Silencing Long Non-Coding RNA ST3GAL6-AS1 Inhibits Adhesion and Migration of Myeloma Cells in Vitro
Authors: Shen Ying , Feng Yuandong , Chen Hongli , Jia Yachun , Peng Yue , Zhang Ru , Yang Yun , Wang Jian-Li , Bai Ju , Wang Fang-Xia , Xu Yan , Hu Jinsong , He Aili 
Published: October 2019

Abstract
Introduction: Multiple myeloma (MM) is a fatal B-cell malignancy characterized by abnormal proliferation of plasma cells (PCs) in bone marrow (BM). Despite the remarkable improvements have been done in knowledge on the pathobiology of MM, treatment and patients care, MM is still an incurable disease. Long non-coding RNAs (lncRNAs) are a new sort of noncoding RNA longer than 200 nucleotides and without the function of translating into canonical functional proteins. Increasing evidence suggests that lncRNAs have huge impact on adjusting gene expression through the processes of transcription and post-transcription regulation, genomic imprinting, and chromatin modification. We have validated several dysregulated lncRNAs in MM by microarray and bioinformatic analysis. Among them, we focused on the function and mechanism of ST3 beta-galactoside alpha-2,3-sialyltransferase 6 antisense RNA 1(ST3GAL6-AS1), which was upregulated markedly in MM patients. In our previous study, we verified the upregulation level and the co-expression relationship of ST3GAL6-AS1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6) in bone marrow samples from MM patients.
Methods: MM cell lines MM1.S and RPMI-8226 were confirmed to have high expression levels of ST3GAL6-AS1 at RNA level compared with healthy controls. Knockdown of ST3GAL6-AS1 was conducted in MM1.S and RPMI-8226 cells using lncRNA Smart Silencer from Ribobio (Guangzhou, China). ST3GAL6-AS1 knockdown and ST3GAL6 expression were confirmed by quantitative real-time PCR (qRT-PCR) at RNA level. We next evaluated the effects of ST3GAL6-AS1 knockdown on MM cell adhesion to human umbilical vein endothelial cell (HUVEC), fibronectin and collagen I coated plates after MM1.S and RPMI-8226 was labeled by CFSE. Migration and invasion to complete medium were assessed using transwell plates comparing ST3GAL6-AS1 knockdown cells to NC controls. Matrigel matrix was coated at transwell plates in invasion analysis. Cell count in adhesion analysis was conducted by Image J software, while migration and invasion analysis were evaluated by flow cytometer.
Results: ST3GAL6-AS1 (Genebank number: NR_046683.1) was a 769bp antisense RNA, which located in chromosome 3. Its expression level was decreased dramatically after transfection of ST3GAL6-AS1 smart silencer or control siRNA into MM1.S and RPMI-8226 cells (Figure 1A). There was a significant reduction in the ability of knockdown MM1.S and RPMI-8226 cells to adhere to HUVEC, fibronectin and collagen I in vitro compared to NC controls (Figure 1B). Migration and invasion ability of MM cells transfected with lncRNA smart silencer in response to complete medium were also decreased obviously (Figure 1C). However, there was no significant difference in the ability of proliferation and apoptosis between ST3GAL6-AS1 knockdown group and NC controls. Furthermore, the expression level of ST3GAL6 was decreased in ST3GAL6-AS1 knockdown MM cells (Figure 1D).
Conclusions: Knockdown of ST3GAL6-AS1 in MM cells significantly inhibits adhesion, migration and invasion in vitro, indicating that ST3GAL6-AS1 may play an important role in the malignant behavior of MM cells. The co-expression between ST3GAL6-AS1 and gene ST3GAL6 has been demonstrated in our previous study, which was further confirmed in present study. Researches are ongoing to address the potential mechanism among them in MM.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.