Non‐SMC condensin I complex subunit H enhances proliferation, migration, and invasion of hepatocellular carcinoma

Abstract Non‐SMC condensing I complex subunit H (NCAPH) is a member of the Barr protein family and part of the condensin I complex. The upregulation of NCAPH is associated with poor prognosis in patients with colon cancer. However, the relationship between NCAPH and hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore NCAPH expression in HCC tissues and to investigate NCAPH functions in HCC cells. In this study, we found that high expression of NCAPH in HCC indicated worse prognosis via bioinformatics analysis. Consistently, quantitative real‐time polymerase chain reaction assays in 20 pairs of HCC specimens and the immunohistochemical analysis of 100 HCC tissues showed the upregulation of NCAPH. We established stable NCAPH‐overexpressing and NCAPH knockdown cell lines. Cell Counting Kit‐8 assays and colony formation assay were performed to analyze cell proliferation. Migration and invasion were analyzed by Transwell assays. Subcutaneous xenograft models were used to explore the role of NCAPH in tumor formation in vivo. Our results showed that NCAPH promoted tumor proliferation, migration, and invasion in vitro and in vivo. In conclusion, our findings indicate that NCAPH could serve as a novel prognostic biomarker and a potential therapeutic target for patients with HCC.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is still the third leading cause of death related to cancer due to the high mortality caused by poor prognosis and frequent metastasis and relapse. [1][2][3] Although HCC patients can be treated by surgery, radiofrequency ablation, and liver transplantation, the 5-year survival rate of HCC patients remains low. 1 Therefore, the identification of genes that may contribute to novel treatments and prolong the survival of HCC patients is urgently needed.
Condensin is important for chromosome assembly and segregation during mitosis and meiosis. 4 Many eukaryotic cells contain condensin I and condensin II, which are composed of sets of subunits. 5 The condensin I complex comprises structural maintenance of chromosome (SMC) proteins and three non-SMC subunits, including non-SMC condensing I complex subunit H (NCAPH), non-SMC condensing I complex subunit G, and non-SMC condensing I complex subunit D2. 6 Previous studies have reported that the abnormal expression of the non-SMC condensin I complex is involved in the progression of human cancer. 7,8 NCAPH encodes a regulatory subunit in the non-SMC condensing I complex and is required for the conversion of interphase chromatin into condensed chromosomes. 9 Previous studies have revealed that abnormal expression of NCAPH is associated with colon cancer and prostate cancer. 10,11 However, there have been no relevant reports about the clinical relevance and functional role of NCAPH in HCC. In this study, we aimed to explore NCAPH expression in HCC tissues and investigate the functions of NCAPH in HCC cells.

| Patients and clinicopathological data
All tissue samples and adjacent nontumor tissue specimens were collected from 100 HCC patients who underwent hepatectomy at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between July 2013 and December 2014. None of the patients received radiotherapy or preoperative chemotherapy before surgery.
All tissues were histopathologically confirmed, and all patients were followed up until December 2019. All fresh samples were stored at −80℃ immediately after resection. Detailed information on the clinical characteristics of all patients is documented in Table 1

| High-throughput data processing
Detailed data on NCAPH expression in HCC were downloaded from The Cancer Genome Atlas (TCGA, http://gdc.cancer.gov/). The data used in the microarray were downloaded from the Gene Expression Omnibus (GEO) datasets GSE6764, GSE29721, GSE45436, GSE62232, and GSE84402 (http://www.ncbi.nlm.nih.gov/geo). The data from TCGA and GEO were log 2 -transformed, and the results were analyzed using Excel 2018 and the GraphPad Prism 6 software.
Gene set enrichment analysis (GSEA) was performed to investigative pathways associated with NCAPH using the data obtained from TCGA. GSEA is supported by the Broad Institute Website (http://software.broadinstitute.org/gsea/index.jsp).

| Hematoxylin and eosin staining
Tissue samples consisting of 100 pairs of HCC tumors together with matched adjacent normal tissues were fixed in formalin and embedded in paraffin for NCAPH immunohistochemistry (IHC).
The scores were evaluated via two scoring systems: the positive cell score and staining intensity score. The NCAPH antibodies used for Western blotting and IHC were obtained from Proteintech (11515-1-AP; Chicago, IL).

| Lentivirus production and stable cell line construction
Lentiviral vectors expressing short hairpin RNA (shRNA) and NCAPH were cotransfected with the packaging vectors psPAX2 and pMD2G (Addgene) into HEK293FT cells for lentivirus production using Lipofectamine 3000 in accordance with the manufacturer's instructions. To establish stable cell lines, cells were transduced by using the above lentiviruses with polybrene (8 mg/mL, Sigma). After incubating for 72 hours, cells were selected with 2 mg/mL puromycin for 3 days.

| NCAPH is upregulated in human HCC tissues and serves as an independent prognostic marker in HCC patients
To examine NCAPH expression in HCC, we analyzed NCAPH expression by using mRNA sequencing or microarray datasets from TCGA and GEO (GSE6764, GSE29721, GSE45436, GSE62232, and GSE84402). The results indicated that NCAPH expression was significantly increased in these datasets ( Figure 1A-F). The qRT-PCR results showed that NCAPH expression was significantly higher in HCC tissues than in normal tissues ( 1 G). In addition, the IHC results demonstrated that the NCAPH immunostaining signals were stronger in HCC tissues than in adjacent normal liver tissues (ANLTs; Figure 2A). compared to those with a low NCAPH level ( Figure 2B). Consistently, the Gene Expression Profiling Interactive Analysis dataset results revealed that patients with a high NCAPH expression level exhibited lower OS and DFS than those with low NCAPH levels ( Figure 2C).

| NCAPH promotes the proliferation of HCC cells in vitro
To explore the biological function of NCAPH, we detected its mRNA levels in HCC cell lines ( Figure 3A). Then, we established a stable knockdown LM3 and Huh7 cell lines and NCAPH-overexpressing PLC/PRF/5 and Hep3B cell lines. Then, the protein levels of NCAPH in the different cell lines were tested by Western blot ( Figure 3B). To determine whether NCAPH plays an important role in HCC cell proliferation, we used a CCK-8 assay and colony formation assay (CFA) assay. CCK-8 and CFA showed that NCAPH knockdown prominently suppressed the proliferation of HCC cell lines ( Figure 3C and 3E), while NCAPH overexpression (OV) promoted the proliferation of HCC cells ( Figure 3D and 3F). Overall, NCAPH promotes the proliferation of HCC cells. These results indicated that NCAPH promoted HCC migration and invasion ( Figure 4C and 4D).

| NCAPH promotes the expression of mesenchymal markers and proliferation markers in HCC
In previous studies, we found that NCAPH promotes proliferation, migration and invasion in HCC. Epithelial-mesenchymal transition (EMT) markers are common markers reflecting cell mesenchymal or epithelial status. In our study, we performed the GSEA analysis and found that NCAPH was associate with the adherens junction pathway ( Figure S1A).
In addition, we found that the expression of NCAPH was associated with slug expression in TCGA ( Figure S1B). Next, we detected the relationship

| Knockdown of NCAPH suppresses tumor growth in vivo
To further validate the tumor-promoting function of NCAPH, we performed an in vivo xenograft assay using nude mice. NCAPH knockdown significantly reduced tumor growth compared with that observed in control cells ( Figure 5A). Tumor weight and volume were significantly decreased in NCAPH knockdown cells ( Figure 5B and 5C).
In addition, IHC staining showed that the expression of the proliferation marker gene Ki-67 was significantly decreased in NCAPH knockdown tumors ( Figure 5D and 5E). These findings indicated that the knockdown of NCAPH inhibited tumorigenesis in vivo.

| DISCUSSION
Increasing evidence confirms that abnormalities in the non-SMC condensin I complex are associated with the progression of HCC. 8 NCAPH is one of the three non-SMC subunits in the condensin I complex and plays an essential role in condensin complex stability and the resolution of sister chromatids. 9 To the best of our knowledge, our study presents the first evidence that NCAPH expression is upregulated in HCC tissues and cell lines, which is consistent with the data from the GEO dataset. In addition, the IHC assay indicated that the expression of NCAPH is significantly associated with clinicopathological features and reduced survival in patients with HCC. These findings suggest that NCAPH may be involved in HCC progression.
The condensin I complex plays an important role in chromosome condensation and segregation. 12 22 In addition, slug, a member of snail, could repress the transcription of E-cadherin. 23 In our study, we found the NCAPH was associated with the adherens junction pathway. And the expression of NCAPH was positive correlated with slug in TCGA datasets. Furthermore, we detected the E-cadherin, N-cadherin, and vimentin expression in HCC tissues. We found that NCAPH expression were negative correlated with E-cadherin, but positive correlated with N-cadherin and vimentin. Moreover, we found that decreased NCAPH levels reduced the expression levels of mesenchymal markers while increasing the expression of epithelial markers in HCC cells. Furthermore, mesenchymal markers were increased but were epithelial markers decreased in the NCAPH-overexpressing cell line. These findings suggest that NCAPH enhances migration and invasion through the induction of EMT in HCC.
In conclusion, our results provide evidence that NCAPH is overexpressed in HCC tissues and associated with poor prognosis. Moreover, NCAPH expression promotes HCC cell proliferation, migration and invasion through EMT. Therefore, NCAPH could be considered a novel prognostic biomarker and therapeutic target for HCC patients.