Cell culture and transfection

Four human NSCLC cell lines (A549, H23, SPC-A1, and NCI-H292) and one human bronchial epithelial cell line (16HBE) were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were incubated in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific) at 37°C in a 5% CO₂ atmosphere.

For knockdown of SNHG8, SNHG8 siRNA (SNHG8 siRNA1, SNHG8 siRNA2, SNHG8 siRNA3) and negative control (NC) SNHG8 were purchased from Thermo Fisher Scientific and kept at −80°C. For overexpression of miR-542-3p, miR-542-3p mimic and NC-miR-542-3p were purchased from Thermo Fisher Scientific, too. After 50%–60% confluence was achieved, cells were transfected with lentivirus, respectively. Then the media were replaced with fresh media after 24 hours. Subsequent experiments were performed after the next 24–96 hours according to the manufacturer’s instructions.

RNA extraction and quantitative real-time-polymerase chain reaction (qRT-PCR) assays

Total RNA was isolated using Trizol (Thermo Fisher Scientific), and reverse transcription was performed using cDNA Reverse Transcription kit (Takara, Dalian, China). qRT-PCR was carried out with SYBR Green Kit (Thermo Fisher Scientific) on ABI 7500 real-time polymerase chain reaction system. The gene expressions were calculated using the 2^−ΔΔCt method, with β-actin as an internal control. All reactions were repeated three times. The sequences of real-time polymerase chain reaction primers were as follows: SNHG8, forward 5′-TGTAAACTCCTTCTCGGGGCG-3′ and reverse 5′-GTCTATTTCGGTGAAATTGG-3′.

Plasmids construction and luciferase assay

Dual-luciferase miRNA target expression vector in SNHG8 containing the predicted miR-542-3p binding site (SNHG8-Wt) and with the mutated miR-542-3p binding site (SNHG8-Mut) were obtained. Then Luc-SNHG8-wt and Luc-SNHG8-mut were co-transfected separately with miR-542-3p into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). The pmirGLO control vector was transfected and it served as a control. Firefly and Renilla luciferase activities were measured in cell lysates 48 hours after transfection following the manufacturer’s protocol (Promega Corporation, Fitchburg, WI, USA).

Western blot analysis

Protein was extracted with RIPA buffer from tissues or cell lysates, and the protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, moved to a polyvinylidene difluoride membrane, and immunoblotted with the following antibodies: CCND1, CDK6, Caspase-3 (1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), or GADPH (1:2,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4°C. After washing, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG goat anti-mouse (1:10,000; Santa Cruz Biotechnology Inc.) or goat anti-rabbit (1:10,000; Santa Cruz Biotechnology Inc.) antibody for 2 hours at room temperature. Protein bands were detected by enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK). GADPH served as a loading control.

Cell proliferation assays

Exactly 1.0×10³ cells per well were plated into 96-well plates (Corning Incorporated, Corning, NY, USA) for 24 hours, and then cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) kit (MedChemExpress, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions. Briefly, 10 μL of CCK-8 was added to each well at 24, 48, and 72 hours after transfection; then, the cells were incubated at room temperature for another 2 hours and the absorbance was measured at 450 nm.

Flow cytometric analysis of apoptosis and the cell cycle

Exactly 1×10⁵ cells per well were seeded in 24-well plates and were fixed with 70% cold ethanol after 48 hours. Two hours later, the fixed cells with 100 μL of RNase (BD Biosciences, San Jose, CA, USA) were incubated at 37°C for 30 minutes. Subsequently, 400 μL of propidium iodide was added and cells were kept in a dark place at 4°C for 0.5 hour. Similarly, 1×10⁵ cells per well were seeded in 24-well plates; after 48 hours, the apoptotic cells were determined using fluorescein isothiocyanate-Annexin V Apoptosis Detection Kit (BD Biosciences). Cell cycle and apoptosis were assayed with flow cytometry (FACScan; BD Biosciences). All experiments were done in triplicate and repeated independently three times.

Tumor xenografts in nude mice

Five-week-old male BALB/c nude mice were obtained from the Southeast University Laboratory Animal Center (Nanjing, China). All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guideline and were approved by the Southeast University Laboratory Animal Center Commission. Exactly 1×10⁷ A549 cells/mL stably transfected with lv-shSNHG8 or negative vector were subcutaneously injected into the left side of the posterior flank of BALB/c nude mice. Mice weight and tumor volume were measured every 7 days. Tumor volumes were calculated as L×W²×0.5, where L is tumor length and W is tumor width. Twenty-eight days after injection, all mice were sacrificed, tumors were weighed and immediately snap frozen in liquid nitrogen, and stored at −80°C until use.

SNHG8 promotes NSCLC cells proliferation

To explore the oncogenic activity and role of SNHG8 in NSCLC, we successfully established NSCLC cells with SNHG8 transient knockdown in mRNA. Interference efficiency of SNHG8 siRNA1, SNHG8 siRNA2, and SNHG8 siRNA3 was evaluated by qRT-PCR after transfection in A549 and H23 cells. We found that both siRNAs obviously downregulated the mRNA level of SNHG8, of which the inhibitory effect of siRNA1 was more significant (P<0.05; Figure 2A). So, we selected siRNA1 for subsequent functional verification. Then, we performed CCK-8 assays which showed that SNHG8 knockdown significantly attenuated the vitality of A549 and H23 cells (P<0.05; Figure 2B). The decrease in proliferation was much higher in A549 cell than in other cells, so we performed the follow-up analysis on A549 cells.

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Figure 2

SNHG8 promotes NSCLC cells proliferation.

Notes: (A) Interference efficiency of SNHG8 siRNA1, SNHG8 siRNA2, and SNHG8 siRNA3 was evaluated by qRT-PCR after transfection in A549 and H23 cells. (B) The effects of SNHG8 knockdown on the vitality of NSCLC cells was measured using the CCK-8 assay. ^*P<0.05, ^**P<0.01, and ^***P<0.001.

Abbreviations: CCK-8, Cell Counting Kit-8; NC, negative control; NSCLC, non-small-cell lung cancer; qRT-PCR, quantitative real-time-polymerase chain reaction.

SNHG8 exerted its function through sponging miR-542-3p

To investigate the underlying mechanism of SNHG8 in NSCLC cells, we examined the potential miRNAs associated with SNHG8 because lncRNAs function mainly as miRNA sponges. With starBase version 2.0,^16,17 miR-542-3p turned out to be the possible target of SNHG8. The putative binding sites of miR-542-3p and wild-type regions of SNHG8 are shown in Figure 3A. Dual-luciferase reporter assay was performed to explore whether SNHG8 was a functional target of miR-542-3p. As shown in Figure 3B, the luciferase activity was significantly reduced in miR-542-3p mimics and SNHG8-Wt co-transfected HEK293T cells, which had no effect on the SNHG8-Mut vector (P<0.05). In addition, qRT-PCR analysis showed that silencing of SNHG8 significantly increased miR-542-3p expression (P<0.001; Figure 3C). However, overexpression of miR-542-3p was not found to affect SNHG8 expression (P>0.05; Figure 3D). Taken together, these results indicated that miR-542-3p was an inhibitory target for SNHG8 in NSCLC progression.

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Figure 3

SNHG8 exerted its function through sponging miR-542-3p.

Notes: (A) Sequence alignment of miR-542-3p with the putative binding sites in the wild-type regions of SNHG8. (B) Dual-luciferase reporter assay showed that miR-542-3p mimics reduced the intensity of fluorescence in HEK293T cells transfected with SNHG8-Wt instead of SNHG8-Mut vector. (C) qRT-PCR analysis of miR-542-3p expression in A549 cells transfected with siRNA-SNHG8 or siRNA-NC. (D) qRT-PCR analysis of SNHG8 expression in A549 cells transferred with miR-542-3p or miR-NC. ^*P<0.05, ^***P<0.001.

Abbreviations: NC, negative control; qRT-PCR, quantitative real-time-polymerase chain reaction.

SNHG8 depletion arrested cell cycle in the G0/G1 phase via targeting miR-542-3p/CCND1/CDK6

We then examined the impact of decreased SNHG8 on cell cycle in A549 cells. Flow cytometric analysis showed a decrease in the percentage of cells in the G2/M phase and a marked accumulation of cells in the G0/G1 phase in the SNHG8 siRNA group compared with control and NC-siRNA groups (P<0.01; Figure 4A). These results indicated that downregulation of SNHG8 expression in A549 cells was arrested in the G0/G1 phase. miR-542-3p has been proved to induce G1 phase arrest of cell cycle in human A549 cells, which seemed to occur by downregulating CDK 6 in the G1/S phase.¹⁸ To analyze whether miR-542-3p regulated the G1 phase cell cycle genes, we transfected A549 cells with miR-542-3p mimics. As Figure 4B shows, overexpression of miR-542-3p led to a significant decrease in endogenous cyclin D1 (CCND1) and CDK6 proteins at the protein level. Meanwhile, knockdown of SNHG8 caused a similar induction of the expression of CCND1 and CDK6 (Figure 4C). These data indicate that miR-542-3p downregulated the expressions of CCND1 and CDK6 at the translational level.

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Figure 4

SNHG8 depletion arrested cell cycle in the G0/G1 phase via targeting miR-542-3p/CCND1/CDK6.

Notes: (A) Depletion of SNHG8 in A549 cells led to an increase of cells at the G0/G1 phase and a concomitant decrease of cells at the G2/M phase. (B) CCND1 and CDK6 proteins in A549 cells were measured by Western blot at 48 hours post-transfection with different treatments (control, NC-miR-542-3p, or miR-542-3p). GADPH was used as an internal loading control. (C) CCND1 and CDK6 proteins in A549 cells were measured by Western blot at 48 hours post-transfection with different treatments (control, NC-siRNA, or SNHG8-siRNA). GADPH was used as an internal loading control. ^**P<0.01.

Abbreviations: NC, negative control; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

SNHG8 depletion reduced cell apoptosis via activation of Caspase-3. In order to determine the effects of SNHG8 depletion on apoptosis, Annexin V/propidium iodide double staining was performed on A549 cells. It was found that depletion of SNHG8 largely promoted late apoptosis (Figure 5). Statistical analysis revealed that ~10-fold and 7-fold increase of late apoptotic populations was detected in SNHG8 siRNA-infected A549 cells (45.1%±0.49%), as compared to NC siRNA-infected and control A549 cells (4.49%±0.08% and 6.46%±0.10%, respectively, P<0.001; Figure 5B). To confirm the apoptosis effect, we examined the protein expression of Caspase-3, which was related to apoptosis, and the results showed that Caspase-3 level was upregulated in SNHG8 siRNA group cells compared to NC-siRNA group cells and control group cells in A549 cells (Figure 5C). These results indicated SNHG8 knockdown could induce apoptosis via activation of Caspase-3 in NSCLC cells.

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Figure 5

SNHG8 depletion reduced cell apoptosis via activation of Caspase-3.

Notes: (A) Apoptotic cells were assessed by Annexin V/PI double staining and then subjected to flow cytometry analysis. Representative images showing results of Annexin V/PI staining in A549 cells with different treatments (control, NC-siRNA, or SNHG8-siRNA). (B) Detection of apoptosis 48 hours after transfection with siRNA using FCM analysis. (C) Caspase-3 proteins in A549 cells were measured by Western blot at 48 hours post-transfection with different treatments (control, NC-siRNA, or SNHG8-siRNA). GADPH was used as an internal loading control. ^***P<0.001.

Abbreviations: FCM, flow cytometry; FITC, fluorescein isothiocyanate; NC, negative control; PI, propidium iodide.