| At present,noncommunicable diseases are the leading cause of death in the global population,and cancer is expected to be the only disease in the 21st century that hinders human life expectancy.Lung cancer is occult,developing rapidly,and has a poor prognosis.The five-year survival rate in the United States is about 16%,and the United Kingdom is less than 10%.It has become one of the most deadly malignant tumors in the world.In China,the incidence(21.9%)and mortality(26.4%)of lung cancer rank first among male cancer patients.Among female cancer patients,morbidity(13.3%)ranks second,followed by breast cancer,and mortality(11.1%)ranks first.This has become a major public health issue in China’s social development Lung cancer is prone to distant metastases at an advanced stage,and bone is one of its common target organs for blood metastasis.Among them,lung adenocarcinoma has the highest incidence of bone metastasis,mainly osteolytic destruction,and is prone to secondary bone-related events,which seriously affects patients’ daily life and survival time.The treatment of bone metastases in lung cancer is still facing great challenges Comprehensive treatments that differ depending on the condition are strongly advocated,and individualized treatment plans are reasonably customized.Seeking the differential genes and diagnostic molecular markers of the disease is helpful to elucidate the relevant molecular mechanism of bone metastasis in lung cancer and promote the process of gene targeted therapy.RNase L is a unique endonuclease in vivo,which is regarded as a negative regulator of cell proliferation and migration.Its mutation increases the risk of malignant tumors.Studies have shown that RNase L is highly expressed in macrophages(osteoclast precursors)and plays an important role in the regulation of osteoclast differentiation and apoptosis.Therefore,it is of great significance to explore the underlying mechanism of RNase L gene in the occurrence and development of osteolytic metastasis of lung cancer.Traditional Chinese medicine has always had a unique and profound theoretical understanding of bone metastases of lung cancer.The clinical compound prescription is widely used.Under the guidance of the overall concept and dialectical treatment,the combination of rationale and prescriptions can achieve comprehensive,phased,multi-target intervention,which can be used throughout the treatment of patients.To explore the mechanism of Chinese medicine against bone metastasis of lung cancer has been in the ascendant,emphasizing the combination with modern medicine to learn from each other,so as to inject new blood into the development of medicine in the motherland.In recent years,the advantages of traditional Chinese medicine in the regulation of tumor metastasis microenvironment have gradually emerged,and the mechanism of bone metastasis has been further explored from the molecular level,so as to better guide clinical application.Through long-term clinical practice,the effectiveness of Guben Jiedu Fang is confirmed,which brings good news for the patients with bone metastasis to alleviate pain and prolong survival period.Therefore,exploring the effect of tumor metastasis microenvironment on osteoclast differentiation,and carrying out the intervention of Guben Jiedu Fang,may provide a new idea for the treatment of tumor bone metastasis.In view of this,this study takes RNase L as the core and Guben Jiedu Fang as the intervention measures to explore the potential molecular mechanism of bone metastasis of lung cancer from different perspectives.The main contents are divided into two parts:1.The potential relationship between RNase L and various tumors was analyzed based on bioinformatics database.2.Preliminary study on the mechanism of RNase L regulating osteolytic metastasis of lung cancer and the intervention effect of Guben Jiedu Fang.1.The potential relationship between RNase L and various tumors was analyzed based on bioinformatics databaseObjective:To explore the variation and expression of RNase L gene in various malignant tumors,construct the RNase L protein interaction network,and perform enrichment analysis of function and pathway,is to provide a new direction for further exploring the potential relationship between RNase L and tumors and lay a theoretical foundation for the next step in vitro cell experiments.Method:The CbioPortal network platform was used to analyze the mutation,deletion and amplification of RNase L in all cancers involved in the TCGA PanCan database,so as to observe the potential relationship between RNase L gene mutation and tumori-genesis.The GEPIA online database analyzes the expression level of RNase L in all cancers and the relationship between RNase L and the overall survival rate of patients,to find tumors that have significant correlation with RNase L.Then,the protein-protein interaction network was constructed by analyzing RNase L related proteins with STRING database.In addition,the top 10 interaction proteins with the highest interaction intensity were selected for GO function enrichment analysis and KEGG pathway enrichment analysis,which mainly involved biological processes,molecular functions,cytological components,and mediated signaling pathways.Result:(1)RNase L gene has gene variation in most tumors.Lung adenocarcinoma and lung squamous cell carcinoma have multiple gene changes,and the variation from high frequency to low frequency is gain,amplification,shallow deletion and mutation.Breast invasive cancer,liver cancer,and ovarian cancer are mainly amplification.Uterine cancer,colon cancer,bladder cancer,and melanoma are mainly mutation.Cervical cancer,testicular cancer,gastric cancer,and uveal melanoma are mainly gain.Sarcoma,head and neck cancer,prostate cancer,and brain lower grade glioma are mainly shallow deletion.Renal papillary cell carcinoma is mainly deep deletion.RNase L protein was significantly lower expressed in endometrial cancer,ovarian serous cystadenocarcinoma and uterine sarcoma,and only significantly higher in pancreatic cancer.In addition,the survival rates of renal clear cell carcinoma,brain lower grade glioma,ovarian serous cystadenocarcinoma,mesothelioma,and sarcoma were significantly correlated with the expression level of RNase L.(2)The top 10 interacting proteins of RNase L protein from strong to weak are ABCE1,OAS3,OASL,ISG15,OAS1,MX1,OAS2,RSAD2,IRF3 and IFIT1.The results of Go functional enrichment analysis showed that RNase L is mainly enriched in biological processes such as type I interferon effect,viral genome replication,negative regulation of viral life cycle,and viral defense response,etc;cell components such as mitochondrial matrix,eukaryotic initiation factor complex,host cells and lipid droplets,etc;molecular functions such as double-stranded RNA binding,adenylate transferase activity,nucleotide transferase activity,and ribonucleoprotein complex binding,etc.The results of KEGG pathway enrichment analysis showed that RNaseL is mainly concentrated in the viral infection pathways such as hepatitis C virus,hepatitis B virus,human herpes virus,and human papilloma virus,etc;signal transduction pathways such as Toll-like receptor signaling pathway,nucleotide binding oligomerization domain-like receptor signaling pathway,and retinoic acid induced gene I-like receptor signaling pathway,etc.Conclusion:This study systematically analyzed the potential link between RNase L gene mutations and the development of various tumors,and found that the expression level of RNase L protein was significantly correlated with the survival rate of some tumor patients.In addition,functional annotation and pathway enrichment of interacting proteins of RNase L further clarified the correlation between RNase L and the pathogenesis of tumor.The results of this study preliminarily prove that RNase L gene mutation increases the risk of tumorigenesis and provides a potential molecular target for clinical anticancer treatment.2.Study on the mechanism of RNase L regulating osteolytic metastasis of lung cancer and the intervention effect of Guben Jiedu Fang.Objective:To investigate the effects of RNase L gene and Guben Jiedu Fang on the proliferation and migration of A549 cells,and the regulatory effect of RNase L gene and tumor metastasis microenvironment on osteoclast differentiation.Method:A549 and A549-RNase L KD cells were cultured in vitro,and Guben Jiedu Fang’s drug-containing serum was prepared for intervention.The effects of RNase L and different concentrations of medicated serum on the proliferation and migration of lung cancer cells were observed by the experimental methods of MTT and scratches,using the absorbed optical density and scratch healing area as measurement indexes.In addition,targeted RNase L-shRNA was constructed and transfected into A549 cells and RAW264.7 cells.Western Blot method was used to verify that it effectively silenced the RNase L gene and inhibited the expression of RNase L protein.Then,the model of RAW264.7 cells differentiation into osteoclasts was established by using Transwell co-culture technique and introducing exogenous RANKL protein.The osteoclasts were counted with TRAP positive staining as the detection index,focusing on the study of the role of RNaseL and tumor metastasis environment in the process of osteoclast differentiation.Result:(1)The proliferation of A549 cells was significantly affected by different drug and cell types(P<0.001).In A549 normal group and A549 KD group,the OD values of traditional Chinese medicine group and western medicine group were significantly different from those of self-control group,P<0.05 in the traditional Chinese medicine group and P<0.01 in the western medicine group.Combined with the inhibition rate,it showed that both traditional Chinese medicine and western medicine could inhibit the proliferation of A549 cells,and the effect of the western medicine group was better than that of the traditional Chinese medicine group.In addition,the inhibition rate of Guben Jiedu Fang’s medicated serum in different doses is ranked as follows:1%>3%>5%>7%>10%>13%,further indicating low dose Chinese medicine serum has a better inhibitory effect on A549 cell proliferation.In addition,for the same drug-containing serum,the OD value of the normal group was higher than that of the RNase L-KD group,and the inhibition rate was lower than that of the RNase L-KD group(P<0.01).It is suggested that RNase L gene may promote the proliferation of A549 cells.(2)The migration of A549 cells was significantly affected by different types of drugs and cells(P<0.05).In A549 normal and A549 KD,there was no significant difference in scratch area among traditional Chinese medicine,western medicine and control group at Oh,but there was significant difference at 6h,12h and 24h(P<0.01).Combined with the healing rate,it can be seen that the drug-containing serum can inhibit the migration of A549 cells,which is western medicine>traditional Chinese medicine>control,from strong to weak.In addition,at the same time,for the same drug-containing serum,the scratch area of the normal group was smaller than that of the RNase L-KD group,and the healing rate was higher than that of the RNase L-KD group(P<0.05),indicating that RNase L gene may play a role in promoting the migration of A549 cells.(3)There were significant differences among RAW264.7+A549 and RAW264.7 KD+A549;RAW264.7+A549 KD and RAW264.7 KD+A549 KD;RAW264.7 and RAW264.7 KD(P<0.05),indicating that the knockout of RNase L gene in mono-nuclear macrophages inhibited osteoclast differentiation.Among RAW264.7 KD+A549 and RAW264.7 KD;RAW264.7 KD+A549 KD and RAW264.7 KD;A549+RAW264.7 and RAW264.7;RAW264.7+A549 KD and RAW264.7,all comparisons were p<0.05,indicating that tumor metastasis microenvironment promoted osteoclast differentiation.The comparison between RAW264.7 KD+A549 and RAW264.7 KD+A549 KD or between RAW264.7+A549 and RAW264.7+A549 KD was P>0.05,indicating that knockout of RNase L gene in tumor cells had no effect on osteoclast differentiation.Conclusion:In this study,four groups of cell lines A549,A549-RNase L KD,RAW264.7,and RAW264.7-RNase L KD were used to carry out MTT,scratches,and Transwell experiments,and Guben Jiedu Fang was used to intervene.The absorption optical density,scratch area and osteoclast count were used as the detection indexes.It was preliminarily confirmed that RNase L could promote the proliferation and migration of A549 cells to a certain extent,and Guben Jiedu Fang can inhibit the proliferation and migration of A549 cells,and the effect of low dose was better.In addition,both RNase L and tumor metastasis microenvironment promoted osteoclast differentiation to varying degrees. |
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