High expression of DHX9 promotes the growth and metastasis of hepatocellular carcinoma

Abstract Background DHX9, an NTP‐dependent RNA helicase, is closely associated with the proliferation and metastasis of some tumor cells and the prognosis of patients, but its role in hepatocellular carcinoma (HCC) is not well‐known. This study was performed to explore the expression and role of DHX9 in HCC. Methods The expression of DHX9 in HCC tissues and cell lines was detected by TCGA database, qPCR, western blotting, and immunohistochemistry. The relationship between the DHX9 expression level and the prognosis of patients with HCC was accessed. Then, the function of DHX9 knockdown in HCC cells was examined by CCK‐8, scratch, Transwell, and apoptosis assays. Epithelial‐mesenchymal transition (EMT) was detected by western blotting. Results DHX9 was highly expressed in HCC tissues by analyzing both TCGA database and clinical samples. High DHX9 expression level was associated with TNM stage, vascular invasion and metastasis of HCC patients, and was an independent adverse prognostic factor. DHX9 knockdown significantly inhibited cell proliferation, migration, invasion and EMT and increased cell apoptosis in HCC cells. Conclusion Our findings suggest that DHX9 participates in the progression of HCC as an oncogene and may be a potential target for the clinical diagnosis and therapy of HCC.

tumor inhibitor genes. 4,5 Exploring new molecular mechanism of the carcinogenesis and development of HCC has an important guiding role in further improving the prognosis of patients with liver cancer. DHX9, also known as RNA helicase A (RHA), is an NTPdependent RNA helicase that belongs to DExD/H-Box superfamily II of helicases. With an ability to unwind DNA and RNA duplexes, as well as more complex nucleic acid structures, DHX9 appears to play a central role in many cellular processes. Its functions include regulation of DNA replication, post-transcriptional activation, posttranscriptional RNA processing, and RNA-mediated gene silencing. 6,7 Overexpression of DHX9 is a characteristic of some cancer types, and closely associated with the proliferation and metastasis of tumor cells and the prognosis of patients. [8][9][10] However, the role of DHX9 in HCC remains unclear.
In this study, the expression of DHX9 in HCC tissues and its function in cells were detected. The correlation between DHX9 level and prognosis of patient with HCC was further analyzed. Our study suggested that DHX9 might be an oncogene and promote cell proliferation, invasion, and metastasis in HCC.

| TCGA data analysis
Expression profile data from TCGA database were used to analyze the expression characteristics of DHX9 mRNA in 369 cases of HCC and 50 cases of non-tumor liver tissue, and the correlation between the DHX9 level and prognosis of HCC patient was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA).

| Clinical specimens and pathological data
From January 2018 to December 2018, tumor samples and adjacent normal tissues from 69 patients with HCC were collected at the First Affiliated Hospital of Zhengzhou University. In the control group, all normal tissues were derived from paracancerous tissues of the same patient (at least 3 cm from the surgical margin). All samples were confirmed by pathological diagnosis.
No radiotherapy, chemotherapy, and hormone therapy were performed before surgery. There were 62 males and 7 females. The patients were aged between 30 and 76 years, with an average age of 58 years. The clinical pathology was classified according to the

| Tissue microarray and immunohistochemical analysis
A tissue microarray containing above 69 paired samples was prepared by Wuhan Servicebio Technology Co. Ltd. The chip was gradually dewaxed and hydrated. Citrate buffer was used for antigen retrieval under high temperature and high pressure conditions. After rinsing with PBS, the chip was blocked with goat serum for 10 min at room temperature (RT) and then incubated with primary antibody against DHX9 (Proteintech) overnight at 4°C. After rinsing, the chip was incubated with biotinylated secondary antibody at RT for 1 h, and HRP-labeled streptavidin and DAB reagent was added after washing with PBS. After washing with tap water, the chip was restained with hematoxylin, dehydrated with gradient ethanol, sealed with gum, and observed under a microscope. The staining intensity was scored by the staining characteristics of the target cells: 0, no staining; 1, yellowish; 2, brownish yellow; and 3, brown. The cell positivity rate was used for scoring: 0, 0%-5%; 1, 6%-25%; 2, 26%-50%; 3, 51%-75%; and 4, >75%. The staining intensity score and the cell positivity rate score were calculated for each tissue on the tissue chip, and the positive comprehensive score was the product of the staining intensity and the positive cell rate.

| Western blotting
The tissue and cell proteins were extracted using RIPA lysis buffer.
The protein concentration was measured by the BCA method. Equal amounts of protein were loaded and separated on 8%-12% SDS-PAGE gels and transferred to nitrocellulose membrane, which were then blocked in 5% skim milk at RT for 1 h. The membrane was then incubated with primary antibodies against DHX9, E-cadherin, Vimentin, matrix metalloproteinase 2 (MMP-2), MMP-9, BAX, BCL-2, Caspase-3, and GAPDH overnight at 4°C. After rinsing three times with TBST, the membranes were incubated with secondary antibodies diluted at 1:2000 at RT for 1 h. After washing three times with TBST, ECL reagent was added to the membranes for visualization, followed by scanning on BIO-RAD Imaging System. The protein expression levels of each sample were calculated by Quantity-One software.

| Cell cultures and transfection
The normal liver cell line L02 and seven liver cancer cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China). These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and were placed in an incubator at 37°C with 5% CO 2 .
DHX9 siRNA and negative control (NC) RNA were transfected to cells using Lipofectamine 3000 reagent according to the instructions of siRNA purchased from Gene Pharma Co., Ltd. The effect of transfection was verified by western blotting.

| Cell Counting Kit-8 (CCK-8) assay
Exponential phase cells were diluted and seeded into 96-well plates at a density of 2.0 × 10 3 cells/well and cultured for 4 days.

| Scratch assay
To detect the migration ability of HCC cells, HCC cells were inoculated in six-well plates at a density of 1 × 10 6 per well. RNA interference was performed when the cells had adhered to the plates and had reached a confluency of approximately 60%. After successful transfection, a vertical scratch was made using pipette tips, and was observed after 0, 24, and 48 h. Five different migration points on both sides of the scratch were selected, and the average distance was measured.

| Statistical analysis
The statistical analysis was performed by SPSS 22.0 software. The Student's t test was used to compare the difference between the two groups. The Kaplan-Meier method and log-rank test were used for survival analysis. The correlation between clinical factors and prognosis was analyzed by univariate and multivariate analysis. All data were expressed as mean ± SD. p < 0.05 was considered statistically significant.

| The expression of DHX9 is increased and correlated with prognosis of HCC patients based on TCGA database
We firstly analyzed the expression of DHX9 in 369 cases of HCC and 50 cases of non-tumor liver tissues based on TCGA database, and found that the expression of DHX9 in HCC was significantly higher than that in non-tumor tissues ( Figure 1A). In addition, survival analysis showed that HCC patients with high DHX9 expression levels showed more adverse overall survival (OS) and disease-free survival (DFS) than patients with low DHX9 expression level ( Figure 1B,C).

| DHX9 mRNA and protein are highly expressed in HCC tissues
We collected 69 cases of clinical samples from our hospital, and the expressions of DHX9 in HCC and paracancerous tissues were detected by qPCR and western blotting. As shown in Figure 2A-C, the mRNA and protein expression of DHX9 in HCC tissues was significantly higher than that in paired paracancerous tissues.
The levels of DHX9 were further detected in a normal immor-

| The expression of DHX9 is correlated with clinicopathological parameters of HCC patients
To further define the correlation between the expression level of DHX9 and clinicopathological parameters in HCC tissues, a microarray containing 69 paired tissues was prepared and analyzed. The results of immunohistochemistry showed that DHX9 protein was mainly expressed in the cytoplasm of hepatocytes, and its expression in HCC tissues was significantly higher than that in paired paracancerous tissues ( Figure 3A). Based on the immunohistochemistry results, DHX9 expression was associated with TNM stage, vascular invasion, and metastasis (Table 1 and Figure 3B

| DHX9 knockdown inhibits HCC cell proliferation, migration, and invasion
To verify the function of DHX9 in HCC cells, we selected MHCC97H cells with high DHX9 expression for siRNA knockdown assay. Western blotting showed that the silencing effect of siRNA was significant when 125 nM siRNA was transfected ( Figure 4A). CCK-8 assay showed that DHX9 knockdown significantly inhibited the proliferation of MHCC97H cells at 48, 72, and 96 h ( Figure 4B). In the scratch assay, DHX9 siRNA-treated cells covered almost 73% of scratches in 24 h and at least 85% in 48 h, while the cells in the NC group covered only 18% in 24 h and 25% in 48 h ( Figure 4C,D). Transwell assays showed that the migration rate of cells in the DHX9 siRNA group was slower than that in the control and NC groups, and the number of invaded cells of the DHX9 siRNA group was significantly increased ( Figure 4E-H).

| DHX9 knockdown promotes apoptosis and inhibits epithelial-mesenchymal transition in HCC cells
The effect of DHX9 knockdown on the apoptosis of HCC cells was assessed by flow cytometry with AnnexinV/PI staining. The results showed that knockdown of DHX9 could significantly promote the apoptosis of MHCC97H cells ( Figure 5A,B). Simultaneously, western

| DISCUSS ION
Because of the central role of DHX9 in gene regulation and RNA metabolism, there are growing implications for DHX9 in human diseases. 6 Much effort has been expended lately in characterizing the association between DHX9 dysregulation and cancer development; however, there is still conflict as to whether it regularly functions as an oncogene or tumor suppressor. 11 The effect of DHX9 on cancers appears to be cell-type specific, dependent on the kind of binding partners. 11,12 Some studies suggested that DHX9 functioned as an oncogene and highly expressed in cancer tissues. 13 Palombo et al. 14 showed that poison-exon inclusion in DHX9 by hnRNPM and SRSF3 reduced its expression and inhibited cell proliferation in Ewing sarcoma malignancy. Can et al. 15 found that DHX9 was overexpressed in the serum and tissues of lung cancer, and promoted the progression of lung cancer. Cheng et al. 16  suggested that DHX9 acted as a p53 IRES trans-acting factor to increase expression and synthesis of p53 and inhibited breast cancer development.
The role of DHX9 in regulating the occurrence and development of HCC remains to be unclear. Wang et al. 19 identified an oncogenic lncRNA in HCC, named lnc-UCID, and disclosed that lnc-UCID enhanced CDK6 expression by competitively binding to DHX9 and sequestering DHX9 from CDK6-3'UTR. Yu et al. 20 discovered a tumor suppressor circRNA-cSMARCA5 that was downregulated transcriptionally by DHX9 in hepatoma cells. Though it has been reported largely that DHX9 can function as a partner to regulate the expression of some gene or non-coding RNA in cancer, the expression and key role of DHX9 itself in HCC remain to be studied further.
In this study, we firstly analyzed TCGA database and found that the expression of DHX9 was upregulated in HCC tissues and associated with the prognosis of the patients. Then, using clinical samples and cell lines collected at our center, we verified that DHX9 mRNA and protein were highly expressed in HCC tissues and some cell lines. In addition, the results of tissue microarray also confirmed that DHX9 expression was associated with TNM stage, vascular invasion, and metastasis. DHX9 high level was an independent adverse  In summary, our results demonstrate that DHX9 is highly expressed in HCC tissues, correlates with the survival and prognosis of patients, and promotes the proliferation, invasion, and metastasis of HCC cells. Therefore, DHX9 has an important role in promoting the occurrence and development of HCC and may be a potential therapeutic target and diagnostic biomarker of HCC.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.