miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN

Retraction in: /10.3892/or.2021.8236

  • Authors:
    • Kai Liu
    • Songyang Liu
    • Wei Zhang
    • Baoxing Jia
    • Ludong Tan
    • Zhe Jin
    • Yahui Liu
  • View Affiliations

  • Published online on: June 4, 2015     https://doi.org/10.3892/or.2015.4030
  • Pages: 1003-1010
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Abstract

MicroRNA-494 (miR-494) acts as an oncomiR and is involved in tumor development, progression and metastasis, and confers resistance to chemotherapeutic drugs by targeting a number of molecules in several human cancers. However, the function and underlying molecular mechanism of miR-494 in hepatocellular carcinoma (HCC) has not been totally elucidated. In the present study, we determined the role played by miR-494 in HCC tissues and HCC cell lines using quantitative RT-PCR (RT-qPCR). The results showed that, miR-494 was significantly upregulated in HCC tissues and HCC cell lines. Additionally, a high miR-494 expression positively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). Luciferase reporter assays confirmed that miR-494 binds to the 3'-untranslated region (3'‑UTR) of the phosphatase and tensin homolog (PTEN) mRNA and represses its translation. Functional analyses indicated that the upregulation of miR-494 promoted cell viability, migration and invasion, decreased cell apoptosis and cell cycle arrest at G1 stage, and conferred sorafenib resistance to HCC cell lines. Underexpression of PTEN by siRNA significantly attenuated the inhibitory effects of anti‑miR-494 on the proliferation, migration and invasion of liver cancer cells. Mechanistic investigations revealed that miR-494 suppressed the expression of PTEN but increased the expression of PI3K and p-Akt, which contribute to the promotion of proliferation, migration and invasion, and increased sorafenib resistance to HCC cell lines. These findings suggested that miR-494 is a potential candidate for HCC therapeutics.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer, causing more than 600,000 mortalites worldwide annually. Although most prevalent in Asia and sub-Saharan Africa, the incidence of HCC in the USA and Europe is on the increase, is the fastest growing cause of cancer-related mortality among men (1). Despite the use of multimodal treatments such as surgical resection, liver transplantation, chemotherapy and radiotherapy or a combination of these options, the prognosis of HCC patients remains poor due to detection of the disease at the late stage, with limited therapeutic options available (2). Chemotherapy is an important therapeutic strategy for patients who are in advanced stages of HCC but are not candidates for surgical resection (3). Sorafenib, a multi-kinase inhibitor, is a recent FDA-approved anticancer drug used for patients with advanced HCC (4). However, high rates of sorafenib resistance in HCC patients often prevent its long-term efficacy (5). Therefore, understanding the underlying molecular mechanisms of HCC is crucial to develop novel strategies for the diagnosis, prognosis and treatment of HCC to overcome drug resistance for the successful treatment of this disease.

MicroRNAs (miRNAs) are an abundant class of endogenous, highly conserved, non-coding 20-23 nt small RNA molecules that have been shown to regulate gene expression by targeting the 3′-untranslated region (3′-UTR) of mRNAs with consequent inhibition of protein translation or degradation of the target mRNA (6). Each miRNA interacts with numerous mRNA transcripts, a promiscuity that indicates the potential of individual miRNAs to serve as ‘master regulators’ mediating complex biological processes, such as, embryonic development, cell proliferation, differentiation, migration, apoptosis and signal transduction (7). Evidence indicates that alterations in miRNA expression are associated with numerous disease states, including human cancer (8). Dysregulated miRNAs have been shown to act as oncogenes by repressing tumor suppressors, or tumor suppressors by negatively regulating oncogenes (9). These miRNAs are associated with different etiologies, metastasis, the propensity for recurrence and prognosis as a molecular signature in human types of cancer (10,11). Recent findings have showed that miRNAs are important in the drug resistance of different tumors (1214). Given the close relationship between miRNAs and the biological progression of various types of cancer, miRNAs are considered potential novel targets for the therapy of various types of cancer (15).

miR-494, which is located on chromosome 14q32.31, has been reported to be highly expressed in acute myeloblastic leukemia (16), bronchial carcinogenesis (17), retinoblastoma (18) and medulloblastoma (19). Accumulating evidence demonstrates that miR-494 plays an important role in proliferation, migration and invasion in several cancer cells by targeting genes (15-19). Notably, miR-494-induced tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance in non-small-cell lung cancer (NSCLC) through the downmodulation of Bim has been previously identified (20). miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the tumor suppressor in mutated colorectal cancer (21). The clinical importance of miR-494 in cancer progression, the detailed function particularly in regards to response to chemotherapy, and the underlying molecular mechanism of miR-494 in HCC have not been fully elucidated.

The present study was therefore undertaken to assess miR-494 expression and its clinical diagnostic significance in patients with HCC and to analyze its role and underlying molecular mechanism on the carcinogenesis of HCC, particularly in regards to the response to sorafenib.

Materials and methods

Clinical samples

Forty paired human HCC and adjacent non-tumoral liver tissues were obtained from the patients who underwent routine curative surgery at the Department of Thoracic Surgery, The First Hospital, Jilin University (Changchun, Jilin, China) between July, 2010 and September, 2014. None of the patients received radiotherapy or chemotherapy prior to surgery. During the surgical procedure, samples of malignant liver tissue and samples from adjacent non-tumoral liver tissue (>5 cm away from the tumor site, cirrhosis tissue was excluded) were taken. Tissue samples were immediately frozen in liquid nitrogen, and stored at −80°C until RNA extraction. Data on all the subjects were obtained from the medical records, pathology reports and personal interviews with the subjects. Data on all the subjects including age, gender, overall survival and HCC features such as tumor number, size and growth phase were collected from the medical records, pathology reports and personal interviews with the subjects. Informed consent was obtained from each patient prior to surgery. The study protocol and consent procedures were approved by the Ethics Committee of the Jilin University.

Cell lines and cultured

The human Huh-7 and HepG2 HCC cell lines, as well as the normal human HL-7702 hepatocyte cell line were purchased from the institute of Cell Biology of the Chinese Academy of Science (Shanghai, China), and originated from ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; HyClone, South Logan, UT, USA) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) at 37°C in a humidified chamber supplemented with 5% Co2.

Cell transfection

The cells were transiently transfected with 100 nmol/l of miR-494 mimics (miR-494), miR-494 inhibitors (anti-miR-494), and miRNA negative control (miR-NC), or 100 nmol/l small interfering RNA (siRNA) specific to phosphatase and tensin homolog (si-PTEN), and scrambled siRNA negative control (si-NC) (all from GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen Life Technologies, grand Island, NY, USA) according to the manufacturer’s instructions. Transfection efficiencies were evaluated in each experiment by RT-qPCR at 48 h post-transfection.

RNA extraction and quantitative PCR

Total RNA containing miRNA and mRNA from tissue samples and cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using a PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. The expression levels of miR-494 were quantified using a Taqman miRNA assay kit (Applied Biosystems, Foster City, CA, USA). The expression of phosphatase and tensin homolog (PTEN) mRNA was detected by quantitative PCR. The primers for PTEN mRNA were: 5′-ACCAGTGGCACTGTTGTTTCAC-3′ (sense), and 5′-TTCCTCTGGTCCTGGTATGAAG-3′ (anti-sense). Quantitative PCR was performed on an ABI 7900 Fast system (Applied Biosystems) using Real-Time PCR mixture Reagent (Takara) according to the manufacturer’s instructions. The relative expression levels of the interest gene were calculated by the 2−ΔΔCt method. U6 and GAPDH were used as internal controls for miRNAs and mRNAs, respectively.

Cell viability assay

Cell viability was assessed using a Cell Counting kit-8 (CCK-8) assay (Dojindo, Japan), was performed. Briefly, 5×103 cells/well were seeded in 96-well plates. The cells were transfected with miR-494 mimic, miR-494 inhibitor (anti-miR-494), or corresponding negative control (miR-NC), or siRNA-PTEN (si-PTEN) and scrambled negative control (si-NC), and cultured for 72 h. The proliferative activity was determined using a CCK-8 assay according to the manufacturer’s instructions. Absorbance was recorded at 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Labsystems, Vantaa, Finland).

Cell migration and invasion assays

The migration and invasion assays were performed using Transwell insert chambers (Costar, Corning, NY, USA). For the migration assay, 1×105 transfected cells were seeded in each well of the upper Transwell chamber in serum-free medium (8-μm pore size; Corning Costar Corp., Acton, MA, USA). DMEM supplemented with 20% FBS was added to the lower chamber. After incubating for 24 h at 37°C with 5% Co2, cells migrating to the lower surface of filter were fixed in 70% ethanol for 30 min and stained with 2% crystal violet for 10 min on a glass slide. The number of cells penetrating across membrane was counted under a microscope in five random visual fields. For the invasion assay, 1×105 transfected cells were seeded in upper chambers precoated with matrigel (BD Biosciences, San Jose, CA, USA) in serum-free medium in triplicate. The following steps were similar to the migration assay. The number of cells invading the Matrigel was counted under a microscope in 5 randomly selected fields.

Cell cycle and apoptosis

Cell cycle and apoptosis were examined by flow cytometry. Briefly, two days post-transfection, the cells were collected for apoptosis and the cell cycle assay. For the cell cycle assay, the cells were collected and fixed in 70% ethanol at 4°C for 16 h and then stained with propidium iodide (PI; Sigma, St. Louis, USA) at 4°C for 30 min in the dark. Cell apoptosis assay was performed using an Annexin V/PI detection kit (KeyGene, Nanjing, China). The apoptotic rate and cycle distribution were measured by flow cytometry (BD Biosciences, Mansfield, MA, USA), and the data were analyzed using CellQuest software (Bd Biosciences San Jose, CA, USA).

Plasmid construction and luciferase reporter assay

The 3′-UTR of PTEN containing the PTEN miR-494 response element was cloned into the pGL3-control vector (Ambion, Austin, TX, USA) at the NheI and XhoI sites. A mutant 3′-UTR of PTEN was produced by PCR and cloned into the pGL3-control vector (Ambion) at the NheI and XhoI sites. HepG2 cells were seeded in a 24-well plate (5×103 cells/well) and transiently transfected with wide PTEN-UTR-pgL3/mute-PTEN-UTR-pgL3, a Renilla luciferase control vector (20 ng) and miR-494 mimic or control-miR. After 48 h, luciferase activity was measured using a dual luciferase reporter assay system, according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Sorafenib sensitivity assay

The relationship between miR-494 expression and cell sensitivity to sorafenib was examined. HepG2 cells were treated with miR-494 mimic, anti-miR-494 (miR-494 inhibitor) or miR-NC, and were divided into 96-well plates for 48 h. The cells were treated with indicated concentrations of sorafenib (10 μm; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Cell viability was assessed using the CCK-8 assay, and cell apoptosis was measured using the Annexin V/PI detection kit.

Western blot analysis

Tissue samples and cells were collected and homogenized with RIPA lysis buffer (Beyotime, Beijing, China). Whole extracts were prepared, and the protein concentration was detected using a bicinchoninic acid (BCA) protein assay kit (Boster, China). Equal amounts of protein lysates (30 μg each lane) were separated by 10% SDS-PAGE gel and then electrotransferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with TBST containing 5% non-fat dry milk for 2 h. The membranes were probed with mouse monoclonal antibodies specific to PTEN (1:400; Santa Cruz Biotechnology, inc.), and rabbit monoclonal antibodies of phospho-Akt (p-Akt) (Ser473) (1:500) and anti-Akt (1:500) (both from Cell Signaling, Boston, MA, USA), anti-PI3K (1:500), and anti-GAPDH (1:1,000) (both from Santa Cruz Biotechnology, inc.) which was used as an internal control for protein loading. The membrane was further probed with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000) or goat anti-rabbit IgG (1:5,000) (both from Santa Cruz Biotechnology, inc.) for 2 h at room temperature. Proteins were visualized with an ECL-chemiluminescent kit (ECL-Plus; Thermo Scientific, Rockford, lL, USA). The integrated density of the band was quantified by Quantity one software (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Data are presented as mean ± standard deviation (SD) of at least three independent experiments of three samples. Statistical analysis between two samples was performed using the Student’s t-test, while for more than two groups one-way ANOVA was used followed by a Tukey’s post hoc test. The relationship between the miR-494 expression level and clinical and pathological variables was analyzed using the Pearson’s χ2 test. The Graphpad Prism 6.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. P<0.05 was considered to indicate a statistically significant result.

Results

miR-494 is upregulated in HCC cell lines and tissues

To determine the expression levels of miR-494 in HCC, RT-qPCR detection was performed in 40 pairs of HCC (T) and adjacent non-tumoral (ANT) tissues. As revealed by RT-qPCR analysis, the expression level of miR-494 was significantly upregulated in tumoral tissues compared to that in the matched non-tumoral tissues (Fig. 1A). We also examined the expression levels of miR-494 in human Huh-7 and HepG2 HCC cell lines and the normal human HL-7702 hepatocyte cell line. It was found that the miR-494 expression level was substantially upregulated according to the RT-qPCR analysis in Huh-7 and HepG2 cells compared to the HL-7702 cells. The median expression level of the miR-494 level (0.87) was used as a cut-off point to divide all 40 patients into two groups: patients who expressed miR-494 at levels less than the median value were assigned to the low expression group (n=19), while those with a miR-494 expression higher than the median value were assigned to the high expression group (n=21). The association between miR-494 expression and the clinicopathological characteristics of the patients, including age, gender, tumor size, TNM stage, differentiation and lymph node metastasis were investigated. As shown in Table I, a high miR-494 expression positively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). There was no correlation between miR-494 expression, and age and gender. These data suggested that miR-494 is involved in HCC initiation and progression.

Table I

Association of miR-494 expression with clinicopathological factors of 40 HCC patients.

Table I

Association of miR-494 expression with clinicopathological factors of 40 HCC patients.

VariablesNo. of casesmiR-494 expression
P-value
Low (n, %)High (n, %)
Age (years)>0.05
 <50189 (50.0)9 (50.0)
 ≥502210 (45.6)12 (54.4)
Gender>0.05
 Male2413 (54.2)1145.8)
 Female166 (37.5)8 (62.5)
TNM stage<0.01
 I-II2818 (64.3)10 (35.7)
 III-IV121 (8.3)11 (91.7)
Differentiation<0.01
 Well/moderate3017 (56.7)13 (43.3)
 Poor102 (20.0)8 (80.0)
Lymph node metastasis<0.01
 No2617 (65.6)9 (34.4)
 Yes132 (15.4)11 (84.6)
Expression of miR-494 affects the proliferation, migration and invasion of liver cancer cell lines

To analyze the function of miR-494 on HCC, we transfected miR-494 mimic or anti-miR-494 into HepG2 cells, and cell proliferation, cell cycle, apoptosis, migration and invasion assays were performed. The RT-qPCR analysis confirmed that miR-494-transfected mimics caused the upregulation of miR-494 expression, while the miR-494-transfected inhibitor resulted in down-regulation of the miR-494 expression (Fig. 2A). The functional analysis demonstrated that the downregulation of miR-494 by anti-miR-494 inhibited the proliferation (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D), and induced cell cycle arrest at G1 stage (Fig. 2E) and apoptosis (Fig. 2F) in HepG2 cells. By contrast the overexpression of miR-494 by miR-494 mimic increased the proliferation (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D), and suppressed cell cyle arrest at G0/G1 stage (Fig. 2E) and apoptosis (Fig. 2F) in HepG2 cells.

miR-494 negatively regulates the expression of PTEN protein by directly targeting PTEN

PTEN has been identified as a direct target of miR-494 in several types of cancer (22,23). However, the association between miR-494 and PTEN in HCC remains to be clarified. To verify whether PTEN is a direct target of miR-494 in HCC, a fragment of the 3-UTR of PTEN containing the conserved putative miR-494 target site was fused to a lucif erase reporter vector, and co-transfected with a control Renilla luciferase reporter construct into HepG2 cells together with miR-494 mimic or control miRNA. It was found that co-transfection with miR-494 mimic and pGL3-PTEN plasmid showed a significant reduction of reporter activity in comparison with cells co-transfected with the control miRNA and pgL3-PTEN plasmid (Fig. 3A). By contrast, the cells co-transfected with anti-miR-494 and pGL3-PTEN-mut plasmid showed no significant difference in reporter activity as compared with cells co-transfected with control miRNA and pGL3-PTEN-mut plasmid (Fig. 3A). To determine whether miR-494 expression suppressed endogenous PTEN expression, synthetic miR-494 mimic, anti-miR-494 or miR-NC were transfected into HCC cells. RT-qPCR and western blot analysis revealed that the overexpression of miR-494 significantly inhibited PTEN expression at the mRNA (Fig. 3B) and protein (Fig. 3C) levels in HepG2 cells, while the underexpression of miR-494 markedly increased PTEN expression at the mRNA (Fig. 3B) and protein (Fig. 3C) levels in Hepg2 cells.

In addition, we determined PTEN protein expression in HCC tissue and cell lines by western blotting. The results of western blotting showed that PTEN protein expression was obviously upregulated in HCC tissue (Fig. 3D) and HCC cell lines (Fig. 3E) as compared to the adjacent non-tumoral tissue and normal human hepatocyte cell line. These results indicated that PTEN is also a miR-494 target in HCC.

Downregulation of PTEN significantly attenuates the inhibitory effects on the proliferation, migration and invasion of miR-494 depletion in liver cancer cells

To assess whether downregulation of PTEN affect HCC cell proliferation, migration and invasion in miR-494 depletion cells, we inhibited PTEN expression with siRNA in miR-494 depletion cells. The RT-qPCR analysis confirmed that transfected si-PTEN significantly inhibited PTEN expression in miR-494 depletion cells (Fig. 4A). Our results showed that the downregulation of PTEN significantly increased the proliferation (Fig. 4B), migration (Fig. 4C) and invasion (Fig. 4D), and suppressed cell cycle arrest at G1 stage (Fig. 4E) and apoptosis (Fig. 4F) compared to si-NC in miR-494-depleted cells, further suggesting that miR-494 affects the proliferation, migration, invasion, cell cycle arrest and apoptosis of HCC cells through the PTEN-mediated signaling pathway.

miR-494 renders HCC cells resistant to sorafenib

Sorafenib is the only oral multi-kinase inhibitor recently approved by the FDA with demonstrated efficacy in enhancing the overall survival of advanced HCC. It is known that some miRNAs can improve or increase the sensitivity of cancer cells to conventional drugs and chemotherapeutic agents. For this reason, we examined whether miR-494 increased the effect of sorafenib on HCC cells. For this purpose, HepG2 cells were transfected with miR-494 mimic or anti-miR-494, and then treated with sorafenib (10 μM), cell viability and apoptosis were determined. The CCK-8 assay showed that sorafenib treatment decreased the viability of HepG2 cells, and HepG2 cells transfected with miR-494 mimic were more resistant to sorafenib treatment than cells transfected with miR-NC (Fig. 5A, P<0.05). By contrast, the cells transfected with anti-miR-494 were more susceptible to the sorafenib treatment than cells transfected with miR-NC (Fig. 5A, P<0.05). In addition, we assessed the effect of expressing miR-494 with the sorafenib on cell apoptosis. The Annexin V assay revealed that the combination of miR-494 mimic with sorafenib decreases HepG2 cell apoptosis compared to sorafenib alone (P<0.05, Fig. 5B), whereas the combination of anti-miR-494 with sorafenib induced cell apoptosis compared to sorafenib alone (P<0.05, Fig. 5B). These findings suggested that elevated miR-494 enhanced HCC cell resistance to sorafenib.

miR-494 promotes cell growth via activation of the PTEN/PI3K/AKT signaling pathway

To determine whether miR-494 promotes the proliferation, migration and invasion of HCC cells via PTEN inhibition, the downstream genes of the PTEN signaling pathway, phosphatidylinositide 3-kinases (PTEN/PI3K)/AKT, were analyzed in miR-494 mimic and anti-miR-494-transfected HepG2 cells. As shown in Fig. 6, after the knockdown of miR-494 in the HepG2 cell line, PTEN gene expression levels increased significantly, and the downstream expression of PI3K and phosphate-AKT (p-Akt) was reduced, while the reverse correlation was found in miR-494 mimic-transfected Hepg2 cells. These findings suggested that miR-494 promotes cell proliferation, migration and invasion via activation of the PTEN/PI3K/AKT signaling pathway.

Discussion

Understanding the precise molecular mechanisms underlying HCC tumorigenesis is crucial in the development of better diagnosis and therapeutic strategy for HCC patients to improve survival. Aberrant miRNA expression is closely associated with various types of cancer, including HCC. Numerous miRNAs play important roles in various processes associated with cancer biology, such as, proliferation, apoptosis, migration, invasion and neoplastic transformation (8,24), suggesting that miRNA acts as a new direction in cancer studies. In the present study, the results showed that miR-494 expression was elevated in most HCC tumoral tissue compared to non-tumoral tissue, and its expression level correlated with key pathological characteristics including tumor differentiation, TNM stage and lymph node metastasis. Our in vitro studies revealed that the overexpression of miR-494 promoted cell proliferation, migration, and invasion, and decreased cell cycle arrest at G1 stage and apoptosis in HepG2 cells. Similarly, knockdown of miR-494 inhibited the proliferation, migration, and invasion and induced cell cycle arrest at the G1 stage and apoptosis in HepG2 cells. These data provide insights into HCC research and therapeutic strategies for malignant HCC.

miR-494 is located in the Dlk1-Dio3 imprinted locus on human chromosome 14q32, a region that contains 54 miRNAs, constituting one of the largest miRNA clusters in the human genome (8) and playing important roles in the development of disease, including cancer (25,26). Several studies showed that miR-494 expression patterns in this cluster are not consistent in different types of tumors. Underexpression of miR-494 was reported in prostate (27,28) and lung cancer (29), gastrointestinal stromal tumors (30), oral cancer (31) and cholangiocarcinoma (13). By contrast, the overexpression of miR-494 was reported in acute myeloblastic leukemia (16), bronchial carcinogenesis (17), retinoblastoma (18), medulloblastoma (19), non-small cell lung (20) and colorectal cancer (23). A recent study demonstrated that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the tumor suppressor of mutated colorectal cancer (21). However, the clinical importance of miR-494 in progression, the detailed function and underlying molecular mechanism of miR-494 in HCC remains unclear. In the present study, we identified miR-494 as an oncomiR that is important in proliferation, migration and invasion. Along with previous studies, our results suggest that the functions of miR-494 in different tumors are diverse, which may rely on the cell context and its potential regulatory targets in different cancer cells.

PTEN, a phosphoinositide phosphatase, was originally identified as a multifunctional tumor suppressor that exhibits frequent loss in various human cancer types, including HCC (33). PTEN acts as a negative regulator of the PI3K/Akt pathway via the dephosphorylation of PI(3,4,5)P3, and is ultimately involved in regulation of the cell proliferation, apoptosis, cell cycle, migration, cell adhesion, invasion as well as metastasis during cancer progression (34). Downregulation of PTEN leads to activation of the PI3K/AKT signaling pathway, promoting anti-apoptosis and cell proliferation in several cancer cells (34-37). PTEN was shown to be a direct target gene for miR-494 in malignant-transformed 16HBE and colorectal cancer cells (22,23). Consistent with those results, in the present study we identified PTEN as a potential target of miR-494 by the luciferase activity assay and point mutation analysis. We also found that the overexpression of miR-494 decreased PTEN expression, but increased PI3K and phosphate-AKT (pAkt) expression, suggesting that miR-494 can promote cell proliferation, migration and invasion, and decrease cell apoptosis by directly targeting PTEN and positively regulating the PI3K/Akt pathway.

Sorafenib, an FDA-approved drug for advanced HCC patients, is an inhibitor of multiple tyrosine kinases that improved the overall survival of HCC patients (4). However, treatment outcomes are poor due to unfavorable pharmaco-kinetics, low tumor accumulation and high rates of sorafenib resistance in HCC patients (5). Evidence has shown that, some miRNAs increased sorafenib sensitivity to HCC cells (13,3840). In addition, inhibition of the activation of Akt may be a valid approach to treating human malignancies and overcoming the resistance of cancer cells to chemotherapy (41). Recent findings showed that activation of the PI3K/Akt-signaling pathways in HCC cells resulted in acquired resistance to sorafenib, and inhibition of the PI3K/Akt-signaling pathways increased sorafenib sensitivity to HCC (13,39,42). Our results show that the downregulation of miR-494 enhanced HCC cell sensitivity to sorafenib, upregulated PTEN expression, and decreased Pi3K and p-AKT expression. These findings suggest that the underexpression of miR-494 enhances the sensitivity of HCC cells to sorafenib by regulating the PI3K/Akt pathway.

In summary, we have identified a critical oncomiR, miR-494 in HCC, which was frequently overexpressed in HCC tissues and cell lines, and its upregulation positively correlated with tumor differentiation, TNM stage and lymph node metastasis. miR-494 promotes cell proliferation, migration and invasion, and decreases cell apoptosis. In addition, it enhancea the resistance of HCC cells to sorafenib by directly targeting PTEN and positively regulating the Pi3K/Akt pathway. These findings suggest that miR-494 is a potential molecular diagnostic marker and therapeutic target for the treatment of patients with HCC.

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August-2015
Volume 34 Issue 2

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Online ISSN:1791-2431

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Spandidos Publications style
Liu K, Liu S, Zhang W, Jia B, Tan L, Jin Z and Liu Y: miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN Retraction in /10.3892/or.2021.8236. Oncol Rep 34: 1003-1010, 2015
APA
Liu, K., Liu, S., Zhang, W., Jia, B., Tan, L., Jin, Z., & Liu, Y. (2015). miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN Retraction in /10.3892/or.2021.8236. Oncology Reports, 34, 1003-1010. https://doi.org/10.3892/or.2015.4030
MLA
Liu, K., Liu, S., Zhang, W., Jia, B., Tan, L., Jin, Z., Liu, Y."miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN Retraction in /10.3892/or.2021.8236". Oncology Reports 34.2 (2015): 1003-1010.
Chicago
Liu, K., Liu, S., Zhang, W., Jia, B., Tan, L., Jin, Z., Liu, Y."miR-494 promotes cell proliferation, migration and invasion, and increased sorafenib resistance in hepatocellular carcinoma by targeting PTEN Retraction in /10.3892/or.2021.8236". Oncology Reports 34, no. 2 (2015): 1003-1010. https://doi.org/10.3892/or.2015.4030