Based On S100A9 And CyPA To Explore The Regulatory Mechanism Of Fuzheng Zengxiao Recipe On Radiosensitization In Lung Adenocarcinoma

In recent years,the incidence and mortality of non-small cell lung cancer in China have increased significantly,and lung adenocarcinoma is a very common subtype.About 70%of patients with primary lung cancer need radiotherapy.Radiosensitizers not only greatly increase the sensitivity of tumor tissue to radiation,but also significantly improve the degree of local control.At the same time,it can also reduce the radiation dose and improve the quality of life of patients.Therefore,it is of far-reaching significance to find a radiosensitizer.In recent years,the combination of targeting and genetic drugs and radiation,through signal transmission,cell cycle regulation,oncogene conversion factors,apoptosis,angiogenesis factors and other ways to improve radiation efficacy,is the current research hotspot.Traditional Chinese medicine has also become the focus of radiosensitizer research because of its high efficiency.Fuzhengzengxiao decoction is indicated for the pattern of toxic heat impairing qi and yin caused by radiotherapy and promoting blood circulation to remove blood stasis.It consists of Honeysuckle Flower,Straight Ladybell Root,Curcumae Rhizome,Chinese Wolfberry Fruit,Raw Astragalus Root,Sappan Wood,Safflower.Previous clinical studies have found that Fuzhengzengxiao decoction can improve the short-term efficacy of radiotherapy for non-small cell lung cancer,reduce the side effects of chemotherapy and increase survival rate.The preliminary study confirmed that Fuzhengzengxiao decoction can reduce the expression level of CyPA and S100A9 in lung adenocarcinoma cells.We used siRNA to initially confirm that S100A9 and CyPA are associated with lung cancer radiosensitivity.In order to in-depth study and clarify the radiosensitization effect and mechanism of traditional Chinese medicine,the subject utilise CRISPR/Cas9 technology to prepare H1975 lung adenocarcinoma cell knock out S100A9 cell line,knock out CyPA cell line,knock out S 100A9-Cy PA cell line,and use lentiviral transfection overexpression technology to prepare CyPA overexpression,S100A9 overexpression,and S100A9-CyPA Overexpressing lung adenocarcinoma cell lines and verify its radiosensitivity,to clarify the relationship between S100A9 and CyPA.To clarify the regulation mechanism of Fuzhengzengxiao decoction on radiosensitivity of lung adenocarcinoma,and determine the predictor of radiosensitivity of lung adenocarcinoma.Purpose:1.To explore the synergy or independent relationship between S100A9 and CyPA in lung adenocarcinoma radiosensitization,and find out the main targets.2.To explore the regulation mechanism of Fuzhengzengxiao decoction on the radiosensitization of S100A9 and CyPA in lung adenocarcinomaMethods:1.Use CRISPR/Cas9 gene knockout technology and gene transfection technology to study the relationship between S100A9,CyPA,S100A9-CyPA non-expression and overexpression and radiotherapy sensitivity.The human lung adenocarcinoma cell line H1975 with high expression of S100A9 and CyPA was selected,and the S100A9,CyPA,and S100A9-CyPA genes were knocked out through CRISPR/Cas9 technology,and then S100A9,CyPA,and S100A9-CyPA were overexpressed through gene transfection technology.The relationship between S100A9,CyPA and radiosensitivity of lung adenocarcinoma was observed through in vivo and in vitro radiobiological experiments.After comparing the over-expression and non-expression of the above-mentioned genes/proteins,the sensitivity of the two cells to radiation can be judged to determine whether the above-mentioned genes/proteins really play a significant role in radiosensitization,and to determine their synergistic or independent relationship to find the main gene.2.Preparation of Chinese medicine extract of Fuzhengzengxiao decoction.By observing the external radiation experiment of Fuzhengzengxiao decoction on lung adenocarcinoma without knocking out related genes and knocking out related genes,we can determine the radiosensitization target ofFuzhengzengxiao decoction on lung adenocarcinoma cells and find the main targets.Results:1.Successfully established lung adenocarcinoma H1975 cells S100A9 gene knockout.CyPA gene knockout,S100A9-CyPA gene knockout,S100A9 gene overexpression,CyPA gene overexpression,S100A9-CyPA gene overexpression cell lines2.CyPA gene knockout cell lines have significantly enhanced radiosensitivity in in vitro experiments.CyPA gene overexpression cell lines have radioresistance,and CyPA expression has a linear relationship with H1975 radiosensitivity.Both S100A9 gene knockout and overexpression showed radioresistance.S100A9-CyPA gene knockout cell lines showed no significant increase in radiosensitivity,but S100A9-CyPA gene overexpression cell lines had significantly enhanced radioresistance.3.CyPA gene knockout group significantly increased the tumor inhibition rate in in vivo experiments,and can promote cell apoptosis.CyPA gene knockout may increase radiosensitivity through G2/M phase block.The S100A9 gene and S100A9-CyPA gene can affect the tumor inhibition rate but not the apoptosis after radiation.4.Fuzhengzengxiao decoction can increase the radiosensitivity of wild-type and S100A9 gene knockout and S100A9-CyPA gene knockout cell lines,and can induce cell cycle G2/M phase arrest,and promote DNA damage and apoptosis.CyPA gene is the main target of Fuzhengzengxiao decoction.Conclusion:1.CyPA gene expression is related to cell radiation tolerance.Decreasing CyPA gene expression can significantly improve the radiosensitivity of lung adenocarcinoma cells,which can be used as a predictive gene of radiosensitivity.S100A9 did not show a modulating effect on the radiosensitivity of lung adenocarcinoma in this experiment.CyPA and S100A9 genes have no synergistic effect of radiation in H1975 cells.2.Fuzhengzengxiao decoction can inhibit cell proliferation and enhance cell radiosensitivity.CyPA gene is one of the main targets of Fuzhengzengxiao decoction to increase radiosensitivity.