Data_Sheet_2_Silencing SCAMP1-TV2 Inhibited the Malignant Biological Behaviors of Breast Cancer Cells by Interaction With PUM2 to Facilitate INSM1 mRNA Degradation.docx

Background: Molecular-targeted therapy plays an important role in the combined treatment of breast cancer. Long noncoding RNA (LncRNA) plays a significant role in regulating breast cancer progression. The present study is to reveal the potential roles and molecular mechanism that the secretory carrier-associated membrane protein 1-transcript variant 2 (SCAMP1-TV2) has in breast.
Methods: Cell Counting Kit-8 (CCK-8), RNA Immunoprecipitation (RIP), and RNA pull-down assays were employed to determine the interactions between SCAMP1-TV2 and Pumilio RNA binding family member 2 (PUM2). The luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays were used to get to know the effect of human insulinoma-associated 1 (INSM1) directly on the SAM and SH3 domain containing 1 (SASH1) promoter.
Results: Silenced SCAMP1-TV2 inhibited the proliferation, migration, and invasion of breast cancer cells, and promoted cell apoptosis. Meanwhile, SCAMP1-TV2 downregulation decreased its binding to PUM2 and increased the binding of PUM2 to INSM1 messenger RNA (mRNA), thus promoting the degradation of INSM1 mRNA. Silencing INSM1 decreased its inhibitory effect on SASH1 transcription and inhibited the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. The xenograft tumor growth in a nude mice was significantly inhibited by the silencing of SCAMP1-TV2 in combination with the overexpression of PUM2.
Conclusions: SCAMP1-TV2/PUM2/INSM1 pathway plays an important role in regulating the biological behavior of breast cancer cells.

背景：分子靶向治疗在乳腺癌联合治疗中发挥重要作用。长链非编码RNA（Long noncoding RNA, LncRNA）在调控乳腺癌进展过程中具有关键意义。本研究旨在揭示分泌载体相关膜蛋白1转录变体2（SCAMP1-TV2）在乳腺癌中的潜在作用及分子机制。
方法：采用细胞计数试剂盒-8（CCK-8）、RNA免疫沉淀（RIP）及RNA下拉实验，检测SCAMP1-TV2与Pumilio RNA结合家族成员2（PUM2）之间的相互作用。通过荧光素酶报告基因实验与染色质免疫沉淀（ChIP）实验，探究人胰岛素瘤相关蛋白1（INSM1）对SAM和SH3结构域包含蛋白1（SASH1）启动子的直接调控作用。
结果：敲低SCAMP1-TV2可抑制乳腺癌细胞的增殖、迁移与侵袭，并促进细胞凋亡。同时，SCAMP1-TV2下调会降低其与PUM2的结合能力，增强PUM2与INSM1信使RNA（mRNA）的结合，进而促进INSM1 mRNA的降解。敲低INSM1可减弱其对SASH1转录的抑制作用，并抑制磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）信号通路的激活。联合敲低SCAMP1-TV2与过表达PUM2，可显著抑制裸鼠体内异种移植瘤的生长。
结论：SCAMP1-TV2/PUM2/INSM1通路在调控乳腺癌细胞的生物学行为中发挥重要作用。