LncRNA MT1JP functions as a ceRNA in regulating FBXW7 through competitively binding to miR-92a-3p in gastric cancer

A total of 80 pairs of matched normal and GC tissues were collected from The Second Affiliated Hospital of Nanjing Medical University between February 2009 and October 2013. Five paired adjacent normal tissues and GC tissues were randomly selected in lncRNA microarrays study, and the remaining 75 paired gastric tissues were applied to qRT-PCR analysis. Additionally, another 330 paraffin-embedded GC tissues and corresponding follow-up information were obtained from Nantong Tumor Hospital between February 2008 and March 2013. All subjects have written informed consent and this study was approved by the Institutional Review Boards of Nanjing Medical University.

The lncRNA expression characteristics of GC were investigated by Arraystar Human LncRNA microarray V2.0, which contains 30,215 coding genes and 33,045 lncRNAs collected from several databases such as UCSC, Ensembl, RefSeq and the lncRNAs reported from literatures were also included. The microarray and data collection were conducted by KangChen Bio-tech (Shanghai, PR China). The details are as mentioned previously [16]. In addition, non-coding RNA profiling GSE53137 from the same platform was downloaded from GEO database, which investigate lncRNAs expression in six pairs of human gastric adenocarcinoma and adjacent normal tissues. Paired t-test was conducted to assess the differentially expressed lncRNAs between tumor and adjacent normal tissues (fold change > 2.0 and P value < 0.05).

The total RNA from GC tissue or cell lines were extracted using Trizol Reagent (Invitrogen, CA, USA) and mirVana miRNA Isolation Kit (Applied Biosystems) according to the manufacturer’s instructions. M-MLV reverse transcriptase (Invitrogen) was used for lncRNA MT1JP reverse transcription. The expression of lncRNA MT1JP and FBXW7 was detected by ABI 7900HT Real-Time PCR System (Applied Biosystem, Foster City, CA, USA), using SYBR Green assays (TaKaRa Biotechnology, Dalian, China) and GAPDH was used as the internal control. The expression of miR-92a-3p was measured using TaqMan MicroRNA Assays (Applied Biosystems) and U6 was treated as an internal control. All the primer sequences were available in Additional file 1: Table S2.

Coding Potential Assessment Tool (CPAT, http://​lilab.​research.​bcm.​edu/​cpat/​) was used to assess the coding capacity of lncRNA MT1JP. The CPAT conducted a logistic regression model by using the sequence features of open reading frame coverage, open reading frame size, hexamer usage bias and Fickett TESTCODE statistic. The CPAT chose 0.364 as a cutoff as human coding probability (CP). CP < 0.364 suggests noncoding sequence, whereas CP ≥ 0.364 indicates coding sequence [17].

Nuclear/cytoplasmic fractionation was conducted by the Protein and RNA Isolation System (Ambion) according to the manufacturer’s protocols. U6 was treated as a nuclear control while GAPDH was a cytoplasmic control.

The Cell Counting Kit 8 (Dojindo) was used to measure cell viability. The spectrophotometric absorbance at 450 nm for each sample was detected using spectrophotometer Infinite M200 (Tecan). All the experiments were repeated three times in six replicates. The transwell assay was used to evaluate cell migration. Cell invasion was assessed using BioCoat Matrigel Invasion Chamber (BD Biosciences Discovery Labware). Cell numbers for cell migration and invasion in three random fields were counted. Cells were stained by Annexin V and propidium iodide using the Annexin V–FITC Apoptosis Detection kit (Invitrogen), and the percentage of apoptosis was examined with flow cytometry (BD Bioscience, San Jose, CA, USA). For detection of cell cycle, cells were stained with PI after 48 h transfection and examination were performed by FACS Calibur system (Beckman Coulter).

The full-length lncRNA MT1JP cDNA was cloned into the BamHI and XhoI enzyme restriction sites of psiCHECK-2 vector (Promega) (psicheck-2-MT1JP-wild vector). The potential miR-92a-3p binding sites were mutated by the QuikChang site-directed mutagenesis kit (Agilent Technologies) (psicheck-2-MT1JP-mut vector). The psicheck-2-MT1JP-wild vector or psicheck-2-MT1JP-mut vector and miR-92a-3p mimics were co-transfected into BGC-823 and SGC-7901 cell by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The wild and mutant miR-92a-3p was cloned into the BamHI and XhoI enzyme restriction sites of psiCHECK-2 vector (Promega). miR-92a-3p-wild or miR-92a-3p-mutant and lncRNA MT1JP overexpression vector were co-transfected into both SGC-7901 and BGC-823 cell by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The luciferase activity was assessed by Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and the firefly luciferase activity was normalized by renilla luciferase activity.

All the cloned sequences were validated by DNA sequencing.

Twenty (10 nude mice in each group) five-week-old female athymic BALB/c nude mice were kept under pathogen-free conditions. BGC-823 cells that were transfected with an empty vector and the MT1JP vector were collected and resuspended at a concentration of 1.5 × 10⁷ cells/mL. Then, 0.1 mL of the cells that were transfected with the MT1JP vector and an empty vector were subcutaneously injected into each posterior flank of the nude mice. The tumor weights and volumes were examined every 3 days. The mice were killed after 16 days post-injection, and the tumors from the mice were measured.

Hematoxylin and eosin (H&E) staining was applied to select representative areas. The antibodies against Ki-67 (Abcam, Cambridge, MA, USA) were applied for IHC. The process of IHC was according to our previous study [18].

Protein was isolated from BGC-823 and SGC-7901 cell lines as previously stated [19].

Forty micrograms of total protein s were run on a 14.7% polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (Hybondenhanced chemiluminescence; Amersham Pharmacia Biotech). The antibodies against FBWX7(1:1000) and β-actin (1:1000) were purchased from Abcam (Abcam, Cambridge, MA, USA). The antibodies against Caspase-9 antibody (1:1000) and Caspase-3 (1:1000) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The protein was measured with a Phototope–horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Inc.), and β-actin was treated as an internal control.

For continuous variables, the results were shown as mean ± SD. Student’s t-test was applied to compare the difference of means between two groups. Differentially expressed MT1JP between gastric cancer and normal tissues from TCGA database was also evaluated by Student’s t-test. We used Kaplan-Meier curve and log-rank test to evaluate the effect of lncRNA MT1JP on survival of GC patients. The relationship between lncRNA MT1JP and FBXW7 expression was assessed by Spearman Pearson correlation analysis. A two-sided P value < 0.05 was considered as statistically significant. All analyses were performed using SAS software (version 9.2; SAS Institute, Inc., Cary, NC, USA).

LncRNA microarray was conducted in five pairs of GC tissues and adjacent normal tissues to investigate the lncRNA expression signatures of GC. Compared with normal tissues, 551 lncRNAs were up-regulated and 278 lncRNAs were down-regulated in GC tissues (fold change > 2.0 and P < 0.05). In order to reduce false positive, GSE53137, lncRNA profile based on the same microarray platform as our study, was download from GEO database. We integrated and selected the consistently differently expressed lncRNAs in both GSE53137 database and our microarray. The top 10 significantly up-regulated and down-regulated candidate lncRNAs are shown in Additional file 1: Table S1. Given that among the differently expressed lncRNAs, the fold change of MT1JP was the most remarkable, therefore, we focused on lncRNA MT1JP in the further study.

Subsequently, the expression levels of lncRNA MT1JP were explored in a large-cohort of 75 pairs of GC tissues and corresponding adjacent normal tissues. Significantly lower MT1JP expression was observed in GC tissues than in adjacent normal tissues (P < 0.001, Fig. 1a).Similar result was also acquired from The Cancer Genome Atlas (TCGA) database (P = 0.018, Additional file 1: Figure S1). We then examined the clinic pathological role of lncRNA MT1JP, and found that higher MT1JP expression was significantly related to lymph node metastasis and advance stage (P = 0.020 and 0.013, respectively, Fig. 1b and c). We further investigate the association between lncRNA MT1JP and survival of 308 GC patients with follow-up data. Kaplan-Meier curve and log-rank results indicated that GC patients with higher lncRNA MT1JP expression had a well prognosis and longer survival (HR = 1.33, 95%CI = 1.02–1.76, log-rank P = 0.031, Fig. 1d).

Consider that lncRNA MT1JP was down-regulated in GC tissues, we next investigated the effects of lncRNA MT1JP overexpression on GC cell phenotypes.

Because the expression of MT1JP was relatively lower in SGC-7901 and BGC-823 cells, we performed overexpression experiments in these two cell lines (Additional file 1: Figure S1B). Constructed pEGFP-N1 vector containing full-length lncRNA MT1JP was transfected into SGC-7901cell and BGC-823 cell, and found that the expression was effectively up-regulated as compare with negative control (NC) (Fig. 2a). Intriguingly, lncRNA MT1JP overexpression markedly inhibit cell proliferation in both SGC-7901and BGC-823 cells (Fig. 2b). Moreover, cell migration and invasion abilities in both GC cell lines were also significantly suppressed by lncRNA MT1JP overexpression (Fig. 2c and d). Flow cytometry analysis demonstrated that compared with NC, lncRNA MT1JP overexpression prominently promoted cell apoptosis in GC cells (Fig. 2e and f).

BGC-823 cells with MT1JP overexpression or negative control were subcutaneously injected into the back flank of nude mice. An effective higher expression level of MT1JP was observed in MT1JP overexpression group than that in negative control group. In line with in vitro analysis, the results indicated that the volume and weight of xenograft tumor were significantly lower in MT1JP overexpression group as compared with negative control group (Fig. 3a-e). Furthermore, H&E and immunohistochemistry for Ki67 was performed to detect the expression of Ki67, and results showed that MT1JP overexpression resulted in a substantial reduce of Ki67 protein expression (Fig. 3f). We also detected the expression of apoptosis mediators by western blot. The results indicated that overepresion of MT1JP was observedcorrelated with the increased cleaved caspase 3 and cleaved caspase 9 (Additional file 1: Figure S2).

The coding capability prediction of MT1JP was determined by online tool CPAT, and the result indicated that MT1JP had no apparent protein-coding ability (Additional file 1: Figure S3A). We next conducted nuclear-cytoplasmic fractionation to evaluate the lncRNAMT1JP subcellular localization, and found that lncRNA MT1JP was primarily detected in the cytoplasm of SGC-7901 and BGC-823 cells (Additional file 1: Figure S3B). To identify the potential miRNA targets of lncRNA MT1JP, in silico analysis was performed by using both FINDTAR3 (http://​bio.​sz.​tsinghua.​edu.​cn/) and RegRNA (http://​regrna.​mbc.​nctu.​edu.​tw/​index1.​php) databases, and jointly predicted that seven miRNAs (miR-24, miR-107, miR-195, miR-298, miR-185, miR-92a-3p and miR-223-5p) may act as biological targets of lncRNA MT1JP (Fig. 4a). We then conducted luciferase reporter assay to validate the binding of candidate miRNAs with lncRNA MT1JP, and selected miR-92a-3p with the mostly decreased luciferase activity into the further study (Fig. 4b). The expression levels of miR-92a-3p was significantly higher in GC tissues than in adjacent normal tissues (P < 0.01, Fig. 4c). In addition, miR-92a-3p mimic prominently decreased luciferase activity in MT1JP-wild not in MT1JP-mut in both SGC-7901 and BGC-823 cell lines, which indicated that miR-92a-3p is a target of lncRNA MT1JP (Fig. 4d). Further prediction of target genes of miR-92a-3p was performed by TargetScan (http://​www.​targetscan.​org/​vert_​71/​) and microRNA.​org (http://​www.​microrna.​org/​microrna/​home.​do), and FBXW7 was selected as the target gene of miR-92a-3p for it acquired consistently highest score in the prediction analysis of both softwares. Luciferase reporter results showed that remarkably reduced luciferase activity was observed in FBXW7-wild not in FBXW7-mut (Fig. 5a). We used RT-PCR to assess the RNA expression of miR-92a-3p and FBXW7 in cells overexpressed MT1JP. The results indicated that the RNA level of FBXW7 significantly increased while no significant change in miR-92a-3p (Additional file 1: Figure S5). Moreover, overepresion of MT1JP markedly declined luciferase activity in miR-92a-3p-wild not in miR-92a-3p-mutant in both SGC-7901 and BGC-823 cell lines (Additional file 1: Figure S4A). Additionally, we detected the expression of miR-92a-3p correlate with MT1JP and FBXW7 in clinical samples. The results showed miR-92a-3p reversely correlate with FBXW7, but not correlate with MT1JP (Additional file 1: Figure S6).

We also found significantly lower expression of FBXW7 in GC tissues, as compared with corresponding normal tissues (P < 0.01, Fig. 5b). Moreover, a positive correlation between MT1JP and FBXW7 expression was detected in GC tissues (r = 0.452, P < 0.001, Fig. 5c). The western blotting and RT-PCR analysis results showed that miR-92a-3p mimic reduced the expression of FBXW7, whereas lncRNA MT1JP overexpression substantially abolished this effect. Additionally, lncRNA MT1JP had no up-regulated effect on FBXW7 with miR-92a-3p inhibitor (Fig. 5d) (Additional file 1: Figure S4B).

Since lncRNA MT1JP played a ceRNA role in regulating FBXW7 expression by binding to miR-92a-3p, we performed rescue assays to validate whether miR-92a-3p and FBXW7 were involved in the lncRNA MT1JP-mediated inhibition proliferation, migration and invasion and promoting apoptosis in GC cells. To determine the effects of miR-92a-3p and FBXW7 in GC cell phenotypes, BGC-823 cell line was transfected with miR-92a-3p mimics and si-FBXW7, respectively. For rescue experiment, lncRNA MT1JP overexpression vector, miR-92a-3p mimics and si-FBXW7 were co-transfected into BGC-823 cell. Intriguingly, both miR-92a-3p mimics and si-FBXW7 remarkably promoted cell prolifereation, migration and invasion, and decreased cell apoptosis, respectively (Fig. 6). Co-transfection group (lncRNA MT1JP overexpression vector, miR-92a-3p mimics and si-FBXW7) could reversed the decrease on cell proliferation caused by lncRNA MT1JP overexpression (Fig. 6a). Moreover, flow cytometry analysis revealed that miR-92a-3p mimics and si-FBXW7 significantly suppress apoptotic cell rate caused by lncRNA MT1JP overexpression (Fig. 6b-c). Cell migration and invasion experiments revealed that miR-92a-3p mimics and si-FBXW7 prominently reversed the effects of lncRNA MT1JP overexpression (Fig. 6d-f).