CircATXN7 Regulates The Proliferation And Invasion Of Esophageal Cancer Cells Through MiR-4319/NLRC5 And The Antineoplastic Mechanism Of Jianpi Xiaoge Recipe

BackgroundEsophageal cancer is one of the most common cancers and the leading cause of cancer death in the world.In China,there are nearly 50%of new cases,which brings considerable burden to patients themselves.The early prognosis of esophageal cancer is good.At present,esophageal cancer screening programs greatly reduces the morbidity and mortality of esophageal cancer,which are actively carried out in high prevalence areas in our country.From the medical point of view,the prognosis of esophageal cancer is still not satisfactory.The prognosis is usually very poor for advanced patients,due to the high degree of malignancy.Studies have shown that medical analysis of missing,mutated and related gene amplification contributes to the occurrence and development of esophageal cancer.Therefore,exploring the key molecular targets affecting the regulation of esophageal cancer will further improve the understanding of the pathogenesis of esophageal cancer.At the same time,by exploring related molecules,powerful and effective diagnostic markers can be found for clinical preventive treatment.Non-coding RNA(such as miRNA,LncRNA and circRNA)without protein coding are a class of RNA transcripts from a medical perspective.Various non-coding RNA have been reported to be involved in the control of multicellular effects such as adenosine production,cell development,autophagy,EMT and cell regulation from a medical perspective.CircRNA is a unique endogenous RNA,which can regulate gene expression through sponge.Sponge absorbs micro-RNA,modifies genes,and regulates transcription and splicing of target genes.From a medical perspective,a large amount of evidence shows that circRNA is involved in multicellular processes and multiple malignancies,including esophageal cancer.In recent years,circATXN7 is an emerging lncRNA,highly expressed in rectal cancer and lung cancer,ovarian cancer,breast cancer,and has an impact on its development.In fact,there are no studies on esophageal cancer.Therefore,this study is the first comprehensive analysis.The biological role and the molecular mechanism of circATXN7 in esophageal cancer were investigated.Chinese herbal medicine contains a variety of active ingredients with anti-tumor potential,good clinical efficacy,and few adverse reactions,which have also attracted extensive clinical attention.The application of Jianpi Powder in the treatment of postoperative patients with esophageal cancer is very reasonable to improve various postoperative complications,and the integration of modern radiotherapy and chemotherapy can reduce postoperative adverse reactions in patients.It improves immunity,anti-cancer activity,survival and quality of life.This study consists of three parts:Part I Expression of circATXN7 in esophageal cancer tissues and its effect on cell proliferation and invasionObjective:To investigate the expression of circATXN7 in esophageal cancer tissues and its effect on cell proliferation,migration and invasion.Methods:1.qRT-PCR was used to the expression level of circATXN7 in Esophageal cancer tissues.The clinicopathological factors significantly related to circATXN7 expression level were analysed.2.Kaplan-meier univariate analysis and COX multivariate analysis were used to select the individual risk factors influencing progression-free survival of esophagealcancer patients.3.qRT-PCR was sued to measure the expression levels of circATXN7 in HET-lA and five esophageal cancer cells(Eca-109,EC-9706,KYSE-30,KYSE-150,TE-1).4.The overexpression circATXN7 vector were synthesized,respectively.Two knockdown vectors(shRNA#1 and shRNA#2)targeting the CircATXN7 sequence and a blank control vector(shRNA control)were designed to transfect esophageal cancer cells.5.The proliferation ability of esophageal cancer cells with stable knockdown of CircATXN7 was detected by MTT and plate colony formation.6.Transwell assay was used to detect knockdown of CircATXN7 effect on the invasion and migration ability of esophageal cancer cells.Result:1.CircATXN7 was highly expressed in esophageal cancer tissues and esophageal cancer cell lines,among which EC-9706 had the highest expression level,followed by KYSE-150,TE-1,KYSE-30 and Eca-109.2.CircATXN7 expression level was significantly correlated with TNM stage and lymph node status(P<0.05).3.There were significant differences in PFS in different differentiation degrees,TNM stage,tumor invasion depth,and circATXN7 expression.4.Overexpression of CircATXN7 can promote the DNA replication ability of Eca-109 cells,that is,the proliferating nuclei are green.Stable knockdown of CircATXN7 significantly inhibited the DNA replication ability of EC-9706 cells,that is,the number of green nuclei was significantly reduced.Stable knockdown of CircATXN7 significantly inhibited the number of clones formed in EC-9706 cells.5.Overexpression of CircATXN7 can significantly inhibit the percentage of Eca-109 cells in G0/G1 phase,and correspondingly significantly promote the percentage of cells in S phase and G2/M phase.Knockdown of the CircATXN7 sequence can significantly promote the percentage of EC-9706 cells in G0/G1 phase,correspondingly significantly inhibit the percentage of cells in S phase,and reduce the ratio of cells in G2/M phase,but there is no statistically significant difference.6.Overexpression of CircATXN7 promoted the ability of Eca-109 cells to reach the lower compartment from the upper compartment through Matrigel,and in addition,overexpression of CircATXN7 significantly promoted the ability of Eca-109 cells to reach the lower compartment directly from the upper compartment through the small hole.Conclusion:CircATXN7 is high expression in esophageal cancer and related to the clinical characteristics of patients.Knockdown of circATXN7 significantly inhibit the proliferation,invasion and migration.Overexpression of circATXN7 had a reverse effect.Part Ⅱ circATXN7 regulates the proliferation and invasion of esophageal carcinoma cells through miR-4319/NLRC5Objective:To explore the molecular mechanism of circATXN7 regulating the proliferation,migration and invasion of esophageal carcinoma cells through mir-4319/NLRC5.Methods:1.CircATXN7 positioning:The localization of circATXN7 in esophageal cancer cells was initially determined by immunofluorescence staining and fluorescence microscope observation.qRT-PCR was used to verify the location of circATXN7 in esophageal cancer cells.2.Screening related miRNA:First,bioinformatics analysis and detection were performed to analyze the miRNA that might be bound in circATXN7 sequence,and then the miRNA with abnormal expression in circATXN7 knockdown esophageal cancer cells were detected by fluorescence quantitative PCR to determine the target miRNA.Mimics and inhibitor of miR-4319 were synthesized and transfected into esophageal cancer cells,respectively.3.RNA binding protein immunoprecipitation test and double luciferase reporter gene were determined the binding activity of circATXN7 to miR-43194.qRT-PCR was detected the expression of miR-4319 after knockdown of circATXN7 and the correlation between circATXN7 and miR-4319 in tissues was analyzed.5.Plate clone formation was used to determine the effect of circATXN7 on the proliferation of esophageal cancer through miR-4319.6.Bioinformatics software was used to analyze the target genes below miR-4319 course from a medical perspective.Luciferase activity and Western blot were used to verify that circATXN7 regulates the expression of NLRC5 through miR-4319.7.Western blot was used to detect the influence of overexpression or inhibition of miR-4319 on NLRC5 expression.Western blot was used to detect the regulation of NLRC5 expression by circATXN7 through miR-4319.8.In vivo was used to observe relevant indicators,and the expression levels of circATXN7 and miR-4319 in the transplanted tumor tissues were detected by fluorescence quantitative PCR.Results:1.Green fluorescent labeled circATXN7 is mainly located in the cytoplasm,and fluorescence quantitative PCR detection results confirm that circATXN7 is mainly distributed in the cytoplasm.2.Online analysis predicted that circATXN7 sequence might contain multiple miRNA:For example,miR-9-5p,miR-3184-5p,miR-4319,miR-423-5p,miR-3196,miR-485-5p,miR-1286,qRT-PCR further demonstrated that overexpression or stable knockdown of circATXN7 could significantly affect miR4319.3.The results of luciferase activity detection and RNA-binding protein immunoprecipitation indicated that there was an interaction between circATXN7 and miR-4319.The expressions of miR-4319 and circATXN7 were negatively correlated,with R2=0.28747 and P value less than 0.001.4.Overexpression of circATXN7 could significantly attenuate the inhibitory ability of overexpression of miR-4319 on Eca-109 cells.Inhibiting the expression of miR-4319 in EC-9706 cells can significantly promote the ability of cells to form clones,and knocking down circATXN7 can significantly attenuate the promoting effect of inhibiting the expression of miR-4319 in cells on the colony formation of cells.5.Overexpression of miRNA-4319 can significantly inhibit the expression of NLRC5,CD 147 and ENTPD4.Among them,the inhibitory effect of NLRC5 was the most significant.Transfection of different concentrations of miR-4319 mimics(30nM and 60nM)could significantly inhibit the expression of NLRC5 in a concentration-dependent manner.In EC-9706 cells,compared with the inhibitor NC group,transfection of different concentrations of miR-4319 inhibitor(50nM and l00nM)could significantly promote the expression of NLRC5 in a concentration-dependent manner.6.Overexpression of NLRC5(Ov-NLRC5)can significantly promote the expression of NLRC5 in esophageal cancer cells.After transfection knockdown NLRC5(sh-NLRC5),the expression level of NLRC5 in EC-9706 cells can be significantly knocked down.Overexpression of NLRC5 significantly promoted invasion and clonogenicity of esophageal carcinoma cells compared with Vector group.7.Overexpression of NLRC5(OV-NLRC5)can significantly inhibit the expression of P21 and promote the expression of Ki-67 and PCNA.8.The expression levels of circATXN7,miR-4319 and NLRC5 in tumor-carrying tissues were detected by qRT-PCR.The results showed that compared with control-shRNA group,the circATXN7 expression level in shRNA#2 group was significantly decreased,while the miR-4319 expression level was significantly increased.These in vivo results suggest that knockdown of circATXN7 in tumor-bearing tissues can significantly promote the expression of miR-4319 and inhibit the expression of NLRC5.Conclusion:In vitro cell and in vivo animal experiments showed that the molecular mechanism of circATXN7 regulating the proliferation,invasion and migration of esophageal cancer was targeting miR-4319/NLRC5 signal pathway.Part III Compound Jianpi Xiaoge recipe can inhibit the proliferation of esophageal cancer cells in vitro and in vivo by activating Caspase-3 and down-regulating NLRC5 expressionObjective:To investigate the molecular target of Jianpi Xiaoge decoction in the treatment of esophageal cancer and its effect on cell proliferation and apoptosis.Methods:1.After the mother liquid of jianpi xiaoge formula extract was prepared in cell culture medium with different concentrations,HET-1A cells and ECA-109,EC-9706 and KYSE-30 cells were treated for 12h,24h,36h and 48h,and the effect of extraction solution of Jianpi Xiaoge Formula on the cells’ proliferation ability was detected by MTT.2.The effects of different(0mg/mL,lmg/ml,2mg/ml and 4mg/ml)on esophageal carcinoma cells ECA-109 and KYSE-30 were determined by flow cytometry.3.Hoechst 33342 was used for staining,and the effect of extraction solution of Jianpi Xiaoge Formula on the nuclear morphology of esophageal carcinoma cells was observed under a microscope.4.Screened the targets of Jianpi Xiaoge prescriptions and esophageal cancer targets by network pharmacology(TCMSP database,GeneCards database and OMIM database).5.GO gene functional annotation and KEGG pathway enrichment were used to analyze the downstream signaling pathways that might be affected by Jianpi Xaoge decoction.6.Apoptosis protein chip was used to detect the effects of extracts from Jianpi Xiaoge prescription on apoptosis related signaling pathways of esophageal cancer cells,and western blot was used to detect the expression of main related proteins.7.A nude mouse model of esophageal cancer was established,and each nude mouse was given 150mg extract of Jianpi Xiaoge Formula twice a day,at 10:00 am and 4:00 PM,by intragastric administration,while the blank control group was given equal volume of PBS buffer for 21 consecutive days.8.After dissection and fixation,HE staining and immunohistochemical analysis of Ki-67 and Cleaved caspase-3 were performed,and morphological changes of heart,liver,spleen,lung,and kidney were also analyzed.Results:1.In Eca-109 cells,compared with blank control(0 mg/ml),the activity of Eca-109 cells cultured for 48h with low concentration of extracts of jianpi xiaoge formula(0.5mg/ml)could be significantly inhibited.According to the corresponding formula,the calculation showed that the half-inhibitory concentration at 36h was 2.09±0.25mg/mL,and the half-inhibitory concentration at 48h was 1.59±0.21mg/mL,Ec-9706 and KYSE-30 had similar test results to Eca-109.2.Compared with blank control(0 mg/ml),low concentration of jianpi xiaoge formula extract(0.5mg/ml)could significantly promote the apoptosis of Eca-109 cells after 48h culture.With the increase of concentration,the effect of extracts of Jianpi Xiaoge Formula(1mg/mL,2mg/mL and 4mg/mL)on promoting esophageal cell apoptosis was also more significant,and showed a concentration dependence.The results of KYSE-30 were similar to those of Eca-109 cells.3.Compared with the blank control(0 mg/ml),low concentration of Jianpi xiaoge formula extract(0.5mg/ml)could induce a small amount of nuclear condensation in Eca-109 cells after 48h culture.With the increase of concentration,the number of nuclear condensation occurred in a concentration-dependent manner,and similar results were observed in KYSE-30 cells.4.A total of 361 targets for Jianpi xiaoge were obtained through TCMSP database and SwissTargetPrediction website.6022 and 561 targets were obtained from GeneCards database and OMIM database,respectively.A total of 19 intersecting targets were obtained after the intersection of Jianpi Xiaoge diaphragm formula with GeneCards and OMIM database,which were the key targets of Jianpi xiaoge diaphragm formula on esophageal cancer.5.Topological properties analysis of the 19 targets protein interaction network diagram showed that CASP8(Caspase-8)with the highest degree value could interact with 11 proteins.6.Compared with blank control(0mg/ml),The extracts of 4mg/ml Jianpi Xiaoge Decoction significantly promoted the expression of Bax,Phospho-Rad17(S635),Claspin,HSP70,HSP27,HSP60,Cytochrome C,Fas,Cleaved caspase-3 and XIAP.Inhibit the expression of Bcl-2.7.Western blot results showed that compared with blank control(Omg/ml),the extractive solution of Jianpi Xiaoge Formula could significantly promote the expression of Bax.8.KEGG Pathway analysis showed that Jianpi Xiaoge decoction could also affect NOD-like receptor signaling pathway,that is,the extract of Jianpi Xiaoge decoction could significantly inhibit the expression of NOD2 and NLRC5 in esophageal cancer cells,but had no significant effect on the expression of NOD1 and NLRP3.9.Compared with the blank control(0mg/ml),the tumor tissue volume was significantly smaller than that of the blank control(150mg)after 21 days,and the growth rate of transplanted tumor was significantly reduced in nude mice.10.In blank control tissue transplanted tumor tissues,cell shapes are diverse,irregular arrangement,nucleo-cytoplasmic ratio is high,and there is a large number of nuclear mitosis,which are typical characteristics of tumor tissues.Immunohistochemical staining showed that ki-67 expression was high in blank control tissues,but Cleaved caspase-3 expression was almost non-expression,and ki-67 expression was low and Cleaved caspase-3 expression was high in transplanted tumor tissues treated with 150mg JianpiXiaoge formula extract.11.HE staining showed that compared with the blank control group,the 150mg extract of Jianpi Xiaoge Formula had no visible changes in the tissues of heart,liver,spleen,lung and kidney in nude mice.Conclusion:Jianpi Xiaoge can inhibit the proliferation of esophageal cancer cells in nude mice and promote the apoptosis of esophageal cancer cells by activating the key apoptotic molecule Caspase-3 and immunomodulatory molecule NLRC5,but has no significant effect on the histomorphology of major organs(heart,liver,spleen,lung,kidney)in nude mice.Summary:(1)CircATXN7 is highly expressed in esophageal cancer tissues,which is significantly correlated with the clinicopathological characteristics of patients,and is involved in the regulation of the proliferation,cloning,cycle,invasion and migration of esophageal cancer cells.Molecular mechanism studies have shown that circATXN7 regulates the proliferation,invasion and migration of esophageal cancer by targeting miR-4319/NLRC5 pathway.(2)Jianpi Xiaoge can inhibit the proliferation of esophageal cancer cells in vitro and in vivo by activating Caspase-3,a key molecule of apoptosis,and down-regulating the expression of NLRC5.