Study On The Regulatory Mechanism Of Aqueous Extract Of Fritillariae Cirrhosae On STAT Signaling Pathway In Non-small Cell Lung Cancer A549 Cells

Lung cancer is one of the fastest growing malignancies in morbidity and mortality,which seriously threatens human health.Drug therapy based on traditional Chinese medicine treatment is a major feature in the field of cancer treatment in China.As a traditional Chinese medicine for cough and phlegm,Fritillaria cirrhosa has been found in recent years that its extract has anti-cancer effect.There are very few studies on non-small cell lung cancer,and its mechanism has not been clarified.Based on this,this study analyzed the antitumor effect of aqueous extract of F.cirrhosa(FC-AE)by in vitro and in vivo experiments,combined with transcriptome sequencing,using molecular biology or cell biology experimental methods such as RT-PCR,western blotting,ELISA et al.explored the anti-lung cancer effect molecular mechanism of FC-AE.The main findings are as follows:1.In vitro cell culture method to verify the anti-cancer effect of FC-AE: After characterized,thin-layer chromatography and PCR-RFLP method for biopharmaceutical identification of F.cirrhosa,FC-AE was prepared by water extraction and alcohol precipitation method.Based on the CCK-8 and plate cloning method,its anti-tumor activity was confirmed at the cell level.The cell cycle detection method found that FC-AE can cause A549 cell cycle arrest in the G2 / M phase,and apoptosis experiments found that FC-AE can induce A549 cell apoptosis.At the same time,western blotting showed that after FC-AE treatment,the expression of pro-apoptotic protein Bax increased,and the expression of anti-apoptotic protein Bcl-2 decreased,indicated that FC-AE may exert an inhibitory effect by inducing A549 cell apoptosis.2.Using transcriptome sequencing to explore the molecular mechanism of FC-AE inhibition in A549 cells: High-throughput sequencing technology was used to analyze the effect of FC-AE on the gene expression profile of A549 cells.Compared with untreated A549 cells,after FC-AE treatment,6983 differentially expressed genes were obtained(3528 up-regulated and 3455 down-regulated).The results of GO and KEGG enrichment analysis showed that most of the differentially expressed genes(DEGs)and immune response,apoptosis and cell migration are involved in cellular processes.The expression levels of eight DEGs related to immunity and apoptosis were identified by RT-PCR,and the expression of STAT1 and STAT4 was significantly up-regulated.STAT1 and STAT4 are members of the STAT signaling pathway.The STAT signaling pathway is closely related to the occurrence and development of non-small cell lung cancer,and is widely involved in biological processes such as cell growth,proliferation,migration,and apoptosis.Based on this,western blotting experiments also found that FC-AE can up-regulate the expression of STAT1,p-STAT1,STAT4,and p-STAT4 in A549 cells.It is speculated that FC-AE may exert its anti-tumor therapeutic effect by promoting the activation of STAT1 and STAT4.3.Evaluation of the in vivo effectiveness of FC-AE on A549 cell inhibition: First,nude mice xenograft model of A549 cells was constructed.The experimental nude mice were divided into a control group(peritoneal injection of normal saline)and a drug treatment group(peritoneal injection of 5 mg / kg,FC-AE).The experimental results show that FC-AE can inhibit the growth of transplanted tumors of A549 cells without causing adverse reactions in nude mice.Immunohistochemical results showed that protein expression of Bax increased and Bcl-2 expression decreased in tumor tissues.In addition,ELISA results also found that the expression levels of IL-12 and IFN? were significantly increased.This shows that STATs play an important role in regulating cytokine-dependent inflammation and immunity by upregulating the expression levels of target genes(IL-12,IFN?)of STAT1 and STAT4.