| Background:Non-Small Cell Lung Cancer(NSCLC)is the most common type of lung cancer,accounting for about 85%of all lung cancers.The disease is insidious,and the early manifestations are not obvious or even asymptomatic.Domestic scholars have confirmed that cryoablation combined with traditional Chinese medicine has achieved good results in the treatment of a variety of solid tumors through a large number of clinical and experimental studies.Yangfei Decoction was developed by Professor XXX based on many years of clinical experience,with the therapeutic principle of invigorating qi,nourishyin,resolving phlegm and detoxifying.Cryoablation(Cry)combined with Yangfei decoction in the treatment of lung cancer can improve the quality of life of patients,effectively prolong the survival time and control postoperative complications,but its specific mechanism is still unclear,it’s worthy of further study.Objectives:1.To observe the therapeutic effect of cryoablation combined with Yangfei decoction on Lewis lung cancer xenografts.2.Based on the whole transcriptome sequencing,the differentially expressed genes in the treatment of Lewis lung cancer with Cry combined with Yangfei Decoction were screened,and the targets and pathways were searched by bioinformatics to explore the molecular mechanism of cryoablation combined with Yangfei decoction in the treatment of lung cancer,so as to provide new ideas for the combined treatment of NSCLC with traditional Chinese medicine.In order to provide a scientific basis for the rational and effective use of Cry combined with Yangfei recipe in clinical practice.Methods:1.The effect of cryoablation combined with traditional Chinese medicine Yangfei decoction on Lewis lung cancer subcutaneous transplantation tumor.C57BL/6J mice were randomly divided into model group,Cry group,Cry+Low-dose YFF group,Cry+medium-dose YFF group,Cry+High-dose YFF group,Cry+DDP group(n=5).The model group was subjected to sham operation,and the other 5 groups were subjected to cryoablation,followed by corresponding intervention for 14 days.The general condition of the mice was observed,and the body weight of the mice and the size of the transplanted tumor were recorded.Two weeks later,the mice were sacrificed,and the tumor tissue,spleen,lung and thymus were taken and weighed.The tumor growth volume,tumor inhibition rate and organ coefficient of each group were calculated to evaluate the therapeutic effect.2.Experimental observation of cryoablation combined with traditional Chinese medicine Yangfei decoction on Lewis mice tumor pathological morphology and immunohistochemistry.HE staining was used to observe the pathomorphological changes of tumor tissues in each group.The expression of Ki-67 in tumor tissues was detected by immunohistochemistry(IHC).3.Whole transcriptome sequencing analysis of subcutaneous xenograft Lewis lung cancer treated with cryoablation combined with Yangfei decoction.Three groups of mouse tumor samples were selected from model group and three groups of cryoablation combined with high dose of Yangfei Decoction group for whole transcriptome sequencing.The differential expression data of non-coding RNA(ncRNA)such as lncRNA,miRNA,circRNA and miRNA were analyzed,and GO and KEGG enrichment analysis were performed.The pathways related to the mechanism of cryoablation combined with traditional Chinese medicine Yangfei decoction in the treatment of lung cancer were explored,and the related differentially expressed genes were screened,and some were randomly selected for qPCR verification.At the same time,according to the joint analysis of ncRNAs and mRNAs,the differential cerans were screened,and the ncRNAs-mRNAs relationship network that may be related to the anti-Lewis lung cancer mechanism of cryoablation combined with Yangfei Decoction was explored.Results:1.Effect of cryoablation combined with Yangfei Prescription on the growth of Lewis lung cancer subcutaneous graft tumor.The statistics of subcutaneous tumor graft volume of Lewis lung cancer mice showed that compared with the model group,the tumor volume of mice in the Cry+DDP group,Cry+Low-dose YFF group,Cry+medium-dose YFF group,Cry+High-dose YFF group decreased,with statistical significance(P<0.05).Analysis of tumor weight and tumor inhibitory rate showed that compared with model group,Cry+DDP,Cry+Low-dose YFF,Cry+medium-dose YFF,Cry+High-dose YFF groups could reduce tumor weight of mice,with tumor inhibitory rates of 78.40%,38.81%,69.14%and 77.15%,respectively.The anti-tumor effect of Cry+High-dose YFF group was similar to that of positive control group.Body weight and visceral coefficient showed no statistical significance among all groups(P>0.05).There was no significant difference in lung index among all groups.Compared with model group,Cry+DDP group,Cry+Low-dose YFF group,Cry+medium-dose YFF group,Cry+High-dose YFF group could increase spleen index and thymus index of mice(P<0.05).The effect of Cry+High-dose YFF group was better than that of Cry+DDP group in increasing spleen index and thymus index of mice(P<0.05).2.Experimental observation of cryoablation combined with traditional Chinese medicine Yangfei decoction on Lewis mice tumor pathological morphology and immunohistochemistry.HE staining showed that the tumor cells in the model group had a clear outline,a high degree of dense growth,large and dark nuclei,different sizes,and closely arranged,without obvious necrosis and apoptosis.Compared with the model group,the cryoablation group and its combination with different doses of Yangfei Decoction showed incomplete structure of tumor cells,irregular arrangement,varying degrees of degeneration and necrosis,and the Cry+High-dose YFF group and the Cry+DDP group showed large necrotic areas.The results of Ki-67 immunohistochemistry showed that compared with the model group,the Ki-67 index of each group was decreased(P<0.05).Compared with the cryoablation group,the Ki-67 index of the Cry+DDP group,Cry+medium-dose YFF group,the Cry+High-dose YFF group were significantly decreased(P<0.001).2.Whole transcriptome sequencing analysis of cryoablation combined with traditional Chinese medicine Yangfei decoction against Lewis lung cancer.In this study,the Cry+High-dose YFF had the most significant anti-tumor effect.Therefore,whole transcriptome sequencing was performed on tumor tissues of Lewis lung cancer mice in the model group and the Cry+High-dose YFF group,and the differential expression data of non-coding RNA(lncRNA,circRNA,miRNA)and mRNA were analyzed.Significantly differentially expressed genes were screened:1160 mrnas(835 up-regulated,325 down-regulated),155 mirnas(121 up-regulated,34 down-regulated),225 lncrnas(108 up-regulated,117 down-regulated),and 45 circrnas(25 up-regulated,20 down-regulated).The top cancer-related genes were selected for qPCR to verify their consistency with sequencing results.GO and KEGG enrichment analysis of differentially expressed genes were performed.Finally,the ceRNA network of ncrNA-mrnas was constructed,and four key candidate mRNAs were found to be related to the mechanism of cryoablation combined with Yangfei Decoction against Lewis lung cancer:Stau2,Zfp704,Inhbb and Flt1.Three mirnas were identified:mmu-miR-136-5p,mmu-miR-150-5p and mmu-miR-326-3p.Three lncrnas:ENSMUST00000152888,ENSMUST00000127010,ENSMUST00000191456.Two circrnas were identified:circ＿001180 and circ＿000413.cryoablation combined raises lung party play a role of cancer may be related to the miRNA-136-5 p/Zfp704/ENSMUST00000191456 regulation there is a close relationship.Conclusions:1.Cryoablation combined with Yangfei Decoction can inhibit the growth of Lewis lung cancer subcutaneous transplantation tumor,and it is positively correlated with the dose of Yangfei Decoction.2.Cryoablation combined with Yangfei Decoction can promote the degeneration and necrosis of Lewis lung cancer cells in mice,reduce the Ki-67 index of tumor tissue,and inhibit tumor proliferation.3.4 mRNA,3 miRNA,3 lncRNA and 2 circRNA were the key candidate genes in the mechanism of cryoablation combined with traditional Chinese medicine Yangfei decoction in anti-Lewis lung cancer.cryoablation combined with Yangfei Decoction may down-regulate Zfp704 to inhibit the proliferation of lung cancer through ENSMUST00000191456 competition for miR-136-5p,and play a role in anti-Lewis lung cancer. |
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