Preparation Of Nano-functional Material Fe₃O₄-CMC-NIM And Its Regulation Of CXCR4 Against Gastric Cancer In Vitro And Its Mechanism

Background and PurposeGastric cancer（GC）is a common malignant tumor in the world,which seriously affects people’s health and economic burden.In China,especially in the"Hexi Zoulang"area of Gansu province,where the incidence of gastric cancer is high and the economy is still underdeveloped.Our team has made long-term efforts to study the epidemiology of gastric cancer in Gansu Province and explore the prevention and control mode through establishing a large-scale gastroscopy screening project of ordinary population in Wuwei city"Natural Population Cohort Study of Wuwei City in Gansu Province".However,as a malignant tumor with multiple pathogenic factors,the pathogenesis of gastric cancer is still unclear,and its treatment is still dominated by surgery,and drug therapy is still very limited.In recent years,with the opening of public databases and the rapid development of bioinformatics and nanomedicine technologies,new ideas and directions have been provided for us to explore the pathogenesis and treatment mode of gastric cancer.Through in-depth mining of TCGA database and GEO database,we screened out gene groups that play a key role in the occurrence and development of GASTRIC cancer,and finally identified CXCR4,the autophagy related gene most closely related to the occurrence of gastric cancer.In the follow-up basic experiments,the database analysis results were analyzed and verified,and the specific action mechanism of key genes was explored.As for the antitumor effect of Fe₃O₄-CMC-NIM,this study took gastric cancer cells as the research object to explore and compare the effects of NIM alone and Fe₃O₄-CMC-NIM on the proliferation,apoptosis and metastasis of gastric cancer cells.CXCR4,as an upstream target gene,was predicted to be closely related to proteins in the PI3K/AKT pathway by bioinformatics enrichment analysis.Later,this study further explored whether Fe₃O₄-CMC-NIM affected autophagy and apoptosis induced by regulating CXCR4 expression through PI3K/AKT signaling pathway,and further revealed the anti-tumor mechanism of Fe₃O₄-CMC-NIM nanoparticles,a novel functional nanomaterials.This study provides theoretical basis for the further development and clinical application of Fe₃O₄-CMC-NIM nanoparticles.Methods（1）Fe₃O₄ nanoparticles were selected,and a new nanometer drug Fe₃O₄-CMC-NIM was constructed by immunocoprecipitating method,which was coated by natural material carboxymethyl chitosan（CMC）and loaded with nimesulide（NIM）.Meanwhile,a series of characterization analysis was conducted on the newly synthesized nanoparticles.X-ray diffraction（XRD）,Specific Surface Area test（BET）,Vibrating Sample Magnetometerr（VSM）,Transmission Electron Microscopy（TEM）,Scanning Electron Microscopy（SEM）,X-ray photoelectron spectroscopy（XPS）and other methods were used for testing.（2）Bioinformatics tools were used to screen autophagy genes associated with gastric cancer prognosis and establish a risk scoring model.Data from TCGA and GEO databases were used to determine the autophagy genes associated with Overall Survival（OS）by Lasso regression,univariate and multivariate Cox regression.To evaluate the predictive value of different autophagy gene models for overall survival of gastric cancer patients.Finally,six autophagy related genes DYNLL1,PGK2,HPR,PLOD2,PHYHIP and CXCR4 were identified by risk scoring model,and survival and prognosis were analyzed based on Kaplan-Meier test and ROC curve.The immune status of the model was evaluated by estimation method,and the function and molecular pathways of the model related genes in gastric cancer were studied by GO analysis and KEGG analysis.To find and identify the key gene CXCR4,which is closely related to the occurrence and development of GC,and to lay a foundation for further research on the anti-tumor effect of Fe₃O₄-CMC-NIM nanoparticles regulating the gene on gastric cancer cells in vitro and related mechanisms.（3）The mRNA expression levels of CXCR4 in gastric cancer cell lines AGS,MGC-803,MKN-45,BGC-823 and HGC-27 and normal gastric mucosa cell lines GES were detected to verify the bioinformatics analysis.The expression of CXCR4 in TCGA was analyzed by GEPIA.The expression of CXCR4 in 66 gastric cancer tissues and adjacent tissues was detected by RT-PCR.Gastric cancer cell lines MGC-803 and BGC-823 were selected as research objects.Fe₃O₄-CMC-NIM nanoparticles and NIM were treated respectively.Nuclear staining,cell scratched migration and Transwell invasion were performed by MTT method,flow cytometry and Hoechst33258.The changes of cell micromorphology were observed under electron microscopy,which jointly verified the antitumor effects of Fe₃O₄-CMC-NIM nanoparticles and NIM in vitro,and the two were compared in detail.Meanwhile,Western Blot was used to detect three apoptosis pathways:Fas/Fas-L pathway,mitochondrial apoptosis pathway and markers in ERS apoptosis pathway.To verify and compare the effect of Fe₃O₄-CMC-NIM nanoparticles and NIM alone on apoptosis of gastric cancer cells.The effect of Fe₃O₄-CMC-NIM nanoparticles and NIM on mitochondrial membrane permeability was verified and compared by Cyt C release assay.（4）Gastric cancer cell line MGC-803 was selected as the object of further study,and the expression of CXCR4 protein in gastric cancer cell line MGC-803 was detected by flow cytometry,RT-PCR and Western Blot.After CXCR4 was silenced,the changes of Bax,Beclin1,Bcl-2 and LC3BⅡ/Ⅰratio,the changes of expression and phosphorylation of related proteins in PI3K/AKT/mTOR signaling pathway,and the changes of autophagy and apoptosis induced by Fe₃O₄-CMC-NIM nanoparticles were detected.After blocking the PI3K/AKT/mTOR signaling pathway with mTOR blocker rapamycin and activating the PI3K/AKT/mTOR signaling pathway with PI3K activator 740 Y-P,The changes of related proteins Bax,Beclin1,Bcl-2 and LC3BⅡ/Ⅰratio,the changes of autophagy and apoptosis induced by Fe₃O₄-CMC-NIM nanoparticles,and the changes of related protein expression and phosphorylation level in gastric cancer cells were detected.Results（1）The effects of concentration of solution,molar ratio of Fe³⁺/Fe²⁺,reaction and curing temperature,pH value of solution and washing mode on particle size,morphology,structure and magnetism of Fe₃O₄ magnetic nanoparticles synthesized by coprecipitation method were systematically studied.The optimal preparation conditions were obtained:The stirring temperature was 25℃,pH 13.11,Fe³⁺/Fe²⁺=0.75.The obtained Fe₃O₄ nanoparticles have a relatively large specific surface area of 135.37 m²/g,mesoporous structure with a pore size of 8.21 nm,coercivity of 0.905 Oe,saturation magnetization of 29 emu/g and 29.026 Emu/g.The agglomeration of Fe₃O₄ nanoparticles was effectively solved by carbon coating,and then the Fe₃O₄ nanoparticles were evenly dispersed.（2）Using TCGA-STAD data set and GEO database data,Lasso and Cox regression analysis,the risk scoring model based on 6 ARGs is established.Based on the analysis of survival and diagnostic performance,this model can be used as a good predictor of gastric cancer prognosis and an independent predictor of OS in gastric cancer patients.CXCR4,as a poor prognostic indicator,is closely related to the occurrence and development of GC.KEGG enrichment analysis showed that CXCR4was positively correlated with PI3K/AKT signaling pathway,which may inhibit the apoptosis of gastric cancer cells and promote the invasion and metastasis of gastric cancer cells.（3）CXCR4 is up-regulated in gastric cancer cells.The expression of CXCR4 in gastric cancer tissues was significantly higher than that in adjacent tissues by GEPIA analysis,and RT-PCR results showed that the expression level of CXCR4 in gastric cancer tissues was higher than that in adjacent tissues.The mean concentration of Fe₃O₄-CMC-NIM nanoparticles and NIM time-dependent inhibited the proliferation,invasion and metastasis of gastric cancer cells and promoted apoptosis.Both Fe₃O₄-CMC-NIM nanoparticles and NIM can induce autophagy and promote apoptosis of gastric cancer cells through three classical apoptosis pathways,and can affect apoptosis of gastric cancer cells by affecting mitochondrial membrane permeability.Compared with NIM,Fe₃O₄-CMC-NIM nanoparticles have more significant anti-tumor effects.（4）Fe₃O₄-CMC-NIM nanoparticles can concentration-dependently reduce CXCR4 protein expression in gastric cancer cells.In the PI3K/AKT/mTOR pathway,Fe₃O₄-CMC-NIM nanoparticles inhibited the PI3K/AKT/mTOR signaling pathway as the inhibitor rapamycin did,and had the effect of inducing autophagy and promoting apoptosis in gastric cancer cell lines.CXCR4 silencing enhanced the ability of Fe₃O₄-CMC-NIM nanoparticles to inhibit PI3K/AKT/mTOR signaling pathway and induce autophagy and apoptosis in gastric cancer cells.Activation of PI3K can reduce the inhibitory effect of Fe₃O₄-CMC-NIM nanoparticles on PI3K/AKT/mTOR signaling pathway,similarly,activation of PI3K/AKT/mTOR signaling pathway can reduce the ability of Fe₃O₄-CMC-NIM nanoparticles to induce autophagy and apoptosis of gastric cancer cells.Conclusion（1）The new synthetic nano functional material Fe₃O₄-CMC-NIM nanoparticles maintain good dispersion and stability,ensuring the best biocompatibility.（2）CXCR4 is a key gene in the occurrence and development of gastric cancer.KEGG enrichment analysis showed that CXCR4 was closely related to the PI3K/AKT pathway（3）The antitumor effect of Fe₃O₄-CMC-NIM nanoparticles is significantly better than that of NIM.Both of them can activate classical apoptosis pathways and induce gastric cancer cell apoptosis in a time-concentration dependent manner,and Fe₃O₄-CMC-NIM nanoparticles have more significant effects.（4）Fe₃O₄-CMC-NIM nanoparticles inhibit the proliferation and metastasis of gastric cancer cells,induce autophagy and promote apoptosis by reducing the expression of CXCR4 and inhibiting the PI3K/AKT/mTOR signaling pathway.