| Objective:Lung cancer is the leading cause of cancer deaths worldwide and the disease with the highest morbidity and mortality in malignant tumors in China.With the continuous emergence of new drugs such as targeted therapy and immunotherapy,the survival of lung cancer has been prolonged to a certain extent in recent years,but the overall prognosis is still unsatisfactory.After years of clinical practice,combining with the core pathogenesis of"phlegm,blood stasis,poison and deficiency",our cancer center created Yiqi Chutan formula(YQCTF)for the treatment of lung cancer.It can prolong the survival of patients with middle stage and advanced stage lung cancer,while maintaining a better quality of life,verified by the national science and technology plan of "Tenth Five-Year Plan" and"Eleventh Five-Year plan".Previous studies have proven that YQCTF could inhibit angiogenesis in lung cancer and has synergistic effects with targeted therapy and immunotherapy,but the molecular mechanism is still unclear.Our study intend to explore the mechanism of YQCTF in inhibiting angiogenesis and regulating tumor immune microenvironment in lung cancer,to explore the possible targets and lay a foundation for the further development.Methods:Part 1:(1)The RNA sequencing,clinical tests and other relevant data of 169 East Asian patients with lung adenocarcinoma were obtained from the international public database,and the data were analyzed using cBioPortal and other platforms.(2)DNA repair related genes were collected through Reactome database to construct gene sets.According to the expression of different genes,the genes with similar expression trend were clustered.(3)Hierarchical clustering analysis were performed on 302 DNA repair genes in each sample,and samples with similar gene expression patterns were classified.(4)The expression of DNA repair related genes among different groups were analyzed by Gene Cluster 3.0 software.(5)The Java Treeview program was used to generate heat maps of the cluster and tumor staging patterns.(6)Protein-protein interaction(PPI)was used to analyze protein interactions.(7)Gene Ontology(GO)was used to analyze the biological process,molecular function,localization and other related information of differential genes.(8)Genes enriched signal pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes(KEGG).(9)GraphPad Prism 8 was used to draw survival curves and compare overall survival to evaluate the relationship between DNA repair genes and the survival of lung adenocarcinoma patients.(10)The overall data of the study were statistically analyzed.Part 2:(1)The human umbilical vein endothelial cell(HUVEC),human lung adenocarcinoma cell A549.human large cell lung cancer NCI-H460 and human small cell lung cancer NCI-H446 were cultured and subcultured in vitro.(2)The A549 cells were divided into four groups:control group,YQCTF group,anlotinib group and cisplatin group.The proliferation of A549 cells in each group was detected by MTT,and the cell growth inhibition rate(IR)was calculated.(3)The apoptosis of A549 cells in each group was detected by flow cytometry.(4)The migration and invasion ability of cells in each group were detected by Transwell.(5)The expressions of tumor immune microenvironment related proteins such as FANCA,BRAC2,PMS2 and XRCC5 were detected by Western blot.(6)The mRNA expressions of FANCA and BRAC2 of each group were detected by fluorescence quantitative PCR.(7)A549,NCI-H460 and NCI-H446 cells in logarithmic growth phase were inoculated subcutaneously into BALB/C mice to construct three kinds of xenograft tumor models.(8)The inoculated mice were randomly divided into 3 groups.The treatment of each group were as follows:Group A was the control group,and normal saline was given by gavage once a day;Group B was YQCTF group,which was given YiqiChutan Formula by gavage once a day at a dose of 3g/kg/d;Group C was anlotinib group,which was given anlotinib by gavage once a day at a dose of 0.8mg/kg/d.All groups were treated for 14 days,and the general conditions of mice in each group were observed daily,such as mental state,diet,activity,defecation,etc.Tumor growth inhibition(TGI)was calculated by measuring tumor volume(V=a×b2/2)in mice every 3 days.TGI=(1-average tumor volume in experimental group/average tumor volume in control group)×100%.On the 15th day,the animals were sacrificed and the tumors were dissected for relevant experiments.(9)T cells,CD4+T cells,CD8+T cells,CD4+/CD8+ratio,NK cells,Ml macrophages,M2 macrophages,M1/M2 ratio and other indicators were detected by flow cytometry of each group.(10)The expressions of proteins such as FANCA,FANCD2(L),FANCD2(S)and HES1 were detected by Western blot.(11)The expression of CD31 was detected by immunohistochemistry and micro vascular density(MVD)were calculated.(12)CD31 expression in each group was detected by immunofluorescence.(13)The expressions of angiogenesis related proteins such as VEGFA,HIF-1α,D114 and Notch-1 were detected by Western blot.(14)The mRNA expression of angiogenesis were detected by fluorescence quantitative PCR.(15)The expression of Notch-1 were detected by immunofluorescence.(16)The expression of Hes-1 were detected by immunofluorescence.(17)Hes1-siRNA was transfected into A549 cells,the migration and invasion ability of cells in each group were detected by Transwell.(18)The proliferation ability of each group was detected by plate cloning.(19)The expression of Hes-1 and Notch-1 in cancer tissues and adjacent tissues of lung adenocarcinoma patients was detected by immunohistochemistry.Results:Part 1:(1)After hierarchical clustering,there are two clusters(Cluster1,Cluster2)and four subclusters(Subcluster 1,Subcluster2,Subcluster3,Subcluster4).There was no significant difference in the prognosis between the two clusters.Among the four subclusters,Subcluster 1(SCI)had the best prognosis,while Subcluster 2(SC2)had the worst prognosis.Compared with the clinical data of the four subclusters,the proportion of smoking patients in SC4 was the lowest,and the proportion of smoking patients in SC1 was the highest.The proportion of moderately differentiated or poorly differentiated SC3 tumors was the highest.The pathological types of mixed subtype adenocarcinoma,papillary adenocarcinoma and alveolar adenocarcinoma showed significant differences among the groups.(2)We selected the group SC2(the worst prognosis group)and SC3(the worst pathological differentiation group)to compare with other patients,and we found 149 differentially expressed genes in SC2 compared with other subclusters,and 213 differentially expressed genes in SC3,107 genes overlaped between the two groups.(3)Through PPI analysis,we found that these differentially expressed genes were functionally related to each other,including XRCC5,BRCA2,FANCA,PMS2,POLE,POLD,etc.(4)Gene Ontology(GO)was used to enrich the function of 107 overlapping genes.Go-biological progress analysis showed that these genes were mainly involved in DNA repair and DNA metabolism,and Go-molecule function analysis showed that these genes were mainly involved in catalytic activity.Go-cell component analysis showed that these genes were mainly distributed in the cytoplasm.(5)KEGG signaling pathway analysis showed that these genes were mainly enriched in Fanconi anemia pathway and homologous recombination repair(HRR)pathway and nucleotide excision repair(NER)pathway.(6)Comparing the immune function related indicators of the two clusters,there were significant differences in interferon,macrophages,monocytes,neutrophils,natural killer cells,total number of T cells and protein translation index.The number of immune cells such as T cells and natural killer cells in Clusterl was higher than that in Cluster2.Comparing the immune function related indicators of each subcluster,we found that tumor purity,interferon,macrophages,monocytes,neutrophils,natural killer cells,total number of T cells,protein translation index and cell proliferation index were significantly different.The number of macrophages,monocytes,neutrophils and natural killer cells in the best prognosis group(SCI)was the highest among the four groups.In the worst prognosis group(SC2),cell proliferation index was significantly increased,protein translation index was decreased,and interferon level was increased.In addition,tumor purity was the lowest,but there was no statistical difference.In the worst differentiated group(SC3),the cell proliferation index increased,the protein translation index and the total number of neutrophils decreased.Part 2:(1)The results of MTT showed that compared with the control group,YQCTF group had obvious inhibitory effect on A549 cell proliferation.the growth inhibition rates(IR)of 24 hours,48 hours and 72 hours after intervention were respectively(13.78±2.61)%,(39.55±4.13)%,(49.07±4.30)%(P<0.01).(2)Theresults of flow cytometry showed that the apoptosis rate of YQCTF group was(22.76± 1.61)%,which was significantly higher than that of anrotinib group and control group(P<0.01),and there was no statistical difference between YQCTF group and cisplatin group.(3)The results of Transwell showed that the number of migrating cells and invading cells in YQCTF group was 103.2±9.28 and 121.4±12.81,which were significantly lower than those in the control group(P<0.01).Compared with the control group,the number of migrating cells(92.4±8.62)and invading cells(104.2±10.16)in the anlotinib group were also significantly decreased(P<0.01),but there was no significant difference between the anlotinib group and the YQCTF group.(4)The results of western blot showed that the protein expressions of tumor immune microenvironment related proteins such as FANCA,BRAC2,PMS2 and XRCC5 were significantly lower than those in control group and anlotinib group(P<0.05),especially in FANCA protein expression(P<0.01).The protein expressions of FANCA,BRAC2,PMS2 and XRCC5 were significantly up-regulated in cisplatin group(P<0.01).There was no significant difference in the expression of the above proteins between the anlotinib group and the control group.(5)The results of quantitative PCR showed that the amplification curve of each index was good,and the primer peak of the melting curve was single.Compared with the control group,the mRNA expressions of FANCA and BRAC2 in YQCTF group were significantly down-regulated(P<0.01),while those of FANCA and BRAC2 were upregulated in cisplatin group(P<0.05).There was no statistical difference between anlotinib group and control group.(6)Three lung cancer xenograft models(A549,NCI-H460,NCIH446)in mice were successfully constructed and grouped according to the experimental requirements.(7)There was no significant difference in the tumor volume of A549 mice at baseline.From the 6th day,the volume of transplanted tumor in YQCTF group and anlotinib group was significantly smaller than that in control group,and it was more obvious with the prolongation of time.On the 15th day,the trmor volume of YQCTF group was 202.12±27.63mm3,and the tumor growth inhibition rate(TGI)was 73.51%.In anlotinib group,the tumor volume was 227.25±42.13mm3 and TGI was 70.21%.The analysis of variance(ANOVA)showed that the tumor volume of YQCTF group and anlotinib group was significantly smaller than that of the control group(P<0.001).The tumor volume of YQCTF group was slightly lower than that of anlotinib group,but there was no significant difference between the two groups.(8)There was no significant difference in tumor volume of NCIH460 mice at baseline among the three groups.The tumor volume of the anlotinib group was significantly smaller than that of the control group and YQCTF group from the 6th day,and the inhibitory effect was more obvious with the extension of time.On the 15th day,the tumor volume was 442.12±30.61mm3.and the TGI was 77.64%.From the 9th day,the average tumor volume of YQCTF group was smaller than that of the control group.On the 15th day,the tumor volume of YQCTF group was 616.12±31.42 mm3,and TGI was 68.83%.(9)There was no significant difference in the volume of NCI-H446 mice at baseline.Compared with the control group,the tumor volume of YQCTF group was significantly reduced from the 9th day.At the 15th day.the tumor volume of YQCTF group was 571.3±28.32mm3,TGI was 30.51%.The anlotinib group showed the most obvious inhibitory effect on NCI-H446 xenografts in mice,and the tumor volume was significantly smaller than that of control group and YQCTF group.On the 15th day.the tumor volume was 217.75±22.48 mm3,and the TGI was 73.52%.(10)The total number of T cells,CD4+T cells,CD4+/CD8+and NK cells in YQCTF group were significantly higher than those in control group and anlotinib group(P<0.05).Compared with YQCTF group and anlotinib group,CD8+T cells in control group were significantly increased,CD4+/CD8+ratio was decreased,and the total number of NK cells was decreased(P<0.05).Compared with the control group and anlotinib group,the proportion of M1-type macrophages in YQCTF group was significantly increased.and the ratio of M1/M2 was increased(P<0.01).(11)Compared with the control group and anlotinib group,the protein expression of FANCA and HES1 and the monoubiquitination level of FANCD2(FANCD2 L/S)in YQCTF group were significantly down-regulated(P<0.01).There was no significant difference in the expression of the above proteins between anlotinib group and the control group.(12)The expression of CD31 in YQCTF group and anlotinib group was significantly lower than that in control group(P<0.01).Micro vascular density(MVD)in YQCTF group was 7.4 ± 1.82,which was significantly decreased compared with the control group(P<0.01).(13)The immunofluorescence expression of CD31 in YQCTF group and anlotinib group was significantly lower than that in control group,and the CD31 expression in YQCTF group was the lowest(P<0.05).(14)The results of western blot of A549 cells showed that the protein expressions of Notch-1 and DLL4 in YQCTF group were significantly lower than those in control group and anlotinib group.The expression of VEGF-A in anlotinib group was lower than that in YQCTF group and control group.The expression of HIF-1α protein in YQCTF group and anlotinib group was lower than that in control group,but there was no significant difference between the two groups.(15)The results of western blot of NCI-H460 cells showed that the protein expressions of DLL-4 and Notch-1 in YQCTF group were significantly lower than those in control group and anlotinib group.The expression of VEGFA in YQCTF group and anlotinib group was significantly lower than that in control group.but there was no significant difference between the two groups.The expression of HIF-1α in YQCTF group and anlotinib group was significantly lower than that in control group,and the anlotinib group showed the lowest expression.(16)The results of western blot of NCI-H446 cells showed that the expression of VEGF-A in YQCTF group and anlotinib group was lower than that in control group,and the expression of VEGF-A in anlotinib group was the lowest.The expressions of DLL-4.Notch-1 and HIF-1α in YQCTF group and anlotinib group were lower than those in the control group,but there was no significant difference in the expressions of these proteins between the two groups.(17)Compared with the control group,the Notch-1 mRNA expression of transplanted tumor in mice of A549.NCI-H460 and NCIH446 in YQCTF group was significantly down-regulated(P<0.01).The Notch-1 mRNA expression in A549 and NCI-H460 tumor mice in YQCTF group was lower than that in anlotinib group(P<0.01),but there was no statistical difference in Notch-1 expression in NCI-H446 mice between the two groups.(18)The results of immunofluorescence showed that Notch-1 expression was significantly lower in YQCTF group and anlotinib group than in control group,and Notch-1 expression was most significantly down-regulated in YQCTF group(P<0.05).(19)Immunofluorescence results showed that the expression of Hes1 in YQCTF group was significantly lower than that in control group and anlotinib group,and the difference was statistically significant(P<0.05).(20)The protein expression of HES1 in the siRNA transfection group was significantly lower than that in the blank control group and the negative control group(P<0.01),and there was no statistical difference between the blank control group and the negative control group,suggesting that HES1-siRNA transfection was effective.Compared with the blank control group and the negative control group,YQCTF group could also significantly down-regulate the expression of HES1 protein,and the expression of HES1 protein in YQCTF+HES1-siRNA group was the lowest(P<0.05).(21)Compared with blank control group and negative control group,HES1-siRNA group,YQCTF group and YQCTF+HES1-siRNA group could significantly reduce the number of migrating cells and invading cells(P<0.01).In addition,the number of migrating cells and invading cells in YQCTF+HES1-siRNA group was the least in each group(P<0.05).(22)The results of plate cloning experiment showed that there was no statistical difference between blank control group and negative control group.Compared with blank control group,HES1-siRNA group,YQCTF group and YQCTF+HES1-siRNA group could significantly reduce the number of clones and the rate of clone formation(P<0.01).Compared with the other 4 groups,the clonal formation rate of YQCTF+HES1-siRNA group was the lowest(P<0.01).(23)The results of immunohistochemistry showed that both Hes-1 and Notch-1 were highly expressed in cancer tissues of lung adenocarcinoma patients,but were low expressed in paracancer tissues.The expressions of HES-1 and Notch-1 in lung adenocarcinoma patients treated with YQCTF were significantly down-regulated compared with the control group(P<0.01).Conclusion:1.DNA repair genes have certain correlation with the prognosis and immune microenvironment of lung adenocarcinoma patients.The genes with high correlation include FANCA,BRCA2,XRCC5,PMS2.POLE and POLD.At the same time,DNA repair genes are closely related to tumor purity,T cells,NK cells,macrophages,interferon and other indicators.Some DNA repair gene and related signaling pathways can increase the number of immune positive cells such as T cells,NK cells and macrophages,which is expected to improve the efficacy of lung cancer immunotherapy.2.YiqiChutan Formula could inhibit the proliferation of lung cancer cells,induce apoptosis,and inhibit cell migration and invasion.YiqiChutan Formula can inhibit the growth of three types of lung cancer(A549,NCI-H460,NCI-H446)xenografts in mice.Its inhibitory effect on A549 cells was the strongest and slightly better than anlotinib.YQCTF can increase the total number of T cells,CD4+T cells and NK cells,CD4+/CD8+ratio and macrophage M1/M2 ratio of lung cancer xenografts,also can down-regulate the expression of FANCA and Hes1 and the monoubiquitination level of FANCD2,suggesting that it can regulate the tumor immune microenvironment and improve the immunosuppressor state,the mechanism may be related to the inhibition of FANCA/Hesl signaling pathway.YQCTF could significantly reduce the microvascular density(MVD)of lung cancer,significantly down-regulate the protein,mRNA and immunofluorescence expressions of Notch-1 and Hes1.These reslts show that the mechanism of inhibiting angiogenesis is mainly related to D114/Notch1/Hes1 signaling pathway,especially the down-regulation of Notch-1 and Hes-1 expression. |
PDF Full Text Request