Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2018.8979 mmr-18-01-0595 Articles Downregulation of microRNA-198 suppresses cell proliferation and invasion in retinoblastoma by directly targeting PTEN WeiDongdong 1 * MiaoYingbin 1 * YuLianxia 2 WangDegong 1 WangYingli 3 Department of Ophthalmology, Dezhou People's Hospital, Dezhou, Shandong 253000, P.R. China Department of Ophthalmology, The Second People Hospital of Dezhou, Dezhou, Shandong 253024, P.R. China Department of Ophthalmology, Yantai Yeda Hospital, Yantai, Shandong 264006, P.R. China Correspondence to: Professor Yingli Wang, Department of Ophthalmology, Yantai Yeda Hospital, 23-1 Huanghe Road, Yantai, Shandong 264006, P.R. China, E-mail: yingli_wang01@163.com

Contributed equally

 072018 07052018 18 1 595 602 02022018 20042018 Copyright © 2018, Spandidos Publications 2018

A number of studies have highlighted that aberrantly expressed microRNAs (miRNAs/miRs) serve crucial roles in the tumorigenesis and tumor development of retinoblastoma (RB). Hence, a full investigation of the biological roles and regulatory mechanisms of miRNAs in RB may provide novel therapeutic targets for patients with this malignancy. miR-198 is frequently abnormally expressed in various types of human cancers. However, the expression level, biological roles and underlying mechanisms of miR-198 in RB remain to be elucidated. In the present study, miR-198 expression was upregulated in RB tissues and cell lines. Silencing of miR-198 attenuated cell proliferation and invasion in RB. In addition, phosphatase and tensin homolog deleted on chromosome ten (PTEN) was predicted as a potential target of miR-198 using bioinformatics analysis. Subsequent luciferase reporter assay indicated that the 3′-untranslated region of PTEN can be directly targeted by miR-198. Furthermore, miR-198 inhibition increased the PTEN expression at the mRNA and protein levels in RB cells. In addition, PTEN mRNA expression was downregulated in RB tissues, and this downregulation was inversely associated with the expression level of miR-198. PTEN knockdown rescued the inhibitory effects of miR-198 underexpression on cell proliferation and invasion in RB. Notably, the downregulation of miR-198 inactivated the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in RB. These results demonstrated that miR-198 may serve oncogenic roles in RB by directly targeting PTEN and regulating the PI3K/AKT signaling pathway. Hence, miR-198 may be a promising therapeutic target for patients with RB.

 microRNA-198 retinoblastoma proliferation invasion phosphatase and tensin homolog deleted on chromosome ten Introduction

Retinoblastoma (RB) develops from immature cells in the retina and is the most common intraocular malignant tumor in childhood (1). It accounts for 2–4% of all childhood malignancies and has an estimated 9,000 novel cases each year worldwide (2). The clinical features of RB include leukocoria, strabismus, nystagmus, red eye, and visual deprivation, and these symptoms mainly depend on the tumor sites (3). Despite recent advancements in surgical resection in combination with chemotherapy and radiotherapy, therapeutic outcomes of patients with RB at advanced stage remain unsatisfactory (4). The unfavorable prognosis of patients with RB is largely attributed to delayed diagnosis and the high incidence rates of tumor metastasis (5). Therefore, an in-depth understanding of the molecular mechanisms underlying RB occurrence and development may provide effective therapeutic strategies for patients with this malignancy.

MicroRNAs (miRNAs) are endogenous, noncoding, and short noncoding RNAs that are considered as novel gene regulators (6). MiRNAs negatively modulate gene expression through partial complementary binding to their target genes in 3′-untranslated regions (3′-UTRs) and cause translational inhibition or mRNA degradation (7). A total of 2,588 mature miRNAs have been validated in the human genome according to miRBase (8). These miRNAs can regulate approximately 30% of human protein-coding genes (9). Dysregulation of miRNAs has been reported in most types of human cancer, such as RB (10), oral squamous cell carcinoma (11), breast cancer (12), and hepatocellular carcinoma (13). Aberrantly expressed miRNAs participate in the formation and progression of RB by regulating various cellular processes, including cell cycle, differentiation, proliferation, metastasis, and apoptosis (14). Hence, miRNAs may be developed as potential therapeutic targets for RB treatments.

MicroRNA-198 (miR-198) is frequently abnormally expressed in various human cancer types, including breast cancer (15), glioblastoma (16), hepatocellular carcinoma (17), and osteosarcoma (18). However, the expression level, biological roles, and underlying mechanisms of miR-198 in RB remain to be elucidated. In this study, miR-198 was significantly upregulated in RB tissues and cell lines. In addition, downregulation of miR-198 inhibited cell proliferation and invasion in RB by directly targeting PTEN and inactivating the PI3K/AKT pathway. These findings may provide novel insight into the molecular mechanism underlying the pathogenesis of RB and suggest that targeting miR-198 may be an effective therapeutic target for patients with this malignant tumor.

 Materials and methods <sec> <title>Tissue collection, cell culture and transfection

A total of 21 RB tissues and 7 normal retina tissues were obtained from patients who underwent surgical resection at Dezhou People's Hospital. These patients were not treated with chemotherapy or radiotherapy before surgery. The present study was approved by the Ethics Committee of the Dezhou People's Hospital, and performed according to principles of the Declaration of Helsinki. Moreover, written informed consent was obtained from all patients with RB who participated in this research.

Three RB cell lines (Y79, SO-RB50, and WERI-RB1) and a normal retinal pigmented epithelium cell line (ARPE-19) were acquired from the Shanghai Institute of Biochemistry and Cell biology (Shanghai, China). All cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS; both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) under a humidified air atmosphere of 5% CO2 at 37°C.

MiR-198 inhibitor and negative control miRNA inhibitor (NC inhibitor), small interfering RNA (siRNA) targeting PTEN (PTRN siNRA) and negative control siRNA (NC siRNA) were synthesized by Shanghai GenePharma Co., Ltd, (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Following transfection 6 h, culture medium was discard and replaced with fresh DMEM medium containing 10% FBS.

 RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from tissues or cells. The concentration of total RNA was evaluated using a Nanodrop 2000 spectrophotometer (Invitrogen; Thermo Fisher Scientific, Inc.). Expression level of miR-198 was quantified using All-in-OneTM miRNA RT-qPCR Detection Kit (GeneCopoeia, Inc., Rockville, MD, USA). To quantify PTEN mRNA level, reverse transcription was conducted using a PrimeScript reverse transcription-PCR kit (Takara Bio, Inc., Otsu, Japan), followed by Real-time PCR with a SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 and GADPH were used as internal control for miR-198 and PTEN mRNA, respectively. Data was analysed using the 2−∆∆Cq method (19).

 Cell counting Kit-8 (CCK-8) assay

Cell proliferation was determined using CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Transfected cells were collected 24 h post-transfection and plated into 96-well plates at a density of 3,000 cells for each well. CCK-8 assay was conducted at 0, 24, and 48 h after transfection. A total of 10 μl CCK-8 solution was added into each well and incubated at 37°C for 2 h. Absorbance at a wavelength of 450 nm was detected with a Spectra Max Microplate® Spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA).

 Transwell invasion assay

Transwell chambers coated with Matrigel (both from BD Biosciences, Franklin Lakes, NJ, USA) were applied to measure cell invasion ability. A total of 48 h after transfection, cells were harvested, washed with PBS and suspended in FBS-free DMEM. A total of 5×104 transfected cells were seeded into the upper chambers, while 500 µl DMEM supplemented with 20% FBS was added into the lower chambers to serve as a chemoattract. Following incubation at 37°C for 24 h, non-invasive cells were gently removed using cotton swabs. Invasive cells were fixed with 100% methanol and stained with 0.1% crystal violet. Finally, the number of invasive cells was counted under an inverted microscope (Olympus Corporation, Tokyo, Japan).

 Luciferase reporter assay

Luciferase reporter plasmids containing wild-type or mutant-binding sites for the miR-198 in the 3′-UTR of PTEN were synthesized and confirmed by GenePharma and labeled ‘pGL3-PTEN-3′-UTR Wt’ and ‘pGL3-PTEN-3′-UTR Mut’, respectively. Cells were seeded into 24-well plates and cotransfected with miR-198 inhibitor or NC inhibitor and luciferase reporter plasmid using Lipofectamine® 2000. Luciferase activities were determined at 48 h after transfection using a Dual-Luciferase® Reporter Assay system (Promega Corporation, Madison, WI, USA), following the manufacturer's instructions. Renilla luciferase activity was used for normalization.

 Western blot analysis

Total protein was extracted from transfected cells at 72 h post-transfection using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred on polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Afterward, the membranes were blocked with 5% non-fat dry milk and subsequently incubated overnight with the primary antibodies: Mouse anti-human monoclonal PTEN (sc-133242; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-human monoclonal PI3K (ab86714; Abcam, Cambridge, UK), mouse anti-human monoclonal p-AKT (sc-81433; Santa Cruz Biotechnology, Inc.), mouse anti-human monoclonal AKT (sc-56878; Santa Cruz Biotechnology, Inc.), and mouse anti-human monoclonal GAPDH (sc-51907; Santa Cruz Biotechnology, Inc.). After incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.), a Tanon High-sig ECL western blotting substrate (Tanon Science and Technology Co., Ltd., Shanghai, China) was employed to detect the protein signals. Protein expression was quantified using Quantity One software v4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as a loading control.

 Statistical analysis

All data were expressed as mean ± standard deviation of at least 3 independent experiments and analyzed with SPSS 19.0 Statistics Software (IBM Corp., Armonk, NY, USA). Data were compared with Student's t-test or one-way analysis of variance (ANOVA) plus multiple comparisons. Student-Newman-Keuls test was used as a post hoc test following ANOVA. Spearman's correlation analysis was applied to determine the correlation between expression levels of mIR-198 and PTEN mRNA in RB tissues. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>MiR-198 expression is increased in RB tissues and cell lines

To reveal the expression pattern of miR-198 in RB, RT-qPCR was utilized to detect miR-198 expression in 21 RB tissues and 7 normal retina tissues. Results showed that miR-198 expression was significantly upregulated in RB tissues compared with that in normal retina tissues (P<0.05; Fig. 1A). Moreover, miR-198 expression levels in three RB cell lines (Y79, SO-RB50, and WERI-RB1) and a normal retinal pigmented epithelium cell line (ARPE-19) were determined using RT-qPCR. Data revealed that miR-198 expression levels were higher in all three RB cell lines than those in ARPE-19 (P<0.05; Fig. 1B). These results suggested that miR-198 may be closely correlated with RB progression.

 Downregulation of miR-198 inhibits RB cell proliferation and invasion in vitro

To explore the biological roles of miR-198 in RB, Y79 and WERI-RB1 cells were transfected with miR-198 inhibitor to knock down its endogenous level. Following transfection, RT-qPCR analysis demonstrated that miR-198 expression was obviously downregulated in Y79 and WERI-RB1 cells transfected with miR-198 inhibitor compared with those transfected with NC inhibitor (P<0.05; Fig. 2A). Subsequent CCK-8 and Transwell invasion assays were employed to determine the effects of miR-198 underexpression on RB cell proliferative and invasive abilities, respectively. Results showed that miR-198 inhibition decreased the proliferation (P<0.05; Fig. 2B) and invasion (P<0.05; Fig. 2C) of Y79 and WERI-RB1 cells. These results suggested that miR-198 may play oncogenic roles in RB progression.

 PTEN is a direct target gene of miR-198 in RB

To elucidate the molecular mechanism underlying the oncogenic effects of miR-198 on RB cells, bioinformatics analysis was used to predict the potential target of miR-198 by using TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdcberlin.de/). PTEN, a well-known tumor suppressor, was predicted as a candidate and selected for further confirmation because that PTEN plays crucial roles in the RB oncogenesis and development (20–22). Two potential binding sites for miR-198 were predicted which were located at 546–552 and 610–617 bp downstream from the 5′ end of the PTEN 3′-UTR (Fig. 3A). To confirm this hypothesis, luciferase reporter assays were performed Y79 and WERI-RB1 cells after cotransfection with miR-198 inhibitor or NC inhibitor and pGL3-PTEN-3′-UTR Wt (1 and 2) or pGL3-PTEN-3′-UTR Mut (1 and 2). As indicated in Fig. 3B, miR-198 downregulation clearly increased the luciferase activities in the pGL3-PTEN-3′-UTR Wt (1 and 2) group (P<0.05), but this effect was not present in the pGL3-PTEN-3′-UTR Mut (1 and 2) group in Y79 and WERI-RB1 cells.

To further illustrate the association between miR-198 and PTEN in RB, we measured PTEN mRNA expressions in 21 RB tissues and 7 normal retina tissues. The RT-qPCR data revealed that the mRNA level of PTEN was significantly reduced in RB tissues relative to that in normal retina tissues (P<0.05; Fig. 3C). Furthermore, an inverse association between miR-198 and PTEN mRNA was validated in RB tissues by using Spearman's correlation analysis (r=−0.5530, P=0.0093; Fig. 3D). Moreover, the mRNA (P<0.05; Fig. 3E) and protein (Fig. 3F) levels of PTEN were upregulated in Y79 and WERI-RB1 cells following transfection with miR-198 inhibitor. In sum, these results demonstrated that PTEN is a direct target of miR-198 in RB.

 PTEN knockdown abolishes the oncogenic effects of miR-198 on RB cells

A series of rescue experiments was performed to further explore whether the oncogenic roles of miR-198 in RB are mediated by PTEN. MiR-198 inhibitor was transfected into Y79 and WERI-RB cells in the presence of PTEN siRNA or NC siRNA. Western blot analysis indicated that the overexpressed PTEN level induced by miR-198 inhibitor was recovered in Y79 and WERI-RB cells after cotransfection with PTEN siRNA (Fig. 4A). Subsequently, functional experiments demonstrated that restored PTEN expression abolished the effects of miR-198 underexpression on proliferation (P<0.05; Fig. 4B) and invasion (P<0.05; Fig. 4C) of Y79 and WERI-RB cells. These results suggested that miR-198 may act as an oncogene in RB, at least partly, by regulation of PTEN expression.

 Downregulation of miR-198 inactivates the PI3K/AKT signaling in RB

PTEN is previously reported as a primary regulator of the PI3K/AKT pathway in RB (21). Hence, we investigated whether miR-198 affects the PI3K/AKT pathway in RB. Y79 and WERI-RB cells were transfected with miR-198 inhibitor or NC inhibitor. Western blot analysis results revealed that miR-198 downregulation reduced the protein levels of PI3K and p-AKT in Y79 and WERI-RB cells (Fig. 5). However, the expression of total AKT was unaffected. These results suggested that miR-198 inactivates the PI3K/AKT signaling pathway in RB cells.

 Discussion

Numerous studies have highlighted that aberrantly expressed miRNAs play crucial roles in the tumorigenesis and tumor development of RB (23–25). Hence, a full investigation of the biological roles and regulatory mechanisms of miRNAs in RB may provide new therapeutic targets for patients with this malignancy. In the present study, miR-198 expression was observed to be significantly upregulated in RB tissues and cell lines. In addition, miR-198 downregulation prohibited cell proliferation and invasion in RB. Furthermore, PTEN was validated as a direct target gene of miR-198 in RB. Moreover, PTEN knockdown abolished the effects of miR-198 underexpression on RB cell proliferation and invasion. Moreover, silencing of miR-198 inhibited the activation of PI3K/AKT signaling pathway in RB. These results suggested that miR-198 possibly plays oncogenic roles in RB and can be identified as a therapeutic target for patients with this disease.

MiR-198 is typically aberrantly expressed in a number of human cancers. For example, miR-198 is downregulated in breast cancer, and this dysregulation is significantly associated with lymph node metastasis (15). In glioblastoma, miR-198 expression level is low in tumor tissues and cell lines. Glioblastoma patients with low miR-198 levels exhibit poorer prognosis than patients with high miR-198 levels (16). In hepatocellular carcinoma, miR-198 expression is reduced in tumor tissues. Decreased miR-198 expression is associated with hepatitis C virus infection, tumor capsular infiltration, metastasis, number of tumor nodes, vaso invasion, and clinical tumor node metastasis stage (17). In osteosarcoma, miR-198 is underexpressed in tumor tissues and cell lines. A low miR-198 expression level is significantly correlated with TNM stage and distant metastasis (18). MiR-198 downregulation is also reported in lung (26) and prostate cancers (27). However, miR-198 expression level is overexpressed in esophageal cancer and significantly associated with the prognosis (28). These findings suggested that miR-198 expression level exhibit tissue specificity and can be investigated as a biomarker for the diagnosis and prognosis of human cancers.

MiR-198 is important in the initiation and progression of multiple types of human cancer. For instance, miR-198 overexpression suppresses cell proliferation and migration and promotes cell adhesion of breast cancer (15). Nie et al (16) found that miR-198 upregulation improves the chemosensitivity of glioblastoma cells to temozolomide both in vitro and in vivo. Tan et al (29) revealed that miR-198 reexpression inhibits cell motility in hepatocellular carcinoma. Zhang et al (18) reported that resumption expression of miR-198 results in the growth reduction and metastasis of osteosarcoma cells. Yang et al (26) and Wu et al (30) revealed that miR-198 prohibits lung cancer cell proliferation, promotes cell apoptosis, and induces cell-cycle arrest. Wang et al (31) demonstrated that enforced miR-198 expression restricts HaCaT cell proliferation and induces cell-cycle arrest in the G1 phase. These findings suggested that miR-198 can be a novel therapeutic target for the treatment of these types of cancer.

Multiple direct targets of miR-198 have been previously identified. These targets include CDCP1 (15) in breast cancer, MGMT (16) in glioblastoma, c-MET (29) in hepatocellular carcinoma, ROCK1 in osteosarcoma (18), and SHMT1 (30) and FGFR1 (26) in lung cancer. PTEN, located at 10q23.3, was demonstrated to be a novel target of miR-198 in RB. PTEN is downregulated in various types of human cancer, such as gastric cancer (32), bladder cancer (33), cervical cancer (34), lung cancer (35), and colorectal cancer (36). PTEN plays tumor-suppressive roles in carcinogenesis and cancer progression and regulates a series of pathological processes, such as cell proliferation, cell-cycle, apoptosis, migration, invasion, metastasis, differentiation, epithelial-mesenchymal transition, and angiogenesis (37–39). PTEN is also lowly expressed in RB and implicated in RB initiation and progression (20–22). Thus, targeting PTEN may provide novel therapeutic opportunities for treating this aggressive cancer.

In conclusion, the present study was the first to demonstrate that miR-198 is upregulated in RB tissues and cell lines. MiR-198 inhibition attenuated cell proliferation and invasion by directly targeting PTEN and regulating PI3K/AKT pathway. Moreover, this study suggests that miR-198/PTEN interaction is a potential therapeutic target for patients with RB.

 Acknowledgements

Not applicable.

 Funding

No funding was received.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

YW designed this research. DoW, YM, LY and DeW performed the functional experiments and analyzed the data. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Dezhou People's Hospital (Shandong, China), and was performed according to principles of the Declaration of Helsinki. In addition, written informed consent was obtained from all patients with RB who participated in this research.

 Consent for publication

Written informed consent was obtained from all patients with RB.

 Competing interests

The authors declare that they have no competing interests.

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MiR-198 is upregulated in RB tissues and cell lines. (A) RT-qPCR analysis was used to detect miR-198 expression in 21 RB tissues and 7 normal retina tissues. *P<0.05, as indicated. (B) Relative miR-198 expression levels in 3 RB cell lines (Y79, SO-RB50, and WERI-RB1) and a normal retinal pigmented epithelium cell line (ARPE-19) were determined using RT-qPCR. Data are expressed as the mean ± standard deviation. *P<0.05 vs. ARPE-19. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA; RB, retinoblastoma.

Downregulation of miR-198 inhibits cell proliferation and invasion in RB cells. (A) Y79 and WERI-RB1 cells were transfected with miR-198 inhibitor or NC inhibitor. Reverse transcription-quantitative polymerase chain reaction analysis was conducted following 48 h of transfection to evaluate transfection efficiency. (B) Effect of miR-198 underexpression on Y79 and WERI-RB1 cell proliferation was assessed using Cell Counting Kit-8 assay. (C) Transwell invasion assay was performed to measure cell invasion capacity in Y79 and WERI-RB1 cells following transfection with miR-198 inhibitor or NC inhibitor (magnification, ×200). Data are expressed as the mean ± standard deviation. *P<0.05 vs. NC inhibitor. NC, negative control; miR, microRNA; RB, retinoblastoma.

PTEN is a direct target gene of miR-198 in RB. (A) Two putative miR-198 binding sites in the 3′-UTR of PTEN and the mutated PTEN 3′-UTR are shown. (B) Luciferase reporter assay was carried out in Y79 and WERI-RB1 cells cotransfected with miR-198 inhibitor or NC inhibitor and pGL3-PTEN-3′-UTR Wt (1 and 2) or pGL3-PTEN-3′-UTR Mut (1 and 2). *P<0.05 vs. NC inhibitor. (C) PTEN mRNA expression was analyzed in 21 RB tissues and 7 normal retina tissues using RT-qPCR. *P<0.05, as indicated. (D) Spearman's correlation analysis of the association between miR-198 and PTEN mRNA in RB tissues. r=−0.5530, P=0.0093. The expression levels of PTEN mRNA and protein were examined in Y79 and WERI-RB1 cells following transfection with miR-198 inhibitor or NC inhibitor using (E) RT-qPCR and (F) western blot analysis, respectively. *P<0.05 vs. NC inhibitor. UTR, untranslated region; NC, negative control; miR, microRNA; RB, retinoblastoma; Wt, wild type; Mut, mutant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; PTEN, phosphatase and tensin homolog.

Recovered PTEN expression rescues the biological actions of miR-198 inhibitor in RB cells. Y79 and WERI-RB1 cells were cotransfected with miR-198 inhibitor and NC siRNA or PTEN siRNA, and were used in the following assays. (A) Following 72 h incubation, the expression level of PTEN protein was quantified by western blot analysis. Cell proliferation and invasion in the indicated cells was determined using (B) Cell Counting Kit-8 and (C) Transwell invasion assays, respectively (magnification, ×200). *P<0.05 vs. NC inhibitor; #P<0.05 vs. miR-198 inhibitor+PTEN siRNA. PTEN, phosphatase and tensin homolog; miR, microRNA; RB, retinoblastoma; NC, negative control; siRNA, small interfering RNA.

Silencing of miR-198 inactivates the PI3K/AKT signaling pathway in RB. Y79 and WERI-RB1 cells were transfected with miR-198 inhibitor or NC inhibitor. Following transfection for 72 h, total protein was extracted and subjected to the detection of PI3K, p-AKT and AKT expression via western blotting. RB, retinoblastoma; NC, negative control; p-, phosphorylated; AKT, protein kinase B; PI3K, phosphoinositide 3-kinase; miR, microRNA.