Research On The Mechanism Of AMMECR1 In A549 Cellline And The Effect Of "Fei Xiao Liu San" On AMMECR1

Objective:To verify whether AMMECR1 is associated with non-small cell lung cancer(NSCLC),to explore the mechanism of AMMECR1 in NSCLC,to clarify whether "FeiXiaoliu San" can inhibit NSCLC cell proliferation,and to clarify whether "FeiXiaoliu San",is related to AMMECR1.Methods:1.Real-time quantitative PCR(qPCR)was used to detect the mRNA expression of AMMECR1 in different tool cells,and A549 cells were selected.2.Using AMMECR1 as a template,the short hairpin RNA(shRNA)interference target sequence was designed to construct AMMECR1 RNA interference(RNAi)lentiviral vector.ShRNA lentivirus was cloned,packaged lentivirus,and infected with A549 by shRNA lentivirus.3.Infected cells were divided into two groups:normal A549 cells with negative control virus infection(shCtrl)and normal A549 cells with AMMECRI gene shRNA lentivirus infection(shAMMECR1).The infected A549 cells were observed under fluorescence microscope 72 hours after infection.If the observation results showed that the infection efficiency of the cells reached more than 80%,it indicated that the cells were in normal state and could be further tested.4.The expression of AMMECR1 mRNA in A549 cells after AMMECR1 gene knockdown was detected by qPCR,so as to determine the interference effect of shRNA lentiviral vector on AMMECR1 gene.5.Three days after Lentiviral infection,the number of cells in each group was recorded by Celigo assay,and the relationship between AMMECR1 gene and A549 cell proliferation was analyzed.The viability of the two groups was detected by MTT,and the effect of AMMECR1 gene on cell proliferation was verified.Flow cytometry was used to detect the number of apoptotic cells in both groups,and to examine the association between AMMECR1 gene and apoptosis.The content of DNA in the two groups was detected to verify the effect of AMMECR1 gene on cell growth cycle.6.Five days after Lentiviral infection,the cloning ability of the two groups of cells on the cell culture plate was detected,suggesting the relationship between AMMECR1 gene and the tumorigenicity of the cells.7.Western Blot method was used to detect the expression of AMMECR1 gene in shCtrl group(normal 293T cells with negative control virus infection group)and shAMMECR1 group(normal 293T cells with AMMECR1 gene shRNA virus infection group).8.A549 cells infected with shRNA lentiviruses were detected by 3'rVT Plus KIT gene chip(RIN?7)and concentration?73 ng/ul),and the genes were screened by IPA analysis.9.Verify the downstream genes obtained by IPA analysis with qPCR.10.Combining with the reading value of OD490,the result of inhibition rate,the mean of inhibition rate and standard deviation,the suitable concentration of Feixiaoliusan was selected.The expression of AMMECR1 was detected by qPCR.Results:1.After shRNA lentivirus infection,the expression of AMMECR1 gene in shAMMECRl group was significantly inhibited.2.Celigo assay showed that the proliferation rate of shAMMECRl group was significantly inhibited,suggesting that AMMECR1 gene was significantly related to the proliferation of A549 cells.3.MTT assay showed that the proliferation rate of shAMMECR1 group was significantly inhibited,suggesting that AMMECR1 gene was significantly related to the proliferation of A549 cells.4.The percentage of cells in S phase in shAMMECRl group was significantly lower than that in shCtrl group,the percentage of cells in G1 phase was significantly higher than that in shCtrl group,and the percentage of cells in G2/M phase was significantly lower than that in shCtrl group,suggesting that AMMECR1 gene was significantly related to A549 cell cycle.5.The number of apoptotic A549 cells in shAMMECRl group was significantly increased,indicating that AMMECR1 gene was significantly associated with the apoptosis of A549 cells.6.The number of A549 cells in shAMMECRl group was significantly decreased,suggesting that AMMECR1 gene was significantly associated with the cloning and forming ability of A549 cells,7.The results of Western Blot showed that shRNA interference target significantly knocked down the exogenous expression of AMMECR1 gene at protein level in 293T cells.8.the results of classical pathway analysis based on IPA suggest that AMMECR1 significantly activates cholesterol synthesis pathway.9.The upstream regulatory analysis based on IPA indicated that TP53 was predicted to be strongly activated.There were 168 uniformly activated genes in TP53,and MYC was predicted to be strongly inhibited.There were 101 uniformly inhibited genes in TP53.10.In-depth analysis showed that AMMECR1 may affect the expression of a series of downstream genes by acting on BIRC5 and RELA,thereby affecting cell proliferation and apoptosis.QPCR verified this result.11.with the increase of the concentration of "Fei Xiao Liu San",the inhibition rate of A549 cells gradually increased.12.we selected 1 g/ml,0.5 g/ml concentration of drug addition cells.The expression abundance of AMMECR1 gene in 0.5g/ml group was 0.984 times higher than that in control group(p>0.05),and that in lg/ml group was 2.511 times higher than that in control group(p<0.05).Conclusion:1.After shRNA lentivirus infection,AMMECRI gene was significantly inhibited.2.AMMECR1 gene promotes the proliferation of A549 cells,promotes the formation of A549 cell clones,and inhibits the apoptosis of A549 cells.3.AMMECR1 was significantly correlated with A549 cell cycle.4.AMMECR1 significantly activated the cholesterol synthesis pathway.5.AMMECR1 may affect the expression of a series of downstream genes by acting on BIRC5 and RELA,thereby affecting cell proliferation and apoptosis.6.In vitro,"lung Xiao Liu San" inhibited the growth of A549 cells.7."Lung Xiao Liu San" can not inhibit the expression of AMMECR1 gene in A549 cells.