Gain of H3K4me3 and H3K27 acetylation-activated lncRPL34-AS1 is down-regulated in ESCC and negatively correlated with poor prognosis
To explore the role of RPL34-AS1 in ESCC, we first assessed RPL34-AS1 expression level in 75 paired primary ESCC and matched adjacent nontumor tissues by RT-qPCR. The results showed that the expression of RPL34-AS1 was significantly reduced in the ESCC tissue samples (Fig. 1a). Moreover, RPL34-AS1 expression was down-regulated in ESCC cell lines compared with Het-1A cells, Among ESCC cell lines, EC109 cells showed the significant downregulation. Thus, we selected EC109 cell line to investigate the downstream regulatory pathway of RPL34-AS1 (Fig. 1b). Furthermore, the results of FISH assay showed that the RPL34-AS1 transcripts were distributed both in the cytoplasm and nucleus of EC109 cells (Fig. 1c), which indicated RPL34-AS1 might function in both cytoplasm and nucleus. To further investigate the expression pattern of RPL34-AS1 in ESCC, we also performed an analysis of RPL34-AS1 expression in a public microarray profile dataset from the Cancer Genome Atlas (TCGA). Consistent with our previous results, RPL34-AS1 expression was downregulated in ESCC tissues (Fig. 1d). Then we divided all ESCC patients into high and low RPL34-AS1 expression level groups according to the median value. Kaplan-Meier survival analysis of ESCC patients with low RPL34-AS1 expression had a significant poorer overall survival than those with high RPL34-AS1 expression (P = 0.01788, Fig. 1e). As shown in Table 1, the correlation analysis between RPL34-AS1 expression and clinicopathologic characteristics of these ESCC patients indicated that low expression of RPL34-AS1 was positively correlated with age (P = 0.008) and hard food (P = 0.042).
To explore the epigenetic modification mechanism of lncRPL34-AS1 in ESCC, firstly, using UCSC Genome Bioinformatics Site (http://genome.ucsc.edu/), we found high enrichment of H3K4me3 and H3K27ac at the promoter of RPL34-AS1. Hence, we speculated that the downregulation of RPL34-AS1 could be attributed to H3K4me3 and H3K27ac at its promoter region. To confirm this hypothesis, we detected the gain of H3K4me3 and H3K27Ac in EC109 cells compared with Het-1A cells at the promoter of RPL34-AS1 (Fig. 1f). Together, the data above confirmed that RPL34-AS1 was frequently reduced in ESCC, histone methylation and acetylation activation of promoter may partly account for this dysregulation.
LncRPL34-AS1 suppresses ESCC cell proliferation, cell-cycle progression, migration, invasion and promotes cell apoptosis in vitro
Given that RPL34-AS1 was down-regulated in ESCC, loss- and gain-of-function approaches were employed to determine the biological function of RPL34-AS1 in ESCC cells. RPL34-AS1-overexpressing EC109 cell line was established by the transfection of pcDNA3.1-RPL34-AS1. In contrast, three siRNAs were designed to silence RPL34-AS1. Real-time PCR analysis confirmed that RPL34-AS1 expression was successfully down-regulated or up-regulated in EC109 cells (Fig. 2a). CCK-8 assays demonstrated that downregulation of RPL34-AS1 significantly enhanced the proliferation viability, whereas the upregulation of RPL34-AS1 exerted opposite effects (Fig. 2b). Colony formation assays further demonstrated that the cell cloning capabilities of EC109 were obviously enhanced by the downregulation of RPL34-AS1 and markedly impaired by the overexpression of RPL34-AS1 (Fig. 2c). Similarly, EdU assays revealed that knockdown of lncRP34-AS1 greatly increased the percentages of EdU-positive cells, which considerably decreased at overexpression of RPL34-AS1 (Fig. 2d). Moreover, flow cytometry assays revealed that the percentage of apoptotic ESCC cells was reduced by RPL34-AS1 knockdown and the overexpression of RPL34-AS1 promoted the ESCC cells apoptosis (Fig. 2e). Depletion of RPL34-AS1 promoted cell cycle progression and upregulation of RPL34-AS1 induced cell cycle arrest at G1/S phase in EC109 cells (Fig. 2f). Furthermore, western blot results showed the consistent trend of cell apoptosis and cycle that RPL34-AS1 knockdown led to increase the levels of Bcl-2, Cyclin D1 and decrease BAX expression, as well as the upregulation of RPL34-AS1 led to opposite effects (Fig. 2g).
Next, transwell assays were carried out to examine the effects of RPL34-AS1 on migration and invasion of EC109 cells. The results indicated that the migratory and invasive capabilities of EC109 cells were remarkably enhanced by downregulation of RPL34-AS1 but significantly suppressed by upregulation of RPL34-AS1 (Fig. 2h, i). These experiments suggested that lncRPL34-AS1 suppressed migration and invasion of EC109 cells.
Overexpression of lncRPL34-AS1 restrains growth and metastasis of ESCC in vivo
To further determine the effects of lncRPL34-AS1 on tumor growth in vivo, EC109 cells transfected with pc-RPL34-AS1 or control vector were subcutaneously injected into BALB/c nude mice. 34 days after injection, the tumors were collected. The tumors formed in the pc-RPL34-AS1 group were substantially smaller than those in the control group (Fig. 3a). The results of tumor growth curves and weights indicated that RPL34-AS1 overexpression obviously reduced tumor growth in mice (Fig. 3b, c). Tumor tissues were harvested for RT-qPCR analysis of RPL34-AS1 and ACAA2. We confirmed that higher expression of RPL34-AS1 and ACAA2 were detected in tumor tissues arising from RPL34-AS1 overexpression group compared to control group (Fig. 3d, e). Furthermore, H&E and IHC for Ki67 were performed to detect the expression of Ki67, and results showed that RPL34-AS1 overexpression caused decreased Ki67 expression (Fig. 3f). To investigate the role of lncRPL34-AS1 in tumor metastasis, EC109 cells transfected with pcRPL34-AS1 or control vector were injected into the tail vein of nude mice. Compared with the control group, the lncRPL34-AS1 over-expression group blunted lower lung metastasis (Fig. 3g). Altogether, these results suggested that RPL34-AS1 upregulation suppressed ESCC tumorigenesis in vivo.
LncRPL34-AS1 serves as a miRNA sponge of miR-575 to regulate ACAA2 expression
To investigate the mechanisms underlying the role of RPL34-AS1 in ESCC, we examined the mRNA expression profiles in EC109 cells after overexpression of lncRPL34-AS1. The cluster analysis, volcano plot, GO and KEGG enrichment of differentially expressed genes show in Additional file 2: Figure. S1. The miRNA information in miRBase was used to perform target prediction based on the RPL34-AS1 sequence and the candidate miRNAs were screened by miRanda and TargetScan algorithm to display the ternary relationship of lncRNA-miRNA-mRNA in the form of a network diagram (Fig. 4a). We used differentially expressed genes of RNA-seq results and competing endogenous RNAs (ceRNA) targets to take the intersection in Venny (Fig. 4b). After DAVID bioinformatics resources functional notes, three metabolic pathways were enriched (Fig. 4c) and 6 targeted genes were selected for subsequent mechanism research in line with fold change and p value. The results of ACAA2 gene expression were proved to show consistent trend with RPL34-AS1 after knockdown and overexpression of RPL34-AS1 in EC109 cells (Fig. 4d, e). Correlated with previous ceRNAs prediction, the candidate miR-575 was selected for targeting ACAA2 and lncRPL34-AS1 via miRbase predicting (Fig. 4f). In brief, the lncRPL34-AS1/miR-575/ACAA2 regulatory network was established for verifying in ESCC.
Next, we detected the expression of miR-575 and ACAA2 in ESCC and matched adjacent normal tissues. Results of RT-qPCR showed significant upregulation of miR-575 and downregulation of ACAA2 in ESCC tissues relative to adjacent normal tissues (Fig. 4g). Furthermore, the results of RT-qPCR in EC109 cells also showed significant upregulation of miR-575 relative to Het-1A cells (Fig. 4h). Correspondingly, we then exemplified the binding relationship between lncRPL34-AS1 and miR-575; we conducted a wild-type (WT) and mutant (MuT) PGL3-CMV-lncRPL34-AS1 vector containing the binding sites of miR-575. The results showed that the luciferase activity of WT lncRPL34-AS1 reporter vector was significantly reduced by miR-575 mimics, compared with the empty vector and mutant reporter vector (Fig. 4i). Followed by an anti‐AGO2 RIP assay was implemented to validate the binding relationship between lncRPL34-AS1 and miR-575, the results of RIP implied that the expression of miR-575 in the lncRPL34-AS1 overexpression group was specifically higher than the NC group (Fig. 4j).
To decipher the regulatory mechanisms of miR-575 on ACAA2, we transfected luciferase reporter vector harboring 3′ UTR (WT and MuT) of ACAA2 into EC109 cells and luciferase activity was then evaluated in the transfection of miR-575 mimics. As compared to the control vector, miR-575 mimics significantly reduced the luciferase activity of the ACAA2 reporter vector (ACAA2 3′ UTR-WT) (Fig. 4k). Furthermore, to confirm the role of lncRPL34-AS1 on regulation of miR-575/ACAA2, we then set up another dual-luciferase (DLR) analysis and divided into two groups: Group 1 (RPL34-AS1 + ACAA2 WT + miR-575 mimics) and Group 2 (NC + ACAA2 WT + miR-575 mimics), the fluorescence intensity in Group 2 was reduced by 31% compared with Group 1 (Fig. 4l). These results enlightened that lncRPL34-AS1 may regulate ACAA2 expression by competitively interacting with miR‐575.
LncRPL34-AS1 suppresses ESCC cell growth and metastasis through lncRPL34-AS1/miR-575/ACAA2 axis
Accordingly, to verify whether RPL34-AS1 served its tumor suppressor function through RPL34-AS1/miR-575/ACAA2 axis, rescue experiments were designed using inhibitors and mimics. As shown in Fig. 5a, knockdown of RPL34-AS1 decreased protein level of ACAA2 in EC109 cells, while upregulation of RPL34-AS1 enhanced the level of ACAA2 in EC109 cells. As few studies had explored the role of miR-575 in ESCC, we began to clarify the mechanism and biological function of miR-575 in EC109 cells. The results indicated that upregulation of miR-575 significantly enhanced the proliferation viability, migratory and invasive capabilities of EC109 cells (Fig. 5b, c).Simultaneously, the target mRNA ACAA2 expression caused by silencing or overexpressing RPL34-AS1 were reversed by miR-575 inhibitor or mimics, respectively (Fig. 5d). Moreover, we attempted to explore whether the biological function of RPL34-AS1 in EC109 cells could also be reversed by miR-575 inhibitor or mimics. The results indicated that the miR-575 mimics reversed the proliferation, migration and invasion inhibiting effects induced by overexpression of RPL34-AS1 in EC109 cells, whereas miR-575 inhibitor counteracted the promoting effects induced by knockdown of RPL34-AS1 in EC109 cells (Fig. 5e-h).
Also we performed the mechanistical function of miR-575 by targeting ACAA2. The results of RT-qPCR and western blot displayed that substantially increased expression of ACAA2 in miR-575 mimics group in EC109 cells (Fig. 5i). Furthermore, the overexpression of ACAA2 (pcACAA2) repressed the proliferation viability, migratory and invasive capabilities of EC109 cells, whereas the above effects were reversed by miR-575 mimics (Fig. 5j-m). In addition, the flow cytometry assays also revealed that the cell cycle progression was promoted after co-transfected by pcACAA2 and miR-575 mimics, as well as the same trend that co-transfected by RPL34-AS1 upregulation and miR-575 mimics (Fig. 5n). In summary, these data strongly suggest that lncRPL34-AS1 suppresses ESCC cell growth and metastasis through lncRPL34-AS1/miR-575/ACAA2 axis.
LncRPL34-AS1 affects biological processes via binding to protein ALOX12B and CAT
To search for the potential interacting molecules of RPL34-AS1 to regulate target genes at distal genomic loci, we purified endogenous RPL34-AS1 complexes using modified ChIRP  that allowed unbiased high-throughput discovery of RPL34-AS1 associated binding proteins, ChIRP-MS was optimized to identify lncRNA‐associated proteins (Fig. 6a). We designed 38 probes (even/odd group) against human RPL34-AS1 RNA (Additional file 1: Table 3). The total TIC peaks of Positive Control (U1 snRNA), Negative control (Ctrl) and Test sample (Lnc) resolved components displayed in Fig. 6b. Protein enrichment classification information for comparison Lnc–Ctrl were displayed as Venn diagram, and the results showed a number of 73 proteins that can bind to RPL34‐AS1 (Fig. 6c). The results of STRINGdb protein-protein network enrichment analysis indicated the correlation between differential proteins (Fig. 6d). The KEGG pathways were enriched that Ribosome and Glycolysis/ Gluconeogenesis exerted significantly effect on interaction between lncRPL34-AS1 and binding proteins (Fig. 6e). According to the iBAQ and fold change, we selected 23 functional proteins for subsequent study (Fig. 6f). As shown in Fig. 6g, the functional protein pathway Glycolysis/ Gluconeogenesis was related to lncRPL34-AS1 in ESCC.
Among all the ChIRP-retrieved proteins, catalase (CAT) and Arachidonate 12-lipoxygenase, 12R-type (ALOX12B) came into notice. The protein ALOX12B catalyzes the region and stereo-specific incorporation of a single molecule of dioxygen into free and esterified polyunsaturated fatty acids generating lipid hydroperoxides . The catalase occurs in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide . The results of MDA and SOD detection confirmed the correlation between lncRPL34-AS1 and cellular oxidative stress (Fig. 6h). To further confirm the relationship, we examined the interaction between RPL34-AS1 and the two proteins by RNA immunoprecipitation (RIP), the results showed significant enrichments of RPL34-AS1 bound to CAT and ALOX12B, compared with the non-specific IgG control (Fig. 6i). Also, western blot showed that CAT and ALOX12B protein were positively proportional to RPL34-AS1 at a posttranscriptional level (Fig. 6j). As previous results shown, the lncRPL34-AS1 regulated target gene ACAA2 was a part of lipid metabolism as well as ALOX12B. In addition, correlation was found between ALOX12B and ACAA2 mRNA in ESCC of TCGA database (Fig. 6k). Altogether, these data showed that RPL34‐AS1 affected biological processes via protein ALOX12B and CAT in ESCC.