miR-148a suppresses human renal cell carcinoma malignancy by targeting AKT2

. MicroRNA-148a (miR-148a) has been reported to be deregulated in different tumor types, whereas the biological function of miR-148a in renal cell carcinoma (RCC) largely remains unexplored. In the present study we investigated the clinical significance, biological effects, and the underlying molecular mechanisms of miR-148 in RCC. Here, we showed that miR-148a was significantly downregulated in RCC tissues and cell lines. Low expression of miR-148a in RCC tissues was associated with large tumor size, advanced TNM stage, and lymph node metastasis. Functional assays revealed that overexpression of miR-148a significantly inhibited RCC cell proliferation, colony formation, migration and invasion in vitro and suppressed RCC xenograft tumor growth in vivo . In addition, using quantitative RT-PCR (qRT-PCR), western blot analysis and luciferase reporter assays, AKT2 was confirmed to be a direct target of miR-148a. AKT2 expression was upregulated, and was negatively correlated with miR-148a expression in RCC tissues (r=-0.641, P<0.001). Silencing of AKT2 phenotypically copied miR-148a-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-148a in RCC cells. Further mechanistic inves-tigations showed that miR-148a exerted its antitumor activity via inhibition of the AKT pathway in vitro and in vivo . Taken together, these findings suggest that miR-148a functions as tumor suppressor in RCC by targeting AKT2.


Introduction
Renal cell carcinoma (RCC) is the most common malignant cancer in the adult kidney, and accounts for approximately 85% of all primary malignant kidney tumors and 3% of all cancers in adults (1,2). Despite increased early detection of RCC and more frequent surgery, 30% of RCC patients develop metastases after surgery, which are associated with poor prognosis (3)(4)(5). Therefore, there is an urgent need to elucidate the mechanisms of RCC which regulate the initiation and progression of RCC to provide useful information for the clinical management of RCC.
MicroRNAs (miRNAs) are small endogenous non-coding RNAs composed of ~19-25 nucleotides that negatively regulate gene expression by degrading target mRNAs or repressing protein translation by binding to the 3' untranslated region (3'UTR) of their target mRNAs (6). Accumulating studies suggest that deregulation of miRNAs is associated with carcinogenesis (7). miRNAs have been shown to function as either oncogenes or tumor suppressors in different types of cancer, which manifest as the regulation of cellular proliferation, cell death and angiogenesis in tumor progression (8,9). Numerous related studies have been carried out in RCC and have shown that dysregulation in miRNAs are involved in the occurrence and progression of RCC by regulating the expression of their target oncogenes and tumor suppressors (10,11), suggesting that miRNAs could serve as diagnostic markers or therapeutic agents for RCC.

Materials and methods
Human RCC clinical specimens. A total of 52 paired clear cell RCC and corresponding adjacent noncancerous tissues (ANT) were obtained sequentially from patients who underwent radical nephrectomy in the People's Hospital of Qinghai (Xining, China) between July 2013 and July 2015. Noncancerous renal tissues were obtained at least 5 cm away from the tumor site. All tissue specimens were snap-frozen in liquid nitrogen until use. The basic clinical characteristics of these patients were collected, and are listed in Table I. The study protocol was approved by the Ethics Committee of the People's Hospital of Qinghai and a written informed consent was obtained from all patients involved in this study.
RNA extraction and real-time PCR assays for miR-148a and AKT2 detection. Total RNA was extracted from the cultured cell and tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA was quantified using NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at 260 nm. For detection of the miR-148a expression level, quantitative PCR was performed using TaqMan miRNA assays with specific primers for miR-148a according to the protocol with endogenous U6 snRNA as the control under an AbI PRISM 7900 sequence detection system (both from Applied biosystems Life Technologies, Foster City, CA, USA). For detection of AKT2 mRNA expression, cDNA was synthesized from 500 ng total RNA using the Primer Script RT reagent kit (Takara bio, Inc., Otsu, Japan). Quantitative PCR analysis of the AKT2 expression at the mRNA level was performed using Fast SYbR-Green Master Mix (Applied biosystems Life Technologies) under AbI PRISM 7900 sequence detection system, with GAPDH used as an internal control. The primers of AKT2 and GAPDH used in this study were previously described (21). The relative miRNA or mRNA expression was quantified by measuring cycle threshold (Ct) values and normalized using the 2 -∆∆Ct method.
Cell proliferation and colony formation assays. Cell proliferation was assessed using a tetrazolium salt (WST-8)-based colormetric assay provided by the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). briefly, transfected cells were seeded into 96-well plates (5x10 3 cells/well) with 100 µl culture medium. At the indicated time-points (24, 48 and 72 h), 10 µl of CCK-8 solution was added to the wells and cultured additionally for 4 h at 37˚C. Cell viability was determined from absorbance readings at 450 nm using a microplate reader (bio-Rad Laboratories, Gaithersburg, MD, USA).
For the colony formation assay, 1x10 3 transfected cells were plated on 6-well plates and incubated for 10 days. Then, the cells were rinsed with PBS and fixed with 1% formaldehyde for 30 min and stained with 1% crystal violet for 10 min. Finally images were captured of the colonies and the number of colonies was counted under a light microscope (Olympus, Tokyo, Japan).
Wound healing assays. For the wound healing assay, transfected cells were seeded on 6-well plates with fresh medium containing 10% FbS at a density of 2x10 5 cells/well. After formation of a confluent monolayer of cells, the membrane was scratched using a sterile 100-µl pipette. The cell culture medium was replaced and images of the wound were taken at different time-points (0 and 24 h after scratching) under a light microscope (Olympus). To assess the migration rate, we measured the fraction of cell coverage across the line.
Invasion assays. A 24-well Transwell plate with 8-µm pore polycarbonate membrane inserts (Corning Costar Corp., Cambridge, MA, USA) was employed to assess invasive capacity of the cells according to the manufacturer's instructions. Briefly, 3x10 4 transfected cells suspended in serum-free medium were added to the upper chamber pre-coated with Matrigel (bD biosciences, bedford, MA, USA). Medium containing 10% FbS was added to the bottom chamber as a Table I. Correlation between clinicopathological features and miR-148a expression in tissues of the RCC cases.
After 48 h of incubation, the cells invading into the lower surface of the membrane were fixed and stained with methanol mixed with crystal violet and then counted under a light microscope (Olympus).
Vector construction and luciferase reporter assay. A luciferase reporter assay was performed using the firefly luciferase-expressing vector psiCHECK2 (Promega Corp., Madison, WI, USA). A wild-type 3'UTR segment of AKT2 mRNA containing the putative miR-148a binding sites was amplified and cloned into the XhoI and NotI sites downstream of the luciferase reporter gene in psiCHECK-2, named as Wt-AKT2-3'UTR. Mutations of their 3'UTR sequence were created using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Palo Alto, CA, USA) following the manufacturer's instructions, referred to Mut-AKT2-3'UTR.
For the luciferase activity assay, cells were seeded into 12-well plates overnight before transfection, and then co-transfected with 100 ng of psiCHECK-2 vectors, which harbored the AKT2 3'UTR wild-type or mutant constructs, 100 ng pRL-TK Renilla luciferase report vector as the internal control, 100 nM of miR-148a or miR-NC. After 48 h, the luciferase activity was measured with a dual-luciferase assay kit (Promega Corp.) according to the manufacturer's instructions. Renilla luciferase activity was normalized to firefly luciferase activity.
Western blot analysis. The cells or tissues were harvested and incubated on ice with lysis buffer (boster Inc., Shanghai, China). Total protein concentration was detected using a bicinchoninic acid (bCA) protein assay kit (boster Inc.); 50 µg of protein was electrophoresed through 10% SDS polyacrylamide gels and were then transferred to a PVDF membrane (Millipore Corp., billerica, MA, USA). Membranes were blocked using 5% non-fat milk for 1 h and blotted in antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), AKT2, AKT, p-Akt (Ser473), mTOR and p-mTOR (Ser2448) at 4˚C overnight. All antibody were purchased from Santa Cruz biotechnology, Inc. (Santa Cruz, CA, USA). After washing with TbST, the membranes were incubated in HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Santa Cruz biotechnology, Inc.) for 2 h at room temperature. Proteins band were visualized with an ECL chemiluminescent kit (ECL-Plus; Thermo Fisher Scientific, Waltham, MA, USA) and exposure to chemiluminescent film. Statistical analysis. All data are expressed as mean ± SD (standard deviation) from at least three independent experiments. Differences were determined by two-tailed Student's t-test by one-way ANOVA. Associations of miR-148a expression and AKT2 expression were estimated using Spearman's correlation analysis. Statistical analysis was performed using the SPSS ® Statistical Package, version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows ® . In all cases, P<0.05 was considered statistically significant.

miR-148a is downregulated in RCC cell lines and tissues.
To investigate the role of miR-148a in RCC development and progression, the expression of miR-148a was examined in four human RCC cell lines (786-O, ACHN, Caki-1, and Caki-2) and a human renal proximal tubule epithelial cell line (HK-2). Results of qRT-PCR showed that expression of miR-148a was decreased in all of the RCC cell lines compared with the HK-2 cells (Fig. 1A). In addition, the levels of miR-148a in 52 RCC tissues and ANT were detected by qRT-PCR. Compared with the noncancerous tissues, the miR-148a expression level was downregulated (P<0.001; Fig. 1b). Clinical patients were divided into two groups according to the median of miR-148a expression in the RCC samples. The downregulation of miR-148a was significantly associated with large tumor size, advanced TNM stage, and lymph node metastasis (Table I).

R E T R A C T E D
Taken together, these results suggest miR-148a as a potential biomarker for prediction of prognosis in RCC.

miR-148a inhibits proliferation, migration and invasion of RCC cells in vitro.
To determine the biological function of miR-148a in RCC progression, we re-introduced miR-148a into 786-O cells, which exploited the lowest expression among the four RCC cell lines (Fig. 1A). miR-148a expression was significantly increased in the 786-O cells transfected with miR-148a mimics compared to those transfected with negative control mimic (miR-NC), as shown by qRT-PCR ( Fig. 2A). CCK-8 assay showed that miR-148a overexpression significantly inhibited cell proliferation (Fig. 2b). Consistently, ectopic miR-148a expression dramatically suppressed colony formation (Fig. 2C). Given that miR-148a expression was associated with lymph node metastasis in RCC patients, we next examined whether miR-148a could affect RCC cell migration and invasion. Results showed that miR-148a overexpression distinctly abrogated the migration and invasion of 786-O cells (Fig. 2D and E). Our results suggest that miR-148a suppresses proliferation, migration and invasion of RCC cells.

AKT2 is the direct target of miR-148a in RCC cells.
According to the bioinformatic analysis, the 3'UTR of AKT2 was complementarily matched to the miR-148a sequence (Fig. 3A).
To test whether miR-148a was able to regulate AKT2 directly, we performed dual luciferase reporter experiments, and found that miR-148a significantly reduced luciferase activity of wildtype AKT2 3'UTR, whereas the luciferase activity of mutant AKT2 3'UTR remained unchanged (Fig. 3b). Furthermore, miR-148a overexpression blocked AKT2 expression at both the mRNA and protein levels in the RCC cells ( Fig. 3C and D). The expression of AKT2 was increased in the RCC clinical samples (Fig. 3E), and was inversely correlated with miR-148a in the RCC tissues (r=-0.641, P<0.001; Fig. 3F). These data supported our hypothesis that miR-148a directly targets the AKT2 gene in RCC.

miR-148a suppresses cell proliferation and migration via the suppression of AKT2.
We assessed whether targeting of AKT2 would mimic miR-148a-induced phenotypes on cell proliferation migration, and invasion. Firstly, we knocked down AKT2 in the 786-O cells by si-AKT2, and found that AKT2 expression was inhibited both at the mRNA and protein levels ( Fig. 4A and b). In addition, we demonstrated that downregulation of AKT2 efficiently inhibited cell proliferation (Fig. 4C), decreased colony formation ability (Fig. 4D), and suppressed migration (Fig. 4E) and invasion (Fig. 4F) in the 786-O cells, and had effects similar to those of the overexpression of miR-148a. Next, we examined whether AKT2 overexpression could rescue the inhibitory effects of miR-148a on RCC cell proliferation, migration and invasion. The overexpressing AKT2 plasmid was introduced in the 786-O cells that had been transfected with the miR-148a mimic and mimic NC. RT-PCR and western blot analysis confirmed that the miR-338-3p mimics markedly and specifically decreased AKT2 expression, whereas transfection of the overexpressing AKT2 plasmid restored AKT2 expression in the 786-O cells ( Fig. 5A and b). Our results also demonstrated that reintroduction of AKT2 significantly abrogated the suppression of cell proliferation, colony formation, migration and invasion in the 786-O cells induced by miR-148a (Fig. 5C-F). Collectively, these results suggest that miR-148a suppresses cell proliferation and migration in RCC cells via the suppression of AKT2.

miR-148a inhibits tumor growth in vivo.
An animal experiment was employed to verify the function of miR-148a in RCC growth. 786-O/miR-148a and 786-O/miR-NC cells were subcutaneously injected into nude mice. Tumor sizes were measured every 5 days until the mice were sacrificed at 30 days

R E T R A C T E D
post-implantation. The results demonstrated that tumor growth was slower in the 786-O/miR-148 group compared with that in the miR-NC group (Fig. 6A). Thirty days after injection, the nude mice were sacrificed, and the tumor tissues were stripped and weighed. The tumor size (Fig. 6b) and weight (Fig. 6C) were significantly decreased in the 786-O/miR-148a group compared with the size and weight in the 786-O/miR-NC group. In addition, we also found that the miR-148a expression level was upregulated (Fig. 6D), while the AKT2 expression at the mRNA level (Fig. 6E) and protein level (Fig. 6F) was downregulated in the 786-O/miR-148a group. These results suggest that miR-148a suppresses RCC tumorigenicity in vivo by repressing AKT2 expression. miR-148a inhibits the Akt pathway in vitro and in vivo. AKT2 belongs to the Akt family that functions as the hub in the

R E T R A C T E D
PI3K/Akt signaling pathway. We therefore assessed the levels of AKT2 downstream proteins including p-Akt (Ser473) and p-mTOR (Ser2448) in the 786-O cells 48 h after transfection with miR-148a or miR-NC, and found that overexpression of miR-148a decreased the phosphorylation levels of Akt and mTOR, whereas the total AKT and mTOR level were not changed (Fig. 7). Moreover, we also determined expression of these proteins in tumor tissues from the RCC xenograft mice.
Our results further showed that p-Akt and p-mTOR expression was decreased in the 786-O/miR-148a group compared to that in the 786-O/miR-NC group (Fig. 7). These results suggest that miR-148a overexpression inhibits the AKT signaling pathway in vitro and in vivo.

Discussion
Recent advances have revealed that dysregulation of miRNAs is involved in the progression and development of cancer (8,9). Modulation of miRNA expression has been proposed to be a feature of various types of cancer, including RCC, which may benefit the development of diagnostic markers and therapeutic agents for RCC (10,11). Here, we found that miR-148a was significantly downregulated in RCC cell lines and tissue specimens relative to normal renal cells and adjacent noncancerous tissues. Meanwhile, we also discovered that miR-148a inhibited RCC growth and metastasis by regulating AKT2 expression. These findings provided a mechanism by which miR-148a suppresses tumorigenesis in RCC.
miR-148a, a member of the miR-148 family, has been reported to correlate with diverse biological functions, including proliferation, migration, invasion, and cell cycle progression (12)(13)(14)(15)(16)(17)(18)(19). In multiple types of tumors, miR-148a functions as a tumor suppressor to block the growth and metastasis of cancer cells (12)(13)(14)(16)(17)(18)(19)(20). On the contrary, in glioblastoma and gastric cancer, miR-148a acts as an oncomiR to promote cancer cell proliferation and survival (15,22). These studies indicated that miR-148a can acts as an oncomiR or tumor suppressor, depending on tumor type. In the present study, we provide the first demonstration that miR-148a expression was downregulated in human RCC tissues and cell lines. We also found that restoration of miR-148a inhibited RCC cell proliferation, colony formation, migration and invasion in vitro, as well as suppressed RCC tumor growth in vivo. Thus, our in vitro and in vivo findings together suggest that miR-148a functions as a tumor suppressor in RCC.
It is well known that miRNAs exert their roles in tumor proliferation, apoptosis, migration, invasion and metastasis through regulation of target gene expression (7). Thus, to explore the mechanism underlying the function of miR-148a in RCC, the miR-148a target was predicted using biological software. We identified AKT2 as a potential candidate. AKT2, an isoform of the AKT family, has been reported to function as an oncogene by primarily enhancing the survival, migration and invasion of cancer cells (23)(24)(25). Recently studies have shown that AKT2 expression was elevated in RCC tissues, and it has an important role in the pathogenesis and progression of RCC (26). In addition, AKT2 has been shown to be regulated by multiple miRNAs, such as miR-29b (21), miR-124 (27), miR-612 (28), miR-137 (29) and miR-615 (30). In this study, we indentified AKT2 as a target of miR-148a in RCC. We also found that downregulation of AKT2 phenotypically copied miR-148a-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-148a in RCC cells. These results suggest that miR-148a exerts tumor suppressor roles in RCC by repressing AKT2.
AKT2 is a significant member of the PI3K/AKT pathway, which is recognized as one of the most frequently activated signaling pathways in human cancers (31,32). This pathway is involved in regulating various cancer processes, such as cell proliferation, apoptosis, migration, and metastasis (33,34). It has been reported that AKT exerts its biological function by phosphorylating its downstream substrates including mTOR (35,36). Therefore, we investigated whether miR-148a affects the AKT pathway and its downstream protein.
We found that overexpression of miR-148a significantly suppressed the phosphorylation of AKT, as well as inhibited its downstream effectors, p-mTOR expression, but not total AKT and mTOR in vitro and in vivo. These findings suggest that miR-148 inhibits RCC growth via regulating the AKT signaling pathway.
In conclusion, the results presented here demonstrated that the miR-148a expression level was downregulated in RCC cell lines and tissues, and its expression level was significantly associated with large tumor size, advanced TNM stage and lymph node metastasis, and that miR-148a acts as a tumor suppressor inhibiting the proliferation, migration and invasion of RCC cells, as well as suppressing tumor growth in vivo by negative regulation of its target AKT2 and regulation of the AKT signaling pathway. Thus, miR-1 may be a potential therapeutic target for RCC treatment.