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. 2019 Jul;110(7):2133-2144.
doi: 10.1111/cas.14037. Epub 2019 May 25.

Lymphoid-specific helicase promotes the growth and invasion of hepatocellular carcinoma by transcriptional regulation of centromere protein F expression

Xuan Yang  1 , Bi-Si Miao  1 , Chuan-Yuan Wei  1 , Rui-Zhao Dong  1 , Ping-Ting Gao  1 , Xin-Yu Zhang  1 , Jia-Cheng Lu  1 , Chao Gao  1 , Xiao-Ying Wang  1 , Hui-Chuan Sun  1 , Jian Zhou  1 , Jia Fan  1 , Ai-Wu Ke  1 , Guo-Ming Shi  1 , Jia-Bin Cai  1
Affiliations

Lymphoid-specific helicase promotes the growth and invasion of hepatocellular carcinoma by transcriptional regulation of centromere protein F expression

Xuan Yang et al. Cancer Sci. 2019 Jul.

Abstract

Lymphoid-specific helicase (LSH) is overexpressed in tumor tissues and its overexpression is associated with poor prognosis in several cancers. However, the role and molecular mechanism of LSH in hepatocellular carcinoma (HCC) remains largely unknown. Herein, we report that LSH was overexpressed in tumor tissues of HCC, and overexpression of LSH was associated with poor prognosis from a public HCC database, and validated by clinical samples from our department. Ectopic LSH expression promoted the growth of HCC cells in vivo and in vitro. Mechanistically, LSH overexpression promoted tumor growth by activating transcription of centromere protein F (CENPF). Clinically, overexpression of LSH and/or CENPF correlated with shorter overall survival and higher cumulative recurrence rates of HCC. In conclusion, LSH promotes tumor growth of HCC through transcriptional regulation of CENPF expression. Therefore, LSH may be a novel predictor for prognosis and a potential therapeutic target for HCC.

Keywords: CENPF; ChIP-seq; HCC; LSH; RNA-seq.

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Figures

Figure 1
Figure 1
Lymphoid‐specific helicase (LSH) is overexpressed in tumor tissues and its expression correlates with overall survival of hepatocellular carcinoma (HCC) patients. A, Results from public database GEPIA indicated LSH expression in HCC is higher than that in para‐tumor tissues. B, Online analysis of overall survival (OS) and disease‐free survival (DFS) shows that higher LSH expression indicates a poorer prognosis. P‐values are shown in the figure. C and D, mRNA and protein expressions of LSH were tested by qPCR and western blot. E, HCC tissue microarrays were tested by immunohistochemistry (IHC) using LSH antibody. F, IHC scores of LSH were analyzed for OS and DFS. *P < 0.05
Figure 2
Figure 2
Knockdown of lymphoid‐specific helicase (LSH) expression inhibits cell growth and invasion of hepatocellular carcinoma (HCC) cells in vitro and in vivo. A, Western blot (WB) and qPCR experiments for testing LSH in HCC cell lines. B, Knockdown and overexpression of LSH confirmed by WB and qPCR. C, CCK‐8 assays show decreased OD450 after LSH knockdown and elevated OD450 after overexpression. D, Colony formation assays for the HCC cell lines used above. E and F, Cell cycle and apoptosis rates tested by flow cytometry. G, Transwell assay carried out in these cell lines. H and I, Results of wound‐healing experiments. J and K, Subcutaneous tumors in nude mice after death. *P < 0.05, **P < 0.01
Figure 3
Figure 3
mRNA sequencing (mRNA‐seq) and ChIP‐seq shows target genes of lymphoid‐specific helicase (LSH). A, Heat map of RNA‐seq after LSH knockdown. B, Gene ontology (GO) and KEQQ analysis of differential genes of RNA‐seq, ChIP‐seq and their overlap. C, Gene set enrichment analysis (GSEA) analysis of differential genes of RNA‐seq. D, Frequencies of included genes in GO and KEGG gene sets
Figure 4
Figure 4
Centromere protein F (CENPF) is overexpressed in hepatocellular carcinoma (HCC) tissues and is positively correlated with lymphoid‐specific helicase (LSH) protein overexpression. A, CENPF and LSH mRNA expression. B, Correlation of mRNA level between CENPF and LSH. C and D, Online analysis for overall survival (OS) and disease‐free survival (DFS) shows that higher CENPF expression indicates a poorer prognosis. E, mRNA and protein levels tested by qPCR and western blot (WB). F, Immunohistochemistry (IHC) test of CENPF protein expression in clinical samples, and the correlation between LSH and CENPF expression. G, IHC scores of CENPF were analyzed for OS and DFS. H, OS and DFS analysis integrated CENPF and LSH IHC scores. *P < 0.05. ***P < 0.001
Figure 5
Figure 5
Lymphoid‐specific helicase (LSH) binds to the transcription start site of cenpf and promotes the growth of hepatocellular carcinoma in a centromere protein F (CENPF)‐dependent way. A, ChIP‐seq visualized by UCSC at the cenpf gene body and transcription start site (TSS). B, ChIP‐qPCR verified for LSH binding. C, Luciferase activity in the indicated cells. D, Western blot results for LSH and CENPF correlations. E, qPCR results for LSH and CENPF correlations. F, Influence on phenotypes of combined CENPF and LSH. **P < 0.01, ***P < 0.001
Figure 6
Figure 6
Schema of lymphoid‐specific helicase (LSH) regulation of centromere protein F (CENPF) by a protein‐DNA interaction. HCC, hepatocellular carcinoma
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