CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway

Abstract

Background: The NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway.

Methods: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/β-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo.

Results: NSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/β-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice.

Conclusion: In conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.

Keywords: CRISPR/Cas9; Human hepatocellular carcinoma; Migration; NSD1; Proliferation; Wnt/β-catenin signaling pathway; Wnt10b.

Fig. 1

High expression of NSD1 in HCC was associated with poor prognosis. a, Expression of NSD1 in normal tissue and primary HCC tumor tissue analyzed by microarray-based analysis (http://ualcan.path.uab.edu/index.html). b, Relative expression of NSD1 in adjacent normal and HCC tissues (n = 63) determined by RT-qPCR. c, Kaplan-Meier survival curve for analysis on the relation between NSD1 expression and prognosis in patients with HCC. * p < 0.05 vs. adjacent normal tissues. Data (mean ± s.d.) between two groups were examined by paired t test

Fig. 2

NSD1 knockout inhibits cell proliferation, migration and invasion abilities in HCC. a, Expression of NSD1 in human immortalized liver cell line (HL-7702) and HCC cell lines (Huh7, Hep3B, SMMC-7721, HepG2, and SK-Hep1) determined by RT-qPCR and Western blot analysis. b, mRNA and protein expression following NSD1 overexpression in HepG2 cell line examined by RT-qPCR and Western blot analysis. c, Proliferation ability of HepG2 cells after overexpression of NSD1 determined by CCK-8 method and monoclonal formation assay. d, Migration and invasion ability of HepG2 cells following NSD1 overexpression determined by Transwell assay (× 100). e, Expression of NSD1 normalized to GAPDH in SK-Hep1 cells after knockout of NSD1 by CRISPR-Cas9 examined by Western blot analysis. f, Proliferation ability of SK-Hep1 cells after knockout of NSD1 measured by CCK-8 method and monoclonal formation assay. g, Migration and invasion ability of SK-Hep1 cells after knockout of NSD1 detected by Transwell assay (× 100). * p < 0.05 vs. the HL-7702 cell line, or the blank group (HepG2/SK-Hep1 cells without any treatment). Data (mean ± s.d.) among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test, while those at different time points were analyzed by repeated measures ANOVA, followed by the Bonferroni’s post-hoc test. The experiment was repeated 3 times independently

Fig. 3

NSD1 regulates H3K27me3 methylation in the Wnt10b promoter region to promote Wnt10B transcription. a, Wnt10b expression in adjacent normal and HCC tissues identified by immunohistochemical staining (× 400). b, Protein expression of Wnt10b normalized to GAPDH in adjacent normal and HCC tissues (n = 63) determined by Western blot analysis. c, Correlation analysis between expression of NSD1 and Wnt10b in HCC. d, Protein expression of Wnt10b normalized to GAPDH in HCC cells following NSD1 knockout determined by Western blot analysis. e, Protein expression of H3K36me2, H3K27me2, H3K27me3 in cells after knockout of NSD1 determined by Western blot analysis. f, Enrichment of H3K27me3 in Wnt10b promoter region in cells after knockout of NSD1 detected by ChIP assay. * p < 0.05 vs. adjacent normal tissues, or the blank group (HepG2/SK-Hep1 cells without any treatment). Data (mean ± s.d.) between two groups were analyzed by paired t test, and those among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test, while data at different time points were analyzed by repeated measures ANOVA, followed by the Bonferroni’s post-hoc test. The experiment was repeated 3 times independently

Fig. 4

Knockout of NSD1 inhibits the Wnt/β-catenin signaling pathway to suppress HCC cell proliferation, migration and invasion. a, Relative TOPFlash luciferase activity after NSD1 knockout. b, Protein expression of key proteins β-catenin, C-myc and CyclinD1 in the Wnt/β-catenin signaling pathway normalized to GAPDH after Wnt10b knockout determined by Western blot analysis. c, Relative TOPFlash luciferase activity after Wnt10b silencing, NSD1 silencing or both NSD1 overexpression and Wnt10b silencing. d, Protein expression of Wnt10b and key proteins, β-catenin, C-myc and CyclinD1 in the Wnt/β-catenin signaling pathway normalized to GAPDH determined by Western blot analysis. e, Proliferation ability of cells in different groups measured by CCK-8 and monoclonal formation assays. f, Migration and invasion ability of cells in differently groups detected by Transwell assay (× 100). * p < 0.05 vs. the blank group (SK-Hep1/HepG2 cells without any treatment). Data (mean ± s.d.) among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test, while data at different time points were analyzed by repeated measures ANOVA, followed by the Bonferroni’s post-hoc test. The experiment was repeated 3 times independently

Fig. 5

Silencing Wnt10b after knockout of NSD1 inhibits the formation and metastasis of tumors by inactivating the Wnt/β-catenin signaling pathway. a, Representative images of tumor formation through nude mouse xenograft model. b, Volume and weight of tumors. c, Pulmonary metastasis (× 200) detected by HE staining. d, TOPFlash luciferase activity in vivo after NSD1 knockout and silencing Wnt10b. e, Positive cell rates of β-catenin, C-myc, and CyclinD1 in tumors (× 400) detected by immunohistochemical staining. * p < 0.05 vs. the vector group (nude mice treated with vector). Data (mean ± s.d.) among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test, while data at different time points were analyzed by repeated measures ANOVA, followed by the Bonferroni’s post-hoc test, n = 12

Fig. 6

The mechanism scheme uncovers that CRISPR/Cas9-mediated knockout of NSD1 promotes H3K27me3 methylation and inhibits Wnt10b transcription, thereby suppressing activation of the Wnt/β-catenin signaling pathway