Identification Of LOX Interacting Proteins And Observation Of LOX Interacting Protein To Affect Migration And Invasion Ablity Of Breast Cancer Cells

Objective1.To verify the interacting proteins of LOX and to observe the interaction location of them.2. To observe the interaction between LOX and XRCC5 protein complexes affect migration and invasion ablity of breast cancer cells.Methods1.We established the lentivirus vector combined LOX with tandem affinity tags (2xstrep II-FLAG) and transfected to MDA-MB-231 cells. The cell line stably expression of fusion protein preliminary detected by realtime fluorescence PCR and western blot.2. Use the Co-IP and immunofluorescence co-localization to verify that LOX interacting with Histone H1.1 and XRCC5 in breast cancer cells.3.We adopt the techniques of overexpression lentivirus virus vector.We respectively established stable LOX and XRCC5 expression vectors. Separate transfection and co-transfection them to low metastasis potential and LOX lower expression of MCF 7 cells. Which into untransfection group, LOX empty vector group, LOX transfection group, XRCC5 empty vector group, XRCC5 transfection group,LOX and XRCC5 empty vector co-transfection group and LOX and XRCC5 co-transfection group. Application fluorescence-activated cell sorting to establish LOX and XRCC5 co-transfection cell and LOX and XRCC5 empty vector co-transfection cell. Western blot and real time-PCR test the protein expression levels of LOX and XRCC5 in the cells of every group. We respectively test the migration and invasion ability of different transfection groups cells by transwell migration and invasion experiments.Results1.Under the condition of the MOI=10, recombinant lentivirus vector expression vectors LOX-SF were transfected into human breast cancer cell line MDA-MB-231,and fluorescence analysis indicated that>90% of cells showed fluorescence signal after 96 hours which could indirect evaluate transfection efficienc. Recombinant protein (LOX-SF) and ontology protein (LOX) were detected in the experimental group and the band for recombinant protein LOX-SF were not seen in non-transfection group or blank transfection. Ontology protein (LOX) was also detected in non-transfection group or blank transfection. The median expression level of endogenous LOX mRNA 2-△△Ct in cells from the untransfected group was set as 1.00±0.00,LOX mRNA in the LOX transfection group and negative vector group were average 15.32±0.12 and 1.18±0.01, respectively. These results showed that the expression levels of LOX mRNA were significant increase in transfected MDA-MB-231 cells (p<0.05) and the expression levels of LOX mRNA in negative vector group were no significant increase in untransfected group (P>0.05).2. Co-IP respectively validation of both Histone H1.1 and XRCC5 could be detected in the anti-LOX immune complexes, and the bands of the Co-IP sample were consistent with those of the input sample. 3. Reverse Co-IP respectively validation of LOX could be detected in the anti-XRCC5 and anti-Histone H1.1 Co-IP immune complexes and the bands of the Co-IP sample were consistent with those of the input sample 4. To examine the localization of the interactions of LOX with Histone H1.1 and XRCC5 in endogenous positioning analysis in MCF-7 and MDA-MB-231 cells, Indirect Immunofluorescence and Confocal Microscopy were performed. Confocal microscopy confirmed the co-localization of LOX and Histone Hl.l in cytoplasm in two cell lines and the co-localization of LOX and XRCC5 were positioned only around the nucleus 5. Under the condition of the MOI=10, LOX expression vectors and XRCC5 expression vectors were co-transfected into human breast cancer cell line MCF-7 cells.Then application fluorescence-activated cell sorting to establish LOX and XRCC5 co-expression stable cell lines and LOX and XRCC5 empty vectors,we sort that LOX and XRCC5 empty vector transfection group selected cell transfection efficiency by 15.37% to 83.94%.LOX and XRCC5 express transfection group of cells streaming fluorescence detection, the transfection efficiency from 45.65% to 70.82%.6. To identify the expression levels of LOX-SF and XRCC5 after transfection, the expression of LOX-SF and XRCC5 was validated by Western blot and RT-PCR. Western blot results can detect LOX protein expression in MCF-7 and for lower expression.Recombinant protein (LOX-SF) and ontology protein (LOX) were detected in the experimental group and the band for recombinant protein LOX-SF were not seen in non-transfection group or blank transfection. Ontology protein (LOX) was also detected in non-transfection group or blank transfection. Compared with the normal control group and negative control group, XRCC5 protein expression was significantly up-regulated in cells transfected with XRCC5 expession vector. Compared with the normal control group and negative control group, XRCC5 protein expression and LOX-SF protein expression were significantly up-regulated in cells co-transfected with LOX and XRCC5 expession vectors. LOX mRNA 2-△△Ct respectively in the LOX transfection group,negative vector group,LOX and XRCC5 co-transfection group, LOX and XRCC5 empty vectors co-transfection group and untransfected group were average 241.04±45.81、2.86±0.41、207.12±23.40、2.06±0.06、1.00±0.00, respectively. These results showed that the expression levels of LOX mRNA were significant increase in LOX transfection group and LOX and XRCC5 co-transfection group cells (p<0.01) and the expression levels of LOX mRNA in negative vector group were no significant increase in untransfected group (P>0.05). XRCC5 mRNA 2-△△Ct respectively in the XRCC5 transfection group,negative vector group,LOX and XRCC5 co-transfection group,LOX and XRCC5 empty vectors co-transfection group and untransfected group were average 6.57±0.85、1.10±0.27、 4.29±0.05、0.76±0.15、1.00±0.00, respectively. These results showed that the expression levels of XRCC5 mRNA were significant increase in XRCC5 transfection group and LOX and XRCC5 co-transfection group cells (p<0.05) and the expression levels of XRCC5 mRNA in negative vector group were no significant increase in untransfected group (P>0.05).7.Transwell migration experiment observed migration cell number, relative to the empty vector group(71.67±3.51), LOX transfection group(358.67±18.58), XRCC5 transfection group (200.67± 11.02) and LOX and XRCC5 co-transfection group (513.33+ 15.28) ability of cell migration were obviously increased (P<0.05). LOX and XRCC5 co-transfection group through the maximum number of cells in total, with separate transfection group to the LOX transfection group and XRCC5 transfection group, significant difference(P<0.05), significantly enhance its capability of cell migration.8. Transwell invasion experiment observed invasion cell number, LOX transfection group (97.00±7.55), XRCC5 transfection group(74.33±6.66) and LOX and XRCC5 co-transfection group(140.00±24.98) ability of cell invasion were obviously increased (P< 0.05) than the empty vector group(14.67± 4.73). What’more, the ability of cell invasion LOX and XRCC5 co-transfection group is stronger than LOX transfection group and XRCC5 transfection group, the difference was statistically significant (P <0.05).Conclusion1.There is a direct or indirect interaction between LOX and XRCC5. 2. There is a direct or indirect interaction between LOX and Histone H1.1. 3. Increasing the expression of LOX and XRCC5 can enhance cell migration and invasive ability in MCF-7 cells.In addiction,LOX and XRCC5 co-transfection can further promote the migration and invasion ability of MCF-7 cells. The LOX-XRCC5 interaction may synergy promotes cell motility and invasion.