The Biological Behaviours And Proteins Expressed Are Changed After The Knock-Down Of UBE3A In MDA-MB-231 Cells

Part 1 The Biological Behaviours are Changed after the Knock-down ofUBE3A in MDA-MB-231 CellsObjective: After the knock-down of UBE3 A by sh RNA, the biological behaviours were observed to clarify the role of UBE3 A in MDA-MB-231 cells.Methods: The vectors of three UBE3A-sh RNAs and negative-sh RNA were constructed; the 239 T cells transformation mediated by lipo2000 was performed, and then MDA-MB-231 cells were transfected; the highest efficiency sh RNA was obtained by q PCR; after the knock-down of UBE3 A, the protein level of UBE3 A was detected by Western blotting, and transwell chamber assay was used to assess invasion of cells, and cell proliferation was determined by Cell Counting Kit-8(CCK-8), and cell cycles were detected by Flow Cytometry; the results were statistically analyzed by SPSS 22.0, P <0.05 regarding as statistically significant difference.Results: The vector of LV-UBE3A-sh RNA was successfully constructed and obtained; comparing to blank control and negative control, the expression of gene and protein of UBE3 A decreased with respective 89.5% and 45.3%(p<0.05), and the proliferation and invasion were interfered(p<0.05), and cell cycle was changed(p<0.05) while most cells stayed on the S phase after the knock-down of UBE3 A. In addition, there was no significant difference between blank control and negative control(p>0.05).Conclusions: After the knock-down of UBE3 A, the biological behaviors of proliferation, cycle and invasion were changed in MDA-MB-231 cells, suggesting that UBE3 A can plays an important role in breast cancer cells, which may be a potential target for treatment of breast cancer.Part 2 The Proteins Expressed are Changed after the Knock-down ofUBE3A in MDA-MB-231 CellsObjective: We observed the differential proteins by proteomics analysis after the knock-down of UBE3 A to study the potential mechanism of effects on the biological behaviours of MDA-MB-231 cells.Methods: The 2-DE was used to separate total proteins, and then the differential proteins were obtained by PDQuest 7.1 and detected by MALDI-TOF-TOF MS/MS; DAVID Functional Annotation and String tools were used to analyze the interactions among the differential proteins, incuding cellular components, biological pathways and molecular function.Results: The 28 differential proteins were identified by MALDI-TOFTOF MS/MS, and 23 proteins were matched in DAVID database including high expression of 4 proteins and low expression of 24 proteins after the knock-down of UBE3A; the 23 protens were classified according to the biological pathway, cellular components and annotation of molecule functions performed by DAVID Functional Annotation with respective 100%, 87.7% and 100.0%, especially, some proteins were classified as which are related with ubiquitin-proteasome system and glucose metabolism; there were direct or indirect relations among the 23 proteins by String tool.Conclusions: There were direct or indirect relations among the 23 proteins, and their functions were changed, especially, both the ubiquitinproteasome system and glucose metabolism, which may be the causes to alter the biological behaviors of breast cancer cells.