Effect Of PpGalNAc-T1 And-T2 On Invasion、Metastasis And Chemosensitivity In Triple-negative Breast Cancer Cells

AbstractObjectivesTriple-negative breast cancer has poor curative effects to endocrine therapy or targeted therapy in the clinic, due to its negative expressed estrogen receptor or progesterone receptor and human epidermal growth factor receptor 2. To improve survival and prognosis rate, finding new target for TNBC pathogenesis and treatment is needed. In this study, differential expression of pp Gal NAc-Ts, α2,3 sialic acids and MMPs were studied in MDA-MB-231(TNBC), MCF-7(Luminal A) and MCF-12 A cells. Then, after knocking-down of highly expressed pp Gal NAc-T1 and-T2 in TNBC, changes of MMP14 and α2,3 sialic acids of O-linked glycan on MMP14 were detected to illustrate the important role of pp Gal NAc-T1 and-T2 in O-glycosylation and invasion of TNBC. And we also investigate chemosensitivity and apoptosis ability affected by the level of O-glycosylation under the combined effect of chemotherapeutic agents and si RNA.Methods(1)The differential expression of pp Gal NAc-Ts, α2,3 sialic acids and MMPs were detected by Real-time PCR, Lectin blots and Western blots in MDA-MB-231, MCF-7 and MCF-12 A cells.(2) α2,3 sialic acids of O-linked glycan on total proteins and MMP14 was detected by Lectin blots when knocking-down of pp Gal NAc-T1 and-T2, respectively. And the invasive ability of differential treated MB-231 cell was detected by transwell methods.(3) After MB-231, MB-231/si-T1 and MB-231/si-T2 treated with chemotherapeutic agents, chemosensitivity and apoptosis ability was detected by MTT and Hoechst 33258 assay. Flow cytometry was used to detect the expression level of α2,3 sialic acids in cell surface.(4) Transwell assay was used to detect the invasive ability under the combined effect of chemotherapeutic agents and RNAi.Results:(1)Higher level of pp Gal NAc-T1,-T2, α2,3 sialic acids and MMP14 was detected in MDA-MB-231 cells when compared to MCF-7 and MCF-12 A cells.(2) α2,3 sialic acids of O-linked glycan on total proteins and MMP14 was inhibited by knocking-down of pp Gal NAc-T1 and-T2. Expression of MMP14 and VEGF was also down-regulated, which eventually affects invasive ability of TNBC.(3) MB-231/si-T1 and MB-231/si-T2 cells have higher chemosensitivity and apoptosis ability than MB-231 cells. And the α2,3 sialic acids of MB-231/si-T1 and MB-231/si-T2 cells in cell surface was down-regulated by chemotherapeutic agents treatment.(4) The invasive ability was inhibited under the combined effect of chemotherapeutic agents and RNAi.Conclusion:(1)Expression of pp Gal NAc-T1,-T2, α2,3 sialic acids and MMP14 in TNBC was co-related with its migration and invasion degree.(2) The down-regulated pp Gal NAc-T1 and-T2 in MDA-MB-231 cells decreased the terminal α2,3 sialic acids of MMP14 and exhibited self-degradation of MMP14, which affects depressed migration and invasion potential. In addition, there is a noticeable down-regulated expression of VEGF was consistent with MMP14 expression.(3) Low level of α2,3 sialic acids may contribute to improve chemosensitivity and apoptosis ability by enhancing drug and tumor contact.(4) The invasive ability was obviously inhibited under the combined effect of chemotherapeutic agents and si RNA.