| Objective: 1.CENP-U-related genes and angiogenesis-related genes in triple-negative breast cancer cell lines were screened by next generation sequencing technology.2.Through in vitro experiments,CENP-U stable expression cell lines were established in MDA-MB-231 and CAL51,and the expression of CENP-U in different cell lines and the regulation of angiogenesis were analyzed.3.The in vivo experiment was used to analyze the correlation between CENP-U gene and angiogenic factors in mouse model and its effect on angiogenesis.Methods: 1.Analyze gene function using next generation sequencing technology;analyze the relationship between genes and diseases;analyze genomic,chemical and systemic function information;analyze human responses and biological pathways.Sequencing analysis: Total RNA was extracted,followed by m RNA enrichment,double-stranded c DNA synthesis,followed by end-repair,addition of A and linker,selection of predicted fragments,PCR amplification,establishment of a library,and then sequenced.2.Real-time quantitative PCR was used to detect the expression of CENP-U in different cell lines.The expression of CENP-U in different cell lines was detected by Western blotting.3.The MDA-MB-231 and CAL51 cell stable lines were constructed by transfecting the plasmid,and the control group(vector group)and the down-expression group(sh CENP-U group)were set.MTT colorimetric assay and plate cloning were used to detect the proliferation and cloning in cell stable lines.4.Using cell wound-healing assay to detect the migration ability of cells in different groups in cell stability.Through tube formation experiments,human umbilical vein endothelial cells were used to detect the formation of small tubes in the control group and the down-expression group in the MDA-MB-231 and CAL51 cell stable lines,respectively.5.The proteins of MDA-MB-231 and CAL51 cells were extracted separately.The protein levels of VEGFA and HIF-1A in the control group and the down-expression group were detected by Western blotting.The real-time quantitative PCR method was used to detect the m RNA level of PTGS2 in the control group and the down-expression group.Western blotting method was used to detect PTGS2 protein level.6.After modeling the tumor formation of nude mice,the expression of VEGFA in the serum of the control group and the down-expression group were detected by ELISA.7.After modeling the tumor formation of nude mice,the protein of solid tumor was extracted,and the expression levels of VEGFA,HIF-1A,p AKT and STAT3 were detected by Western blotting.8.After modeling the tumor formation of nude mice,the tumors of the mice were taken and the expression of VEGFA,HIF-1A,CD31 and CD34 in the tumor tissues were detected by immunohistochemistry.Results: 1.A total of 3223 genes related to down-expression of CENP-U were screened,266 genes related to the over-expression of CENP-U,and 105 overlapping genes were selected:IL1RL1/RP11-875O11.3/HIST1H4H/RP11161I2.1/ABCC9/RELB/HIST1H2AC/CA9/ATF3/TNFRSF9/IL32/HIST1H2BJ/EGR2/IL1A/MUC5B/TNFAIP3/AQP1/PLA2G4C/NEURL3/RND1/FILIP1L/LINC00944/CENPU/HIST1H2BC/ZNF641/EF TUD1P1/OSGIN1/MIR210HG/MZT1/ALOXE3/VLDLRAS1/PPFIA4/NR1D1/GGT5/HIST1H2BD/CPLX1/HSPB8/LUCAT1/CYP1B1/HIST1H1C/SSC5D/GADD45G/P TGS2/INSC/EGR1/BTF3L4/HIST2H2BE/ITGB3/INHBB/SSPO/FGF21/LFNG/NCF2/ARC/HIST1H2BN/GSKIP/RP11467L13.5/ARHGDIB/PIWIL2/OLFML2A/P4HA3/RP11301G19.1/FLT1/ANKFN1/SPANXB1/SPNS3/TRAF1/BIRC3/RP111055B8.4/CD22/LINC00622/NTM/IFIT2/MRPS24/SCD/OVGP1/CD163L1/BMP6/NGF/ALG5/ANGPT2/LURAP1LAS1/PAIP1/TOLLIPAS1/MIPEPP3/AFP/MYO5B/NFKBIE/L GALS9C/CYP4F26P/RP11430C7.5/DDX60/RAET1L/SREBF1/S100A1/FAM71F2/SNX16/LAIR2/RNF224/GRIK5/PLCXD1/AKNAD1/RP11-88I21.1/HKDC1/ITGB2.Nine genes were associated with down-expression and over-expression of CENP-U: CD22,PTGS2,IFIT2,NR1D1,NGF,DDX60,The remaining 2 genes are non-significant genes and another is CENP-U.2.Through enrichment analysis,106 genes related to angiogenesis were found to change after CENPU changes: HSPG2/AQP1/EPHB2/TGFB2/NRP1/CAV1/ITGB3/ADGRG1/ADGRA2/NOTCH3/THBS1/FGF18/CSPG4/NOV/PLXND1/LOXL2/COL8A1/ADGRB2/PRKCA/JAG1/IHH/F3/ADM2/PLAU/BRCA1/COL4A2/FLT1/C5/CCBE1/MMP19/EMP2/APOLD1/HIF-1A/FN1/CYP1B1/TGFBI/E2F7/RAMP1/COL4A1/SOX18/MCAM/PNPNA6/A DRB2/EFNB2/DLL4/HIPK2/ATPIF1/VEZF1/TBX1/BMPER/SASH1/EDN1/E2F8/FGF8/PDGFRB/HIPK1/GATA2/ANGPT2/MTDH/C5AR1/ELK3/SPARC/IL1A/GNA13/ANGPTL4/NFATC3/SEMA3E/WNT7B/SPHK1/PAXIP1/BAK1/ROBO1/CRHR2/NFATC4/NR4A1/WNT5A/EPHB3/LAMA5/LEF1/ADAM8/PTK2/UBP1/CD34/GT F2I/MAP2K5/ROCK1/WARS/ARHGAP22/PTGS2/KLF5/HPSE/TEAD2/PIK3CB/N OTCH4/FMNL3/PTPRM/VEGFA/ITGB1/EIF2AK3/EGFL7/TNFRSF12A/PLXDC1/TNFAIP3/ERAP1/BMP4/PIK3 CG.3.The CENP-U primers were constructed and verified by real-time quantitative verification in multiple cell lines.Real-time quantitative PCR were performed in multiple cell lines to find that CENP-U was highly expressed in MCF-7,CAL-51 and MDA-MB-231 cell lines.Western blotting assays were performed in multiple cell lines to find that CENP-U was highly expressed in MCF-7,CAL-51,MDA-MB-231 and CAL/DOX cell lines.4.The MTT colorimetric assay was performed in the CAL-51 and MDA-MB-231 stable lines.After Measured OD value,the cell growth curves of the control group(vector group)and the down-expression group(sh CENP-U group)were plotted.The OD value in the down-expression group was significantly lower than that in the control group,and the cell proliferation ability in the down-expression group was significantly decreased.The plate cloning experiments were performed in the stable lines of CAL51 and MDA-MB-231,respectively.After cell counting,the cell growth curves of the control group and the down-expression group were plotted respectively.The results showed that the number of cell clones in the down-expression group was significantly lower than that in the control group.The cell cloning ability of the down-expression group was significantly decreased.5.The cell wound-healing assay was performed in the CAL-51 and MDA-MB-231 stable lines,respectively,and the results showed that the cell migration ability in the down-expression group was significantly slower than that in the control group.Tube formation assay results: the length of the tube formation branch in the downexpression group was significantly lower than that in the control group in the CAL51 stable cell line;the tube formation length in down-expression group was significantly lower than that in the control group in the MDA-MB-231 stable cell line,After the expression of CENP-U decreased,the ability of tubule formation was significantly weakened.6.Western blotting was performed in the stable lines of CAL-51 and MDA-MB-231,respectively.The results showed that the expression of VEGFA and HIF-1A was down-regulated after CENP-U decreased.Real-time quantitative PCR was performed in the stable lines of CAL-51 and MDA-MB-231,respectively.The results showed that the m RNA expression level of PTGS2 was also decreased after the m RNA expression level of CENP-U was decreased.Western blotting was performed in the stable lines of CAL-51 and MDA-MB-231,respectively.The results showed that the protein expression level of PTGS2 was also decreased after the protein expression level of CENP-U was decreased.7.After the nude mice were successfully modeled,the growth curve was drawn and the results showed that the growth rate of the tumor in the down-expression group was slower than that in the control group.The results showed that the tumor volume of the down-expression group was lower than that of the control group.After the expression of CENP-U decreased,the proliferation of tumor cells was significantly reduced.The serum of the living mice was tested by ELISA,and the expression level of VEGFA of down-expression group was significantly lower than that of the control group,After the expression of CENP-U decreased,the expression level of VEGFA decreased,too.8.The immunohistochemistry of the tumor tissues in mice showed that the positive expression of VEGFA in the down-expression group(sh CENP-U)was significantly lower than that in the control group.The positive expression of HIF-1A in the down-expression group was significantly lower than that in the control group,the positive expression of CD31 was significantly lower than that of the control group,too.The positive expression of CD34 in the down-expression group was significantly lower than that of the control group.The protein of tumor tissues in mice was extracted and measured by Western blotting,The results showed that the expression of VEGFA,HIF-1A,p AKT and STAT3 which are related to angiogenesis were also down-regulated after CENP-U decreased.Conclusion: 1.Using next generation sequencing technology,through enrichment analysis,it was found that CENP-U is closely related to tumor angiogenesis.2.CENP-U can affect the expression of angiogenic factors in triple-negative breast cancers cell lines and in animal model of triple-negative breast cancer.3.CENP-U can affect angiogenesis in triple-negative breast cancers. |
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