MicroRNAs (miRNAs) have been found to play fundamental roles in the pathogenesis of hepatocellular carcinoma (HCC). Previous miRNA array data showed that miR-519a was upregulated in HCC tissues compared to adjacent non-tumor tissues. However, the functional role of miR-519a in HCC remains unexplored. In this study, we demonstrated that the expression of miR-519a was elevated in both HCC tissues and cell lines. Clinical association analysis revealed that high expression of miR-519a was correlated with adverse clinicopathological characteristics including large tumor size, high Edmondson-Steiner grading, advanced tumor-node-metastasis (TNM) tumor stage and venous infiltration. Furthermore, high expression of miR-519a conferred a reduced 5-year overall survival and disease-free survival of HCC patients. Moreover, we disclosed that miR-519a overexpression promoted, but miR-519a silencing reduced, HCC cell proliferation and cell cycle progression
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third leading causes of cancer-related death (
MicroRNAs (miRNAs) are endogenous non-coding 20–22 nucleotide RNAs that regulate protein expression by interacting with complementary sites within the 3′-untranslated region (UTR) of target mRNAs (
miR-519a, which belongs to a large cluster of miRNAs, C19MC, plays a critical role in the pathogenesis of human cancers. It played an oncogenic role and could serve as a diagnostic and prognostic biomarker in ovarian epithelial tumors (
In this study, we demonstrated that upregulation of miR-519a was associated with poor prognostic features and reduced 5-year survival of HCC patients. Gain- and loss-of-function studies revealed that miR-519a promoted HCC cell proliferation and cell cycle progression. Finally, we identified PTEN and PI3K/AKT pathway as direct targets of miR-519a.
Tumor samples and matched tumor-adjacent tissues were obtained from 116 patients undergoing curative resection of their primary HCC in the Department of Hepatobiliary Surgery at the First Affiliated Hospital of Xi'an Jiaotong University during January 2006 to December 2009. Clinical sample was used after obtaining informed consent from each patient. None of the patients had received any perioperative chemo- or radiotherapy. The demographic and clinicopathological data of all enrolled patients were obtained through review of hospital records, and are presented in
The human immortalized normal hepatic cell line LO2 and HCC cell lines (HepG2, Hep3B, Huh7, SMMC-7721 and Bel-7402) were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C with 5% CO2. Akt inhibitor MK-2206 (1 μM, Selleck Chemicals, Houston, TX, USA) was used to treat HCC cells following the manufacturer's instructions.
Total RNA was extracted from clinical specimens or HCC cells using TRIzol reagent (Invitrogen) following manufacturer's instruction. PCR amplification was performed using a TaqMan human miRNA assay kit (Applied Biosystems, Foster City, CA, USA) and a SYBR® Premix Ex Taq™ II (Perfect Real-Time) kit (Takara Bio Inc., Shiga, Japan) with an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). qPCR primer against mature miRNA hsa-miR-519a-3p (HmiRQP0588), Homo sapiens snRNA U6 qPCR Primer (HmiRQP9001), PTEN (HQP015535) and GAPDH (HQP006940) were purchased from Genecopoeia (Guangzhou, China).
Total protein was extracted from HCC cells and 40 μg of isolated protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with the following primary antibodies: Akt (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), p-Akt (1:1000, Cell Signaling Technology, Inc.), PTEN (1:1000, Cell Signaling Technology, Inc.), Cyclin D (1:1000, Cell Signaling Technology, Inc.), and p27 (1:1000, Cell Signaling Technology, Inc.) overnight. Then the membranes were incubated with the HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (ZSGB-BIO, China). Protein bands were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).
Immunohistochemistry was performed on paraformaldehyde-fixed paraffin sections. PTEN (1:100, Cell Signaling Technology, Inc.) antibody was used in immunohistochemistry by a streptavidin peroxidase-conjugated (SP-IHC) method. Detailed procedure of immunohistochemistry was performed as previously reported (
miRNA vectors, including miR-519a expression vector (HmiR0342-MR04), the control vector for miR-519a (CmiR0001-MR04, miR-control), miR-519a inhibitor (HmiR-AN0588-AM04, anti-miR-519a) and the negative control for the miR-519a inhibitor (CmiR-AN0001-AM04, anti-miR-NC), and PTEN expression plasmid (EX-I0450-M02-5) were purchased from Genecopoeia (Guangzhou, China). The PTEN siRNA duplex sequence, 5′-GUU AGC AGA AAC AAA AGG AGA UAU CAA-3′ (sense)/5′-UUG AUA UCU CCU UUU GUU UCU GCU AAC-3′ (antisense) and a nonspecific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Cells were transfected with oligonucleotides using Lipofectamine 2000 Reagent (Invitrogen Life Technologies) following the manufacturer's instructions.
Cells were seeded in triplicate in 24-well plate and allowed to settle for ~12 h. pGL3-PTEN was co-transfected into HCC cells with TK-Renilla plasmid as control signals using Lipofectamine 2000. Luciferase and control signals were measured at 48 h after transfection using the Dual Luciferase Reporter Assay kit (Promega, Madison, WI, USA), according to a protocol provided by the manufacturer. Three independent experiments were performed and the data are presented as the mean ± SD.
At 48 h after transfection, 5×103 cells were seeded into 96-well plates and stained with 0.5 mg/ml sterile MTT (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C. Then the culture medium was discarded and an extra 150 μl DMSO (Sigma-Aldrich) were added. The absorbance at 490 nm was measured at 24, 48 and 72 h after transfection. The colony formation assay was performed as previously described. Briefly, 500 cells per well were seeded on six-well plates. After 2 weeks, the colonies were stained with 1% crystal violet and the number of colonies was counted.
At 48 h after transfection, HCC cells were collected for cell cycle analysis. After being washed with PBS three times, the cells were fixed with 80% ethanol overnight at −20°C, and were subsequently treated with RNaseA (Sigma) for 30 min at 37°C, followed by incubation in 20 μg/ml of propidium iodide (Sigma) for 20 min at room temperature. After incubation, the cells were subjected to flow cytometry analysis using a FACS Calibur (BD Biosciences, Bedford, MA, USA). For proliferation, 5-bromo-deoxyuridine (BrdU) labeling and immunofluorescence was used. Cells grown on cover slips (Fisher Scientific, Pittsburgh, PA, USA) were incubated with BrdU for 1 h and stained with anti-BrdU antibody (Sigma) according to the manufacturer's instruction. Gray level images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).
Data are presented as the mean ± SD from at least three independent replicates. SPSS software, 16.0 (SPSS, Inc, Chicago, IL, USA) was used to conduct the analysis, and a two-tailed Student t-test was employed to analyze the differences between two groups. Pearson's correlation analysis was used to analyze the correlation between two indices. Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Differences were considered statistically significant at P<0.05.
We investigated the expression level of miR-519a in 116 pairs of HCC tissues and matched tumor-adjacent tissues using qRT-PCR. The results showed that the mean level of miR-519a expression in HCC tissues was significantly higher than that in the non-tumor tissues (P<0.05,
We determined the mean level of miR-519a as a cut-off value to investigate the correlation between miR-519a level and the clinical features and prognosis of HCC patients. As shown in
To investigate the biological function of miR-519a in the development and progression of HCC, we transfected HCC cell line Hep3B with miR-519a mimic (P<0.01,
Loss-of-function studies were further performed to confirm the biological function by anti-miR-519a vector. The expression of miR-519a was significantly decreased in SMMC-7721 cells (P<0.01,
To further explore the underlying mechanisms by which miR-519a exerted its functional effects on HCC, we used TargetScan to search for potential downstream targets of miR-519a. As shown in
To evaluate whether PTEN could mediate the biological function of miR-519a in HCC, we inhibited PTEN expression with siRNAs in miR-519a-suppressive SMMC-7721 cells (P<0.05,
Previous studies have demonstrated that PTEN was a negative regulator of PI3K/Akt signaling (
Increasing evidence demonstrates that aberrant expression or function of miRNAs plays a crucial role in carcinogenesis (
In the present study, we identified that miR-519a was upregulated in HCC tissues compared to matched tumor-adjacent tissues, and this was also confirmed in HCC cells. Elevated expression of miR-519a was significantly correlated with clinicopathological features of HCC, including large tumor size, high histological grade and TNM stage and venous infiltration. In addition, patients with higher miR-519a had a poorer prognosis. Functionally, ectopic expression of miR-519a promoted cell viability, colony formation, proliferation and cell cycle progression in HCC cells. Moreover, we identified PTEN as a novel direct target of miR-519a. The functional effects of miR-519a alteration on HCC cells could be reversed by PTEN modulation. Moreover, the expression of PTEN was downregulated in HCC cases and its expression level was inversely correlated with miR-519a. Additionally, we found miR-519a led to upregulation of cell cycle regulator Cyclin D1 and downregulation of p27 through activation of PI3K/Akt pathway. Collectively, our results demonstrate that miR-519a promotes HCC progression through activating Akt signaling pathway by inhibiting PTEN.
Numerous studies showed that activation of the PI3K/Akt signaling pathway was essential to the development and progression of HCC and could modulate the malignant behavior of HCC (
In conclusion, we demonstrated that miR-519a was frequently overexpressed in HCC tissues and cell lines. miR-519a played a crucial role in the malignant progression of HCC cells through activation of PI3K/Akt by inhibiting PTEN. Therefore, miR-519a has the potential to be a valuable diagnostic and prognostic biomarker and a novel therapeutic target for HCC.
This study was supported by grants from the National Natural Science Foundation of China (no. 81402039) and the Natural Science Basic Research Plan in Shaanxi Province of China (Program no. 2015JM8409).
The expression of miR-519a is significantly elevated in HCC. (A) Relative miR-519a expression levels in HCC tissues and matched adjacent normal tissues were determined by qRT-PCR (n=116). (B) The expression of miR-519a in five HCC cell lines was significantly increased compared with that in the LO2 cells. U6 snRNA was used as internal control. *P<0.05, **P<0.01.
The prognostic value of miR-519a for HCC patients. HCC patients with higher expression of miR-519a had worse (A) overall survival and (B) disease-free survival. **P<0.01.
Overexpression of miR-519a promotes the growth of Hep3B cells. (A) The expression of miR-519a was measured in Hep3B cells transfected with miR-519a mimics or control vector. (B) Cell viability was evaluated by the MTT assay at the indicated days. (C) Representative results for colony formation by the indicated cells. (D) Representative micrographs (left) and quantification (right) of BrdU-incorporating cells of indicated Hep3B cells. (E) Effects of miR-519a overexpression on the cell cycle progression of Hep3B cells measured by flow cytometric analysis. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P<0.05.
Downregulation of miR-519a inhibits the growth of SMMC-7721 cells. (A) The expression of miR-519a was measured in SMMC-7721 cells trans-fected with miR-519a inhibitor or scramble vector. (B) Cell viability was evaluated by the MTT assay at the indicated days. (C) Representative results for colony formation by the indicated cells. (D) Representative micrographs (left) and quantification (right) of BrdU-incorporating cells of indicated SMMC-7721 cells. (E) Effects of miR-519a downregulation on the cell cycle progression of SMMC-7721 cells measured by flow cytometric analysis. Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P<0.05.
PTEN is a direct target of miR-519a in HCC cells. (A) The putative binding sequence of miR-519a in the 3′-UTR of PTEN. (B) qRT-PCR analysis of PTEN mRNA expression in Hep3B cells transfected with miR-519a mimics or control vector and in SMMC-7721 cells transfected with miR-519a inhibitor or negative control vector (NC); n=3 repeats with similar results. (C) Overexpression of miR-519a reduced the expression of PTEN protein in Hep3B cells and knockdown of miR-519a increases the level of PTEN protein in SMMC-7721 cells; n=3 repeats with similar results. (D) miR-519a significantly suppressed the luciferase activity of PTEN. Anti-miR-519a led to a notable increase in the luciferase activity of 3′-UTR of PTEN. miR-519a-mut had no influence on the luciferase activity of PTEN; n=3 repeats with similar results. (E) The expression of PTEN mRNA in miR-519a high-expressing tumors was significantly lower than that in miR-519a low-expressing tumors. (F) A significant inverse correlation between the mRNA levels of miR-519a and PTEN was observed in HCC tissues. *P<0.05, **P<0.01.
The expression of PTEN protein in HCC tissues. (A) The expression of PTEN protein in miR-519a high-expressing tumors was significantly lower than that in miR-519a low-expressing tumors. (B) Representative immunostaining showed negative expression of PTEN in miR-519a high-expressing HCC tissue and positive expression of PTEN in miR-519a low-expressing tumor. *P<0.05.
PTEN is the functional mediator downstream of miR-519a in HCC cells. (A) miR-519a-suppressive SMMC-7721 cells that were transfected with control siRNA or PTEN siRNA and miR-519a-overexpressing Hep3B cells that were transfected with EV or PTEN expression plasmid were subjected to western blot analysis for PTEN; n=3 repeats with similar results. Altering PTEN expression partly abolished the functional effect of miR-519a on (B) cell viability, (C) colony formation, (D) cell proliferation and (E) cell cycle progression; n=3 independent experiments. *P<0.05, **P<0.01.
Activation of PI3K/Akt pathway is essential for the biological functions of miR-519a in HCC. (A and B) Western blotting analysis of p-Akt (Ser473), total Akt, Cyclin D1 and p27 protein levels in indicated cells. α-tubulin was used as a loading control. Quantification of cell viability (C), colony formation (D), cell proliferation (E) and cell cycle (F) in indicated HCC cells treated with Akt inhibitor (0.5 μM). (G) The protein expression level of Cyclin D1 and p27 by western blot analysis in indicated HCC cells treated with Akt inhibitor (0.5 μM). Experiments were repeated at least 3 times with similar results, and error bars represent ± SD. *P<0.05.
Correlation between the clinicopathological characteristics and miR-519a expression in HCC (n=116).
Expression level | ||||
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Clinical parameters | n | miR-519ahigh (n=64) | miR-519alow (n=52) | P-value |
Age (years) | ||||
<65 years | 61 | 34 | 27 | 0.897 |
≥65 years | 55 | 30 | 25 | |
Gender | ||||
Male | 89 | 49 | 40 | 0.964 |
Female | 27 | 15 | 12 | |
Tumor size (cm) | 0.005 | |||
<5 | 78 | 36 | 42 | |
≥5 | 38 | 28 | 10 | |
Tumor number | 0.972 | |||
Solitary | 98 | 54 | 44 | |
Multiple | 18 | 10 | 8 | |
Edmondson | ||||
I+II | 71 | 33 | 38 | 0.018 |
III+IV | 45 | 31 | 14 | |
TNM stage | 0.013 | |||
I+II | 93 | 46 | 47 | |
III+IV | 23 | 18 | 5 | |
Capsular infiltration | 0.599 | |||
Present | 70 | 40 | 30 | |
Absent | 46 | 24 | 22 | |
Venous infiltration | 0.005 | |||
Present | 19 | 16 | 3 | |
Absent | 97 | 48 | 49 | |
AFP (ng/ml) | 0.947 | |||
<400 | 42 | 23 | 19 | |
≥400 | 74 | 41 | 33 | |
HBV | 0.873 | |||
Positive | 110 | 60 | 50 | |
Negative | 6 | 4 | 2 |
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; AFP, α-fetoprotein; TNM, tumor-node-metastasis.
Statistically significant.