The Mechanism Research Of New Houttuyfonate Sodium Against Non-small Cell Lung Cancer

BackgroundLung cancer ranks first standard in cancer incidence and mortality in China,which is comparable to that in developed countries.Among them,non-small-cell lung cancer(NSCLC),as the main type of lung cancer,accounts for 80%to 85%of the total incidence of lung cancer,and its 5-year survival rate is less than 15%.As a treatment method with multi-component.multi-target and multi-channel advantages,TCM has a good effect on restricting the proliferation of NSCLC patients,improving their clinical symptoms,preventing recurrence and improving their quality of life.However,due to the lack of basic research and the differences between the theoretical system of traditional Chinese medicine and Western medicine,a large number of empirical formulas of traditional Chinese medicine lack the support of modern medical evidence.Houttuynia cordata Thunb.is a dry aboveground part and fresh whole grass of Houttuynia cordata Thunb.It has many functions,such as clearing heat and detoxification,diuresis,hemostasis,relieving coughs and analgesia,etc.It is commonly used in the folk for pulmonary carbuncle,vomiting pus or phlegm,heat dysentery,heat gonorrhea,carbuncle and sore.In an ancient formula of traditional Chinese medicine,Houttuynia cordata Thunb.was even used to treat pulmonary carbuncle(a disease similar to modern medical lung cancer)alone.Zhou Zhongying,a State Medical Master in China,has also been used in his formula of "Kejinyan"for the treatment of lung cancer.There are many studies on the extracts of Houttuynia cordata Thunb.and its research directions are also diverse.In this study,we focused on the mechanism of Sodium New Houttuyfonate(SNH)on the mechanism of anti-NSCLC.Endogenous competitive RNA(ceRNA)theory is one of the most popular research methods in modern medical research.In this study.ObjectiveWe tried to explore the mechanism of SNH acting on NSCLC from the perspective of the combination of Chinese and western medicine(especially on CeRNA).MethodIn this study,the cytotoxicity of SNH on NSCLC cells was investigated by CCK8 assay,and the effect of SNH on the proliferation of NSCLC cells was explored by clone formation assay.HOCHEST33258 nuclear staining and Annexin V/PI flow cytometry confirmed the effects of SNH on apoptosis of NSCLC cells.Western Blot was used to explore apoptosis-related signaling pathways.Secondly,the effects of SNH on the migration and invasion ability of NSCLC cells were explored by cell scratch test and Transwell invasion test.QRT-PCR,Western Blot and immunofluorescence test identified the mechanisms of migration and invasion.Then,the most relevant signaling pathways were analyzed by transcriptome high-throughput sequencing,and the most possible mode of action and target gene was identified as Linc00668.After clarifying the relationship between Linc00668 and cancer in bioinformatics,we constructed its overexpression and interference model and clarified its mechanism.Linc00668 competitive target gene and intermediate microRNAs were analyzed by bioinformatics,and the effects of microRNAs and target genes on signaling pathways were identified.Finally,competitive binding sites were identified by double luciferase reporter analysis.In vivo model was used to evaluate and validate the signaling pathways of SNH treating NSCLC found in vitro experiments by the methods of bioluminescence,H&E staining,immunofluorescence and immunohistochemistry.ResultThis study first confirmed that SNH can promote apoptosis,inhibit proliferation,invasion and migration of NSCLC cells,and its apoptotic mechanism is achieved by shearing Caspase 3 and PARP proteins,while its mechanism of inhibiting cell invasion and migration is to inhibit the process of EMT.Then,transcriptome sequencing revealed that SNH might inhibit the EMT process of NSCLC cells by regulating Linc00668.And the role of Linc00668 could promote the migration/invasion ability of NSCLC cells was confirmed by overexpression and interference Linc00668.It was confirmed that Linc00668 regulates the EMT process by competing with slug’s mRNA in the 3’UTR binding to miR-147a.Finally,it was confirmed with animal model that SNH inhibits metastasis of NSCLC through Linc00668/microRN A-147a/slug axis.ConclusionIn this study,we found that SNH can inhibit proliferation,promote apoptosis,inhibit migration/invasion in NSCLC.We further studied that SNH may regulate EMT signaling pathway through the Linc0668/miR-147a/slug axis.In this study,the mechanism of anti-NSCLC action of SNH is explained through the current research focus ceRNA theory,which is an innovative step in the theoretical research of traditional Chinese medicine and provides a new way for the basic research of traditional Chinese medicine.