LncRNA SNHG14 regulates the DDP-resistance of non-small cell lung cancer cell through miR-133a/HOXB13 pathway

The parental NSCLC cell (A549) and DDP-resistant NSCLC cell (A549/DDP) were purchased from Cell bank of Chinese Academy of Sciences (Shanghai, China). Both A549 and A549/DDP cells were maintained in DMEM supplemented with fetal bovine serum (10%, Sigma, USA), penicillin (100 U/ml, Sigma) and streptomycin (100 mg/ml, Sigma) under 5% CO2 and 95% air. Thee specific sh-SNHG14, miR-133a mimics, miR-133a inhibitor, pcDNA3.1-HOXB13 (pc-HOXB13), and corresponding negative control (NC) were obtained from GenePharma (Shanghai, China). All these oligonucleotides were transfected into A549/DDP cells using Lipofectamine 3000 (Invitrogen, USA) following the instructions of manufacturers. After 48 h of transfection, cells were harvested for further research.

TRIzol reagent (Invitrogen) was adopted to extract RNAs from indicated cells were prepared following the manufacturers’ protocol. The RNA quality was determined via a NanoDrop 2000 instrument (Thermo Scientific, USA), and 5 μg RNA was used as template to synthesis cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA). SYBR Premix Ex Taq Kit (Takara, Tokyo) was adopted to conduct qRT-PCR on an ABI Prism 7700 system (PE Applied Biosystems, USA). Fold changes in RNA relative expression was counted through the 2^–ΔΔCt method. Primers were summarized as follows:

After 48 h of transfection, exponentially growing A549/DDP cells (1 × 10⁵ cells/well) were harvested and plated into 96-well plates supplemented with DDP (0, 2, 4, 6, AND 8 μg/ml), and then the proliferation viability of A549/DDP cells were tested by the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) and colony formation assays after 24 h of DDP incubation. For CCK-8, the CCK-8 solution (200 μl) was added to the each well and then incubated at 37 °C for 2 h. The absorbance was measured by a microplate reader (Bio-Rad Laboratories, USA) at 450 nm. For colony formation assay, indicated A549/DDP cells (2000 cells/dish) were seeded into 35-mm culture dish and maintained at 37 °C. After 2 weeks, the visible colonies were fixed in 4% paraformaldehyde for 0.5 h, stained with 0.1% crystal violet solution for 10 min, and counted using a microscope.

The A549/DDP cell cycle and apoptosis were estimated using flow cytometry. In brief, 48 h after transfection, cells were harvested for DDP treatment for additional 24 h. For the cell cycle assay, A549/DDP cells were fixed in 75% alcohol for 30 min, and then stained with propidium idoide (Sigma, MO, USA) for 15 min in dark. For cell apoptosis analysis, A549/DDP cells were washed with PBS followed by staining with Annexin V/PI kit (Vazyme, Nanjing, China) following the instructions of manufacturer. Finally, a BD Biosciences FACSCalibur Flow Cytometer (BD Biasciences, USA) was applied to detect the cell cycle and apoptosis.

The wide type and mutant type miR-133a binding sequence of lncRNA SNHG14 and HOXB13 mRNA 3′-UTR were sub-cloned into the pmirGLO vector (Promega, Madison, USA) and named as SNHG14-WT, HOXB13-WT, SNHG14-MUT, and HOXB13-MUT, respectively. A549/DDP cells (1 × 10⁶ cells/wel) seeded in 96-well plates were co-transfected with constructed recombinant luciferase vectors and miR-133a mimics or mimics NC using Lipofectamine 3000 (Invitrogen). Luciferase intensity of A549/DDP cell was examined using the Dual-Luciferase Reporter Assay System (Promega) after 48 h of co-transfection.

Total proteins of indicated A549/DDP cells were isolated using the RIPA buffer (Beyotime, Beijing, China) contained protease inhibitors (Roche, Germany). Protein samples (50 μg) were isolated via 10% SDS-PAGE and then transferred into polyvinylidene difluoride (PVDF) membranes (Millipore, UK). After incubated in 5% skimmed milk for 2 h, the membranes were subjected for probe of primary antibodies that against Bcl-2 (1:2000, ab491583, Abcam), Bax (1:1000, ab199677, Abcam), cleaved-caspase-3 (1:1000, orb227889, Biorbyt) and HOXB13 (1:2000, ab53931, Abcam) overnight. Subsequently, the membranes were washed with PBS and then probed with corresponding secondary antibodies conjugated with horseradish peroxidase for 2 h. The signals were visible using an enhanced chemiluminescent reagent (ECL, Germany).

To investigate whether lncRNA SNHG14, HOXB13 and miR-133a play roles in the chemo-resistance of NSCLC, we firstly examined their expression in parental NSCLC cell (A549) and DDP-resistant NSCLC cell (A549/DDP) using qRT-PCR. As results indicated that lncRNA SNHG14 and HOXB13 were remarkably increased, while miR-133a was significantly decreased in A549/DDP cells compared with A549 cells (Fig. 1a). The dysregulation of lncRNA SNHG14, HOXB13 and miR-133a in A549/DDP cells implied that they may be involved in the chemo-resistance of NSCLC.

To further investigate the functional roles of lncRNA SNHG14 in the development of DDP resistance of A549/DDP cells, we silenced its expression in A549/DDP cells followed by the examination of cell viability, proliferation cycle and apoptosis. Results from qRT-PCR A549/DDP indicated a high knockdown efficiency of sh-SNHG14 in A549/DDP cells (Fig. 2a). The effects of A549/DDP cell viability under the treatment of DDP with different concentration (0, 2, 4, 6 and 8 μg/ml) were evaluated by CCK-8. The DDP-induced downregulation of A549/DDP cell viability was augmented in sh-SNHG14 group (Fig. 2b). Moreover, results from colony formation experiment demonstrated that lncRNA SNHG14 knockdown significantly reduced the A549/DDP cell number under the treatment of DDP (1 μg/ml) (Fig. 2c). In addition, flow cytometry was conducted in lncRNA SNHG14 silenced A549/DDP cells to estimate the influences of SNHG14 knockdown on A549/DDP cell cycle and apoptosis. In the presence of 1 μg/ml of DDP, lncRNA SNHG14 silencing resulted in a significant upregulation of cell number in G0/G1 phases and a remarkable downregulation of S phase (Fig. 2d). The A549/DDP cell apoptosis rate was significantly increased in sh-SNHG14 transfected group (Fig. 2e). In the western blot detection of apoptosis-related protein expression, we found that sh-SNHG14 transfection significantly decreased Bcl-2 expression, while increased Bax and cleaved-caspase-3 expression (Fig. 2f). Taken together, these findings strongly supported that lncRNA SNHG14 knockdown enhanced the sensitivity of A549/DDP cells to DDP.

We then explored the functional roles of miR-133a in the development of DDP resistance of NSCLC by overexpressing its expression in A549/DDP cells, and then estimated the cell viability, proliferation, cycle and apoptosis of miR-133a overexpressed A549/DDP cells. The overexpression efficiency of miR-133a in A549/DDP cells was determined by qRT-PCR (Fig. 3a). CCK-8 was utilized to detect the influences of miR-133a overexpression on A549/DDP cell viability in the presence of different concentrations of DDP (0, 2, 4, 6 and 8 μg/ml). As results showed that the A549/DDP cell viability of miR-133a mimics group was significantly lower than that of sh-NC group (Fig. 3b). Forthermore, miR-133a overexpression led to a significant reduction of A549/DDP cell number (Fig. 3c). In addition, the influences of miR-133a mimics treatment on A549/DDP cell cycle and apoptosis in the presence of 1 μg/ml of DDP were estimated via flow cytometry. As results demonstrated that miR-133a overexpression remarkably increased the A549/DDP cell number of G0/G1 phases, while reduced the A549/DDP cell number of S phase (Fig. 3d). Moreover, miR-133a overexpression led to a significant upregulation of A549/DDP cell apoptosis rate induced by DDP (Fig. 3e). Similar to lncRNA SNHG14 knockdown, we also found that miR-133a overexpression significantly downregulated the protein expression of Bcl-2 while upregulated the protein expression of Bax and cleaved-caspase-3 in A549/DDP cells treated with DDP (Fig. 3f). These data highly suggested that miR-133a overexpression increased the DDP sensitivity of A549/DDP cells.

Numerous studies have shown that lncRNAs participated almost in every aspect of the biological processes of tumor progression by competitively binding to specific miRNAs and thus releasing target genes. Therefore, bioinformatics analysis was performed to predict the target miRNAs which could interact with lncRNA SNHG14. We found that lncRNA SNHG14 possessed complementary sequence of miR-133a. Moreover, HOXB13 was also found to possess the complementary sequence of miR-133a (Fig. 4a). To validate the direct binding between lncRNA SNHG14 and miR-133a, as well as miR-133a and HOXB13, dual-luciferase reporter assay was conducted in A549/DDP cells. Results demonstrated that co-transfection with SNHG14-WT or HOXB13-WT and miR-133a mimics caused a significant repression of luciferase activity, while co-transfection with SNHG14-MUT or HOXB13-MUT and miR-133a mimics exhibited no obvious effects on the luciferase activity (Fig. 4b). Besides, we found thatA549/DDP lncRNA SNHG14 knockdown resulted in an upregulation of miR-133a in A549/DDP cells (Fig. 4c), and miR-133a overexpression caused a downregulation of HOXB13 (Fig. 4d). Together, these results indicated that lncRNA SNHG14 competed with HOXB13 for miR-133a binding.

To investigate whether HOXB13 plays a role in the enhanced effects of miR-133a on the sensitivity of A549/DDP cell to DDP, we treated A549/DDP cells with pc-HOXB13 and miR-133a mimics followed by the examination of cell viability, proliferation and apoptosis in the presence of DDP. The results indicated that overexpression of HOXB13 dramatically increased A549/DDP cell viability and colony-formation ability. However, co-transfection of pc-HOXB13 and miR-133a mimics reversed the upregulation of cell viability and colony-formation ability induced by pc-HOXB13 transfection in A549/DDP cells (Fig. 5a-b). Moreover, by using flow cytometry analysis, we revealed that pc-HOXB13 transfection sharply reduced A549/DDP cell apoptosis, while co-transfection of pc-HOXB13 and miR-133a mimics abolished the reduction of A549/DDP cell apoptosis induced by pc-HOXB13 transfection (Fig. 5c). Additionally, results from western blot indicated that overexpression of HOXB13 dramatically increased the expression of HOXB13 and Bcl-2 while decreased the expression of Bax and cleaved caspase-3 in the A549/DDP cells, however, co-transfection of pc-HOXB13 and miR-133a mimics abolished the dysregulation of these molecules (Fig. 5d). These findings suggested that overexpression of HOXB13 repressed the sensitivity of A549/DDP cell to DDP through antagonizing miR-133a.

Considering the interaction among lncRNA SNHG14, HOXB13 and miR-133a in A549/DDP cells, we further investigated whether miR-133a is involved in the process of lncRNA SNHG14 regulating the DDP-resistance of NSCLC through HOXB13 pathway. We have shown that sh-SNHG14 transfection could significantly decrease the A549/DDP cell viability in the presence of DDP while this repression of cell viability caused by sh-SNHG14 treatment was reversed by the miR-133a inhibitor (Fig. 6a). Similarly, in colony formation assay, the sh-SNHG14 treatment induced reduction of A549/DDP cell number was recovered by the co-transfection of sh-SNHG14 and miR-133a inhibitor (Fig. 6b). Moreover, the sh-SNHG14 treatment induced upregulation of G0/G1 phases cell number and downregulation of S phase cell number were counteracted partially by inhibition of miR-133a (Fig. 6c). The co-transfection of sh-SNHG14 and miR-133a inhibitor also abolished the promotive effects of sh-SNHG14 transfection on A549/DDP cell DDP-inducted apoptosis (Fig. 6d). In the western blot assay, we demonstrated that co-transfection of sh-SNHG14 and miR-133a inhibitor could reverse the sh-SNHG14 transfection induced Bcl-2 downregulation and Bax, cleaved-caspas-3 upregulation (Fig. 6e). In addition, we found that lncRNA SNHG14 knockdown significantly reduced the epression of HOXB13, however, co-transfection of sh-SNHG14 and miR-133a inhibitor abolished this effect (Fig. 6e). These results implied that miR-133a/HOXB13 pathway involved in the lncRNA SNHG14 regulation of A549/DDP cell sensitivity to DDP.