The Role And Mechanism Of MicroRNA-199a-3p Regulating PI3K/AKT/mTOR Signaling Pathway In Phenotype Transformation Of Human Embryonic Lung Fibroblasts Cells

Research ObjectiveTo explore the role and mechanism of microRNA-199a-3p(miR-199a-3p)regulating PI3K/AKT/mTOR signaling pathway in phenotype transformation of Human Embryonic Lung Fibroblasts(HELF)cells.Research Methods1.The experiment was divided into two groups: Control group and TGF-?1 group.TGF-?1 group was given 5ng/ml TGF-?1 for 72 hours;Control group was given the same volume of medium.The protein of ?-SMA protein was detected by Western Blot.2.We transfected respectively in HELF cells with mir-199a-3p inhibitors NC and mir-199a-3p inhibitors.The cells was divided into six groups:control group?TGF-?1group ? miR-199a-3p inhibitors NC group ? miR-199a-3p inhibitors NC+TGF-?1 group?miR-199a-3p inhibitors group?miR-199a-3p inhibitors+TGF-?1 group.3.QRT-PCR was used to measure the expression of miR-199a-3p in the above six groups.4.The expression of ?-SMA?CollagenI?PI3K?Akt?P-Akt(S473)?P-Akt(T308)?mTOR?P-mTOR(S2448)protein in the above six groups was detected by Western Blot.Research Results1.The results of Western Blot showed that the expression level of ?-SMA protein was significantly increased when HELF cells were treated with TGF-?1(5ng/ml)for 72 h,which showed that the in vitro model was successfully established.2.The expression of mirR-199a-3p in each group was detected by qRT-PCR.It was found that the expression level of miR-199a-3p in HELF cells induced by TGF-?1 was significantly higher than that in the control group.The results showed that mirRNA-199a-3p expression level have no significant differences in the group of TGF-?1 ? miR-199a-3p inhibitors NC+TGF-?1.The expression level of mirR-199a-3p in HELF cells induced by miR-199a-3p inhibitors NC was significantly higher than that in the mirR-199a-3p inhibitors group.The expression level of mirR-199a-3p in HELF cells induced by miR-199a-3p inhibitors+TGF-?1 was significantly lower than that in the mirR-199a-3p inhibitors NC+TGF-?1 group.MiR-199a-3p inhibitors could inhibit the reduction of miR-199a-3p by TGF-?1.3.The results of Western Blot showed that the expression level of ?-SMA and CollagenI protein was significantly higher when HELF cells were treated with TGF-?1 than that in the control group.The results showed that ?-SMA and CollagenI protein expression expression level have no significant differences in the group of TGF-?1?miR-199a-3p inhibitors NC+TGF-?1.While expression level of ?-SMA and CollagenI protein in the group of miR-199a-3p inhibitors+TGF-?1 was significantly lower than that in the mirR-199a-3p inhibitors NC+TGF-?1 group.Which indicated that miR-199a-3p played an important role in phenotype transformation of HELF cells.4.The results of Western Blot showed that the expression level of P-Akt(5473)?P-Akt(T308)?P-mTOR(52448)protein was significantly higher when HELF cells were treated with TGF-?1 than that in the control group,while there was no significant change in the expression of PI3K?Akt and mTOR.The results showed that P-Akt(5473)?P-Akt(T308)?P-mTOR(52448)protein expression expression level have no significant differences in the group of TGF-?1?miR-199a-3p inhibitors NC+TGF-?1.Which indicated that in the TGF-?1-induced phenotype transformation PI3K/Akt/mTOR pathway was activated.The expression level of P-Akt(5473),P-Akt(T308)? P-mTOR(52448)protein in the group of miR-199a-3p inhibitors+TGF-?1 was significantly lower than that in the mirR-199a-3p inhibitors NC+TGF-?1 group,while there was no significant change in the expression of PI3K?Akt and mTOR.Which indicated that miR-199a-3p inhibitors could inhibit the effect of TGF-?1 in phenotype transformation and PI3K/Akt/mTOR pathway.Research Conclusion1.The expression of miR-199a-3p was increased in TGF-?1-treated HELF cells?2.MiR-199a-3p inhibitors could inhibit the phenotypic transformation of HELF cells by TGF-?1?3.MiR-199a-3p played an important role in the phenotypic transformation of HELF cells by regulating PI3K/Akt/mTOR signaling pathway?...