(1)The recombinant pcDNA3/mTOR plasmid was identified with double digestion and nucleic acid sequencing,which proved the transfected plasmid was corrected.(2)After transfection with mTOR gene,the results of expressions of gene and protein were as follows.The gray scale values of mTOR/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.68±0.03 and 0.38±0.03 respectively with RT-PCR(P＜0.01), and 0.79±0.04 and 0.57±0.01 respectively with Western blotting(P＜0.01). The gray scale values of FN/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.63±0.07 and 0.39±0.03 respectively with RT-PCR(P＜0.01),and 0.77±0.07 and 0.55±0.04 respectively with Western blotting(P＜0.01).The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.41±0.07 and 0.39±0.02 respectively with RT-PCR(P＞0.05),and 0.65±0.02 and 0.63±0.03 respectively with Western blotting(P＞0.05). The gray scale values of p7.0S6K/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.69±0.03 and 0.34±0.02 respectively with RT-PCR(P＜0.01),and 0.75±0.06 and 0.53±0.05 respectively with Western blotting(P＜0.01).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in control group(all P＜0.01).(3)After treated with rapamycin,the results of expressions of gene and protein were as follows.The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.40±0.06,0.39±0.02,0.41±0.05 and 0.39±0.04 respectively with RT-PCR(P＞0.05),and were 0.66±0.02,0.63±0.04,0.67±0.07 and 0.64±0.05 respectively with Western blotting(P＞0.05).The gray scale values of p70S6K/GAPDH of NIH3T3 fibroblasts in transfected group, control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.70±0.03,0.34±0.04,0.35±0.02 and 0.33±0.06 respectively with RT-PCR(P＜0.01),and were 0.76±0.05,0.54±0.04,0.52±0.01,0.53±0.03 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The gray scale values of FN/GAPDH of N/H3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.64±0.04,0.40±0.05,0.40±0.03 and 0.41±0.03 respectively with RT-PCR(P＜0.01),and were 0.76±0.03,0.53±0.01,0.54±0.02 and 0.54±0.05 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).Conclusions:(1)Transfection of mTOR gene increases the proliferation of fibroblasts and secretion of FN.(2)The effect of mTOR gene probable was by means of the Akt-mTOR-p70S6K signal pathway,but does not depend on the activation of Akt.(3)The Rapamycin suppresses the proliferation of fibroblasts and secretion of FN probable by means of inhibiting mTOR-p70S6K signal pathway.