Tissue specimens

In total, 60 matched hepatocellular carcinoma specimens of patients with pathological diagnosis of hepatocellular carcinoma and corresponding adjacent non-tumor tissues were recruited from the Clinical Laboratory of Hainan General Hospital between October 2015 and September 2017. Immediately after the surgery, all tissue samples from patients with hepatocellular carcinoma were treated in liquid nitrogen, and then stored at –80°C until use. Clinical characteristics of patients with hepatocellular carcinoma included in the present study are presented in Table I. The medical ethics committee of Hainan General Hospital approved this research. A letter of written agreement consistent with the principles of the Helsinki Declaration was obtained from each participant before the study.

Cell culture and transfection

Cells from the hepatocellular carcinoma cell lines Huh7 and HepG2 and the immortalized human fetal liver cell line L02 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Briefly, cells were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% Gibco fetal bovine serum (FBS) (GE Healthcare Life Sciences, Logan, Ut, USA) under 37°C with 5% CO₂. The HCC cells were transfected with SNHG1si-RNA, si-Negative Control (si-NC) or si-SNHG1 combined with miR-195 inhibitor (10 nmol/l for each) using Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Si-RNA of SNHG1 was synthesized by Guangzhou ribobio Co. Ltd. China with the sequence of si-5′-CUUAAAGUGUUAGCAGACATT-3′. miR-195 inhibitor was purchased from Shanghai Gene Pharma Co., Ltd, China with the sequence of 5′-GCCAAUAUUUCUGUGCUGCUA-3′. The negative control was a blank vector while the control group was without any treatment.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated with the TRIzol (Invitrogen, America) reagent according to the manufacturer’s instructions. Approximately 1 ng of RNA was reverse transcribed using the Reverse EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Trangene, Beijing, China) following the manufacturer’s instructions. Real-time polymerase chain reaction (RT-PCR) was performed using the SYBR Green Master Mixture (Roche, America) reagent in an ABI 7500 Real-time PCR instrument. The following primers were used to detect the expression: the stem-loop RT primer for miR-195, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCCAAT-3′ and the amplifying primers for miR-195, forward 5′-CGTAGCAGCACAGAAAT-3′ and reverse 5′-GTG CAGGGTCCGAGGT-3′; SNHG1, forward 5′-AGGCTGAAGTTACAGGTC-3′ and reverse 5′-TTGGCTCCCAGTGTCTTA-3′; Astrocyte elevated gene 1 (AEG-1), forward 5′-AAATGGGCGGACTGTTGAAGT-3′ and reverse 5′-CTGTTTTGCACTGCTTTAGCAT-3′; GAPDH, forward 5′-AAGGTGAAGGTCGGAGTCA-3′ and reverse 5′-GGAAGATGGTGATGGGATTT-3′. The relative expression level of miRNAs was normalized to U6 small nucleolar RNA. All qRT-PCR runs were repeated in three replications. The results were calculated with the 2^–ΔΔCt method. GAPDH served as an internal control.

MTT assay

After 48 h after transfection, the effect of lncRNA SNHG1 on the proliferation of Huh7 and HepG2 cells was measured by MTT assay. The cells were seeded at a density of 1 × 10⁵ cells per well in 96-well plates and transfected with 50 nM Ln cRNA mimics. 1 × 10⁴ cells per well were cultured in 96-well plates. MTT (100 μl) was added and incubated for an additional 4 h. Next, 150 μl of DMSO was added and the absorbance was read at 570 nm using a microplate reader (Thermo Scientific).

Cell migration and invasion test

For the migration assay, 1 × 10⁵ Huh7 and HepG2 cells were seeded in six-well plates and cultured at 37°C. A linear wound was carefully made in the middle of the plates, and cell debris was removed and incubated with serum-free medium. The wounded monolayers were then photographed at 0 and 24 h after wounding. For the invasion assay, 3 × 10⁵ cells were seeded in FBS-free DMEM in 24-well transwell chambers pre-coated with Matrigel (BD Bioscience, USA). The lower chamber was filled with DMEM supplemented with 20% FBS. After 24 h of incubation, cells in the upper chamber were removed, and the cells that had traversed the membrane were stained with crystal violet. All experiments were performed at least three times in triplicate.

Western blotting analysis

Briefly, total protein extracted from cells was quantified with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Inc.), then loaded on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibody at 4°C overnight, and subsequently incubated with the corresponding secondary antibodies at 37°C for 45 min. The target bands were scanned using Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. GAPDH served as an internal control.

Dual luciferase reporter assay

The miR-195 full length 5′ lncRNA SNHG1-binding seed region or mutant (Mut) was amplified and sub-cloned into the pGL3-basic luciferase vector (Promega, USA). Huh7 and HepG2 were co-transfected with either the wild-type or Mut SNHG1-binding seed region, together with lncRNA SNHG1 or pcDNA6.2-control. After 48 h of transfection, relative luciferase value was measured and normalized to the values of Renilla luciferase activity using the Bright-Glo Luciferase Assay System (Promega, Cat. E2610, Promega).

Statistical analysis

All data were expressed as mean ± SD. Comparison between two groups was performed using Student’s t-test. It was considered to be statistically significant when the p-value was less than 0.05. All calculations were made using SPSS 18.0.

Long non-coding RNA SNHG1 was up-regulated in HCC cell lines and tissues

The expression level of SNHG1 was detected in two hepatocellular carcinoma cell lines, Huh7 and HepG2. Generally, a significant increase in SNHG1 expression was observed in cancer cell lines compared to normal liver cell lines (Figure 1 A, p < 0.001). Additionally, the levels of SNHG1 expression were detected in 60 paired hepatocellular carcinoma tissues and normal adjacent liver tissue. Consistent with this observation, SNHG1 was significantly up-regulated in hepatocellular carcinoma tissues in comparison to the levels in adjacent non-tumor tissues (Figure 1 B, p < 0.001). These results indicated that SNHG1 expression was impaired in HCC and may be involved in HCC progression.

Figure 1

Long non-coding RNA SNHG1 was up-regulated in HCC tissues and cell lines. A – Expression of SNHG1 in cell lines HepG2 and Huh7, and a normal human hepatic cell line (L02); ***p < 0.001 vs. control. B – The levels of SNHG1 were quantified by qRT-PCR analysis in tumor tissues and normal tissues (n = 60)

Knockdown of SNHG1 inhibits HCC cell proliferation and induces apoptosis

To determine the function of SNHG1 in hepatocellular carcinoma tumor genesis, Huh7 and HepG2 were transfected with si-SNHG1 and the scramble negative control si-Scramble. The results showed that compared with si-Scramble, the levels of SNHG1 in Huh7 and HepG2 cells transfected with si-SNHG1 were significantly down-regulated (Figure 2 A, p < 0.01). We then performed an MTT assay to determine cell proliferation, and as a result, knockdown of SNHG1 was scrambled by the negative control (p < 0.05) (Figures 2 B, C) compared to the control group. As shown in Figures 2 D and E, inhibition of SNHG1 expression reduced the invasive ability of Huh7 and HepG2 cells (all p < 0.01). These results indicate that inhibition of si-SNHG1 can significantly inhibit migration and invasion of hepatocellular carcinoma cells.

Figure 2

Silencing SNHG1 could significantly inhibit migration and invasion of HCC cells. A – The interfering effects of si-SNHG1 were evaluated in Huh7 and HepG2 cells after transfection with si-SNHG1 or si-Scramble; B, C – the Huh7 and HepG2 cells were transfected with si-SNHG1 or si-Scramble and the MTT assay was used to detect the cell proliferation; D, E – results of transwell assay in Huh7 and HepG2 cells transfected with si-RNA and si-NC. The invasion of HCC cells was significantly inhibited by si-SNHG1

*P < 0.05, **p < 0.01, compared with the si-Scramble group.

Silencing SNHG1 could significantly inhibit expression of AEG-1 protein in HCC cells

To further investigate the influence of SNHG1 on hepatocellular carcinoma cells, expression of AEG-1 protein was quantified using modern molecular techniques of qRT-PCR and Western blotting assay. The results showed that after silencing SNHG1 in both Huh7 and HepG2 cells, expression of AEG-1 significantly decreased at both mRNA and protein levels compared with the control (p < 0.05, Figure 3).

Figure 3

Silencing SNHG1 could significantly inhibit expression of AEG-1 protein in HCC cells. Protein expression of AEG-1 in Huh7 and HepG2 cells transfected with si-RNA and si-NC by Western blotting

*P < 0.05, compared with the si-NC group.

SNHG1 and miR-195 interact with and repress each other

LncRNA acts as a miRNA sponge, and mRNA has been reported to reduce regulatory effects. This function introduces an additional layer of the composite layer in the miRNA target interaction network. Major mRNA and non-coding RNA are miRNAs that may be affected by methods, including cancer cell proliferation, apoptosis, and invasion of cancer. In addition, miR-195 was confirmed by luciferase assay to regulate SNHG1. A luciferase reporter vector (MUT-SNHG1 includes a predicted binding site mutation of 8BP in WT-SNHG1 and miR-195) with wild-type and mutant SNHG1 was constructed (Figure 4 A). These indicated vectors were co-transfected into HEK293 cells with miR-195 mimics or miR-195 NC; the luciferase activity was then monitored. The results showed that the luciferase activity of wt- SNHG1 vector could be significantly suppressed by miR-195 mimics; after mutation in the predicted binding sites of miR-195 in SNHG1, the effect of miR-195 mimics or miR-195 NC on luciferase activity was abolished (Figure 4 B), indicating that SNHG1 could directly bind to miR-195.

Figure 4

LncRNA SNHG1 as an endogenous sponge of miR-195. A – The lncRNA SNHG1-binding sequence in the 5′ region of miR-195; B – the relative luciferase activity of SNHG1 wild-type or mutant 3’-UTR in HEK293 cells following transfection with the miR-195 mimic or corresponding mimic NC; C – the relative expression levels of miR-195 were quantified using qRT-PCR in the Huh7 and HepG2 cells; D – the relative expression of miR-195 in tumor tissues and normal tissues; E – the negative correlation between SNHG1 and miR-195 levels in the HCC patients (R² = –0.6625, p < 0.001)

*P < 0.05; **p < 0.01 vs. control.

In addition, the level of miR-195 was significantly down-regulated compared to the normal human liver cell line L02, indicating that there is SNHG1 expression in the HCC cell line (p < 0.01; Figure 4 C). Subsequently, qRT-PCR was performed to determine the level of miR-195 in tumor tissues. The results indicate that miR-195 is shown to be much lower in tumor tissues than in corresponding normal tissues (p < 0.01; Figure 4 D). Furthermore, correlation analysis indicated that there was a strong negative correlation between SHHG1 expression and miR-195 expression in HCC tissues (Figure 4 E). These data indicate that interaction inhibition occurs between SNHG1 and miR-195.