HJURP Promotes Epithelial-to-Mesenchymal Transition via Upregulating SPHK1 in Hepatocellular Carcinoma

Cell migration and invasion assays

Cell migration and invasion assay were performed by Millicell Cell Culture Inserts (8μm pore size, Millipore, USA). Briefly, 5×10⁴ cells were seeded in serum-free medium into upper insert with (invasion assay) or without (migration assay) Matrigel Matrix (BD, USA). The subjacent compartment was added with 10% FBS medium. After incubation for 48h, cells on the lower surface of insert were fixed with 4% paraformaldehyde and stained using 0.5% crystal violet solution.

Western-blot

All cell lysates were extracted with RIPA (Cell Signaling Technology, USA) and proteinase inhibitor (Cell Signaling Technology, USA), which were mixed in advance. The supernatant was collected from samples after centrifugation at 12,000 g for 15min. Protein concentrations were measured using the BCA protein assay kit (Thermo Scientific, USA) according to the manufacturer's instructions. Equal amounts of protein (30ug) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) (Thermo Scientific, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked with Tris- buffered saline plus Tween 20 (TBST; 0.1% Tween-20) containing 5% non-fat dry milk at room temperature and incubated with corresponding primary antibodies overnight at 4°C. Primary antibodies include mouse anti-human GAPDH monoclonal antibody, HJURP (Abcam, UK), E-Cadherin, N-cadherin, Vimentin, and SPHK1 (Cell Signaling Technology, USA). After washing with TBST, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1 : 5000) for 1h at room temperature. The membranes were detected by enhanced chemiluminescence method (Thermo scientific, USA).

Co-Immunoprecipitation (Co-IP)

Cell samples were lysed in a IP lysis buffer containing 1mM MgCl₂, 100 mmol/L NaCl, 1mM DL-dithiothreitol (DTT) (Life Technology, USA) and protease inhibitor cocktails (Cell Signaling Technology, USA). The homogenates were incubated on ice for 30-45 min. Subsequently, cell samples were centrifuged at 2600g for 15 min at 4°C. The total cell lysates were used for immunoprecipitation with HJURP and SPHK1 primary antibodies on protein A/G mix beads (Thermo Scientific, USA) overnight. After overnight incubation, the immunoprecipitates were collected and washed three times and prepared for western-blot analysis.

RT-qPCR

Total RNA was extracted from HCC cell lines or tissue samples using Trizol Reagent (Life, USA) according to the manufacturer′s instruction. To analysis the expression of mRNA, total RNA was reverse transcribed to cDNA using Prime Script RT master mix (Takara, Japan), after which Quantitative Real Time-PCR (RT-qPCR) was performed with SYBR Premix Ex Taq (Takara, Japan). It was calculated by using the 2-ΔCT method and GAPDH was taken as the endogenous control. The relative expression of HJURP between HCC cell lines was calculated using the 2-ΔΔCT method. The sequences of primers used were listed in Table ​Table11.

Immunofluorescence assay

For immunofluorescence assay, 5×10⁴ stable transfected tumor cells were seeded in 2 mm confocal plate (Nunc, USA) for culture in indicated incubator. Cultured cells were fixed with 4% paraformaldehyde and then permeabilized by 0.25% TritonX-100. After blocking with 1% bovine serum albumin (BSA), primary antibodies against E-cadherin (1:50) and Vimentin (1:100) (Cell Signaling Technology, USA) were added into each plate for an overnight incubation at 4°C. A secondary antibody (1:200, Sigma-Aldrich, USA) was incubated at 37°C for 30 min and DAPI was used to stain the nuclei at room temperature for 15 min. The images were captured by a fluorescence microscope (Olympus BX53, Japan).

In vivo metastasis assays

To establish in vivo metastasis assays,1×10⁶ tumor cells per mouse were suspended in 50 μl PBS and injected via tail vein. After 10 weeks, liver and lung were harvested for H&E staining and calculation of metastasis lesions.

HJURP promotes cell migration and invasion by activating Epithelial-to-Mesenchymal Transition in vitro and facilitates tumor metastasis in vivo

For more biological functions of HJURP, transwell assay was used to validate the migration and invasion capabilities of HCC cells. HJURP knockdown dramatically suppressed Huh7 and HCC-LM3 cells invasion and migration. Inversely, overexpression of HJURP in Huh7 and HCC-LM3 cells promoted its invasion and migration (Figure ​(Figure2A2A and B, Supplementary Figure 1A, 1B). Moreover, we established an in vivo tumor metastasis model to study the effect of HJURP on pulmonary metastasis. In consistent with results of in vitro experiments, HJURP down-expression HCC-LM3 cells caused less lung metastasis clusters than its control group (Figure ​(Figure2C).2C). Subsequently, we studied EMT proteins to understand the mechanism in relation to HJURP mediated aggressive effect. And E-cadherin was significantly upregulated whereas Vimentin were downregulated in HJURP-knockdown HCC-LM3 cells. And the same results were mirrored in HJURP overexpression group (Figure ​(Figure2D).2D). Immunofluorescence assay revealed that HJURP silence significantly enhanced the expression of E-cadherin and inhibited the expression of vimentin. Besides, HJURP overexpression in Huh7 cells resulted in E-cadherin decrease and vimentin enhancement (Figure ​(Figure22E).

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Figure 2

HJURP promotes cell migration and invasion by activating Epithelial-to-Mesenchymal Transition in vitro and facilitates tumor metastasis in vivo. (A) The invasion and migration abilities were significantly decreased in HJURP-knockdown group (HJURP-KD) HCC-LM3 and Huh7(B) cells compared to the control group (NTC). (C) Representative pictures H&E staining from HJURP-KD and NTC group (n=4). Lung metastasis lesions were shown by black arrows. (D)EMT related protein (E-cadherin, Vimentin) were detected by western-blot. (E) E-cadherin (red) and Vimentin (green) were analyzed by immunofluorescence in HCC-LM3 cells (HJURP-knockdown) and Huh7 cells (HJURP-overexpression).

HJURP facilitates EMT via upregulating Sphingosine Kinase 1

To further investigate the mechanism of HJURP induced EMT, we performed a microarray analysis in Huh7 cells (Figure ​(Figure3A).3A). Based on the results of microarray, 85 genes were upregulated and 79 genes were downregulated. In those 164 differential expressed genes, 20 EMT-related genes were tested by RT-qPCR. Dramatically, only Sphingosine Kinase 1 (SPHK1) changed with HJURP knockdown or overexpression (Figure ​(Figure3B).3B). And the KEGG pathway analysis proved that HJURP regulated sphingosine metabolites process (Figure ​(Figure3C).3C). According to this result, we wondered whether HJURP induced EMT via SPHK1. To address that hypothesis, we detected the expression of SPHK1 in HJURP knockdown and overexpression cells respectively. Western-blot showed that HJURP silence inhibited the expression of SPHK1. Conversely, HJURP overexpression increased the expression of SPHK1 (Figure ​(Figure3E).3E). Moreover, SPHK1 overexpression significantly reversed the invasion abilities and EMT markers (E-cadherin, Vimentin and N-cadherin) in HJURP- knockdown Huh7 and HCC-LM3 cells (Figure ​(Figure4A4A and B). To further elucidate whether HJURP interacts with SPHK1, Co-IP assay was performed to detect endogenous HJURP and SPHK1 in HCC-LM3 cells. As shown in Figure ​Figure3F,3F, HJURP was found to be co-precipitated with SPHK1.

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Figure 3

HJURP facilitates EMT via upregulating Sphingosine Kinase 1. (A) Microarray analysis of NTC and KD group in Huh7 cells. (B) EMT-related genes were analyzed by RT-qPCR in HCC-LM3 and Huh7 cells. Relative mRNA levels of 20 genes (left) and SPHK1 (right) are shown. (C) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways enriched in 164 genes whose expression is correlated with HJURP. The sphingolipid metabolic progress is marked with “*”. (D) Scatter plots shows a positive correlation between HJURP and SPHK1 at the mRNA level. Data were from TCGA database. (E) The protein level of HJURP and SPHK1 were detected by western-blot in HCC-LM3 and Huh7 cells. (F) Co-Immunoprecipitation assay shows HJURP interacts with SPHK1 in HCC-LM3 cells.

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Figure 4

The effects of SPHK1 overexpression on HJURP induced EMT. (A) Compared to HJURP KD group, SPHK1 overexpression (KD+SP) significantly enhanced the invasive ability in HJURP-knockdown Huh7 and HCC-LM3 cells. (B) SPHK1 overexpression in HJURP knockdown group increased the expression of E-cadherin but decreased the expression of N-cadherin and Vimentin.

The expression of HJURP is positively correlated with SPHK1

We next determined the correlation between HJURP and SPHK1. Data from The Cancer Genome Atlas (TCGA) database showed that mRNA of SPHK1 was correlated with that of HJURP (Figure ​(Figure3D).3D). Furthermore, immunohistochemistry of HCC tissues revealed that 72.86% HCC specimens with high HJURP expression were accompanied with high SPHK1 expression. Similarly, 71.43% of samples with low HJURP expression displayed weak staining of SPHK1 (Figure ​(Figure5A5A and B).

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Figure 5

The expression of HJURP is positively correlated with SPHK1 and high HJURP expression predicts poor disease-free survival rate of HCC patients. (A) and (B) Immunohistochemical staining of HCC tissues showed that high HJURP expression is correlated with high SPHK1 expression (magnification: 200X). (C) Kaplan-Meier analysis showed that HCC patients with low HJURP expression had a better disease-free survival rate than patients with high HJURP expression.

This research was funded by National S&T Major Project (No. 2017ZX10203205-003-002), Innovative Research Groups of National Natural Science Foundation of China (No.81721091) and Zhejiang International Science and Technology Cooperation Project (No.2016C04003).