Background
Hepatocellular carcinoma (HCC) is one of the most deadly tumors in the world, especially in China [
1] due to the high hepatitis B virus (HBV) infection rate [
2‐
4]. Surgery and some other interventional therapies have greatly improved in recent years, but the outcomes of HCC patients remain poor [
5]. Because of frequent recurrence and metastasis, HCC patients usually have poor prognosis [
6]. Thus, exploring the mechanisms of HCC development is important for optimizing early diagnosis and treatment [
7,
8].
Recent research has shown that the aberrant expression of noncoding RNAs (ncRNAs) is ubiquitous in different types of cancers, suggesting that ncRNAs play a key role in human carcinogenesis [
9]. NcRNAs of less than 200 nucleotides are considered as small ncRNAs, i.e., microRNAs, while ncRNAs of more than 200 nucleotides are considered as long ncRNAs, i.e., lncRNAs [
10]. Growing evidence suggests that the abnormal expression of lncRNAs is implicated in a variety of diseases, including cancer [
11‐
13], and that some tumor-associated lncRNAs play key roles in the development and metastasis of HCC [
14‐
16]. For example, lncRNA HULC [
17], lncRNA EGFR [
18], lncRNA HOST2 [
19] and lncRNA Tim3 [
20] accelerate HCC tumorigenesis and metastasis. However, lncRNA FTX [
21] has been reported to inhibit HCC development and proliferation. lncRNA beta-Catm [
16] is essential for the self-renewal of hepatocellular carcinoma stem cells and the proliferation of HCC tumors. MicroRNAs (miRNAs) constitute a group of small RNAs containing 18–25 nt. There is growing evidence that miRNAs are involved in various types of biological processes such as self-renewal, survival and tumor progression [
22,
23]. In various studies, several miRNAs, such as miR-451, miR-128, miR-34 and miR-203, have been suggested to regulate cancer stemness and drug resistance in different types of cancer [
24]. By targeting the 3′-untranslated regions (UTRs) of mRNAs, the expression of the target gene can be regulated posttranscriptionally, thereby affecting the regulation of cell proliferation, differentiation and apoptosis [
1].
In the current study, we hypothesized that lncRNA X inactive-specific transcript (XIST) targets specific miRNAs and proteins to regulate HCC proliferation and migration, resulting in a poor prognosis in HCC patients. The biological roles of miR-497-5p in HCC development were explored, and we found that miR-497-5p was increased in both HCC tissues and cells, whereas lncRNA XIST was significantly decreased. We also observed that silencing of miR-497-5p could inhibit HCC progression in vitro. In addition, using bioinformatics methods, programmed cell death 4 (PDCD4) was predicted to be the target of miR-497-5p. Thus, we propose that lncRNA XIST inhibits HCC progression by targeting miR-497-5p and PDCD4 in vitro.
Materials and methods
Patient specimens
In total, 77 patients with pathological diagnosis of HCC and who underwent hepatectomy at the 1st Affiliated Hospital of Sun Yat-sen University (SYSU) between January 2004 and December 2008 were included in this study. All samples were immediately frozen in a liquid nitrogen tank. The inclusion criteria were as follows: (1) radical resection; (2) no chemotherapy before surgery; (3) no distant metastasis; (4) survival for over 1 month after hepatectomy surgery; and (5) complete clinicopathological and follow-up data are available. In our study, the tumor-node-metastasis (TNM) staging was evaluated based on the American Cancer Joint Commission (AJCC) Cancer Staging Manual, 7th Edition. The basic clinical information of the 77 HCC patients is shown in Table
1. All procedures carried out in studies involving human participants met the ethical standards of the Ethics Committee of the 1st Affiliated Hospital of Sun Yat-sen University and the 1964 Declaration of Helsinki and its subsequent revisions and amendments.
Table 1Correlation between miR-497-5p expression and clinicopathological features of patients with HCC
Gender |
Male | 66 | 47 | 19 | |
Female | 11 | 6 | 5 | 0.269 |
Age (years) |
< 50 | 33 | 20 | 13 | |
≥ 55 | 44 | 33 | 11 | 0.177 |
AFP (ng/mL) |
Low, < 200 | 39 | 24 | 15 | |
High, ≥ 200 | 38 | 29 | 9 | 0.162 |
Cirrhosis |
Yes | 34 | 20 | 14 | |
No | 43 | 33 | 10 | 0.092 |
HBsAg |
Negative | 11 | 8 | 3 | |
Positive | 66 | 45 | 21 | 0.761 |
Tumor size (cm) |
< 5 | 28 | 16 | 14 | |
≥ 5 | 49 | 37 | 10 | 0.019 |
Tumor number |
Solitary | 54 | 34 | 20 | |
Multiple (≥ 2) | 23 | 19 | 4 | 0.111 |
PVTT |
No | 63 | 43 | 20 | |
Yes | 14 | 10 | 4 | 0.545 |
TNM stage |
I/II | 47 | 27 | 20 | |
III/IV | 30 | 26 | 4 | 0.011 |
Edmondson grade |
I/II | 30 | 23 | 7 | |
> II | 47 | 30 | 17 | 0.026 |
Cell culture
Human hepatic carcinoma cell lines (HepG2, HepB3, Huh7, SMMC-7721, MHCC-97L and Bel-7402), an immortalized hepatocyte cell line (LO2) and HEK293T cells were used in this study. All cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (HyClone, Shanghai, China), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) or Dulbecco’s modified Eagle’s medium (DMEM, Sigma) was used as the cell culture medium, and all cells were cultured in a humidified chamber containing 5% CO2 at 37 °C.
Lentiviral vector transfection
The human XIST full complementary DNA (cDNA) was amplified from HCC cells. The shRNA-luciferase (shluc) sequence was designed as a negative control. The target product was subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) using a lentivirus packaging vector and pMD2.G. In the medium containing 800 µg/mL G418 (Sigma-Aldrich), cells stably expressing XIST were cultured. Lipofectamine 2000 (Invitrogen) was used to introduce miR-497-5p mimics, inhibitors or negative controls into cells.
CCK8 assay
The cells were inoculated in 96-well plates overnight and infected for 48 h with a miRNA-497-5p inhibitor, an empty lentivirus vector, lentivirus (LV)-XIST or a LV negative control (NC) using Dojindo Molecular Technologies (Tokyo, Japan) on days 0, 1, 2, 3 and 4 with a 100-µL cell counting Kit-8 (CCK8). After incubating the cells with the CCK8 reagent for 4 h, the absorbance was measured at 450 nm by enzyme labeling (Bio-Tek, Winooski, VT).
Transwell invasion assay
A 200-µL cell suspension was loaded into the upper chamber of 24 transwell permeability support chambers with 8-micron pores coated with 1 mg/mL Matrigel (Corning Incorporated, NY). The basement is equipped with 600 µL of RPMI-1640 containing 10% FBS. After that, the cells on the filter surface were fixed with 4% formaldehyde for 15 min, stained with 0.5% crystal violet for 30 min, and then observed using a microscope.
Scratch wound assay
The cells were inoculated in a 6-well plate, scraped through each hole with the tip of a sterile 10-µL pipette and washed with phosphate buffered saline to remove any debris. After 24 h, the cells that migrated to the empty space were observed.
qRT-PCR
RNAiso Plus (TaKaRa Biotechnology, Dalian, China) was used to extract total RNA. Prime Script™ RT Master Mix was used to perform RNA reverse transcription. SYBR Premix Ex Taq II (TaKaRa Biotechnology) was used for qPCR. The primers used were as follows: for XIST (sense, 5ʹ-AGCTCCTCGGACAGCTGTAA-3ʹ; antisense, 5ʹ-CTCCAGATAGCTGGCAACC-3ʹ); for PDCD4 (sense, 5ʹ-TCG TCGTTACGATTGGTTAGTC-3ʹ; antisense, 5ʹ-GAAAAATCTCTA ACCCTTCTCGC-3ʹ); for miR-497-5p: (sense, 5ʹ-CCTTCAGCAGCACACTGTGG-3ʹ; antisense, 5ʹ-CAGTGCAGGGTCCGAGGTAT -3ʹ); for U6: (sense, 5′-CTCGCTTCGGCAGCACA-3′; antisense, 5′-TGGTGTCGTGGAGTCG-3′). An Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) was also utilized. The 2−∆∆Ct method was employed to analyze the relative gene expression levels.
Western blot analysis
Total protein was isolated from the cell lines and then resolved by 10% SDS-PAGE. Isolated proteins were transferred using a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was incubated with a primary antibody, followed by incubation with secondary antibodies. The main antibodies included anti-PDCD4 (1:2000; Abcam of Cambridge University, Britain) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Abcam).
Flow cytometry
Cells were digested, washed with cold PBS, fixed with 70% cold ethanol and stored at − 20 °C for at least 48 h. Before flow cytometry determination, fixed cells were washed and resuspended in 1 mL of PBS containing 10 mg/mL RNase A and were then incubated for 1 h at 37 °C. Cell suspensions were stained with propidium iodide solution (100 µg/mL) in the dark for 30 min. For each sample, 10,000 events were acquired, and cell cycle determinations were made by a FACS flow cytometer.
Luciferase reporter gene assay
For the luciferase reporter gene assay, 5 × 105 HEK293T cells were inoculated in a 24-well plate overnight. pmirGLO-PDCD4-WT or pmirGLO-lncRNA XIST-WT reporter plasmids (150 ng each) and their mutant vectors were cotransfected into cells with 50 nM mimic of miRNA-497-5p using Lipofectamine 2000 reagent. After 36 h of cell culture, the firefly and Renilla luciferase activities were determined by a double Luciferase Reporter Analysis System (Promega) based on the manufacturer’s instruction manual. The relative luciferase activity was calculated based on the firefly/Renilla fluorescence ratio.
Immunohistochemistry
The tissue was fixed by 4% formalin and embedded in paraffin. The endogenous peroxidase activity was blocked, and each slide was subjected to antigen retrieval after peeling and rehydration. The slides were incubated overnight with antibodies against Ki67 (1:500, #ab15580, Abcam) and PDCD4 (1:500, #ab80590, Abcam) at 4 °C. Slides were then incubated with a second antibody coupled with horseradish peroxidase (HRP) at 37 °C for 1 h. The positive immune response rate was determined according to the ratio of positive cells.
Detection of the xenotransplantation of tumors
The animal experiment procedure was approved by the Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. One-month-old female BALB/c thymus-free nude mice were purchased from Shanghai Pharmaceutical Research Institute (Shanghai, China). HepG2 cells transfected with LV-XIST or LV-NC were subcutaneously implanted into the lateral abdomen of each nude mouse. After 1 week, the volume of the tumor was estimated using a caliper once a week for 5 weeks. The volume was calculated using the following formula: (mm3) = 0.5 × length × width2. All mice were euthanized, and the weights of the tumors were measured after 5 weeks. The levels of PDCD4 in the resected tumors were analyzed by Western blot and immunohistochemistry analyses. For immunohistochemical staining, two slices were stained with terminal TUNEL (Yeasen, Shanghai, China) according to the manufacturer’s protocol. The cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma). TUNEL-stained slides were visualized under a fluorescence microscope (Zeiss Axio Observer. Z1).
Statistical analysis
Student’s t-test was used to analyze the differences between the two experimental groups. One-way ANOVA was used to analyze the differences among more than two different groups. Pearson’s correlation coefficient was used to evaluate the correlations between different groups. χ2 test or Fisher’s exact test were used to determine whether the target was correlated with the clinical pathological parameters. SPSS 24.0 software (Chicago, IL, USA) was used for the statistical analysis. A p < 0.05 was considered statistically significant.
Discussion
The prognosis of HCC patients is typically poor. The main cause of the poor prognosis of these patients is the high possibility of metastasis and recurrence after operation. Previous studies have shown that advanced TNM staging is a vital independent predictor of poor prognosis in HCC. In this study, we also found that a late TNM stage and high expression of miRNA-497-5p were independent prognostic factors of OS and DFS in HCC through a Cox proportional hazard analysis. Additionally, the results of Kaplan–Meier curve analysis showed that patients with high levels of miRNA-497-5p had a worse prognosis than those with low miRNA-497-5p expression. In addition, a higher level of miR-497-5p was associated with malignant behavior in HCC (Table
1), including a larger tumor size, later TNM stage, and higher PVTT and Edmondson grade (Table
2). These findings suggest that the highly expressed miRNA-497-5p is involved in the progression of HCC.
Recent studies have shown that ncRNAs could serve as molecular markers of cancers, including HCC [
25,
26]. Therefore, it is important to clarify the roles of miRNAs and lncRNAs in the prognosis of HCC. In this study, miRNA-497-5p was upregulated in HCC cells, while lncRNA XIST was downregulated. The overexpression of XIST could inhibit the development of HCC and rescue the miR-497-5p mimic-induced progression of HCC in vitro. In addition, using bioinformatics methods, we predicted that PDCD4 is a target of miRNA-497-5p. Therefore, we propose that the XIST/miR-497-5p/PDCD4 axis participates in the development of HCC. We found that the level of lncRNA XIST was lower in HCC cells and that overexpression of lncRNA XIST inhibited the progression of HCC. The above results indicate that lncRNA XIST plays a major role in inhibiting HCC development.
Studies have demonstrated that XIST could modulate proliferation and apoptosis in osteoarthritis chondrocytes [
27]. In addition, it has been reported that the level of lncRNA XIST in many cancer patients is reduced. For example, in ovarian cancer, the upregulation of lncRNA XIST has anticancer effects due to the inverse downregulation of has-miR-214-3p [
28]. In breast cancer, XIST could inhibit proliferation and migration by activating MSN-c-Met and reprogramming microglia to promote brain metastasis [
29]. Importantly, the elevation in miR-497-5p may contribute to the XIST-mediated inhibition of liver cancer cell growth. In the current study, our findings provide novel evidence to support this action. This affirmation stems from several findings: (1) XIST is negatively associated with miR-497-5p levels in human HCC tissues and cells; (2) XIST suppressed miR-497-5p expression by targeting PDCD4; and (3) XIST increased the expression of PDCD4 by decreasing miR-497-5p in human liver cancer tissues.
PDCD4 is involved in regulating apoptosis, is located on human chromosome 10q25.2 and is considered a novel tumor suppressor gene [
30]. While the PDCD4 protein is usually located in the nucleus, when the cell microenvironment changes, such as during malignant proliferation, PDCD4 can be transferred to the cytoplasm through nuclear export signals [
31]. PDCD4 can also bind ribosomes directly, affect the posttranscriptional translation process, and lead to cell apoptosis in cancer [
32]. In this study, we predicted that PDCD4 was a target gene of miRNA-497-5p by bioinformatics and that the expression level of PDCD4 could be inhibited by miR-497-5p. In addition, we found that XIST could attenuate the level of PDCD4 and that PDCD4 expression could be inhibited by overexpressed miR-497-5P in HCC cells.
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