HCC samples

Pair-matched tumor tissues and adjacent non-tumorous tissues from 35 patients undergoing resection of HCC were obtained from Tongji Hospital, Huazhong University of Science and Technology in 2016. Informed consent was obtained from all of the patients. The study was performed according to the guidelines of the Ethics Committee of the Tongji Hospital and approved in accord with the ethical standards of World Medical Association Declaration of Helsinki.

Cell culture

The cell lines of human HCC including Huh7, MHCC-97H, Hep-G2, Hep-3B, HCCLM3, MHCC97-L and L02 liver cells were kept in the Institute of Liver Diseases (Tongji Hospital, Wuhan, China) and cultured after resuscitation using DMEM supplemented with fetal bovine serum (10%, Gibco, Grand Island, NY, America) in a 37 incubator with 5% CO2.

Plasmids, virus, and transfection

Transfection of the plasmid of luciferase reporter was carried out using the reagent of Lipofectamine 3000 (Invitrogen, Carlsbad, America) following the instructions from the manufacturer. For overexpression of LINC01503, human full-length LINC01503 gene was acquired using PCR assay and inserted into the pMSCV plasmid. To downregulate LINC01503, shRNA sequence which targeted LINC01503 was inserted into the pSuper-puro plasmid. The transfection assay was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, America). The target cells were infected using retroviruses generated by pMSCV-puro-LINC01503 and pSuper-puro-LINC01503-shRNA for 72 h. Puromycin (0.6 μg/mL) was employed to screen the stable cell lines for 9 days.

Extraction of RNA and real-time PCR assay

Trizol reagent (Invitrogen, Carlsbad, America) was used to extract total RNA, and the manufacturer manual was followed. The complementary DNA which was reversely transcribed was synthesized by Prime Script RT Reagent Kit (Takara, Tokyo, Japan). Real-time PCR assay was carried out as what was mentioned previously ¹⁵. The 2-ΔΔCt method was applied to determine the difference between multiple samples. LINC01503 primer sequence: sense strand, 5'-TCTTCGTGTTCATCATCAGTCCC-3'; antisense strand, 5'-CTGAAAGAAACTCATTGCATCGTG-3'.

Histology

After the mice were sacrificed, the tumor samples were obtained and fixed with paraformaldehyde (4%), followed by paraffin embedding. The sections were prepared and stained by H&E. Furthermore, tumor tissues were applied to conduct immunohistochemistry assay for Ki67. The procedures were carried out as what were described previously ¹⁶.

Western blot analysis

Western blot assay was conducted as mentioned previously ¹⁶. The primary antibodies were listed below: anti-BCL2 apoptosis regulator (BCL2) antibody (Proteintech, 12789-1-AP, 1:1000), anti-BCL2 associated X, apoptosis regulator (BAX) antibody (Proteintech, 50599-2-Ig, 1:1000), anti-ERK1/2 antibody (CST, 4695, 1:1000), anti-phosphoERK1/ 2 (p‐ERK1/2) antibody (CST, 4370, 1:1000), anti-JNK antibody (CST, 9252, 1:1000), anti-phosphoJNK (p‐JNK) antibody (CST, 9251, 1:1000), anti-P38 antibody (CST, 8690, 1:1000), anti-phosphoP38(p-P38) antibody (CST, 4511, 1:1000) and anti-α-tubulin antibody (CST, 2125, 1:1000). Anti-rabbit IgG (Jackson ImmunoResearch Laboratories, America, 1:2000) was used as the secondary antibody. The protein bands were scanned and analyzed by using ImageJ software (NIH, America).

CCK8, clone formation, and cell apoptosis experiments

Cell proliferation ability was measured by the Cell Counting Kit-8 (CCK-8, Promotor, China) following the instructions. After addition of CCK-8 reagent into the 96-well plates, the cells were cultured for two hours. The absorbance at 450 nm (OD450) was recorded. Clone formation assay was used to evaluate the clone formation capability of HCC cells. Cells (1 × 10³/well) were seeded in 6-well plates. Afterwards, the cells were maintained in DMEM (Promoter, Wuhan, China) which was added with fetal bovine serum (10%, Gibco, Grand Island, NY, America). After 2 weeks, the colonies were fixed by using paraformaldehyde (4%, Servicebio, Wuhan, China) in a 37 °C incubator for four hours, and then stained by crystal violet (0.5%, Promoter, Wuhan, China) for 2 h at 37 °C. The colony number was counted under a light microscope. Annexin V-FITC / propidium iodide (annxinV/PI) apoptosis detecting kits (Nanjing KeyGen Biotech, Nanjing, China) were used to determine the cell apoptosis, and the cells were stained. Flow cytometer (FACSCalibur, BD Biosciences) was carried out to determine the apoptosis ability and the results were analyzed with CellQuest software. SCH772984 (1μM, a highly selective and ATP-competitive ERK inhibitor, Medchem Express, America) was used to inhibit ERK1/2 in this study.

In vivo tumor xenograft model

BALB/c nude mice (male; age, 3-4 weeks; body weight, 12-14g) were purchased from Beijing HFK Bioscience. Mice were maintained in a pathogen-free environment (relative humidity, 60% ± 5%; temperature, 22 ± 2°C) with a light / darkness cycle of 12 h/12h. The mice were allowed free access to common chow diet and water in the Center of Animals in Tongji Hospital. All in-vivo experiments were performed in adherence to the Guide for the Care and Use of Laboratory Animals of Tongji Medical College. HCC cells (5×10⁶) were suspended in PBS (0.1 mL) and injected subcutaneously into the mice from the flank of both sides at the dorsal region to establish a hepatoma xenograft model. Tumor size was monitored by measuring the length (L) and width (W) with digital caliper, and tumor volume (V) was calculated based on the equation V = 1/2 × L × W². On the 21^st day, the mice were injected with luciferin (150 mg/kg) in the intraperitoneal cavity. The tumors were observed using a Lago X imaging system (SI Imaging) about 5 minutes post luciferin injection, excised and weighed.

The expression of LINC01503 is relatively high in HCC cell lines

To further investigate the expression and effect of LINC01503 in HCC cells, we detected the expression levels of LINC01503 by using real-time PCR assay. The results showed that LINC01503 expression levels were upregulated in HCC cell lines (especially in MHCC-97H cell lines) compared with normal liver cells (L02 cell) (P<0.05) (Figure ​(Figure2A).2A). The endogenic LINC01503 expression was downregulated by shRNA in MHCC-97H cell line, which had a high basal expression of LINC01503. Meanwhile, LINC01503 was overexpressed in Huh7 cell line with pMSCV plasmids, which had a relatively low basal expression of LINC01503 (P<0.01) (Figure ​(Figure22B).

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Figure 2

The LINC01503 expression and regulation in HCC cell lines. (A) Relative expression levels of LINC01503 in HCC cell lines (HCCLM3, MHCC-97H, MHCC97-L, Huh7, Hep-3B and Hep-G2) and L02 liver cells measured by using real-time PCR. (B) The regulation of LINC01503 levels in HCC cells. MHCC-97H cells were transfected by shRNA targeting LINC01503 (shLINC01503), and Huh7 cells were transfected by pMSCV plasmids encoding LINC01503 (LINC01503). Cells were transfected by scrambled shRNA (scramble) or pMSCV (vector) and served as negative control, respectively. LINC01503 was measured by real-time PCR. Data were shown as mean ± SD, n = 3. *, P<0.05, **, P<0.01. shRNA, short hairpin RNA.

LINC01503 promotes cell proliferation and clone formation in HCC cells

Since LINC01503 is highly expressed in HCC tissues and cell lines, we then wanted to study its potential biological functions involved in HCC pathogenesis. GSEA v4.0.1 was applied to perform GO analysis among the HCC samples in TCGA database. It demonstrated that high expression of LINC01503 was mainly involved in positive regulation of cell proliferation (Table ​(Table4)4) (Figure ​(Figure3A).3A). To confirm this, we performed CCK8 assay. As expected, cell proliferation ability was decreased when LINC01503 was silenced in MHCC-97H cell line but significantly enhanced when LINC01503 was upregulated in Huh7 cell line (P < 0.01) (Figure ​(Figure3B).3B). Consistently, similar results were found in the clone formation abilities of HCC cells (P < 0.01) (Figure ​(Figure3C).3C). Taken together, LINC01503 could positively regulate cell proliferation and clone formation in HCC cells.

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Figure 3

LINC01503 promoted HCC cells proliferation. (A) The correlation between LINC01503 expression levels and proliferation in HCC was indicated by GO analysis. (B) Cell proliferation was assayed in MHCC-97H-scramble, MHCC-97H-shLINC01503, Huh7-vector, and Huh7-LINC01503 cells using CCK8. (C) The clone formation was detected by crystal violet staining. Data were shown as mean ± SD, n = 3. **, P<0.01. GSEA, Gene Set Enrichment Analysis.

LINC01503 inhibits cell apoptosis in HCC cells

GO analysis also indicated that LINC01503 expression levels were significantly correlated with the negative regulation of apoptosis (Table ​(Table4)4) (Figure ​(Figure4A).4A). We also tested this by apoptosis assays in vitro. Flow cytometry assay implied that silence of LINC01503 promoted apoptosis of MHCC-97H cells. On the contrary, overexpression of LINC01503 inhibited cell apoptosis in Huh7 cell line (P<0.01) (Figure ​(Figure4B).4B). Similar results were found by western blot assay, which demonstrated a decreased BCL2 expression, an anti-apoptotic protein, and an increased BAX level, the pro-apoptotic protein, in LINC01503 silence group compared to the control. There was an increased BCL2 expression and decreased BAX expression in the LINC01503 upregulation group in comparison with the control (P<0.01) (Figure ​(Figure4C).4C). These results implied that LINC01503 inhibited apoptosis of HCC cells.

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Figure 4

LINC01503 inhibits HCC cells apoptosis. (A) The correlation between LINC01503 expression levels and apoptosis in HCC was indicated by GO analysis. (B) Apoptosis of MHCC-97H and Huh7 cells assessed by flow cytometry. (C) The expression of BAX and BCL2, apoptosis-related proteins, was measured by western blot. Data were shown as mean ± SD, n = 3. **, P<0.01. GSEA, Gene Set Enrichment Analysis; BCL2, BCL2, apoptosis regulator; BAX, BCL2 associated X, apoptosis regulator.

LINC01503 promotes xenograft tumor growth in vivo

Since LINC01503 could promote proliferation and inhibit apoptosis of HCC cells in vitro, we wanted to learn whether it could play a consistent role on tumor growth in vivo. Xenograft mouse models were established by subcutaneously inoculating control cells and cells with upregulated or silenced LINC01503 respectively into the left and right flank at dorsal region. After 21 days, Lago X imagining system demonstrated that the group with silencing of LINC01503 showed weaker fluorescence signals compared to the control. Conversely, LINC01503 overexpression group showed stronger fluorescence signals compared with the control group, as shown in Figure ​Figure5A.5A. Consistent with the above data, knockdown of LINC01503 in MHCC-97H cells ameliorated the tumor growth rate compared with the control cells, whereas LINC01503 upregulation in Huh7 cells showed an opposite effect (P<0.01) (Figure ​(Figure55B).

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Figure 5

LINC01503 promotes tumor growth of implanted HCC cells in vivo. (A) Representative luciferase signal images of the tumor-bearing mice on the 21^st day post-implantation. (B) The growth curves of xenografts via measuring the volume of the tumor every 3 days after implantation. (C) Images of dissected tumors on the 21^st day post-implantation. (D) Tumor weight analysis. (E) Representative images of H&E (400×) and Ki67 IHC staining (400×) for tumor tissues. *, P<0.05, **, P<0.01.

On the 21^st day following inoculation, the tumors were excised, photographed, and weighed after the mice were sacrificed, as shown in Figure ​Figure5C.5C. LINC01503 knockdown in MHCC-97H cell line apparently decreased the tumor weight compared with the control group while LINC01503 overexpression in Huh7 cell line showed converse effects (P<0.05) (Figure ​(Figure5D).5D). Moreover, the protein expression of Ki67 in tumors was determined by immunohistochemistry. Compared with the control group, MHCC-97H derived tumors which were transfected by shLINC01503 exhibited lower levels of Ki67. On the contrary, more Ki67 positive signals were observed in LINC01503-overexpressing tumors implanted by Huh7 compared with the control group (Figure ​(Figure5E).5E). These data implied that LINC01503 could promote tumor growth of HCC in vivo.

This work was supported by the State Key Laboratory of Cancer Biology (Fourth Military Medical University) [grant number CBSKL201720]; the Natural Science Foundation of Hubei Province [grant number 2017CFB457]; and the National Natural Science Foundation of China [grant number 81372663].