The Study Of Metformin Inhibiting The M2-type Polarization Of Microglia Through MiR-1966-3p/SCD1/AMPK And Then Affecting The Invasion Of Glioma

Glioma is the most common primary malignant tumor in the skull.Because of the strong invasiveness,glioma is difficult to be completely removed by surgical treatment,and it is easy to recur after surgery.Microglia is an important part of the microenvironment of glioma and plays an important role in the invasion process of glioma.Microglia have strong plasticity and can be polarized into two distinct functional groups influenced by many factors in the microenvironment of glioma,namely M1-type which inhibits the proliferation of glioma,and M2-type which promotes the invasion and metastasis of glioma.In recent years,the anti-tumor effect of metformin has received extensive attention from researchers.Previous studies on metformin’s anti-glioma effects mostly focused on the tumor cells themselves,and there is still a lack of research on its effect on microglia in the tumor microenvironment.In this study,starting from the treatment of glioma,metformin’s inhibition of the M2-type polarization of microglia in the microenvironment of glioma and its influence on the invasion of glioma were explored.The specific research includes the following four parts:Part Ⅰ:Metformin inhibits M2-type polarization of microglia.Objective:The M1 and M2 polarization models of microglia were constructed by classical methods.The effect of metformin on the polarization of M1 and M2 microglia was preliminarily explored.Methods:(1)Use Lipopolysaccharide(LPS)and Interleukin-4(IL-4)to induce the M1 and M2 polarization models of BV-2 microglia,respectively.Observe cell morphology changes through a microscope,quantitative Reverse Transcription Polymerase Chain Reaction(qRT-PCR)to detect M1(iNOS,CD86,TNF-α)and M2(Arg-1,YM1,FIZZ1)type specific marker gene transcription level changes,Western Blot(WB)detects the changes in the expression levels of M1(iNOS,IL-6)and M2(Arg-1,CD206,IL-10)type specific marker proteins,to determine the construction of the microglia polarization model.(2)The CCK-8 experiment was used to screen the appropriate concentration of metformin to stimulate BV-2 cells.On the M1-type polarization model induced by LPS,qRT-PCR and WB experiments were used to detect the expression of specific markers in the M1 polarization model of BV-2 cells under the action of metformin,to observe the effect of metformin on the M1 polarization of microglia.(3)On the IL-4 induced M2-type polarization model,qRT-PCR,Immunofluorescence(IF)and WB experiments were used to detect the expression of specific markers in the M2-type polarization model of BV-2 cells under the action of metformin,to observe the effect of metformin on the M2-type polarization of microglia.Results:(1)Under the microscope,the resting BV-2 cells in the blank group showed elongated and elongated processes,while the M1 and M2 polarized BV-2 cell processes induced by LPS and IL-4 were significantly shortened,resembling amoebiforme.M2-type polarized microglia had fewer processes and a more nearly round cell body than M1-type polarized microglia.The transcription and expression levels of specific markers are significantly increased,which is consistent with the inducible M1 and M2 polarization models described in the related research results.(2)In this study,the concentration of 1 mM metformin that does not affect proliferation was selected as the subsequent concentration.QRT-PCR and WB experiments showed that metformin had no significant effect on the transcription and expression levels of M1-type specific markers.(3)M2-type polarized BV-2 cells were given the effect of metformin,qRT-PCR experiments showed that the transcription levels of M2 specific marker genes Arg-1,YM1,CD206,FIZZ1,CCR2,IL-10 decreased,IF and WB experiments showed that the expression of M2-type specific marker proteins Arg-1 and CD206 decreased.The changes of M2-type polarization markers detected by different experimental methods are basically the same.Conclusion:LPS and IL-4 can induce resting microglia to form M1 and M2 polarization models,respectively.Metformin has no obvious effect on LPS induced M1-type polarization of microglia.Metformin inhibits IL-4 induced M2-type polarization of microglia.Part Ⅱ:Metformin inhibits the invasion of glioma by inhibiting the M2-type polarization of microglia.Objective:In vitro and in vivo experiments were conducted to investigate the effect of metformin on inhibiting the invasion of glioma by inhibiting M2-type polarization of microglia cells.Methods:(1)Use IL-4 and metformin to intervene BV-2 cells alone or together to make Conditioned Medium(CM).CCK-8 and cell apoptosis(flow cytometry)experiments were used to detect the effects of CM on the proliferation and apoptosis of glioma GL261 cells.The effect of CM on the invasion of glioma cells was detected by wound healing,Transwell,Enzyme linked immunosorbent assay(ELISA)and WB experiments.(2)Use Clodronate liposome(Clo)and stereotactic instrument to establish SD rat microglia-free and glioma models respectively.First,check the tumor formation by Magnetic Resonance Imaging(MRI)and Hematoxylin-eosin staining(HE).The expression of microglia specific marker Iba-1 was detected by IF method to observe the construction of microglia-free model.Then the expression levels of CCL-2 and IL-10 were detected by ELISA.Finally,Immunohistochemistry(IHC)and WB experiments were used to detect the expression of M1,M2 and invasion marker proteins.Results:(1)The results of CCK-8 and cell apoptosis experiments suggest that M2-type polarized BV-2 cells under the action of ImM metformin have no significant effect on the proliferation and apoptosis of glioma GL261 cells.The results of cell scratching and transwell invasion experiments suggest that the ability of M2-type polarized BV-2 cells to promote the invasion of GL261 cells decreased under the action of metformin.ELISA results indicated that metformin decreased the secretion of glioma promoting factor CCL-2 and IL-10 in M2-type polarized microglia CM,and the WB results suggest that the expression levels of the marker proteins MMP2 and MMP9 related to tumor invasion have also decreased.(2)The results of MRI,HE and IF indicated that the stereotactic intracranial glioma and microglia-free were successfully constructed in SD rat.ELISA results suggested that metformin down-regulated the expression of M2-type polarization promoting glioma invasion factor CCL-2 and IL-10 in plasma microglia.IHC assay indicated that metformin significantly inhibited the expression of invasion marker MMP2 and MMP9.WB experiment showed that metformin had no significant effect on M1-type polarization and significantly inhibited the expression of M2-type polarization and invasion marker protein.The above ELISA,IHC and WB experiments showed that the inhibition of microglia was significantly weakened after the removal of microglia by Clo,suggesting that microglia played an important role in this process.Conclusion:Metformin inhibits the invasion of glioma by inhibiting M2-type polarization of microglia.Part Ⅲ:Analysis of the differentially expressed RNAs in the inhibition of metformin in the M2-type polarization of microglia.Objective:In order to explore the expression characteristics of RNAs in metformin suppressed M2 polarized microglia,we introduced transcriptome sequencing methods and bioinformatics analysis.From the perspective of RNAs,this part lays the foundation for the fourth part to study the molecular mechanism of metformin inhibiting the M2-type polarization of microglia.Methods:(1)In this study,IL-4 induced M2-type polarization model was used as the control group,and 1mM metformin was given as the experimental group.Each group randomly selected 3 bottles of the same processed samples for transcriptome sequencing,screened differentially expressed circRNAs,miRNAs and mRNAs,and used qRT-PCR to complete the preliminary verification of some significantly differentially expressed RNAs.(2)With the help of Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,Protein-protein Interaction(PPI)protein interaction,MCODE screening of key genes,ceRNA network construction and other bioinformatics technologies,the results of sequencing are significant differentially expressed RNAs were analyzed.Results:(1)According to the criteria of P<0.05 and |log2Fold Change|>1,the differentially expressed RNAs in the sequencing results were screened,and 15 circRNAs,10 miRNAs,and 8 mRNAs with significant differential expression were obtained.Select some significantly differentially expressed up-regulated genes(Dusp8,Bche,Sgpp2,circ-001083,miR-1966-3p)and down-regulated genes(Jam2,SCD1,F13a1,circ-010517,circ-005090,miR-6933-3p,miR-3110-5p)completed qRT-PCR verification.The results are basically consistent with the sequencing results,indicating that the sequencing results of this study are highly reliable.(2)Complete analysis of significantly differentially expressed RNAs through bioinformatics technology:KEGG enrichment analysis results mainly include insulin secretion,leukocyte transendothelial migration,vascular smooth muscle contraction,cell adhesion,necroptosis,AMPK and other pathways.SCD1 is enriched in AMPK,a common pathway of metformin.PPI was used to construct a protein interaction map consisting of 84 nodes and 96 edges,including SCD1 and Arg-1.Among them,1 significant Module and 9 key genes were obtained by MCODE screening.A ceRNA network composed of 33 circRNA-miRNA-mRNA axes was constructed,of which the miR-1966-3p/SCD1 attracted attention.Conclusion:Metformin induced partial differential expression of RNAs in M2-type polarized microglia,and miR-1966-3p/SCD1/AMPK may play an important role in this process.Part Ⅳ:Metformin inhibits M2-type polarization of microglia through miR-1966-3p/SCD1/AMPK axis.Objective:Through the third part of transcriptome sequencing and bioinformatics analysis,some significantly differentially expressed RNAs were obtained.This part aims to further explore whether the miR-1966-3p/SCD1/AMPK axis plays a role in the process of metformin inhibiting the M2-type polarization of microglia.Methods:(1)QRT-PCR and WB were used to detect the transcription and expression changes of miR-1966-3p and SCD1 in IL-4-induced M2-type polarization of BV-2 cells.(2)After intervention with SCD1 inhibitor and overexpression plasmid,the expression of SCD1,P-AMPK/AMPK,M2-type polarization marker protein was detected by WB method.To study the involvement of SCD1/AMPK in the process of metformin inhibiting the M2-type polarization of microglia.(3)Dual luciferase gene reporter assay was used to verify the binding between miR-1966-3p and SCD1.(4)First,intervene with miR-1966-3p-inhibitor and miR-1966-3p-mimics plasmids.Then,the changes of miR-1966-3p and SCD1 transcription levels were detected by qRT-PCR experiment.The expression of SCD1,P-AMPK/AMPK,and M2 polarization marker proteins was detected by WB method to investigate the involvement of miR-1966-3p/SCD1/AMPK in the process of metformin inhibiting the M2 polarization of microglia.Results:(1)The transcription level of miR-1966-3p was significantly reduced,and the transcription and expression levels of SCD1 were significantly increased,in the M2-type polarization of BV-2 cells induced by IL-4.(2)SCD1 inhibitors have basically the same effect as metformin,and both can decrease the expression of SCD1 and M2-type polarization marker proteins,and the expression of P-AMPK/AMPK will increase significantly.Overexpression of SCD1 inhibited the above-mentioned effects of metformin treatment on M2-type polarization of BV-2 cells.(3)The results of the dual luciferase gene report experiment suggest that miR-1966-3p and SCD1 can directly target binding.(4)After miR-1966-3p-inhibitor plasmid was down-regulated,the transcription and expression levels of SCD1 were up-regulated,P-AMPK/AMPK expression was decreased,M2 polarization marker protein expression was increased,and the above effects disappeared after combined with metformin or SCD1 inhibitor.After miR-1966-3p-mimics plasmid up-regulated miR-1966-3p,the transcription and expression levels of SCD1 were down-regulated,P-AMPK/AMPK expression was increased,and M2 polarization marker protein expression was decreased.The above effects did not change significantly after combined with metformin,while the above effects disappeared after combined with SCD1 overexpression plasmid.Conclusion:Metformin inhibits M2-type polarization of microglia through the miR-1966-3p/SCD1/AMPK axis.