Invasion Of BT-549Cells Is Affected By The Level Of AnnexinA2and The Change Of Proliferation And Apoptosis Mediated By UBE3A In Breast Cancer

Background:UBE3A (ubiquitin protein ligase E3A or human papilloma virusE6-associated protein, E6-AP) is an important member of ubiquitinproteasome systems, and E6/E6-AP complex targets p53for ubiquitinationand degradation via the UPP (ubiquitin-proteasome pathway) in cervicalcancer. It has been reported that abnormal expression of UBE3A was found ina variety of tumors such as prostate cancer, cervical cancer and breast cancer.But how the loss of function of UBE3A causes disease pathogenesis is poorlyunderstood now. We previously reported that UBE3A was over-expressed inbreast cancer and related with its pathological mechanism, so we infer thatUBE3A may be play an important role in breast cancer as an ubiquitin-proteinligase activity.Recently, a variety of proteins were found to be degraded by the mannermediated by UBE3A, including BAK (Bcl-2homologous antagonist killer),c-MYC, p27, Mcm-7(minichromosome maintenance protein7, which isinvolved in DNA replication), PR-B (progesterone receptor-B protein) andannexinA1protein. So far these cellular substrates of E6-AP have beenidentified and characterized, however, the significance of these interactionsinto disease context is unclear.Annexin A2, a member of annexins family, was reported to beabundantly expressed in many cancer tissues and is believed to play an important role in tumorigenesis and cancer progression. It is reported thatannexin A2was inhibited by RNAi to decrease metastasis and invasion ofbreast cancer cells. In addition, we verified that annexin A2wasoverexpressed and ubiquitinated in breast cancer tissues[14], so annexin A2may contribute to pathological pathway of breast cancer mediated byubiquitination, and it may be a novel substrates of E6-AP.It was not clear that the function of UBE3A was associated withproliferation, invasion and apoptosis in breast cancer, so in this study we usedRNAi to knock down UBE3A in breast cancer cell BT-549, and then toresearch the function of UBE3A in breast cancer cells.Objective: RNA interference(RNAi) was used to study the expression ofannexinA2affected by UBE3A, the proliferation, apoptosis and invasion ofBT-549cells.Methods: Three pairs of small interfering RNA fragments and onenegative small interfering RNA fragement targeted to the UBE3A weredesigned by the siDirect Version2.0and transported by theLipofectamine2000to the BT-549cells. The effect of knockdown wasdetected by RT-PCR and Western blotting, meanwhile, the expression ofannexinA2was detected too. Cell proliferation was determined by CellCounting Kit-8(CCK-8); flow cytomerey and transwell chamber assay wereemployed to assess cell apoptosis and invasion, respectively.Results: After treatment with three specially siRNAs of UBE3A for72h,the mRNA expression level of UBE3A were down-regulated compared to thenegative siRNA, Lipofectamine2000and the blank control group, andsiRNA1was the most effective siRNA for UBE3A.The trend of proteinexpression levels were consistent with the mRNA expression levels of UBE3A, similarly, the expression of annexinA2were down-regulated.Cellular proliferation and invasion of the siRNA1group was inhibitedcompared to the negative siRNA and the blank control group, and Cellularapoptosis of UBE3A-siRNA1group was increased compared to the blankcontrol group and the negative-siRNA.Conclusion: After knockdown of UBE3A, expression of annexinA2wasdown-regulated, meanwhile, proliferation and invasion were inhibited, butapoptosis promoted in BT-549cells. UBE3A may regulate the level ofannexinA2, and results in promotion of proliferation and invasion andsuppression of apoptosis in BT-549cells.