Downregulation of the lncRNA ASB16-AS1 Decreases LARP1 Expression and Promotes Clear Cell Renal Cell Carcinoma Progression via miR-185-5p/miR-214-3p

Clear cell renal cell carcinoma (ccRCC) comprises approximately 75% of renal cell carcinomas, which is one of the most common and lethal urologic cancers, with poor quality of life for patients and is a huge economic burden to health care systems. It is imperative we find novel prognostic and therapeutic targets for ccRCC clinical intervention. In this study, we found that the expression of the long noncoding RNA (lncRNA) ASB16-AS1 was downregulated in ccRCC tissues compared with non-diseased tissues and was also associated with advanced tumor stage and larger tumors. By constructing cell and mouse models, it was found that downregulated lncRNA ASB16-AS1 enhanced cell proliferation, migration, invasion, and promoted tumor growth and metastasis. Furthermore, by performing bioinformatics analysis, biotinylated RNA pull-downs, AGO2-RIP, and luciferase reporter assays, our findings showed that downregulated ASB16-AS1 decreased La-related protein 1 (LARP1) expression by inhibiting miR-185-5p and miR-214-3p. Furthermore, it was found that overexpression of LARP1 reversed the promotive effects of downregulated ASB16-AS1 on ccRCC cellular progression. Our results revealed that downregulated ASB16-AS1 promotes ccRCC progression via a miR-185-5p-miR-214-3p-LARP1 pathway. We suggest that this pathway could be used to monitor prognosis and presents therapeutic targets for ccRCC clinical management.


INTRODUCTION
Renal cell carcinoma (RCC) is one of the most common types of urologic cancers accounting for more than 90% of renal malignancies (1,2). Clear cell renal cell carcinoma (ccRCC) comprises approximately 75% of RCCs and is the most lethal pathological subtype of RCC (3). More than 30% of ccRCC patients are diagnosed with metastasized disease and have a 13month median survival time (4). There are many risk factors such as dietary habits, occupational exposure, and physical inactivity that can lead to ccRCC tumorigeneses. The inefficiency of treatment and limitations in diagnosing ccRCC contribute to the poor quality of life for patients and the huge economic burden this disease has on health care systems (5,6). Therefore, it is imperative we find novel diagnostic and therapeutic targets for clinical management and intervention.
Long noncoding RNAs (lncRNAs) are one type of noncoding RNA consisting of more than 200 nucleotides (7). The underlying molecular mechanisms of lncRNA have been revealed in the past decades. For example, LncRNAs can act as a molecular sponge for microRNAs (miRs) (8) or interact with proteins, modulating their functions (9). LncRNAs are involved in many biological functions, especially tumorigenesis (10)(11)(12). Recently, the roles of lncRNAs in ccRCC tumorigenesis have been partly demonstrated; He et al. found that MEG3 regulates ccRCC progression via sponging miR-7 (13); Yang FQ et al. investigated the role of HOXA11-AS in ccRCC progression via promoting ccRCC growth and invasion ability (14); Qi Y et al. demonstrated that PENG suppresses ccRCC proliferation via sponging miR-15b (15); and the tumor-suppressing role of HOTAIRM1 in ccRCC has recently been demonstrated (16). These studies suggest that lncRNAs play essential roles in ccRCC development.
ASB16-AS1 is localized to 17q21, and is approximately 2275 bp. Previous studies have reported that lncRNA ASB16-AS1 functions as a microRNA sponge and regulates cell proliferation, migration, invasion, and apoptosis in several cancers including hepatocellular carcinoma, glioma, non-small lung cancer, and cervical cancer (17)(18)(19)(20), indicating that ASB16-AS1 play its crucial role in tumorigenesis. However, whether ASB16-AS1 exerts its function in ccRCC progression is poorly understood.
We hypothesized that ASB16-AS1 is involved in ccRCC progression and regulates ccRCC cell functions. Firstly, we tested ASB16-AS1 expression in ccRCC tissues. We then constructed in vitro and in vivo models to demonstrate the biological functions of ASB16-AS1 in ccRCC. Furthermore, we conducted bioinformatic analysis, AGO2-RIP, biotinylated RNA pull-downs, and luciferase reporter assays to elucidate the underlying molecular mechanisms of ASB16-AS1. Collectively, our data suggest that ASB16-AS1 could be used to monitor prognosis and presents therapeutic targets, altogether providing new insights regarding ccRCC basic research.

Clinical Samples
ccRCC tumors and adjacent non-diseased tissues were collected from 42 patients with ccRCC who received operative treatment at the Department of Urology, Shanghai General Hospital between August 2012 to December 2013. The specimens were collected from a tumor and a region at least 5 cm away from the tumor in each patient. The histological diagnosis was confirmed by two pathologists using hematoxylin and eosin stained sections. Following the American Joint Committee on Cancer (AJCC) guidelines, the pathological stage of each tumor was also determined by two pathologists. All patients provided informed consent.

Cell Culture and Transfection
The ccRCC cell lines A498, 786-O, 769-P, CAKI-1, OS-RC-2, ACHN, and the human kidney proximal tubular epithelial cell line, HK-2 and 293T, were purchased from The American Type Culture Collection (ATCC, USA). DMEM (Gibco, USA) with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) was used to culture cells at 37°C in a humidified atmosphere. The plasmids and short hairpin RNAs (shRNAs) used in this study were synthesized by and purchased from GenePharma (Shanghai, China). All transfections were conducted using lipofectamine 3000 or RNA iMax (Invitrogen, US) following the manufacturer's instructions.

5-Ethynyl-2′-Deoxyuridine (EdU) Incorporation
Cell proliferation was determined using the Cell-Light EdU DNA Cell proliferation kit (RiboBio, China) following the manufacturer's instructions. Two days after transfection, 50 mM EdU was applied to cells an incubated for 2 h. DAPI was used to stain nucleic acids and Apollo Dye Solution used to stain cells. The cell proliferation rate was calculated using Image J software (NIH, USA).

Cell Invasion and Migration Assays
Cell invasion and migration assays were performed in 24-well transwell plates filled with a polycarbonate membrane (pore size, 8 mm) (Corning, US), and Matrigel basement membrane matrix (1 mg/ml) (BD Biosciences, US) was used to fill the membranes. Briefly, 100 ml of serum-free media suspension was used to fill the upper chamber and 600 ml DMEM with 10% FBS was used to fill the lower chamber. After 24 h, the membranes of chambers were treated with crystal violet staining and observed under the microscope. Six fields of view were randomly chosen and cell numbers were recorded. Experiments were repeated three times.

RNA Fluorescent In Situ Hybridization (FISH)
The FISH kit (Ribibio, China) was used to detect the location of ASB16-AS1 in the ccRCC cell line 786-O following the manufacturer's protocol. Briefly, 786-O cells were incubated with pre-hybridization solutions for 30 min. Probes were treated with 20 mM hybridization solution and allowed to hybridize for 12 h. Next, saline sodium citrate was used to wash slides three times before treating with DAPI for 20 min. Results were visualized using a confocal microscope.

RNA Immunoprecipitation
Anti-AGO2 (#03-110, Millipore, Germany) was used to perform RNA immunoprecipitation (RIP) by using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Germany). To analyze the RNA bound complexes qRT-PCR assay was performed. Anti-IgG was used as an isotype control.

In Vivo Mouse Xenografts
The Committee for Animal Care and Use of Shanghai general Hospital approved our animal experiments. Six-week old nonobese severe diabetic/severe combined immunodeficiency (NOD/SCID) mice were randomly divided into two groups (n=5 each). The ccRCC 786-O cells pre-transfected with Sh-NC or Sh-ASB16-AS1 were then subcutaneously inoculated into the NOD/SCID mice (1×10 7 cells per tumor). From day 25 post inoculation, tumor volumes were measured every five days until day 45.

Immunohistochemistry
After surgery, all specimens were collected and fixed in formalin immediately. Then, specimens were all subjected to the process of dehydration, paraffinization, and embedded in paraffin blocks. Subsequently, specimens were cut into sections at 4 µm and dried in air 12 h. Tissue sections were undergoing the process of dehydration, paraffinization once again, and subjected to antigen retrieval with sodium citrate buffer upon heat stimulation. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 5 min. Next, tissue sections were incubated with primary antibody (LARP1; Abcam; 1:200; ab245635), and biotinylated goat anti-mouse IgG. Results were visualized using the VECTASTAIN ABC kit (Vector Laboratories) according to manufacturer's protocol.

Statistical Analysis
All experiments were carried out at least three times unless otherwise stated. Statistical analysis was performed using SPSS 19.0 (IBM, USA). The differences between two groups were tested using a Student's t-test; whereas, a one-way ANOVA was used to analyze the difference between three or more group. Data are presented as mean ± SD, and P < 0.05 was considered as statistically significant.

The Expression of ASB16-AS1 in ccRCC Tissues
In order to investigate whether ASB16-AS1 plays a role in ccRCC progression, we tested ASB16-AS1 expression in 42 pairs of ccRCC tumor tissues and adjacent non-diseased tissues. We found that ASB16-AS1 expression in tumors was significantly lower than in adjacent non-diseased tissues ( Figure 1A). Moreover, ASB16-AS1 was abundantly expressed in later stage and larger tumors ( Figures 1B, C), suggesting that ASB16-AS1 might be involved in ccRCC initiation and progression. Next, we measured ASB16-AS1 expression in different ccRCC cell lines and the human proximal tubular epithelial cell line HK-2. The results showed that ASB16-AS1 was abundantly expressed in 786-O cells and lowly expressed in 769-P cells ( Figure 1D). Fluorescent in situ hybridization (FISH) shows that ASB16-AS1 is mainly located in the cytoplasm of 786-O cells ( Figure 1E).

ASB16-AS1 Downregulation Promotes ccRCC Proliferation, Migration, and Invasion
Next, we determined the biological function of ASB16-AS1 in ccRCC progression. Cell models were generated by transfecting sh-NC or sh-ASB16-AS1 into 786-O cells, and OE-NC or OE-ASB16-AS1 into 769-P cells, respectively. Transfection efficiency is shown in Figure 2A.

ASB16-AS1 Downregulation Promotes ccRCC Cell Growth and Metastasis In Vivo
In vivo experiments were applied to further assess the biological functions of ASB16-AS1 in ccRCC progression. Nonobese severe diabetic/severe combined immunodeficiency (NOD/SCID) mice (6 weeks old) were subcutaneously inoculated with 786-O cells (1×10 7 per tumor) which were pre-transfected with sh-NC or sh-ASB16-AS1. The representative images of excised tumors are shown in Figure 3A. comparative statistics of final tumor weights are shown in Figure  3C. it was suggested that downregulation of ASB16-AS1 significantly inhibits tumor growth compared with control levels of ASB16-AS1. Lung tissues from xenografted mice using hematoxylin and eosin staining showed that downregulation of ASB16-AS1 enhanced tumor metastasis ( Figure 3D).

DISCUSSION
Despite the improvement of RCC clinical management in the past decades, RCC still is the sixth most frequently diagnosed cancer in men and tenth in women, which has been a great threaten to people health (23). As one most lethal pathological subtype of RCC, the diagnosis and intervention of ccRCC present great challenge to urologist. While, its underlying mechanisms still remain unclear. The clinical intervention of ccRCC demands novel targets more than ever.
In the current study, our results demonstrate the role of ASB16-AS1 in ccRCC progression. By performing in vitro and in vivo experiments, we found that ASB16-AS1 expression was downregulated in ccRCC tissues, which was also associated with a later tumor stage and larger tumors. Subsequently, our findings demonstrated the biological functions of ASB16-AS1 in ccRCC progression. Downregulation of ASB16-AS1 promoted cell proliferation, migration, and invasion, regulated EMT associated genes in ccRCC cells and promoted tumor growth and metastasis in a xenograft mouse model. Furthermore, it was found that ASB16-AS interacts with miR-185-5p and miR-214-3p. It has been reported that miR-185-5p is involved in cancer development and regulates tumorigeneses via its involvement in cell proliferation, migration, invasion, and apoptosis (24)(25)(26). Additionally, miR-214-3p is involved in osteosarcoma, breast cancer, endometrial cancer, and lung cancer (27)(28)(29)(30). These studies suggest that miR-185-5p and miR-214-3p play important roles in tumorigeneses and tumor development. However, neither have been studied in ccRCC.
Here, our findings suggested that ASB16-AS1 acted as a molecular sponge for miR-185-5p and miR-214-3p. Furthermore, the promotive effects of ASB16-AS1 on cell proliferation, migration, invasion, and EMT associated gene expression were rescued by miR-185-5p and miR-214-3p inhibition. While, the relationship between miR-185-5p and miR-214-3p, and their contribution in ccRCC cellular progression upon ASB16-AS1 downregulation still need further exploration. Moreover, the downstream transcriptional targets of ASB16-AS1 were not the final answer for its role in ccRCC biological progression. Therefore, bioinformatic tools, biotinylated RNA pull-down and luciferase reporter assays were used to identify its post-transcriptional target. Our findings found that LARP1 was targeted by miR-185-5p and miR-214-3p in 786-O and 293T cells.
LARP1, as one RNA binding protein, has been in-depth studied recently due to its capability to interact with mammalian target of rapamycin complex 1 (mTORC1) and act as a key repressor of ribosomal protein mRNA translation (31)(32)(33)(34). The biological role of LARP1 was investigated in non-small cell lung cancer, ovarian cancer, and hepatocellular carcinoma (35)(36)(37). While, the molecular mechanisms of LARP1 in ccRCC progression still uncovered. Since the crucial role of LARP1 in ribosome production, which is an essential unit for cellular progression in all living organisms (38). We presumed that the biological effects of ASB16-AS1/miR-185-5p/miR-214-3p in ccRCC progression were functioned through regulating LARP1. Our results showed that overexpression of LARP1 reversed the promotive effects of downregulated ASB16-AS1 on ccRCC cellular progression and EMT phenotype. Furthermore, the expression ASB16-AS1 and LARP1 in ccRCC tumor tissues were suggested strongly correlated. Although, the role of ASB16-AS1/miR-185-5p/miR-214-3p/LARP1 pathway in ccRCC progression has been partially demonstrated. While, in the contrast with previous findings, our results implicated that LARP1 exerts its tumor suppressive effect in ccRCC cellular progression, which might owe to the timepoint-dependent feature of LARP1 knockdown (39). Furthermore, since the role of mTOR-LARP1 axis in cancer cellular progression is universally agreed, we assumed that LARP1 might exert its function in ccRCC through this axis, which requires in-depth investigation in our future study.

CONCLUSION
In the current study, we demonstrate the interaction of miR-185-5p and miR-214-3p with LARP1 and show the role of the ASB16-AS1-miR-185-5p/miR-214-3p-LARP1 pathway in ccRCC progression. Collectively, our data determined the role of ASB16-AS1, miR-185-5p, and miR-214-3p in ccRCC progression. Moreover, we identified a mechanism of upstream regulation of LARP1 in ccRCC. We therefore provide new insights into ccRCC basic research, and present potential prognostic and therapeutic targets for ccRCC clinical management.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are available from the corresponding author upon request.

ETHICS STATEMENT
The animal study was reviewed and approved by Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University. Expression of LARP1 in four ccRCC tumor tissues and its adjacent normal tissues, which were randomly selected, were detected using western-blot. (K) Expression of LARP1 in two ccRCC tumor tissues and its adjacent normal tissues, which were randomly selected, were assessed using IHC assay (Scale bars -50 mm). (L) The expression correlation between ASB16-AS1 and LARP1 were analyzed using spearman statics. *P < 0.05, **P < 0.01.
Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.