MSC-AS1 induced cell growth and inflammatory mediators secretion through sponging miR-142-5p/DDX5 in gastric carcinoma

Emerging studies have noted that dysregulated lncRNAs are implicated in cancer progression and tumorigenesis. We first showed that MSC-AS1 was overexpressed in gastric cancer (GC) cells (HGC-27, MKN-45, SGC-7901 and MGC-803 cells) compared with GES cells. We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens. MSC-AS1 increased cell growth and cycle progression. Moreover, the overexpression of MSC-AS1 enhanced the secretion of the inflammatory mediators IL-1β, IL-6 and TNF-α. We found that the overexpression of MSC-AS1 inhibited the expression of miR-142-5p in HGC-27 cells. We noted that DDK5 was a target gene of miR-142-5p. The overexpression of miR-142-5p suppressed the luciferase activity of wild-type DDX5, but the luciferase activity of the mutant DDX5 was not changed. We showed that miR-142-5p was downregulated in GC specimens compared with paired normal specimens. MSC-AS1 expression was inversely correlated with miR-142-5p expression in GC specimens. MSC-AS1 induced cell growth, cell cycle progression and inflammatory mediator secretion by modulating DDX5. These results showed that MSC-AS1 functions as a key oncogene in the development of GC.


INTRODUCTION
Gastric cancer (GC) is the 3 rd most common cause of tumor-associated death and the 3 rd most commonly diagnosed tumor worldwide [1][2][3][4]. The cause and pathogenesis of this disease are complex and associated with many factors [5][6][7][8]. Despite the great achievements that have been made in GC therapeutics, the survival rate of GC patients remains unsatisfactory [9][10][11][12]. The major challenge in the treatment of advanced GC is the manifestation of peritoneal, distal organ and lymphatic metastases [13][14][15]. Therefore, a detailed and improved understanding of the molecular mechanisms underlying GC progression and development is greatly needed.

MSC-AS1 was upregulated in GC specimens
We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens ( Figure 2A). The level of MSC-AS1 was upregulated in 29 GC specimens (72.5%, 29/40) compared to their paired normal specimens ( Figure 2B).

MSC-AS1 induced cell growth, cell cycle progression and inflammatory mediator secretion by modulating DDX5
The level of DDX5 was significantly decreased in HGC-27 cells after treatment with DDX5 siRNA ( Figure 6A). Knockdown of DDX5 suppressed the expression of Ki-67 ( Figure 6B) and CKD2 ( Figure 6C) in MSC-AS1-overexpressing HGC-27 cells. Inhibition of DDX5 expression decreased the cell cycle ( Figure  6D) and growth ( Figure 6F
Found that MSC-AS1 enhanced hepatocellular carcinoma progression by enhancing PGK1 expression. Yao et al. [30]. showed that MSC-AS1 increased nasopharyngeal carcinoma development by regulating miR-524-5p/NR4A2. Hu et al. [28]. showed that MSC-AS1 regulated renal carcinoma cell migration and growth by modulating the miR-3924/ WNT5A/Wnt/β-catenin axis. In the present study, we conducted tests to examine the functional role of MSC-AS1 in GC. We first showed that MSC-AS1 was overexpressed in GC cells (HGC-27, MKN-45, SGC-7901 and MGC-803 cells) compared to GES cells. We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens. MSC-AS1 increased cell growth and cell cycle progression. Moreover, the overexpression of MSC-AS1 enhanced the secretion of the inflammatory mediators IL-1β, IL-6 and TNF-α.
In summary, we observed that MSC-AS1 was overexpressed in GC cells and specimens and that ectopic expression of MSC-AS1 enhanced cell growth, cell cycle progression and inflammatory mediator secretion by modulating miR-142-5p/DDX5. These results showed that MSC-AS1 acts as a key oncogene in the development of GC.

Luciferase assays
The DDX5 3'-UTR and mutated DDX5 3'-UTR were cloned into the pGL3 plasmid as wild-type or mutant type 3'-UTRs, respectively. GC cells were treated with the wild-type or mutant DDX5 3'-UTR together with miR-142-5p scramble or mimic using a Lipofectamine kit. After transfection for 2 days, the luciferase activity was determined by Dual-Glo luciferase analysis (Promega, WI, USA).

CCK-8 assay, cell cycle analysis and ELISA
Cell growth was detected with a CCK-8 assay kit (Dojindo, Japan) according to the manufacturer's instructions. These cells were plated in 96-well plates and cultured for 0, 24, 48 and 72 hours. Ten microliters of CCK-8 were added to each well, and the cells were cultured for an additional 3 hours. The absorbance was detected at 450 nm at different time points. To analyze cell cycle progression, GC cells were stained with cell cycle reagent (Thermo) following a standard protocol. The cell cycle was measured with flow cytometry on a Beckman flow cytometer (Dickinson, USA). The protein levels of IL-1β, IL-6 and TNF-α in the cell suspension were detected by ELISA following the manufacturer's protocol.

RNA Immunoprecipitation (RIP) analysis
The RIP assay was conducted utilizing the Magna RNA-Binding Protein Immunoprecipitation of the RIP Kit (Millipore) following standard instructions. The cells were harvested and then lysed in RIP lysis buffer containing RNase and protease inhibitor, and then, the lysates were treated for 2 hours with buffer containing magnetic beads coated with antibodies against Ago2. IgG served as the negative control. Coprecipitated RNAs were determined by RT-qPCR assay.

Statistical analysis
All the statistical assays were analyzed using SPSS 19.0 (Chicago, IL, USA), and the graphs were generated by Prism 5.0. Student's t-test was used to compare significant differences between two groups, and the correlation between miR-142-5p and MSC-AS1 in GC was analyzed by Pearson correlation assay. p<0.05 was defined as statistically significant.

Editorial note
& This corresponding author has a verified history of publications using a personal email address for correspondence.

AUTHOR CONTRIBUTIONS
Yan Liu, Lin Li, Xiaoxu Wu, Haiyan Qi, Yan Gao, Yanqi Li, Da Chen collected the related paper, drafted and wrote the manuscript. Yan Liu Yanqi Li, Da Chen and participated in the design of experiments. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.