The Role And Mechanism Of LncRNA N341773 In Phenotype Transformation Of Human Embryonic Lung Fibroblasts Cells

Research Objectives:1.To explore the expression of lncRNA n341773 in TGF-?1-induced human embryonic lung fibroblast(HELF)cells.2.To verify the role of lncRNA n341773 in the human embryonic lung fibroblast cells phenotype transformation.3.To explore the molecular mechanism by which lncRNA n341773 regulates the phenotypic transformation of human embryonic lung fibroblast cells.Research Methods:1.The experiment was divided into two groups: Control group and TGF-?1group.TGF-?1 group was given 5ng/ml TGF-?1 for 72 hours;Control group was given the same volume of medium.The expression of ?-SMA protein was detected by Western Blot,and the expression of lncRNA n341773 gene was detected by qRT-PCR.2.Based on the HELF cell phenotype transformation model,we used lentiviral to intervene the expression of lncRNA n341773.The cells was divided into ten groups:control group,LV-RNAi-NC group,LV-RNAi-lncRNA n341773 group,LV-oe-NC group,LV-oe-lncRNAn341773 group,TGF-?1group,LV-RNAi-NC+TGF-?1group,LVRNAi-lncRNA n341773+TGF-?1 group,LV-oe-NC+TGF-?1 group,LV-oe-lncRAN n341773+TGF-?1 group.The interfering ? overexpressing lentivirus and their respective empty lentiviruses were transfected into HELF cells,to establish in-vitro overexpressing or silencing model.Cells were collected after treatment with 5 ng/ml of TGF-?1 for 72 h.3.QRT-PCR was used to measure the expression of lncRNA n341773 in the above ten groups.4.EdU test was used to measure the proliferation of the above ten groups.5.Cell cycle assay was used to measure the cell cycle distribution of the aboveten groups.Research Results:1.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)for72h,which showed that the in vitro model was successfully established.The results of qRT-PCR showed that the level of lncRNA n341773 was significantly decreased when HELF cells were treated with TGF-?1.2.The expression of lncRNA n341773 in each group was detected by qRT-PCR.The results showed that the expression level of LV-oe-lncRNA n341773 was significantly increased.While expression level of LV-RNAi-lncRNA n341773 was significantly decreased.The results showed that the lncRNA n341773 expression level have no significant differences in the group of Control ? LV-oe-NC and LV-RNAi-NC.LV-oe-lncRNAn341773 could inhibit the reduction of lncRNA n341773 by TGF-?1.LV-RNAi-lncRNA n341773 could promote the reduction of lncRNA n341773 by TGF-?1.3.The results of Western Blot showed that the expression level of ?-SMA and Collagen?protein was significantly increased when HELF cells were treated with TGF-?1.LV-oe-lncRNAn341773 could inhibit the up-regulation effect of ?-SMA and Collagen?protein by TGF-?1,while LV-RNAi-lncRNA n341773 could promote the up-regulation effect of ?-SMA and Collagen ? protein by TGF-?1,which indicated that lncRNA n341773 played an important role in the phenotypic transformation of HELF cells.4.The results of Western Blot showed that the expression level of P-Akt(S473)?P-Akt(T308)?P-mTOR(S2448)protein was significantly increased,while there was no significant change in the expression of PI3K?Akt and mTOR when HELF cells were treated with TGF-?1,which indicated that in the TGF-? 1-induced phenotypic transformation PI3K/Akt/mTOR pathway was activated.LV-oe-lncRNAn341773 could inhibit the effect of TGF-? 1 on phenotypic transformation and PI3K/Akt/mTOR pathway,while LV-RNAi-lncRNA n341773 could promote the effect of TGF-?1.The results of EdU tests : the capacity of cell proliferation was significantly increased when HELF cells were treated with TGF-?1.LV-oe-lncRNAn341773 could inhibit the proliferation effect of TGF-?1,while LV-RNAi-lncRNA n341773 could promote the proliferation effect of TGF-?1.Theresults of Western Blot showed that the expression level of PCNA protein was significantly increased when HELF cells were treated with TGF-?1.LV-oe-lncRNAn341773 could inhibit the up-regulation effect of PCNA by TGF-?1,while LV-RNAi-lncRNA n341773 could promote the up-regulation effect of PCNA by TGF-?1,The expression of PCNA protein was consistent with the results of EdU tests.The results of Cell cycle test : the proportion of cells in G1/G0 phas was significantly decreased and cells in S phase ? G2/M phase were significantly increased when HELF cells were treated with TGF-?1.LV-oe-lncRNAn341773 could inhibit the up-regulation effect cell proportion by TGF-?1,while LV-RNAi-lncRNA n341773 could promote the up-regulation effect cell proportionby TGF-?1.The results of Western Blot showed that the expression level of Cyclin E protein was significantly increased and Cyclin B protein was significantly decreased when HELF cells were treated with TGF-?1.LV-oe-lncRNAn341773 could inhibit the up-regulation effect of Cyclin E protein and down-regulation effect of Cyclin B by TGF-?1,while LV-RNAi-lncRNA n341773 could promote the up-regulation effect of Cyclin E protein and down-regulation effect of Cyclin B by TGF-?1,Which was consistent with the results of Cell cycle tests.All of these indicated that lncRNA n341773 played an important role in the phenotypic transformation of HELF cells by regulating PI3K/Akt/mTOR signaling pathway and cell proliferation,Research conclusion:1.The expression of lncRNA n341773 was decreased in TGF-?1 treated HELF cells.2.LV-oe-lncRNAn341773 could inhibit the phenotypic transformation by TGF-?1,while LV-RNAi-lncRNA n341773 could promote the phenotypic transformation by TGF-?1.3.LncRNA n341773 played an important role in the phenotypic transformation of HELF cells by regulating PI3K/Akt/mTOR signaling pathway and cell proliferation.