Abstract:Aim To explore the association between mammalian target of rapamycin (mTOR) gene polymorphism and genetic susceptibility to coronary heart disease in Han population in South China, and provide new ideas for early prevention and intervention of coronary heart disease. Methods Polymerase chain reaction-ligase detection reaction (PCR-LDR) technology was used to classify mTOR gene polymorphisms in 804 patients with coronary heart disease and 979 control subjects. The genotypes of rs2295079 and rs1883965 of mTOR gene were detected. The association between each polymorphic site and susceptibility to coronary heart disease was analyzed by unconditional Logistic regression. Results There was no statistical difference in the distribution of alleles and genotype frequencies of rs2295079 and rs1883965 between the coronary heart disease group and the control group (P＞0.05); the distribution frequencies of the haplotype rs2295079C-rs1883965A constructed by these two loci in the coronary heart disease group and the control group were 8.3% and 6.5%, respectively, and the coronary heart disease group was significantly higher than the control group; individuals carrying rs2295079C-rs1883965A haplotype had a significantly increased risk of developing coronary heart disease (OR＝1.29, P＝0.047), this haplotype had a more significant risk of developing coronary heart disease in people aged ≤60 years old (OR＝1.73, P＝0.009). Conclusion The haplotype rs2295079C-rs1883965A of the mTOR gene is closely related to the genetic susceptibility to coronary heart disease, and is more obvious in the population aged ≤60 years old, suggesting that this haplotype may be an important rick factor in increasing the risk of coronary heart disease in the Han population in South China.

Abstract:Aim To construct the lentivirus expression vector of overexpression for RagB gene, realize the overexpression of RagB gene in C2C12 cells, test the regulation of mammalian target of rapamycin (mTOR) activation mediated by Ursolic acid. Methods The lentivirus expression vectors, Flag pLJM1 RagBWT, Flag pLJM1 RagB54L and Flag pLJM1 RagB99L were employed in the experiment. Using PCR to amplify the full length of Flag-RagBWT, Flag-RagB54L and Flag-RagB99L, then they were subcloned into the pWPI lentivirus vector through SwaI restriction site and identified by bacteria liquid PCR. The cloned lentivirus vectors(pWPI-RagBWT, pWPI-RagB54L and pWPI-RagB99L) were packaged with packaging vectors (psPAX2 and pMD2.G) via specific ratio and transfected into 293T cells. 48 hours after the transfection, the media was collected and used to infect C2C12 cells. RagB99L-overexpressed C2C12 myoblasts were treated individually or jointly with leucine and ursolic acid, mTOR activation was detected by Western blot. Results The efficiency of infection observed by fluorescence microscope was almost 100%, the expression of RagB in C2C12 cells proved by Western blot was enhanced significantly in RagB overexpression group. Ursolic acid inhibited significantly mTOR activation in RagB99L-overexpressed C2C12 myoblasts. Conclusion The results indicate that RagB can be overexpressed in C2C12 cells by constructing the lentivirus expression vector, RagB plays a critical role in ursolic acid-inhibited mTOR activation.