A total of 50 pairs of human T-LBL tissues and 38 pairs of adjacent normal tissues were surgically collected from patients in Chengdu Military General Hospital of PLA. The specimens were collected and rapidly frozen in liquid nitrogen and stored at -80°C until use. The study was approved by the Human Ethics Committee/Institutional Review Board of Chengdu Military General Hospital of PLA and was fully informed consent was obtained from all the patients before the sample collection.

The T-LBL cell lines (CCRF-CEM, Jurkat and SUP-T1) and human T-cell line H9 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All the cell lines were cultured in RPMI-1640 media, (cat. no. 11875-093; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Inc., Grand Island, NY, USA). The cells were grown in humidified air at 37°C with 5% CO₂.

Total RNA from related tissues and cell lines was harvested using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed using RT-PCR kits (Applied Biosystems, Foster City, CA, USA) with an oligo d(T) according to the manufacturer's protocol. The RT-PCR primers for MEG3 and miR-214 were purchased from GeneCopoeia, Inc. (San Diego, CA, USA). The specific primers were as follows: MEG3 (forward, 5′-CCTGCTGCCCATCTACACCTC-3′ and reverse, 5′-CCTCTTCATCCTTTGCCATCCTGG-3′); miR-214 (forward, 5′-AGCATAATACAGCAGGCACAGAC-3 and reverse, 5′-AAAGGTTGTTCTCCACTCTCTCAC-3′). GAPDH was used as the internal control of the mRNA or miRNA, respectively. Fold-change of MEG3 or miR-214 was calculated by the equation 2^−ΔΔCt.

Mimics/inhibitors specific for miR-214 and LncRNA-MEG3 were designed and purchased from Invitrogen. The mock and fragments were designed as the negative control of miR-214 and UCA1, respectively. The Jurkat and SUP-T1 cells were seeded in 24-well plates at 1×10⁵ cells/well. LncRNA-MEG3 and MEG3 scramble was amplified using PrimerSTAR premix (Takara) and cloned into lentivirus plasmid according to the manufacturer's protocol. Jurkat and SUP-T1 cells were transfected with recombinant lentivirus. Mimics/inhibitors specific for miR-214 and mock were transfected into Jurkat and SUP-T1 cells using Lipofectamine 2000 (Invitrogen). Cells were harvested for subsequent experiments after transfection for 24 h.

Cell proliferation was assayed using the Cell Counting kit-8 (CCK-8; Dojindo Laboratories, Tokyo, Japan) according to the manufacturer's protocol. Firstly, Jurkat and SUP-T1 cells were pretreated with LncRNA-MEG3 or MEG3 scramble. A total of ~5×10³ Jurkat and SUP-T1 cells were seeded onto 96-well plates and were transfected with LncRNA-MEG3 or MEG3 scramble, respectively. Twenty-four hours later, cells were incubated with CCK-8 solution for 3 h at 37°C. Absorbance was determined at 570 nm using multifunctional microplate reader SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA) at indicated time-points. All experiments were repeated in triplicate. The cell proliferation trends were depicted according to the absorbance at each time-point.

The expression levels of MEG3 and miR-214 in T-LBL samples, adjacent normal tissues, T-LBL cell lines and human T-cell lines H9 were further determined by Northern blot assay. Northern blot analysis was performed according to the previously described procedures (28).

Total protein was extracted from related tissue and cells and then protein concentrations were measured using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equivalent amounts of protein were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked in phosphate-buffered saline (PBS) with 0.1% Tween-20 containing 5% non-fat milk for 2 h at room temperature, and then incubated with the primary antibodies: anti-Ki-67, anti-proliferating cell nuclear antigen (PCNA), anti-caspase-3, anti-caspase-9, anti-p27, anti-cyclin A, anti-AIFM2 and anti-GAPDH (Abcam, Cambridge, UK) and the corresponding HRP-conjugated secondary antibodies, followed by detection and visualization using a ChemiDoc XRS imaging system and analysis software (Bio-Rad Laboratories, San Francisco, CA, USA). GAPDH (Abcam) was used as endogenous references.

SUP-T1 cells were seeded in 6-well plates and treated with LncRNA-MEG3 or MEG3 scramble for 24 h. Cell apoptosis were detected by the Annexin V apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China) following a previous study (29). Cell apoptosis percentage was reflected by Annexin V/PI ratio, detected by a flow cytometry (BD Biosciences, Shanghai, China).

SUP-T1 cells were harvested after 24 h of treatment with LncR-MEG3 or MEG3 scramble, respectively. After washing with ice-cold PBS, the cells were then harvested and fixed in 70% ethanol overnight at 4°C. After that, the cells were washed with PBS, re-suspended in a staining solution containing 20 µl RNase A solution and 400 µl propidium iodide staining solution (Vazyme Biotech, Co., Ltd., Nanjing, China). Then, cell cycle distribution was assessed using a fluorescence-activated cell sorter (BD FACSCalibur). Results are presented as the percentage of cells in each phase.

The full-length 3′-UTR segments of MEG3 mRNA containing the miR-214 binding site was amplified by chemical synthesis and inserted into the luciferase reporter vector (pGL3) and named MEG3 WT. The mutation of MEG3 in the seed sequence was synthesized using a Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). Then, 1×10⁶ SUP-T1 cells were co-transfected with 0.1 µg Luc-MEG3-WT or Luc-MRG3-MUT, together with 40 nM miR-214 mimic/mimic control or 40 nM pGL3 for 24 h using Lipofectamine 2000. Then luciferase activity was measured using the Dual-luciferase assay system (Promega, Madison, WI, USA) and normalized to Renilla luciferase activity.

Specific pathogen-free (SPF) athymic nude mice (male, 6 to 8 weeks of age) were housed and manipulated according to the protocols approved by the Experimental Animal Center of Chengdu Military General Hospital of PLA. For investigating tumorigenicity of MEG3 in vivo, xenograft mouse model was created by subcutaneous injection of 1×10⁷ SUP-T1 cells transfected with LncRNA-MEG3 or MEG3 scramble to SPF nude mice. After development of a palpable tumor, the tumor volume was monitored every 5 days for a month and assessed by measuring the 2 perpendicular dimensions using a caliper and the formula (a × b²)/2, where a is the larger and b is the smaller dimension of the tumor. Then the mice were sacrificed and the tumor weights were assessed. Tumors from each mouse were randomly selected for immunohistochemical (IHC) analysis. All the animal experiments were performed according to relevant national and international guidelines and were approved by the Animal Experimental Ethical Committee.

Briefly, 5 µm-thick paraffin-embedded tumor tissue sections were deparaffinized in xylene, rehydrated in graded ethanol gradually and were rinsed twice with PBS. In order to quench the activity of the endogenous peroxidase, the tissue sections were incubated in 30% H₂O₂ for 30 min. After antigen retrieval in heated 10 mM citrate buffer for 10 min, the tissue sections were incubated with mouse anti-human Ki-67 and PCNA primary antibody overnight at 4°C. Corresponding mouse horseradish peroxidase (HRP)-conjugated secondary antibody was added for 1 h at room temperature. The images were viewed under a light microscope.

The significance of differences between 2 groups was determined by the Student's t-test. All data were expressed as the mean ± standard deviation (SD) of at least three independent experiments performed in triplicate. All of the P-values were 2-sided and differences were considered statistically significant at P<0.05.

To investigate the roles of MEG3 in T-LBL, the expression of MEG3 was detected in T-LBL tissues compared with adjacent non-cancer tissues. We found that the level of MEG3 was conspicuously lower in T-LBL tissues compared with normal tissues (P<0.01; Fig. 1A). Besides, the level of MEG3 was significantly decreased in T lymphocyte leukemia cell lines (CCRF-CEM, Jurkat and SUP-T1) compared with human T-cell line H9 (P<0.05, P<0.01; Fig. 1B). The expression of MEG3 in the above tissues and cell lines was confirmed by western blot analysis. As shown in Fig. 1C, the result displayed that the expression of MEG3 was strongly suppressed in T-LBL tissues compared with control groups. Similarly, an obvious downregulation of MEG3 was detected in T-LBL cell lines compared to that of T-cell line H9 (Fig. 1D). Collectively, these results suggest that the decreased expression of MEG3 may be involved in the development of T-LBL.

To better understand the functional role of MEG3 in T-LBL, we used a lentiviral vector (lv-LncRNA-MEG3) to exogenously upregu-late the expression of MEG3. Two T-LBL cell lines, Jurkat and SUP-T1, were used in this experiment. Upregulation of MEG3 in Jurkat and SUP-T1 cells was verified through qRT-PCR (P<0.01; Fig. 2A). Next, CCK-8 assays were performed to detect cell proliferation. The results showed that cell proliferation was significantly inhibited in Jurkat and SUP-T1 cells after transfection with LncRNA-MEG3 lentivirus (Fig. 2B; P<0.01). The expression of proliferation marker proteins Ki-67 and PCNA was also detected. The result exhibited that the level of Ki-67 and PCNA was both restrained by LncRNA-MEG3 transfection (Fig. 2C and D; P<0.01). The results above indicate that overexpressed MEG3 suppresses cell growth in T-LBL.

Having established that overexpression of MEG3 suppressed cell growth in T-LBL, flow cytometric analysis was conducted to explore whether MEG3 has a pro-apoptosis effect on T-LBL cells. The results indicated that overexpressed MEG3 markedly promoted tumor cell apoptosis in SUP-T1 cells transfected with lv-LncRNA-MEG3 compared with the scramble group cells (Fig. 3A and B; P<0.01). We further analyzed the effect of MEG3 on cell cycle distribution through flow cytometry in SUP-T1 cells. In comparison with the scramble group cells, lv-LncRNA-MEG3 transfected cell induced an obvious cell cycle arrest in G2/M phase (Fig. 3C and D; P<0.01). The expression of apoptosis-related proteins (caspase-3 and caspase-9) was markedly enhanced under the treatment of LncRNA-MEG3 (Fig. 3E). Moreover, the expression of cell cycle marker protein p27 and cyclin A was detected through western blotting. Upregulated expression of p27 and decreased cyclin A in SUP-T1 cells transfected with lv-LncRNA-MEG3 further identified that overexpressed MEG3 inhibited cell proliferation (Fig. 3F). The above results demonstrate that overexpressed MEG3 induces cell apoptosis and G2/M cell cycle progression in T-LBL cell lines.

Considering the effect of MEG3 overexpression in inhibiting cell viability of T-LBL, we then explored the underlying molecular mechanisms. According to bioinformatics predictions, MEG3 RNA contains the complementary sequences of miR-214 (Fig. 4A). After that, relative expression of miR-214 in T-LBL tissues and cell lines was detected through qRT-PCR and Northern blotting. As shown in Fig. 4B, relative expression of miR-214 was largely upregulated in T-LBL tissues compared with normal tissues. Similarly, remarkable difference was measured between the expression of miR-214 in T-LBL cell lines (CCRF-CEM, Jurkat and SUP-T1) and that in human T-cell lines H9 (Fig. 4C; P<0.01). Additionally, a significantly increased level of miR-214 was found in T-LBL tissues compared with the normal tissues through Northern blotting (Fig. 4D). As expected, the expression of miR-214 in related T-LBL cell lines was drastically increased compared with the control cell line (Fig. 4E). All the experiments above reveal that the level of miR-214 is elevated in T-LBL.

To further assess the potential relationship between miR-214 and MEG3, qRT-PCR and Northern blotting were conducted in SUP-T1 cells transfected with lv-LncRNA-MEG3 or lv-MEG3 siRNA in combination with miR-214 mimic/miR-214 inhibitor/miR-214 mock. As shown in Fig. 5A, the upregulated level of miR-214 in SUP-T1 cells transfected with miR-214 mimic was down-regulated by co-transfection with lv-LncRNA-MEG3 (P<0.05). Similarly, the decreased level of miR-214 was elevated by adding miR-214 inhibitor in SUP-T1 cells transfected with lv-MEG3 siRNA (Fig. 5B; P<0.05). A similar result was reflected through Northern blotting. miR-214 mimic reversed the weakening effect of LncRNA-MEG3 on the expression of miR-214 as shown in Fig. 5C. Likewise, the upgrading functions of MEG3 siRNA on the level of miR-214 was weakened by miR-214 inhibitor (Fig. 5D). In addition, the wild-type and mutant 3′-UTR of MEG3 were, respectively, cloned into the luciferase plasmid (pGL3) and co-transfected with miR-214 mimic or miR-214 inhibitor into SUP-T1 cells. The luciferase assay showed that miR-214 mimic effectively inhibited the luciferase activity of the MEG3-WT reporter but that of the MEG3-Mut reporter was unaffected, suggesting that miR-214 is a direct target gene of MEG3.

The target sequences of miR-214 in the 3′-UTR region of AIFM2 was predicted through bioinformatics analysis (Fig. 6A). In order to further investigate the regulatory relationship between MEG3, miR-214 and AIFM2, Jurkat and SUP-T1 cells were transfected with lv-LncRNA-MEG3 or lv-control and then co-transfected with miR-214 mimic or miR-214 mock. As shown in Fig. 6B and C, the RNA level of AIFM2 was strongly upregulated by MEG3 and was suppressed by miR-214 detected through qRT-PCR (P<0.05). Similarly, the result of western blotting exhibited that the levels of AIFM2 in Jurkat and SUP-T1 cells was significantly elevated by MEG3 and was reduced by miR-214 (Fig. 6D and E).

Furthermore, the impact of miR-214 on cell proliferation, cell apoptosis and cell cycle was adverse to that of MEG3. Compared with the miR-214 mock group, cell proliferation was promoted by miR-214 mimic and the facilitating role was weakened by MEG3 (Fig. 6F; P<0.01). Moreover, the increased cell apoptosis rate by MEG3 was downregulated by co-transfecting with miR-214 mimic in SUP-T1 cells (Fig. 6G; P<0.01). Compared with the control group, cell cycle was arrested in G2/M phase by MEG3 and the regulating role was abolished by miR-214 mimic (Fig. 6H; P<0.01).

The expression of related proteins in SUP-T1 cells was detected through western blotting. Compared with the control group, the level of proliferation marker proteins Ki-67 and PCNA was inhibited by MEG3 and was elevated by miR-214 mimic (Fig. 6I). As expected, the level of apoptosis marker proteins (caspase-3 and caspase-9) was enriched by MEG3 and was decreased by miR-144 mimic (Fig. 6J). The opposite effect on the regulation of cell cycle marker proteins p27 and cyclin A was seen between miR-214 and MEG3 through western blotting (Fig. 6K). Taken together, our results above reveal that AIFM2 is upregulated by MEG3 by targeting miR-214.

Knowning that the level of AIFM2 was upregulated by MEG3 and was suppressed by miR-214 in T-LBL cell lines, further research was conducted to explore the relationship among the expression level of MEG3, miR-214 and AIFM2 in T-LBL tissues. RNA was extracted in T-LBL tissues donated from Chengdu Military General Hospital of PLA (n=38). Relative expression of MEG3, miR-214 and AIFM2 was detected through qRT-PCR. As shown in Fig. 7A and B, the expression of miR-214 varied inversely with the expression of MEG3 and AIFM2, respectively. Besides, relative expression of AIFM2 was positively related to the level of MEG3 (Fig. 7C). Thus, we conclude that the expression of AIFM2 was upregulated by MEG3 and was downregulated by miR-214 in vivo.

We then set out to explore the effect of exogenous MEG3 over-expression on T-LBL in vivo. SUP-T1 cells were transfected with lv-LncRNA-MEG3 or lv-scramble for 24 h. Then, the cells were collected and inoculated into female athymic nude mice subcutaneously. Average tumor volume in LncRNA-MEG3 group was much smaller than the scramble group (Fig. 8A and B). In order to investigate the effect of MEG3 on the expression of miR-214 and AIFM2 in vivo, qRT-PCR and western blotting were separately conducted on SUP-T1 cells transfected with lv-LncRNA-MEG3 or lv-scramble. The relative expression level of MEG3 and AIFM2 was largely increased by LncRNA-MEG3 (Fig. 8C and E). By contrast, the expression of miR-214 was apparently suppressed by LncRNA-MEG3 (Fig. 8D). Consistent with this, the western blotting results exhibited that overexpressed MEG3 raised the expression of AIFM2 and reduced the level of miR-214 (Fig. 8F). Moreover, the upregulated expression of proliferation marker protein Ki-67 and PCNA in LncRNA-MEG3 group also verified that MEG3 promoted cell proliferation in T-LBL (Fig. 8G). Based on the above results, we deduce that overexpressed MEG3 inhibits tumor growth in vivo.