3.1 ABHD11-AS1 is highly expressed in GC tissues
To prove oncogenic role of lncRNA ABHD11-AS1 in GC, we firstly analyzed the relationship between the expression level of ABHD11-AS1 and survival duration of GC patients in open databases (Figure1A,B). We found a worse prognosis of gastric cancer patients was correlated with a higher level of ABHD11-AS1.
Based on the analysis, we further explore whether the level of ABHD11-AS1 was significantly increased in GC specimens and cell lines. Firstly, we determined the expression level of ABHD11-AS1 in GC tissues and adjacent normal tissues with RT-PCR analysis, and the gene expression was normalized to GAPDH. As expected, the expression level of ABHD11-AS1 was significantly upregulated in GC tissues compared with normal counterparts (*p<0.01, Firgure 1C). Moreover, to further validate whether the expression level of ABHD11-AS1 was significantly increased in GC, we measured the expression of ABHD11-AS1 in various GC cell lines by RT-PCR analysis and normalized the gene expression to β-actin. Compared to normal gastric epithelium cell line (GES-1), two cell lines (BGC-823, MGC-803) exhibited significant higher levels of ABHD11-AS1(*p<0.01,Figure1 D). Therefore, we focused on these two cell lines to explore the biological functions of ABHD11-AS1 in GC. In short, the aberrant overexpression of ABHD11-AS1 may be related to GC.
3.2 Knock-down of ABHD11-AS1 could suppress the aberrant proliferation of GC cells.
Yang et al has proved that the overexpression of ABHD11-AS1 could promote the proliferation of GC cells. To explore the biological effects of ABHD11-AS1 knock-down on GC cells, we knocked-down ABHD11-AS1 with siRNA in two GC cell lines (BGC-823, MGC-803 ) and evaluated the effects on cell proliferation as well as apoptosis. The knock-down efficiency of ABHD11-AS1 was confirmed with RT-PCR compared with Scramble (*p<0.01, Firgure2 A). CCK8 assay revealed that the reduced expression of ABHD11-AS1 significantly suppressed the cell proliferation in GC cell lines compared to the si-Scramble (*p<0.01, Figure 2B,C). Likewise, colony formation assay showed that the reduced expression of ABHD11-AS1 markedly decreased the clonogenic survival percent of GC cell lines (BGC-823, MGC-803) compared to si-Scramble ( *p<0.01, Figure2F,G).
Meanwhile, the results of Annexin V-FITC staining assay proved that the knock-down of ABHD11-AS1 promoted the apoptosis percent of GC cell lines compared to si-Scramble ( *p<0.01, Figure2H,I). Since apoptosis-promoting gene Bax and enzyme caspase-3 could mediate the process of cell apoptosis[23-25], their protein levels were measured by RT-PCR and Elisa assay in our study. The results of Elisa assay suggested that inhibition of ABHD11-AS1 enhanced the level of apoptosis-promoting enzyme caspase-3 compared to si-Scramble (*p<0.01 ,Figure2J ) and the RT-PCR analysis showed that Bax was remarkably higher in ABHD11-AS1 knock-down group comapred to si-Scramble ( *p<0.01, Figure2k ).
In conclusion, ABHD11-AS1 could be a potential therapeutic target to suppress the growth of GC.
3.3 LncRNA-ABHD11-AS1 served as a sponge for miR-1301-3p in GC cell
Growing number of evidences have demonstrated that ABHD11-AS1 could regulate the expression of miR-133a in colorectal cancer cell (CRC). We hypothesized that another miRNA interacting with ABHD11-AS1 might exist in GC cell lines. Indeed, we noticed that the expression of miR-1301-3p was significantly upregualted after the knockdown of ABHD11-AS1 in GC cell lines (BGC-823, MGC-803) compared to si-Scramble ( *p<0.01,Figure3A,B ). Therefore, we suspected miR-1301-3p as a potential target molecule interacting with ABHD11-AS1 in this research. To further explore whether ABHD11-AS1 functioned by sponging miRNA or acting as competing endogenous RNAs (ceRNAs), bioinformatics tools (microRNA.org and miRcode) were utilized to identify the microRNA binding sites of ABHD11-AS1(Figure 3C). qPCR assay showed miR-1301-3p expression level was significantly decreased in GC tissues compared with adjacent normal tissues (*p<0.01 , Figure 3D ).
To determine whether ABHD11-AS directly interacted with miR-1301-3p, dual-luciferase reporter assay demonstrated that miR-1301-3p could significantly weaken the luciferase activity of the WT-ABHD11-AS1 compared with mock, while similar observation was not found for MUT-ABHD11-AS1 (*p<0.01, Figure 3E,F ). To further investigate whether RNA-induced silencing complex (RISC) was involved in the mutual suppression between ABHD11-AS1 and miR-1301-3p, RNA immunoprecipitation assay (RIP) were conducted with antibody against Ago2, a major component of RISC complex. ABHD11-AS1 and miR-1301-3p were remarkably higher in pellet pulled-down by Ago2 antibody in GC cell lines (BGC-823, MGC-803) compared with IgG (*p<0.01, Figure 3G,H). Eventually, biotin-labeled pull-down assay further demonstrated miR-1301-3p could directly interact with ABHD11-AS1 compared with Bio-control ( *p<0.01,Figure3I ).
3.4 miR-1301-3p/ABHD11-AS1 regulated the expression of PDPK1 in GC cells
By searching public database (starbase), Pearson correlation analysis suggested that there was a strong negative correlation between miR-1301-3p and PDPK1 expression in GC tissues (Firgure 4A). Bioinformatics analysis by TargetScan and miRBase predicted that PDPK1 had matched binding bases with PDPK1 (Figure 4B). Luciferase reporter assay revealed that the over-expression of miR-133a remarkably suppressed PDPK1 3’-UTR activity while the activity of the mutant PDPK1 3’-UTR was unaffected compared with mock (*p<0.01 , Figure 4C ). Moreover, the up-regulation of miR-133a had a significant negative effect on the expression of PDPK1 at the levels of both mRNA and protein, compared to mock treatment (*p<0.01 , Figure4D,E ). To determine whether ABHD11-AS1 competitively inhibited the binding of miR-1301-3p with PDPK1, luciferase reported assays were conducted. The results suggested that ABHD11-AS1 overexpression could offset the suppressing effect of miR-1301-3p on endogenous ABHD11-AS1 in GC cell lines (*p<0.01,Figure H,I ). In addition, q-PCR analysis and western blot assays demonstrated that there was a mutual antagonism between miR-1301-3p and ABHD11-AS1 on binding to PDPK1 (Figure 4J,K,L ). In short, miR-1301-3p/ABHD11-AS1 behave as counteract regulators on the expression of PDPK1 in GC lines.
3.5 miR-1301-3p/ ABHD11-AS1 co-mediated proliferation and apoptosis of GC cell
Finally, we showed that ABHD11-AS1 could promote GC through regulating the binding between miR-1301-3p and PDPK1 mRNA. The blockage of miR-1301-3p counteracted the inhibitor effects of ABHD11-AS1 knock down on PDPK1 expression in GC cells (*p<0.01 ,Firgure5A-C ). Moreover, simultaneous suppression of miR-1301-3p and ABHD11-AS1 expression could significantly enhance the expression of PDPK1. Additionally, CCK8 assay and colony formation assay demonstrated that the negative effect of ABHD11-AS1 down-regulation on proliferation of GC cells could be neutralized by miR-1301-3p inhibitor (*p<0.01,Figure5D,E,F). Meanwhile, Annexin V-FITC staining assay and caspase-3 activity assay showed that miR-1301-3p knockdown could partially abolish the pro-apoptotic effect of ABHD11-AS1 down-regulation in GC cells ( Figure G, H, p<0.01 ).
In conclusion, the check and balance relationship between miR-1301-3p and ABHD11-AS1 could mediate the expression of PDPK1, disorder of which may contribute to the development of GC.