The Study Of The Role And Mechanism Of CDK2 In Gastric Cancer

1.Background and ObjectiveGastric cancer is one of the most common cancers in the world,It ranks second in global cancer-related deaths.Although significant progress has been made in diagnosing and treating cancer,some progress has been made in improving the mortality associated with gastric cancer.In addition,it is estimated that the mortality associated with gastric cancer will continue to increase in the future.Therefore,it is urgent to identify new biomarkers that can be used to predict the prognosis and clarify potential molecular mechanisms.In recent years,abnormal tumor cell cycle and differentiation play a decisive role in the maintenance of tumor malignancies,and the cycle and differentiation of targeted cancer cells are considered as new targets for the treatment of cancer.Changes in the genes involved will cause the cell cycle to run out of control and to run supernormal,leading to tumors.In recent years,the research on the relationship between cell cycle regulation gene,cell differentiation and cancer has become the focus.CDK2 and CDX2 to participate in the regulation of cell cycle and cell differentiation,cell differentiation and cell cycle regulation are two independent processes,but they are closely related,as the stagnation of the cell cycle is a precondition for terminal differentiation,the transition between the two play an important role in the process of cell growth.This section observation build good slow virus carrier of gastric cancer cells after transfection,the gastric cancer cell proliferation,migration and invasion ability,the influence of the cell cycle and apoptosis,clear purpose CDK2 and CDX2 biological effect of gastric cancer cells.Application of real-time quantitative PCR and Western Blot protein imprinting experimental detection target genes and its downstream gene mRNA and protein level of change,and the purpose of target genes and CDK2 and CDX2 expression level of correlation analysis,further clarify the mechanism of action of CDK2 and purpose of CDX2 in gastric cancer.CDK2 is a cell cycle dependent kinase that normally regulates cell cycle progression,DNA damage response,and is reported to increase in many cancers.However,its role in cancer cell metabolism is rarely discussed.In this study,we proved that silent CDK2 inhibited the aerobic glycolysis ability of gastric cancer cell lines.Mechanism exploration shows that silent CDK2 can increase the expression of SIRT5 tumor suppressor genes.Finally,the physiological role of SIRT5 in the process of proliferation and glycolysis was studied in gastric cancer cells.To sum up,this study reveals the new role of CDK2/SIRT5 axis in gastric cancer and elucides the future research on the metabolism of gastric cancer cells.In order to verify the accuracy of the account of the above assumptions,this study proposed by i TRAQ technology screening of different proteins,by building a slow virus vector and transfection gastric cancer cells to establish a stable cell lines,and through the fluorescence quantitative polymerase chain reaction(PCR)to detect the expression of expression in gastric cancer cell lines.Objective to observe the function of the target gene in vitro and in the tumor cells of gastric cancer,and to detect the target gene by using the real-time quantitative PCR and Western Blot.The effect of CDK2 on the aerobic glycolysis of gastric cancer was investigated by using colony cell formation experiment and apoptosis detection technology.Cell glycolysis ability was determined by extracellular acidification rate(ECAR).Mitochondrial respiration was measured by oxygen consumption(OCR).To clarify the relationship between CDK2,CDX2 and SIRT5 in gastric cancer,and to clarify the function and mechanism of CDK2 in the development of gastric cancer.2 Methods 2.1 identification and verification of gastric cancer cell screening differential protein based on iTRAQ technology transfection and silent expression of CDK2.The gastric cancer cells with CDK2 expression and silencing expression were extracted by cell lysis solution,and the protein concentration was determined.Through the enzymatic hydrolysis of the protein,the liquid chromatography mass spectrometry was analyzed,labeled peptides and salt removal,and the high PH reversed-phase liquid chromatography was separated in the labeled peptides,and the mass spectrometry was finally detected.After the qualitative and quantitative analysis of protein,the mass spectrogram was input into the Proteome Discoverer software(PD)(version 1.4.0.288,thermo Fisher Scientific),the software first screened the mass spectrogram.By quantitative and qualitative Mascot search repository PD after extraction of the spectra with Mascot(version 2.3.2,Matrix Science)to search,search after the PD software according to the Mascot search results and the spectra of the first step after screening for quantitative analysis.2.2 construction of fluorescence lentivirus vectors containing the target CDK2,CDX2 and ck2-CDX2 overexpression targets,and constructing the fluorescence lentiviral vector containing the expression of CDK2 expression.The virus particles of CDK2,CDX2 and ck2-CDX2 were collected and reconstructed by the positive recombinant cloning and sequencing,and the target virus particles were collected and reorganized to express the silent CDK2.Transfected virus vectors successfully obtained mgc-823-cdk2,mgc-823-CDX2 and mgc-823-cd2-CDX2(or mgc-823-oe group),and successfully constructed mgc-823-nc as the negative control group.Transfection was successful in mgc-803-cdk2 rnai and scg-7901-CDK2 RNAi,and the negative control group was constructed respectively.The expression of CDK2,CDX2 and ck2-CDX2 in transfected cell lines was detected by puromycin,and mRNA expression of CDK2 was detected by fluorescence quantitative PCR.2.3 the effects of CDK2,CDX2 and ck2-CDX2 on the proliferation capacity of gastric cancer cells were detected by cck-8 detection.Transwell method was used to test the influence of CDK2,CDX2 and ck2-CDX2 on the invasion ability of gastric cancer cells,and to identify the migration and invasion ability of different gastric cancer cells.Flow cytometry was used to detect the effects of CDK2,CDX2 and ck2-CDX2 on gastric cancer cell cycle and apoptosis.The expression of mRNA in mgc-823-cdk2,mgc-823-CDX2,mgc-823-cdk2-CDX2 and mgc-823-nc was determined by fluorescence quantitative PCR.The protein expressions of mgc-823-cdk2,mgc-823-CDX2,mgc-823-cd2-CDX2 and mgc-823-nc were detected by Western blot.With stable cell line cell suspension 200 mu l(7 x 106 / ml)on the left side of the axillary subcutaneous in nude mice subcutaneous injection and build one stomach cancer nude mice transplantation tumor model,measuring every 3 days time,tumor length to diameter and short diameter(b)(a)measured with vernier caliper,the calculation of relative volume of tumor(RTV)and tumor growth curve drawing,the mice were euthanized at cervical dislocation method(3 weeks),full cut in tumor tissue and HE staining confirmed the tumor tissues,and CDX2 line and CDK2 antibody immunohistochemical determination of CDX2 and expression of CDK2 in tumor volume;With stable cell line cell suspension 200 mu l(6 x 105 / ml),caudal vein in nude mice were injected into transfer model and build the stomach nude mice,mice were euthanized at cervical dislocation method(5 weeks),taking nude mice liver and lung tissue,complete with HE staining detection of liver metastasis of gastric cancer.2.4 construction of the virus vector with silent CDK2 expression and SIRT5 overexpression,transfected to scg-7901 and MGC803 cells and established stable strains;The mRNA content of stable strain was detected by quantitative PCP.The protein content of stable strain was detected by western blot.The cell value was measured by CCK-8 reagent.The cell cloning of SIRT5 overexpression group was detected by using colony forming experiment.The apoptosis of SIRT5 overexpression group was detected by flow cytometry.Cell apoptosis was used to detect the effect of silencing CDK2 and SIRT5 on aerobic glycolysis of gastric cancer cells.3.Results 3.1 through the iTRAQ technology out differences in protein: guanine nucleotide binding protein G,tail protein,72 kda ? collagenase,ubiquitin carboxyl terminal hydrolase 38 11 genes,etc.Through the data analysis of Gene Ontology,it was found that in the proteins we screened,the unfold-protein response and the related multiple pathways were found to be significant in many cases.The results showed that 0004674 protein serine/threonine kinase,0045095 keratin silk,0005882 intermediate filaments,0045111 middle wire cytoskeleton,cell membrane bound organelles,0043232,0043228 of membrane bound organelles in the two groups the largest genome differentially expressed.The expression of CDK2 was found to be maximal in differentially expressed protein.The Pathway analysis method was used to find that the cytotoxic effect mediated by natural killer cells,autoimmune thyroid disease,antigen processing and presentation were the most relevant.It is suggested that CDK2 is closely related to the morphological maintenance,differentiation and immunity of gastric cancer cells.3.2 fluorescence quantitative PCR and western blot test showed that CDK2,CDX2 and cdk2-CDX2 were successfully constructed to express lentiviral vectors and silent CDK2 expression of lentiviral vectors;It also showed that CDK2,CDX2 and cdk2-CDX2 overexpression of gastric cancer cell lines were successful and could be used for the next experiment.It also showed that the expression of silent CDK2 expression of gastric cancer cell line was successful and could be used for the next experiment.3.3 CCK-8 test results showed that the proliferation ability of mgc-823-oe and mgc-823-CDX2 cells was significantly reduced compared with the negative control group.The proliferation ability of mgc-823-CDK2 cells was significantly increased(P = 0.05).Transwell test results showed that compared with the negative control group,mgc823-cdk2 cell migration and invasion ability were stronger,and mgc823-oe cell migration and invasion ability were weaker than mgc823-CDX2.Mgc823-CDX2 cell migration and invasion and mgc823-nc,mgc-823-oe were weaker(P<0.05).Flow cytometry instrument testing results show that compared with negative control group,the expression of CDX2 and OE experimental gastric cancer cell line MGC-823 cell cycle arrest in G0 / G1 phase and S phase cell proportion significantly reduced,the difference was statistically significant(P < 0.05).The cell cycle of gastric cancer cell line mgc-823 was significantly reduced in G0/G1 phase,and the proportion of S phase cells increased significantly(P<0.05).The apoptosis rate of mgc823-cdk2 was decreased,and the apoptosis rate of mgc823-CDX2 and mgc823-oe cells increased(P <0.05).Fluorescence quantitative PCR experiments show that compared with negative control group,experimental group target base MGC-CDK2 by CDKN1 A,TP53I3,increased Rock1 mRNA expression,the experimental group target MGC-CDX2,MGC-OE CDKN1 A,TP53I3,increased Rock1 mRNA expression;Western blot results showed that compared with the negative control group,the expression of MGC823CDK2 protein was increased in the experimental group,and the protein expression of MGC823CDX2 and MGC823 OE decreased.Nude mice subcutaneously transplanted tumor model of tumor volume measurement results show that compared with negative control group,MGC-823-CDX2 and MGC-823-OE set can express experimental gastric cancer cell lines in nude mice subcutaneous transplantation tumor model of tumor(P?0.05)with minor volume,MGC-823-CDK2 set can express experimental gastric cancer cell lines in nude mice subcutaneous transplantation tumor model of larger tumor size(P?0.05);HE staining confirmed that the subcutaneous transplanted tumor tissue was malignant gastric cancer tissue;Compared with the negative control group,the expression rate of CDK2 in the subcutaneous transplanted tumor tissues of mgc-823-cdk2 group was significantly increased(P<0.05).The expression rate of CDK2 in mgc-823-CDX2 and mgc-823-oe group was significantly reduced(P<0.05).The expression rate of CDX2 in the subcutaneous transplanted tumor tissues of mgc-823-CDX2 group was significantly decreased(P<0.05).The expression rate of CDK2 in the subcutaneous transplanted tumor tissues of mgc-823-CDX2 and mgc-823-oe group was significantly increased(P<0.05).The results of pathological examination of liver and lung tissues of metastatic tumor model showed that the hepatic metastasis ability of mgc-823-CDX2 overexpression group was significantly decreased(P<0.05).Mgc-823-cdk2 expression group was significantly enhanced(P = 0.05).3.4 CDK2 on aerobic glycolysis has a positive role in regulation of gastric cancer: silence expression of CDK2 inhibit ECAR levels,shows that CDK2 is aerobic glycolysis is regulatory factors,CDK2 in the silence of the SGC-7901 and MGC-803 cells,the results show that the CDK2 in aerobic glycolysis positive role in restructuring,aerobic glycolysis GLUT1 gene,HK2,LDHA and PDK1 in real time quantitative PCR results showed that CDK2 knockout result in the expression of these genes to reduce(P?0.05);SIRT5 expression of CDK2 in gastric cancer cells negatively regulated: quantitative PCR results showed that the CDK2 reduces the SGC-7901 and MGC-803 cells SIRT5 expression of mRNA level(P?0.05)and western blot results showed that the expression of CDK2 reduces the SGC-7901 and protein level of SIRT5 MGC-803 cells(P?0.05).SIRT5 inhibited the proliferation of gastric cancer cells: CCK-8 cell viability assay,and the results showed that SIRT5 had negative regulation on the cell activity of gastric cancer cells(P < 0.05).Cloning experiments: the expression of SIRT5 in sgc-7901 and mgc-803 cells decreased(P < 0.05),and the overexpression of SIRT5 increased the apoptosis of sgc-7901 and mgc-803 cells(P < 0.05).4 conclusion 4.1 It was found that CDK2 expressed the largest amount of differentially expressed proteins through the iTRAQ technology.Pathway analysis showed that CDK2 was closely related to the morphological maintenance,differentiation and immunity of gastric cancer cells.4.2 It was test by fluorescence quantitative PCR and western blot and it showed that CDK2,CDX2,cdk2-cdx2 and SIRT5 were successfully constructed to express gastric cancer cell lines and silent CDK2 expression of gastric cancer cell lines.4.3 CDK2 on gastric cancer cell has a positive regulatory role,CDX2 has negative regulation effect to gastric cancer,double OE turn expression on gastric cancer cell has a negative regulatory role,in cell function experiment and mechanism were confirmed;CDX2 expression of CDK2 in expression group was obviously lower,the amount of expression of CDK2 in CDX2 expression group was obviously lower,prompt CDK2 and CDX2 exist mutual regulation during the cell cycle and differentiation of the tumor target provide new targets for gene therapy.4.4 Nude mice subcutaneous tumor transplantation tumor model,according to the results compared with negative control group,MGC-823-CDX2 and MGC-823-OE group overexpression of gastric cancer cell lines tumor growth inhibition of nude mice subcutaneous transplantation tumor model of MGC-823-CDK2 set can express the subcutaneous transplantation tumor model of experimental gastric cancer cell lines to promote nude mice tumor growth.4.5 CDK2 aerobic glycolysis of gastric cancer has positive role is to adjust,CDK2 SIRT5 expression showed a negative regulation of gastric cancer cells,SIRT5 inhibiting gastric cancer cell proliferation,SIRT5 inhibiting gastric cancer cell aerobic glycolysis,SIRT5 inhibiting gastric cancer cell tumor formation ability.The new role of CDK2/SIRT5 axis in gastric cancer was revealed,and future research on the metabolism of gastric cancer cells was eluciated.The innovation points: 1 It was based on the iTRAQ technology expression and silent expression of CDK2 in transfection gastric cancer cell screening and validation verification,differences in protein interaction network building is found find CDK2 differences in protein expression of the maximum,Pathway analysis,found CDK2 in the shape of the gastric cancer cells,differentiation and immune biology behavior are closely related.2.Based on cell function experiment,mechanism experiment and animal experiment,the mechanism of interaction between CDK2 and CDX2 in the cycle and differentiation mechanism of gastric cancer was suggested.CDK2 and CDX2 are regulated in the cell cycle and differentiation process,providing new targets for tumor target gene therapy.3 Silent information regulator SIRT5 in mitochondria may be CDK2 in aerobic glycolysis regulation of the downstream effects,SIRT5 in cell proliferation and negative role in the process of glycolysis,the expression of CDK2 inhibit SIRT5,inhibit the negative regulation of aerobic glycolysis,and has carried on the positive adjustment to aerobic glycolysis.The new role of CDK2/SIRT5 axis in gastric cancer was revealed,and a new target was provided for future research on the metabolism of gastric cancer cells.