| Oral cancer is a common malignant tumor in the human body and is one of the top ten tumors in the world,with the increase of about 350,000 people each year and 170,000 people died according to statistics.In China,there are about 48,000 new patients each year and the number of deaths is about 22,000,and the incidence rate is increasing year by year and the age of onset tends to be younger.Oral squamous cell carcinoma(OSCC)is one of the most common tumors in head and neck cancer,accounting for about 80%of head and neck tumors.High malignancy,easy metastasis,invasion of surrounding tissues and poor prognosis are some of the characteristics of the disease,and the 5-year survival rate is only about 50%.Moreover,about 20%of the patients have local recurrence and lymph node metastasis within two to three years after initial treatment,which greatly reduced the patient’s survival time and bringing great pain to the patients.Angiogenesis is a key process in human growth and development,and occurs in many physiological processes like wound healing,menstrual cycles,and pregnancy.When the balance between the angiogenesis-related promoting and inhibiting factors is broken,the process of angiogenesis will be disordered,causing a variety of diseases including tumors,which seriously threatens human health.Angiogenesis is a recognized basis for the growth of solid tumors including OSCC,which is essential for proliferation,invasion and metastasis of tumor,and is one of the most important features of tumor.Therefore,in recent years,much attention has been paid to angiogenesis,exploring the mechanism of angiogenesis and regulating the generation of new blood vessels,which has become one of the main goals and key research of cancer treatment.With the deepening of understanding of angiogenesis,it has been found that immune cells such as monocytes/macrophages,neutrophils,eosinophils,mast cells,dendritic cells and natural killer cells,are heavily involved in tumor angiogenesis,in addition to the known immune function.Among them,the monocytes subgroup has attracted much attention because of its pivotal role in regulating angiogenesis and remodeling.A large number of studies have confirmed that in physiological and pathological conditions,especially in tumors,monocytes are important cells that migrate from the blood to tissues through autochemokine receptors and pathogen recognition receptors,and participate in the regulation of angiogenesis.A subset of monocytes can express the angiogenin receptor TIE2,which is called TEM(TIE2-expressing monocytes).TEM are able to accumulate around the blood vessels in inflammation and tumors,to express pro-angiogenic factors,to degrade the basement membrane,to promote vascular sprouting and endothelial cells migration,and directly promote angiogenesis.Furthermore,even TEM itself can be transformed into blood vessels,forming a capillary-like structure by expressing a marker similar to endothelial cells and reacting with VEGF,which can replace the real endothelial cells and eventually form mature blood vessels.Macrophages are the first line of defense against infection in organisms,and can eliminate dead cells during development and repair.Regulating angiogenesis is one of the important functions of them.A large number of studies have confirmed that,from the development of the body to the regeneration of adult tissues to the development of diseases,macrophages exist and participate in various stages of angiogenesis of them.Among them,monocyte that penetrates into the tumor is called tumor-associated macrophage(TAM).A large amount of TAM infiltration can be found in OSCC,and they are involved in angiogenesis.TAMs are also immune cells that exist earlier in the tumor site,and are "switches" for tumor neovascularization.TAMs express a variety of factors,such as VEGF-A,TNF-a,IL-8,etc.,which promote proliferation of tumor cells,angiogenesis,tumor deterioration and metastasis.Based on the important role in angiogenesis,monocytes/macrophages have become an important target in tumor therapy.However,the therapeutic strategy for targeting monocyte-macrophage angiogenesis has not yet had substantial progress.One of the important reasons is that its regulatory factors and mechanisms have not yet been fully elucidated.Therefore,exploring the mechanism of monocytes/macrophages regulating angiogenesis and deepening the understanding of angiogenesis will help to provide powerful theoretical support for the treatment of various diseases including tumors with monocytes/macrophages as target cells.In recent years,more and more studies in vivo and in vitro have confirmed that kinesin superfamily proteins(KIFs)are involved in the process of capillary formation and angiogenesis.KIFs are molecular motor proteins that form a unique transport system in cells,mainly located in eukaryotic cells,using microtubules as "orbits" to transport "cargos".They use the chemical properties of ATP to drive the generation of motor forces,carry out intracellular transportation,and sustain the survival of living organisms.Kif4,a member of the KIFs,which is involved in chromosome stabilization,DNA repair,cytoplasmic division,neuronal survival and spontaneous contraction,and play an important role in the development of tumors,has shown an angiogenic regulatory function that has attracted researchers’ interest.Recent studies have found that Kif4 is involved in angiogenesis and regulation.With the stimulation of VEGF-A,Kif4 expression was significantly up-regulated in vascular endothelial cells.In human hepatocellular carcinoma,the up-regulation of Kif4 expression is associated with tumor vascular localization and is most expressed in the vascular invasion sites of tumors.In monocytes/macrophages,various KIF family members has been found expressed,and they play an important role in the regulation of biological behavior such as migration and invasion of monocytes/macrophages.However,the function of Kif4 in monocytes/macrophages and whether it is involved in the regulation of angiogenic activity of monocytes/macrophages has not been reported yet.Therefore,clarifying its role and mechanism will provide strong theoretical support for the treatment of various diseases including tumors with monocytes/macrophages as target cells.Based on the above phenomena and problems,our group intends to explore the function of Kif4 in monocytes/macrophages,to discover whether Kif4 regulates the expression of angiogenic genes in RAW264.7 cells,and to preliminary discuss the molecular mechanism of angiogenesis,by using gene silencing technology,genome microarray screening technology,morphological observation,flow cytometry,RT-PCR,Western Blotting and enzyme-linked immunosorbent assay(ELISA).Through this study,we hope to have a deeper understanding of the role of monocytes/macrophages in angiogenesis,and look forward to a new understanding of the function of Kif4 and providing new trategies for tumor treatment.Part I Genome expression microarray reveals novel roles for Kif4 in RAW264.7 monocytes/macrophagesObjective:To screen out the most effective small RNA for inhibiting the Kif4 gene in RAW264.7 monocytes/macrophages.Using genomic expression microarray,and compared with the si-NC group,the differential gene of RNA in the si-Kif4 group was functionally analyzed and pathway analysis by genome expression microarray to reveal the roles of Kif4 in RAW264.7 monocytes/macrophages.Metheds:1.Culture of RAW264.7 cells:The murine monocyte/macrophage cell line RAW264.7cells preserved in liquid nitrogen were resuscitated and cultured in an incubator at 37 C,95%humidity,and 5%CO2 until the cells were in logarithmic growth phase to be used in the experiment.2.Detection of the transfection efficiency of small interfere RNA(siRNA):The siRNA transfection was performed in Green fluorescent FAM labeled negative control siRNA respectively by cationic liposome and transferred into RAW264.7 cells,according to the Invitrogen company LipofectamineTM RNAi-MAX reagent for manufacturer’s instructions.After 6h,compared the bright field and the fluorescence field in the same field of view using a fluorescence inverted microscope,and determined whether the siRNA was transfected into the cells by observing whether there was green fluorescence in the cells.3.Detection of the efficiency of Kif4 siRNA knocking down Kif4 mRNA in cells:RAW264.7 cells were transfected with Kif4-siRNA#1,Kif4-siRNA#2,Kif4-siRNA#3,the control group was treated with Mock group(added Lipo-fectamineTM 2000 only)and si-NC(transfected in negative control small interfering RNA).After 24h of transfection,total RNA was extracted from each group.The knockdown rate of Kif4 gene was detected by RT-PCR,and the Kif4 siRNA with the highest knockdown efficiency(defined as si-Kif4 group)was selected for subsequent experiments.4.Quality detection of extracted RNA:Total RNA of si-NC group and si-Kif4 group was isolated by TRIzol RNA Isolation Reagent.OD230,OD260 and OD280 values were obtained on NanoDrop ND-1000 spectrophotometer to measure RNA quantity and quality.Agarose electrophoresis was used to detect the quality of the extracted RNA(repeated three times).5.Analysis of differential genes using genome expression microarray:Differential genes were identified using the Mouse Gene Expression 4×44K Microarray V2 chip,and analyzed the acquired chip images using Agilent Feature Extraction software(version 11.0.1.1),using the GeneSpring GX v12.1 software package for quantile normalization and subsequent data processing.The judging criteria for differentially expressed genes was:gene difference folds≥ 1.5 and the P-value was<0.05.A box plot was used to show the intensity distribution of all samples,while scatter plots and volcano plots showed differentially expressed genes.Gene function was described by Gene Ontology(GO).Kyoto Encyclopedia of Genes and Genomes(KEGG)Pathway analysis was used to determine the genes in different biological pathways.Results:1.After transfected with FAM-labeled si-NC for 6 hours,green fluorescent spots in RAW264.7 cells were observed under the fluorescence light microscope,indicating that FAM-labeled si-NC with green fluorescent label had been transfected into the cells.2.The knockdown effect of siRNA on Kif4 mRNA was determined by RT-PCR.The results showed that Kif4 gene expression was significantly inhibited in cells transfected with Kif4-siRNA#3(defined as si-Kif4 group)(P<0.01),which were used in the following experiments.3.NanoDrop ND-1000 spectrophotometer analysis showed that all samples had an OD A260/A280 ratio between 1.8 and 2.0,and the ODA260/A230 ratio was greater than 1.8.The 28S and 18S bands in the electropherogram were clear and free of DNA.The intensity distribution of the samples in each group was similar,indicating that the RNA samples were of good quality and suitable for chip analysis.4.Data analysis showed that there were 1216 genes differentially expressed between the si-NC group and the si-Kif4 group,of which 240 were up-regulated and 976 were down-regulated.It was enriched to 805 items of GO-Biological Process,93 items of GO-Cellular Component,163 items of GO-Molecular function and 23 items of KEGG pathway,which is mainly concentrated in cell growth,survival,immune response,lipid metabolism,cell engulfment and lysosome metabolism,etc.5.VEGF-A,which is associated with regulation of angiogenesis in monocyte macrophages was down-regulated by 1.63-fold(P=6.29E-04),while VEGFR1 was down-regulated by 1.82-fold(P=0.0052),found in the differentially expressed genes,and the AKT signaling pathway in the "pancreatic cancer pathway"(ID:mmu05212)in cells was significantly inhibited(P=0.0315),which may be related to the involvement of Kif4 in the regulation of angiogenesis in monocytes/macrophages.These differential gene and signal pathway changes will provide an information basis for subsequent experiments.The results of genomic expression microarray suggested that Kif4 could participate in the regulation of various biological functions of RAW264.7 monocytes/macrophages,such as cell growth,survival,innate immunity and antigen presentation,lipid metabolism and metabolic function of the lysosome in phagocytosis,etc.,and changes in VEGF-A,VEGFR1 gene and AKT signaling pathways associated with angiogenesis were found and would be verified in subsequent experiments.Part II Effect of Kif4-mediated RAW264.7 monocytes/macrophages on the tube formation of Human Umbilical Vein Endothelial CellsObjective:To investigate the effects of Kif4 on the morphology,proliferation and expression of cell surface costimulatory molecules CD11c and CD80 in RAW264.7 monocytes/macrophages,and to observe the effect of supernatent of RAW264.7 cells’ transfected with si-Kif4 on tube formation of human umbilical vein endothelial cells.Metheds:1.Observing the changes of RAW264.7 cells morphology after transfection of si-Kif4:RAW264.7 cells were transfected with si-NC and si-Kif4 for 24h and 48h respectively.The morphology of the cells was observed under an inverted microscope.2.Detection of changes in the expression of costimulatory molecules CD11c and CD80 on the cell surface of each group:3.After transfecting RAW264.7 cells for 48h,1-2×104 cells of the Mock,si-NC and si-Kif4 groups were collected respectively,and washed with cold PBS.Monoclonal CD11c or CD80 antibody was added to the cells.The expression of CD11c and CD80 on the cell surface was detected by Flow cytometry(FCM).4.MTT assay for proliferative activity of each group of cells:The cell proliferation of the Mock,si-NC and si-Kif4 groups was detected by MTT assay respectively on days 1,2,3 and 4 after RAW264.7 monocytes/macrophages transfection.5.HUVECs tube formation experiment:A 96-well plate was used,and 50μl of Matrigel gel was added to each well.After the Matrigel gel was coagulated,HUVECs were seeded at a density of 2×104 cells/well in 96-well plates.After transfecting RAW264.7 monocytes/macrophages for 48 hours,the supernatants of the Mock,si-NC and si-Kif4 groups were collected,and added to 96-well plates at 200μl/well respectively.Continued to culture them in a 37 ℃,5%CO2 incubator.After 8 hours,the tube formation of different groups of HUVECs was observed.Results:1.After transfection with si-Kif4 for 24h and 48h,the morphology of the RAW264.7 cells showed no significant change compared with the si-NC group.2.After transfecting RAW264.7 cells for 48 hours,the expression of co-stimulatory molecules CD11c and CD80 on the surface of each group were detected by flow cytometry.The results showed no significant changes of CDllc and CD80 between groups.3.MTT results showed that there was no significant difference in the proliferation activity among the Mock,si-NC and si-Kif4 groups on the first day after transfection(P>0.05).On days 2,3 and 4,the cell proliferation activity of the si-Kif4 group was significantly lower than that of the Mock group and the si-NC group(P<0.05).There was no significant difference between the Mock group and the si-NC group(P>0.05).4.HUVECs tube formation experiment showed that the cell supernatant of si-Kif4 group significantly enhanced the tube formation of HUVECs compared with the Mock group and si-NC group,and the difference was statistically significant(P<0.01).These results suggested that Kif4 is able to promote RAW264.7 monocytes/macrophages proliferation,and the cell supernatant can inhibit the tube forming ability of HUVECs,but the effect on the morphological changes of RAW264.7 monocytes/macrophages and on the expression of cell surface CD11c and CD80 is not significant.Part Ⅲ Regulation of Kif4 on VEGFR1 expression in RAW264.7 monocytes/macrophages and its signaling pathwayObjective:To investigate the regulation of Kif4 on VEGFR1 expression and its signaling pathway in RAW264.7 monocytes/macrophages.Metheds:1.Detection of the effect of Kif4 on the expression of VEGF-A in RAW264.7 cells:At 24h and 48h following transfection of the RAW264.7 cells,the expression of VEGF-A mRNA in the Mock,si-NC and si-Kif4 groups was detected.At 48h,the cell supernatants of the Mock group,the si-NC group,and the si-Kif4 group were collected,and the VEGF-A enzyme-linked immunosorbent assay(ELISA)kit was used to detect the VEGF-A protein expression in the supernatant of each group.2.Detection of the effect of si-Kif4 on the expression of VEGFR1 and sVEGFR1 in RAW264.7 cells:At 24h and 48h following transfection of the RAW264.7 cells,the total RNA of each group was extracted,and the expression of VEGFR1 in the Mock,si-NC and si-Kif4 groups was detected.At 48h,total protein in cells and cell supernatant of each group were collected,and change of the expression of VEGFR1 in the transmembrane form(mVEGFR1)of the Mock,si-NC and si-Kif4 groups,and the expression of total VEGFR1 protein in each group were analyzed.The secretion of sVEGFR1 protein in cell supernatant was detected by ELISA.3.To detect whether si-Kif4 regulates VEGFR1 expression via AKT signaling pathway:At 24h following transfection of the RAW264.7 cells,RT-PCR was used to detect the expression of Akt mRNA in Mock,si-NC and si-Kif4 groups.At 48h post transfection,the total protein was collected and the expression of Akt and phosphorylated Akt(p-Akt)in Mock,si-NC and si-Kif4 groups were detected.At 48h,the AKT signaling pathway agonist insulin-like growth factor(IGF-1)(100 ng/ml)was added to the si-NC and si-Kif4 groups.Western Blotting was used to analyze the expression of Akt and phosphorylated Akt(p-Akt)at Omin,15min,30min,and 60min.At 24h following transfection of the RAW264.7 cells,IGF-1(100ng/ml)was added to si-NC and si-Kif4 groups.After 36h,the total RNA was collected,and the expression of VEGFR1 mRNA was analyzed.Meanwhile,the total protein of the cells was collected to analyze the expression of VEGFR1,and the supernatant of the cells was collected to detect the secretion of sVEGFR1.4.Effect of AKT pathway on the expression of VEGF-A after activation:At 24h following transfection of the RAW264.7 cells,IGF-1(100ng/ml)was added to the si-NC and si-Kif4 groups,respectively.After 36h,total RNA was collected,and the mRNA expression of VEGF-A was analyzed by RT-PCR.Then the supernatant of the cells was collected and the expression of VEGF-A protein was examined by ELISA.Results:I.At 24h following transfection of the RAW264.7 cells,the expression of VEGF-A mRNA was significantly decreased compared with the control group(P<0.05).At 48h following transfection,there was no significant change in the expression of VEGF-A mRNA(P>0.05).The expression of VEGF-A in the supernatant of si-Kif4 group was significantly higher than that of the control group(P<0.05).2.The expression of VEGFR1 mRNA was significantly decreased in RAW264.7 monocytes/macrophages after 24h transfection with si-Kif4(P<0.05).At 48h following transfection,the expression of VEGFR1 mRNA was not significantly changed(P>0.05).There was no significant change in the expression of VEGFR1 on cell membrane,while the expression of total VEGFR1 protein was decreased(P<0.05),and the secretion of sVEGFR1 in the cell supernatant was decreased(P<0.05).3.At 24h and 48h following transfection with si-Kif4 into the RAW264.7 cells,the expression of Akt mRNA among the groups was not significantly changed(P>0.05).After 48 hours of transfection,the expression of Akt protein in each group showed no significant change(P>0.05),but the expression of p-Akt in si-Kif4 group was significantly lower than that in the control group.After 30 min of IGF-1 addition,the level of p-Akt in the si-Kif4 group began to increase,and there was still agonism after 60 min,but the change of Akt was not significant(P>0.05).4.After 36h of IGF-1 addition,the expression of VEGFR1 mRNA was significantly increased in the si-Kif4 group(P<0.05),and the expression of VEGFR1 was increased(P<0.05).The expression of sVEGFR1 protein in the supernatant of the cells was also higher than that in the control group,and the expression of mVEGFR1 on the cell surface was significantly increased(P<0.05).5.After 36 hours of IGF-1 addition,there was no significant difference in the expression of VEGF-A mRNA between the si-NC and si-Kif4 groups(P>0.05),and the expression of VEGF-A in the supernatant was decreased(P<0.05).The results indicate that in RAW264.7 monocytes/macrophages,Kif4 promotes the expression of VEGFR1 mRNA and protein and the secretion of sVEGFR1 by activating the AKT signaling pathway,which reduces the concentration of extracellular free VEGF-A and inhibits the regulation of angiogenesis of RAW264.7 cells.Conclusion:Kif4 promotes the proliferation of RAW264.7 monocytes/macrophages and is involved in the regulation of physiological functions such as cell survival,innate immune response,lipid metabolism and phagocytosis.Through the AKT signaling pathway,Kif4 promotes the expression of VEGFR1 mRNA and protein and the secretion of sVEGFR1 in RAW264.7 cells,thereby reducing the concentration of extracellular free VEGF-A and inhibiting the regulation of angiogenesis of monocytes/macrophages. |
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