Human renal cell carcinoma (RCC) is the most common type of kidney malignancy in adults accounting for 2–3% of all adult malignancies. In China, RCC accounts for ~0.5% of all cancer-associated mortalities, ranking 16th among all cancer types. For early-stage RCC, surgery is the recommended treatment. Molecularly targeted therapy is the preferred first-line treatment for clear-cell RCC. However, more potential targets are required. MicroRNA-338-3p (miR-338-3p) functions as a tumor suppressor in various cancers, but has not been studied in RCC. Accordingly, the present study investigated the role of miR-338-3p of RCC. It was demonstrated that miR-338-3p was present at low levels in RCC tissues. Also, overexpression of miR-338-3p inhibited cell proliferation and promoted cell apoptosis, and downregulation of miR-338-3p promoted cell proliferation. The 3′ untranslated region of AKT serine/threonine kinase 3 was targeted by miR-338-3p. In conclusion, the data of the present study revealed the inhibitory function of miR-338-3p in RCC and suggested that miR-338-3p is novel therapeutic target for RCC, but further investigation is needed.
Renal cell carcinoma (RCC), also called kidney cancer, is a heterogeneous disease. RCC begins in the lining of the proximal convoluted tubule, a component of the very small tubes in the kidney which transport primary urine. RCC is the most common kidneys malignancy in adults (
MicroRNA (miRNA), is a small endogenous non-coding RNA, which negatively regulates gene expression by inhibiting translation of messenger RNA (mRNA) or targeting mRNAs for degradation (
MicroRNA-338-3p (miR-338-3p) functions as a tumor suppressor in various cancers, including thyroid cancer (
However, the role of miR-338-3p in RCC remains unknown. In the present study, we investigated the function of miR-338-3p in RCC, and found that miR-338-3p is an inhibitor of tumor growth. We anticipate that our data may provide a potential molecular target for the treatment of RCC, but further investigation is needed.
Twelve RCC tissues samples were collected from the Department of Urology, West China Hospital of Sichuan University (Chengdu, China). The use of human tissues in the present study was evaluated and approved by the Ethics Committee of Sichuan University. Written informed consent were obtained from all patients enrolled in the presnet study and all specimens were handled and made anonymous as required according to the legal standards of China. All RCC tissues samples were evaluated and confirmed by a senior pathologist at the Sichuan University Cancer Center.
The twelve RCC tissues were processed in standard protocol for H&E staining. Briefly, 4-µm-thick sections were cut. After deparaffinization and hydration, the slides were stained in hematoxylin for 3–5 min and washed in running water for 5 min. After being differentiated in 1% acid alcohol, the slides were stained in 1% Eosin Y for 10 min. Then these slides were dehydrated in increasing concentrations of alcohol and cleared in xylene prior to observation.
The RCC cell lines Caki-1 and 786-O cells, and human proximal convoluted tubule epithelial cell line HK-2, were purchased from the Cell bank of Sichuan University. All cells were maintained cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) in 6-well plate (Shengong, Shanghai, China).
The expression of miR-338-3p in tissues samples and cells were analyzed by RT-qPCR. Briefly, the total RNA from tissue samples or cells were extracted using the TRIzol reagent according to the manufacturers's protocol (Invitrogen, Carlsbad, CA, USA). The level of miR-338-3p was determined by the TaqMan miRNA Assay (Thermo-Fisher, Waltham, MA, USA). The U6 snRNA was used as an internal loading control. RT-qPCR was performed on an ABI 7900HT instrument (Applied Biosystems, Foster City, CA, USA). The primers were synthesized and tested by the ShengRui Company (ShengRui, Chengdu, China) (
The miR-338-3p levels in Caki-1 and 786-O cells was increased and decreased by miR-338-3p mimics and miR-338-3p antisense oligonucleotides (ASO), respectively. The miR-338-3p mimics and miR-338-3p ASO were both purchased from JingHong Biotechnoloy (Chengdu, China). Before transfection, the cells were cultured overnight (1×106 per well). The cells transfection was performed with Lipofectamine 2000 (Invitrogen), as recommended by the manufacturer's instructions.
The cellular growth was analyzed by the MTT assay. Briefly, cells were placed into 96-well plates at a density of 5×105/well for overnight. Then remove the medium and replace it with 100 µl of fresh culture medium, and the MTT reagent was added into the medium at a final concentration of 0.1 mg/ml. Then the plates were incubated for 4 h at 37°C. Next medium from each well was carefully removed and 100 µl DMSO were added and incubated at 37°C for 15 min. OD was measured on a microplate reader with a 570 nm filter (
Cells (5×105 cells/ml) were suspended in the Annexin V-FITC (Abcam, Cambridge, UK) binding buffer. Then, Annexin V-FITC was added and the suspension was incubated for 15 min at room temperature. Afterwards, propidium iodide (PI; Abcam) was added to each sample. Next the samples were analyzed on a FACS analyzer instrument using the 488 nm excitation line (Argon-ion laser or solid state laser) and emission was detected at 530 nm (green, FITC) and 575–610 nm (orange, PI).
The Targetscan software (
Cells were seeded at 1×105 per well and were serum-starved for 6 h pre-transfection. The 3′untranslated region (3′UTR) of Akt3 and mutated controls were cloned and inserted into the reporter plasmid (500 ng) and the pGL3-control (100 ng; Promega, Madison, WI, USA). MiR-338-3p p mimics were then transfected into the Caki-1 cells containing the wild-type or mutant 3′UTR plasmids with Lipofectamine 2000 (Invitrogen). Cells were harvested and luciferase activitity was measured after 24 h using the Dual-Luciferase Reporter Assay System (Huijun Company, Guangzhou, China). Mutant of Akt3 3′UTR were generated using the Site-Directed Mutagenesis kit (Promega).
Cells were frozen and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCI, 1% Triton X-100 and 0.1% SDS) with the protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and phosphatase inhibitor cocktail. For Akt3 western blotting, an anti-Akt3 antibody (Abcam) were used at a dilution of 1:1,000, followed by detection with a peroxidase-linked antibody to rabbit antibody IgG (1:2,000 dilution, Abcam). Proteins were detected with the ECL Western Blotting Detection Reagents (GE Healthcare, Chicago, IL, USA). Images were analyzed using Image J (NIH, Bethesda, MD, USA).
All experiments were repeated three times. Data are shown as mean ± SD. Two-tailed Student's t-test was used to analyze the mean value between two groups; ANOVA was used to test the mean value among three or more than three groups. P<0.05 was considered to indicate a statistically significant difference. All calculations were performed using SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA).
Initially, we collected 12 RCC tissues and the pathological images are shown in
We assayed the miR-338-3p levels in RCC cell lines (Caki-1 and 786-O) by RT-qPCR analysis. The human proximal convoluted tubule epithelial cells, HK-2 were used as blank control. We found that the miR-338-3p levels in Caki-1 and 786-O cells was lower than in HK-2 cells (
We also transfected miR-338-3p ASO into Caki-1 and 786-O cells to decrease the miR-338-3p levels. The miR-338-3p levels in cells were measured 24 h after miR-338-3p ASO transfection. We found that miR-338-3p was inhibited by miR-338-3p ASO (
In the present study, we examined the role of miR-338-3p in RCC and found that miR-338-3p exhibited a tumor suppressor activity in RCC. Additionally, we determined that the direct target gene of miR-338-3p is
Previous studies have shown that miR-338-3p functions as a tumor suppressor in various cancers, including thyroid cancer (
AKT3 is a member of Akt kinase family, also called PKB, serine/threonine protein kinase family. Akt kinases are regulators of cell signaling in response to insulin and growth factors. Akt kinases play a role in cell proliferation, differentiation, apoptosis, and tumorgenesis (
In conclusion, our data revealed the suppressive role in miR-338-3p in RCC. We hope our findings may provide a new therapeutic target for further investigation.
The present study was supported by a grant from Department of Sichuan Science and Technology (grant no. 2017FZ0057).
RCC tissue samples showed low levels of miR-338-3p. A total of 12 RCC tissues were collected from the West China Hospital of Sichuan University. The H&E staining images are shown (A). The miR-338-3p levels in the 12 RCC tissues samples were analyzed by RT-qPCR (B). The mean values of miR-338-3p expression in 12 RCC tissues and their matched adjacent normal tissues were calculated (C). The data are represented as the mean ± SD. Each experiment was repeated at least three times. *P<0.05. miR, microRNA; RCC, renal cell carcinoma.
Overexpression of miR-338-3p inhibited Caki-1 and 786-O proliferation and promoted cells apoptosis. The miR-338-3p levels in normal renal tissues, Caki-1 and 786-O cells were analyzed by RT-qPCR. The miR-338-3p levels in HK-2 cells were arbitrarily defined as 100% (A). Caki-1 and 786-O cells were seeded separately and then each was transfected with miR-338-3p mimics and NC mimics separately and 24 h later, the miR-338-3p expressions was evaluated by RT-qPCR (B). Following miR-338-3p mimics transfection, the cellular proliferation was assessed by the MTT assay (C). After 48 h miR-338-3p mimics transfection, Caki-1 and 786-O cells were stained with Annexin V-FITC and PI, and then the cell apoptosis rate was evaluated by FACS analysis (D). Caki-1 cell morphological changes following 24 h miR-338-3p mimics and miR-338-3p ASO transfection (magnification, −200) (E). The data are represented as the mean ± S.D. Each experiment was repeated at least three times. *P<0.05. miR, microRNA; NC, negative control; ASO, antisense oligonucleotides.
Suppression of miR-338-3p levels promoted Caki-1 and 786-O proliferation. Caki-1 and 786-O cells were seeded separately and then each was transfected with miR-338-3p ASO and 24 h later, the miR-338-3p expression was tested by RT-qPCR (A). After miR-338-3p ASO transfection, the cellular proliferation was analyzed (B). The data are represented as the mean ± S.D. Each experiment was repeated at least three times. *P<0.05. miR, microRNA; NC, negative control; ASO, antisense oligonucleotides.
AKT3 was targeted by miR-338-3p in Caki-1 cells. The binding sites and its mutated version in AKT3 and miR-338-3p are shown (A). MiR-338-3p mimics and plasmid containing wild-type or mutated 3′UTR sequence of AKT3 were transfected into Caki-1 cells and 48 h later, the luciferase activity was analyzed (B). miR-338-3p mimics was transfected into Caki-1 cells, and the abundance of AKT3 protein was determined by western blotting (C). Each experiment was repeated at least three times. *P<0.05. UTR, untranslated region; Wt, wild-type; miR, microRNA; NC, negative control; ASO, antisense oligonucleotides.