LncRNA TNFRSF10A-AS1 promotes gastric cancer by directly binding to oncogenic MPZL1 and is associated with patient outcome

Background: LncRNA is closely associated with the progression of human tumors. The role of lncRNA TNFRSF10A-AS1 (T-AS1) in gastric cancer (GC) is still unclear. We aim to investigate the functional significance and the underlying mechanisms of T-AS1 in the pathogenesis and progression of GC. Experimental Design: The clinical impact of T-AS1 was assessed in 103 patients with GC. The biological function of T-AS1 was studied in vitro and in vivo. T-AS1 downstream effector were identified by RNA sequencing and RNA pulldown assay. Results: T-AS1 was upregulated in GC cell lines and GC tissues as compared to adjacent non-cancer tissues (n = 47, P < 0.001). Multivariate analysis showed that GC patients with T-AS1 high expression had a significantly shortened survival (n=103, P < 0.05). T-AS1 significantly promoted GC cell proliferation, cell-cycle progression, and cell migration/invasion abilities, but suppressed cell apoptosis. Silencing of T-AS1 in GC cells exerted opposite effects in vitro. Knockout of T-AS1 significantly inhibited xenograft tumor growth in nude mice. Mechanistically, T-AS1 directly bound to Myelin Protein Zero Like 1 (MPZL1). MPZL1 showed an oncogenic function in GC by promoting cell proliferation, migration and invasion but inhibiting cell apoptosis. High expression of MPZL1 was associated with poor survivor of GC patients. Knockdown of MPZL1 could abrogate the effect of T-AS1 in the tumor-promoting function. Conclusions: T-AS1 plays a pivotal oncogenic role in GC and is an independent prognostic factor for GC patients. The oncogenic function of T-AS1 is dependent on its direct downstream effector MPZL1.


Colony formation and cell growth curve assays
Cells were plated in 6-well plates at 1,000 cells per well in complete DMEM.

Immunocytochemistry staining
Paraffin slides from xenograft were used. Ki-67 signal was assessed. The proliferation index was determined by counting the numbers of positive staining cells of Ki-67 as percentages of the total number of colon cells. At least 1000 cells were counted each time.

Migration and invasion assays
For the "Transwell" migration assay, 3×10 4 cells with applied genetic modification in 200 μL serum-free medium were seeded onto the upper chamber of an 8-μm Transwell filter (Corning, 3422, Shanghai, China). In the lower chamber, 600 μL complete medium containing 20% FBS was added. After incubation, cells in the lower surface were fixed by methanol, stained with 1% crystal violet, and visualized under a microscope. For the "Transwell" invasion assays, matrigel-coated chambers (Corning, 354480, Shanghai, China) were used. For all "Transwell" assays in this study, five random views were included to calculate the average number of migrated/invaded cells.

Apoptosis analyses
Cells were plated in 12-well plates and serum-starved overnight. Annexin V Apoptosis Detection Kit APC (Invitrogen, Thermo Fisher) was used to determine cell apoptosis. The experiments were conducted three times in triplicates.

Cell cycle analysis
BGC823 and GES1 cells that were stably transfected with TNFRSF10A-AS1 or empty vector were plated in a 6-well plate, while MGC803 and AGS cells were transfected with siTNFRSF10A-AS1 or siNC. After 48h of transfection, the cells were fixed in ice-cold 70% ethanol for 24h before staining with 50μg/ml propidium iodide (BD Biosciences, Franklin Lakes, NJ). The cells were sorted by BD AccuriTM C6 (BD Biosciences), and cell cycle distributions were analyzed using the ModFitLT 5.0 software (Verity Software House, Topsham, ME). All experiments were conducted three times in triplicates.

Wound-healing assay
Confluent cultures in 6-well plates were scratched with sterile P-200 pipette tips, washed, and cultured in DMEM containing 2% FBS. Cells were photographed after 0, 24, and 48 hours, respectively. The cells migrated across the gap wound were observed and documented using an inverted microscope. Distance of the gap was quantified using Image J.

At 6×10 4 cells per well, GC cells were initially seeded into six-well plates in
polybrene-containing complete medium. The lentivirus was added to GC cells for 48h. Afterwards, cells were cultured in 1 puromycin (3 μg/mL)-containing medium for five more passages (10-12 days). In stable cells, expression of targeted gene was assessed by qRT-PCR.