microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis

Abstract

Background & aims: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues.

Methods: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay.

Results: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice.

Conclusions: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

Keywords: Gene Regulation; Liver Cancer; Systems Biology; Translation.

Figure 1.

MicroRNA-193a-5p is downregulated in hepatocellular carcinoma in mice and human. (A) Schematic diagram showing the experimental set-up of 750 miRNA array in three experimental HCC mouse models DEN / Untreated (n = 6 per group), AlbLTα/β / Untreated (n = 3 per group) and Tet-O-Myc/Untreated (n = 4 per group). (B) Venn diagram representing the principle of selecting functionally unknown miR-193a-5p. (C) qRT-PCR analysis of miR-193a-5p in four different genetic HCC mouse tumor models: Mcl-1^Δhep (n = 5), TAK1-KO (n = 11), TRAF2-RIPK1-KO (n = 7), Mdr2-KO (n = 7) compared to either nontumoral liver (NTL) or Wild type (WT) mice. (D) qRT-PCR of miR-193a-5p expression in normal liver (n = 4), non-tumor liver (n = 17) and HCC tissues (n = 125) obtained from patients. (E) miR-193a-5p log₂ expression in HCC patients with different TNM stages: Stage I (n = 169), Stage II (n = 83), Stage III (n = 82), Stage IV (n = 4) and normal liver (n = 50) obtained from TCGA HCC datasets. (F) Kaplan-Meier curves for the overall survival of HCC patients (n = 372) plotted against time (months) based on miR-193a-5p expression levels (obtained from the TCGA HCC datasets). Results are represented as mean ± SEM. ns nonsignificant, * p<0.05, *** p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc test (D and E); 2- tailed, unpaired t test (C); Log-Rank with Mantel-Cox test (F).

Figure 2.

Overexpression of miR-193a-5p suppresses the progression of cell cycle in vitro. (A) Huh7 cells were transfected with miR-193a-5p mimic and Control siRNA and cell viability was analyzed by CCK-8 assay (n = 4 per group). (B) Representative immunofluorescence images and quantification of Ki67 staining of Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group) (bar = 50 μm). (C) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p mimic or Control siRNA (n = 4 per group) (bar = 1 cm). (D) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p Antagomir and Control (miScript Inhibitor Negative Control) (n = 4 per group) (bar = 1 cm). (E) Representative images and quantification of BrdU⁺ Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group). (F) Huh7 cells were transfected with miR-193a-5p for 72 h and analyzed with flow cytometry (FACS) by DAPI staining (n = 6 per group). The percentage of cells in sub-G1, G₁, S and G2/M phases of the cell cycle are indicated. (G) Huh7 cells were transfected with miR-193a-5p and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G₁, S and G2/M phases of the cell cycle are indicated. (H) Western Blot showing the protein expression of the indicated proteins in Huh7 cells transfected with either miR-193a-5p mimic or Control siRNA (n = 3 per group). Results are represented as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 by 2- tailed, unpaired t test.

Figure 3.

Overexpression of miR-193a-5p increases apoptosis in vitro. (A) Cell death (TUNEL staining (upper row); cl. Casp-3 staining (lower row)) was analyzed in Huh7 cells transfected with miR-193a-5p and Control siRNA for 72h. (bar = 50 μm). (B) Apoptosis of Huh7 cells transfected with miR-193a-5p was determined by Annexin V/Propidium iodide staining and flow cytometry (n = 3 per group). (C) Transwell migration assay (upper row) or Transwell coated with Matrigel invasion (lower row) assay for Huh7 cells was determined after transfection with miR-193a-5p mimic or Control siRNA for 72 h (n = 3 per group) (bar = 50 μm). (D) Wound-healing assay was performed on Huh7 cells transfected with miR-193a5p mimic and wound closure was monitored over the indicated time post-transfection after treatment with 10 ¼g/ml Mitomycin C for 2 h (n = 3 per group) (bar = 200 μm). Results are represented as mean ± SEM. ns non-significant, * p<0.05, ** p<0.01, *** p<0.001 by 2- tailed, unpaired t test.

Figure 4.

NUSAP1, a direct target of miR-193a-5p, is upregulated in mouse and human HCC tissues. (A) Venn diagram showing the overlap between predicted targets of miR-193a-5p and genes (threshold: fold change > 2, p value < 0.01) obtained from mRNA-array of DEN, AlbLTα/β and Tet-O-Myc mouse HCC models. (B) Relative expression of NUSAP1 in human HCC (n = 57) compared to normal liver (n = 5) from Affymetrix array. (C) Kaplan-Meier curves for the overall survival of HCC patients (n = 242) plotted against time (months) based on NUSAP1 expression levels. (D) Expression levels of NUSAP1 in 371 HCC patients and solid normal tissue (n = 50) are shown (primary data obtained from TCGALIHC datasets). (E) Relative expression of Nusap1 was measured in non-tumoral liver (NTL) and HCC tumors of mice from DEN, AlbLTα/β and Tet-O-Myc in comparison with untreated mice. (F) Relative expression of NUSAP1 in mouse (Hepa1–6 and Hepa1c1c7) and human (Huh7 and HepG2) HCC cell lines was quantified by qRT-PCR (n = 3 per group). (G) Relative luciferase activity was quantified in Huh7 cells co-transfected with reporter constructs containing either WT or Mutant NUSAP1 together with miR-193a-5p mimic or control (n = 3 per group). (H) Relative expression of NUSAP1 in Huh7 cells transfected with miR-193a-5p mimic was quantified by qRT-PCR and Western Blot (n = 3 per group). Results are represented as mean ± SEM. ns non-significant, * p<0.05, ** p<0.01, *** p<0.001 by 2- tailed, unpaired t test (B, D, G and H); Log-Rank with Mantel-Cox test (C); 1-way ANOVA with Newman-Keuls post-hoc test (E and F).

Figure 5.

NUSAP1 knockdown suppresses the cell growth and increases apoptotic cell death in Huh7 cells. (A) qRT-PCR and Western blot analyses of NUSAP1 in Huh7 cells transfected with siRNA against NUSAP1 or Control siRNA (n = 3 per group). (B) Quantification of colonies from Huh7 cells transfected with NUSAP1 or Control siRNA for 72 h (n = 3 per group) (bar = 1 cm). (C) Levels of cell-cycle proteins (Cyclin D1, Cyclin E1, Cyclin B1, Cyclin A2, p21) were detected in cell lysates of NUSAP1 siRNA transfected Huh7 cells by Western blot with GAPDH as a loading control (n = 3 per group). (D) Huh7 cells were transfected with siNUSAP1 for 72 h and analyzed with flow cytometry by DAPI staining (n = 6 per group). The percentage of cells in the Sub-G1, G₁, S and G2/M phases of the cell cycle are indicated. (E) Huh7 cells were transfected with siNUSAP1 and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G₁, S and G2/M phases of the cell cycle are indicated. (F and G) Representative images and quantification of TUNEL (F) and cl. Casp-3 (G) stainings in Huh7 cells transfected with NUSAP1 siRNA for 72 h (n = 3 per group) (bar = 50 μm). (H) Transwell migration assay for Huh7 cells was determined after transfection with NUSAP1 siRNA or Control (n = 3 per group) (bar = 50 μm). Results are represented as mean ± SEM. ns non-significant, * p<0.05, *** p<0.001, by 2- tailed, unpaired t test.

Figure 6.

Overexpression of miR-193a-5p and knockdown of NUSAP1 reduce tumor growth in vivo. (A) Huh7 cells transfected with either miR-193a-5p mimic or control siRNA were injected subcutaneously to the right flank of nude mice to obtain tumor xenografts. Tumor weight was measured at 28^th day after injection when the mice were sacrificed (n = 5 per group). Tumor volume was measured every 7 days. (B) Huh7 cells transfected with either NUSAP1 siRNA or Control siRNA was injected subcutaneously to the right flank of nude mice to obtain tumor xenografts. Tumor weight was measured at 28^th day after injection when the mice were sacrificed (n = 5 per group). Tumor volume was measured every 7 days. (C) Huh7 cells were injected when tumor growth reached 0.5 cm, Control siRNA or miR-193a-5p mimic was injected intra-tumorally three times per week for additional 2 weeks. (D) Tumor weight was measured after 28^th day of injection when the mice were sacrificed. Tumor volume was also calculated using a digital caliper (n = 5 per group). (E) Amounts of NUSAP1 protein from intra-tumoral parts were measured by Western blot (n = 5 per group). Results are represented as mean ± SEM. ** p<0.01, *** p<0.001 by 2- tailed, unpaired t test.

Figure 7.

Knockdown of NUSAP1 limits liver cancer development in a transposon- based endogenous mouse HCC model (A) Representative images of mouse livers that developed c-myc/Akt-1 driven liver cancer 8 weeks after hydrodynamic injection (n = 5) (bar = 1 cm). qRT-PCR showing the relative expression of miR-193a-5p and NUSAP1 in normal livers, non-tumoral liver (NTL) and liver tumors isolated from c-Myc/Akt-1 injected mice. (B) Top panel: The efficiency of each shRNA used for HDTV experiments was determined by qRT-PCR in retroviral transfected liver cancer cells isolated from c-Myc/Akt-1 driven tumors of p19^−/− mice. Lower panel: Schematic diagram illustrating the HDTV injection vector containing either shREN713 (Control) or the varying sequences of shNUSAP1 along with cMyc and Akt-1. (C) Representative images of tumor burden 7 weeks after delivery of transposon vectors containing either shREN713 as a control or the respective sequences bearing shNUSAP1 (shNUS1919, shNUS1933, shNUS2260, shNUS2906) (n = 4 per group) (bar = 1 cm). (D) Western Blot of NUSAP1 and cyclins (CyclinE1, CyclinA2, CyclinB1 and p21) with protein extracts from c-Myc/Akt-1/shRNA HDTV-injected livers (n = 3 per group) corresponding to the respective liver number (1–3) as indicated in Figure 7C. Results are represented as mean ± SEM. ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc test (A and B).