Long noncoding RNA NEAT1 promotes cell proliferation and invasion by regulating hnRNP A2 expression in hepatocellular carcinoma cells

Figures & data

Figure 1 The expression of NEAT1 is upregulated in HCC tissues and cell lines.

Notes: (A) Quantitative analysis of the expression levels of NEAT1 in samples normalized to GAPDH according to the 2^−ΔΔCt method. Three adjacent normal tissues, 12 HCC tissues and the HCC cell lines HepG2, SMMC-7721 and HCCLM3 were analyzed. (B) The statistical graph shows the quantitative analysis of the expression levels of NEAT1 in adjacent normal tissues, low-grade HCC (I–II) and high-grade HCC (III–IV). The significance of differences in each group was measured with a one-way ANOVA followed by Dunnett’s post hoc multiple comparison test. *P<0.05, ***P<0.001.
Abbreviations: ANOVA, analysis of variance; HCC, hepatocellular carcinoma; GAPDH, lyceraldehyde 3-phosphate dehydrogenase; NEAT1, nuclear-enriched abundant transcript 1.

Figure 2 Knocking down NEAT1 inhibited the proliferation of the hepatocellular carcinoma cell lines HepG2 and SMMC-7721.

Notes: (A) HepG2 and SMMC-7721 cells were treated with 100 nM si-NEAT1 or si-NC for 6 h. Total RNA was extracted 48 h later, and the expression levels of NEAT1 in each group were quantitatively analyzed and normalized to the GAPDH expression according to the 2^−ΔΔCt method. The significance of differences between groups was measured with a paired-sample t-test. ***P<0.001. (B) A total of 2×10⁵ HepG2 or SMMC-7721 cells labeled with 2 μM CFSE were seeded in six-well plates 48 h after si-NEAT1 or si-NC treatment. The cells were collected, and the CFSE intensity was detected by flow cytometry to measure cell proliferation 0 and 72 h after seeding. The data represent at least three independent experiments. (C) A total of 2×10³ HepG2 and SMMC-7721 cells were seeded in a 96-well plate 48 h after si-NEAT1 or si-NC treatment. Ten microliters of CCK-8 was then added to each well 0, 24, 48 and 72 h after seeding and the plate was incubated for 1 h at 37°C. The cell viability was then assessed based on the OD450 value using an automatic microplate reader. Data are presented as the mean ± SD from three independent experiments. The significance of differences between groups was measured with a two-way ANOVA. **P<0.01.
Abbreviations: ANOVA, analysis of variance; CCK-8, cell counting kit-8; CFSE, cell tracer: carboxyfluorescein diacetate, succinimidyl ester; GAPDH, lyceraldehyde 3-phosphate dehydrogenase; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; OD, optical density; SD, standard deviation; si, small intefering.

Figure 3 Knocking down NEAT1 inhibited the migration and invasion of the hepatocellular carcinoma cell lines HepG2 and SMMC-7721.

Notes: (A) A total of 2×10⁵ HepG2 and SMMC-7721 cells treated with si-NEAT1 or si-NC were suspended in 200 μL of serum-free DMEM in the upper chamber, which contained a porous membrane coated with Matrigel for the Transwell invasion assay; the membrane was uncoated for the migration assay. Subsequently, 15% serum was added to the lower chamber as a chemoattractant. After migration for 24 h or invasion for 48 h, cells that had penetrated the filters were fixed in dry methanol, stained with 0.1% crystal violet and then photographed. The scale bar represents 100 μm. (B) In each group, cells on the filter were detached with trypsin, and the number of cells was counted and is presented in the statistical graph. Data are presented as mean ± SD from three independent experiments. Significant differences from each group were measured by paired sample t-test. ***P<0.001.
Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; SD, standard deviation; si, small intefering.

Figure 4 Knockdown of NEAT1 altered global gene expression patterns in HCC cells.

Notes: HepG2 cells were treated with si-NEAT1 for 6 h, and RNA was extracted 48 h later. Gene sequencing assays were performed to screen for global transcriptional changes. (A) Hits for different genes and associated biologic processes and molecular function are presented based on the GO analysis of NEAT1 knockdown gene sequencing data. (B) Hits for different genes and associated pathways are presented based on the KEGG pathway analysis of NEAT1 knockdown gene sequencing data.
Abbreviations: CCR; C chemokine receptor; GO, gene ontology; HCC, hepatocellular carcinoma; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; NTP, nucleoside triphosphate; si, small interfering.

Figure 5 Knockdown of NEAT1-regulated genes which were related with HCC progression.

Notes: The expression levels of genes selected for further validation were normalized to the expression of GAPDH according to the 2^−ΔΔCt method. The significance of differences between groups was assessed with a paired-sample t-test. *P<0.05, **P<0.01. The data shown represent three independent experiments.
Abbreviations: GAPDH, lyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; NEAT1, nuclear-enriched abundant transcript 1; si, small interfering.

Table 1 Selected transcriptional changes

Selected gene name	Fold changes	P-value
FASN (fatty acid synthase)	0.352775678	0.000588873
TXNRD1 (thioredoxin reductase 1)	0.421050498	0.009009959
AR (androgen receptor)	0.443871707	0.005314096
SRXN1 (sulfiredoxin 1)	0.451058201	0.007842611
hnRNP A2 (heterogeneous nuclear ribonucleoprotein A2/B1)	0.478395062	0.016578563
FUS (FUS RNA-binding protein)	0.483988604	0.011259985
IQGAP1 (IQ motif containing GTPase activating protein 1)	0.498584211	0.014995828
DDX58 (DEXD/H-box helicase 58)	4.630358625	0.000000012
HERC5 (HECT and RLD domain containing E3 ubiquitin protein ligase 5)	3.336812332	0.000000271
STAT1 (signal transducer and activator of transcription 1)	3.668795232	0.004716635
OSMR (oncostatin M receptor)	2.154985755	0.007187685
NAMPT (nicotinamide phosphoribosyltransferase)	2.079423868	0.011377416
IGFBP3 (insulin-like growth factor binding protein 3)	2.007540475	0.014632835

Figure 6 U2AF65 binds to NEAT1 and mRNA hnRNP A2.

Notes: (A) RIP experiments were performed using the U2AF65 antibody and IgG control antibody. HepG2 or SMMC-7721 cell lysate (input control) was incubated with beads coated with U2AF65 antibody or IgG control antibody for 6 h at 4°C. Proteins eluted from the beads with SDS in each group were subjected to gel electrophoresis. The gel was stained with Coomassie blue and photographed. The highlighted region represents U2AF65 protein. (B) Beads attached to an antibody–protein–RNA complex were incubated with proteinase K at 55°C for 30 min, and the RNA was extracted by the TRIzol:chloroform method. RT-qPCR was then performed using specific primers to detect lncRNA-NEAT1, hnRNP A2 or GAPDH. The RT-qPCR product of each group was subjected to electrophoresis and photographed. (C) The quantitative expression levels of NEAT1 and hnRNP A2 were normalized to GAPDH expression in each group according to the 2^−ΔΔCt method. RIP enrichment was assessed based on RNA associated with the U2AF65 protein, which was compared with the IgG control group. The fold enrichment of NEAT1 and hnRNP A2 in the anti-U2AF65 group is presented in the statistical graph. The data represent the average and standard deviation of three independent experiments. (D) Full-length NEAT1 was biotinylated and targeted with streptavidin beads and then incubated with whole HepG2 extracts (input control) for 1 h at 4°C and washed. Streptavidin beads without biotinylated RNA are shown as a control. Associated protein eluted from the beads was subjected to gel electrophoresis, stained with Coomassie blue and photographed. (E) The highlighted regions were subjected to Western blotting using U2AF65 antibody for region 1, β-actin antibody for region 2 and hnRNP A2 antibody for region 3. The bands in each group were visualized by exposure for 20 s under a CCD camera. (F) RNAs corresponding to the different fragments (1–5) of NEAT1 were biotinylated and incubated with HepG2 whole cell extracts, targeted with streptavidin beads and washed. Bound U2AF65 protein was detected by Western blotting (n=3).
Abbreviations: CCD, charge coupled device; GAPDH, lyceraldehyde 3-phosphate dehydrogenase; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; IgG, immuglobulin G; NEAT1, nuclear-enriched abundant transcript 1; RIP, RNA immunoprecipitation; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SDS, sodium dodecyl sulfate; U2AF65, U2 small nuclear RNA auxiliary factor 2.

Figure 7 Knocking down NEAT1 and U2AF65 downregulated hnRNP A2 expression.

Notes: (A) NEAT1 was knocked down in HepG2 cells using si-NEAT1 or si-NC (control). HepG2 cell extracts were obtained 48 h after transfection. The extracts from each group were subjected to Western blotting to detect U2AF65, hnRNP A2 and β-actin. (B) An EMSA competition experiment was performed using 1 nM of the biotinylated lncRNA-NEAT1 fragment 2, a 1,000-fold molar excess of unlabeled hnRNP A2 mRNA and whole HepG2 extracts in a total volume of 20 μL. Different ratios of biotinylated RNA, protein and unlabeled RNA were incubated. The masses of the 20 μL system were resolved using 6% native polyacrylamide gel electrophoresis, and the proteins were then transferred to a nylon membrane and crosslinked. Representative images obtained with a CCD camera are shown. ^aUnlabeled hnRNP A2 mRNA; ^bBiotin lncRNA-NEAT1 fragment 2; ^cHepG2 extract. (C) U2AF65 mRNA was knocked down in HepG2 cells using si-U2AF65 or si-NC (control). HepG2 cell extracts were obtained 48 h after transfection. The extracts from each group were subjected to Western blotting to detect U2AF65, hnRNP A2 and β-actin. (D) Slides of HepG2 cells were incubated with hnRNP A2 antibody at 4°C for 12 h after si-NEAT1, si-U2AF65 or si-NC treatment, and the slides were then incubated with secondary HRP-antibody, colored with a DAB kit and photographed. The scale bar represents 100 μm.
Abbreviations: CCD, charge coupled device; DAB, diaminobenzidine; EMSA, electrophoretic mobility shift assay; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; HRP, horseradish peroxidase; mRNA, messenger RNA; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; si, small interfering; U2AF65, U2 small nuclear RNA auxiliary factor 2.

Figure 8 The overexpression of hnRNP A2 rescued the proliferation and invasion of HCC cells expressing low levels of lncRNA-NEAT1.

Notes: (A) NEAT1 was knocked down in HepG2 cells using si-NEAT1 or si-NC (control). The overexpression of hnRNP A2 in cells transfected with retroviruses encoding empty vector or hnRNP A2 was analyzed by Western blotting for hnRNP A2. β-actin was used as a loading control. (B) The cells from each group described in A were seeded in 96-well plates and cultured with 100 μL of DMEM containing 10% serum for 72 h. Ten microliters of CCK-8 was added to each well and incubated for 1 h at 37°C. The cell viability was then assessed based on the OD450 value using an automatic microplate reader. Number (C) and representative images (D) of invasive cells per filter in each group described in A. Transwell assays were performed according to the method described in Figure 2D. The significance of differences between groups was assessed with a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test. *P<0.05, ***P<0.01.
Abbreviations: ANOVA, analysis of variance; CCK-8, cell counting kit-8; DMEM, Dulbecco’s Modified Eagle’s Medium; HCC, hepatocellular carcinoma; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; lncRNA, long noncoding RNAs; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; OD, optical density; si, small interfering.

Figure 9 Knocking down NEAT1 inhibited HepG2 cell growth and downregulated hnRNP A2 expression in vivo.

Notes: (A) The axillary fossae of male athymic nude mice aged 4–6 weeks were bilaterally inoculated with 2×10⁶ cells/0.1 mL per site. HepG2 cells were transfected with si-NEAT1 and expressed a lower level of NEAT1 than the control group. Tumors were taken and size was monitored 7 and 14 days later (n=4). **P<0.01. (B) Total RNA was extracted from the tumors 14 days later and the expression levels of hnRNP A2 in each group were quantitatively analyzed by normalizing them to the GAPDH level according to the 2^−ΔΔCt method (n=3). ***P<0.001. (C) The tumor extracts from each group were subjected to a Western blot analysis to detect U2AF65, hnRNP A2 and β-actin. (D) A representative IHC image obtained using hnRNP A2 antibody as the primary antibody is shown. The scale bar represents 100 μm.
Abbreviations: GAPDH, lyceraldehyde 3-phosphate dehydrogenase; hnRNP A2, heterogeneous nuclear ribonucleoprotein A2; IHC, immunohistochemistry; NC, normal control; NEAT1, nuclear-enriched abundant transcript 1; si, small interfering; U2AF65, U2 small nuclear RNA auxiliary factor 2.