miR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A

The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/tcga) was used to load data on miR-30c-2-3p and TOP2A expression in RCC and neighboring tissues. Correlations among miR-30c-2-3p, TOP2A, and RCC development were analyzed using Prism 7.0 (GraphPad Software) [19]. Simultaneous analysis of the correlation between miR-30c-2-3p and TOP2A was conducted. Additionally, to determine the binding connections between miR-30c-2-3p and TOP2A UTRs, TargetScan (http://www.targetscan.org) was used [19].

Twenty-five paired RCC and adjacent nontumor kidney tissues were obtained from patients of the Affiliated Hospital of Yan’an University, Shaanxi, China. All patients were clinically diagnosed with RCC based on the 2015 Chinese Society of Clinical Oncology Guidelines for the Treatment of Renal Carcinoma[20]. All procedures conducted in this study were approved by the Ethics Committee of Yan’an University (approval number 2019104). Written informed consent for tissue usage and research publication was obtained from all patients.

RCC cell lines 786-O, Caki-1, ACHN, and normal renal tubular epithelium cell line (HK-2) were derived from Medical Research and Experiment Center of Yan’an University. HK-2 and 786-O cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Caki-1 cells and ACHN cells were cultured in McCoy's 5A medium and Eagle's minimum essential medium, respectively. All of the above media were obtained from Biological Industries (Beit Haemek, Israel) and contained 10% fetal bovine serum. Cells were cultured in 5% CO₂ at 37 °C.

Homo sapiens (hsa)-miR-30c-2-3p (miR-30c-2-3p) mimics, hsa-miR-30c-2-3p inhibitor (miR-30c-2-3p inhibitor), and their negative controls (NCs) (miR-30c-2-3p mimic NCs and miR-30c-2-3p inhibitor NCs) were synthesized by Gene Pharma Biotech. The TOP2A interference vectors (short hairpin [sh]TOP2A-1 and shTOP2A-2) and the empty vector (shTOP2A NC) were synthesized by Genechem. Cells were transfected with jetPRIME (Polyplus) according to the manufacturer's instructions [19].

Total RNA was isolated from cells using TRIzol reagent (TransGen Biotech), in accordance with the manufacturer's instructions. miRNAs were extracted using a miRcute miRNA extraction kit (Tiangen Biotech). For cDNA fragment synthesis, EasyScript One-Step gDNA Transcription Kit (TransGen Biotech) was used. cDNA fragments for protein-coding genes were synthesized using the RNA templates with Oligo dT primer, while U6 and miR-30c-2-3p cDNA were synthesized using the miRNA template and gene-specific primers. Relative gene expression of miR-30c-2-3p and TOP2A was detected using the Cobas z480 analyzer (Roche Molecular Diagnostics) and the KAPA SYBR FAST Universal reagent (Sigma-Aldrich). All primers used are listed in Table 1. The relative quantification of miR-30c-2-3p and TOP2A expression in different samples was performed by the 2^−ΔΔCt method. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the internal reference genes for the miRNA and genes of interest, respectively. The above experiment was repeated 3 times.

TOP2A expression was examined in tissues using a streptavidin–horseradish peroxidase (HRP) kit (ZSGB-BIO) and rabbit anti-human TOP2A polyclonal antibody (1:500; Proteintech; research resource identifier (RRID): AB_10664923). After deparaffinization, the slides were subjected to antigen retrieval by autoclaving in 0.01 M ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.5) for 3 min 15 s, followed by incubation in 3% H₂O₂ for 10 min to quench endogenous peroxidase. Sections were incubated overnight at 4 °C with rabbit anti-human TOP2A polyclonal antibody. Slides were washed with phosphate-buffered saline (PBS) 3 times and incubated with secondary antibody (PV9000; ZSGB-BIO) at room temperature for 0.5 h. Staining was carried out by incubating the slides in 3,3′-diaminobenzidine, followed by counterstaining with hematoxylin and dehydration in gradient ethanol and xylene. Protein expression level was analyzed through ImageJ (Rawak Software) assessment of the average gray value of positive cells. TOP2A is localized in the nuclei of tumor cells, so we selected the nuclear staining mode.

RIPA buffer with protease inhibitors (protease inhibitor cocktail; MCE) was used to obtain whole-cell protein extracts. The bicinchoninic acid (BCA) Protein Assay Kit (Solarbio) was used to determine the protein concentration. The protein extract was electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane after separation using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Antibodies against TOP2A (#20233-1-AP; 1:800; Proteintech), Fas (#4233S; 1:1,000; Cell Signaling Technology), FasL (#5237S; 1:1,000; Cell Signaling Technology), caspase 9 (#9502T; 1:1,000; Cell Signaling Technology), caspase 8 (#13423-1-AP; 1:1,000; Proteintech), Bcl-2 (#12789-1-AP; 1:1,000; Proteintech), caspase 3 (#10380-1-AP; 1:1,000; Proteintech), b-tubulin, and b-actin (TRAN) were used for detection with a G: BOX Chemi xx9 system (Syngene). HRP-labeled goat anti-rabbit IgG (#A0208; 1:2,000) and HRP-labeled goat anti-mouse IgG (#A0216; 1:2,000) were purchased from Beyotime.

We constructed 2 vectors: one consisting of the fragments of the TOP2A 3′-UTR region (blinded by the miR-30c-2p binding site), which was inserted into the pmirGLO vectors; the other consisting of the mutated TOP2A 3′-UTR region of miR-30c-2p binding sites, which was also inserted into the pmirGLO vectors, named pmirGLO-TOP2A-WT (TOP2A W) and pmirGLO-TOP2A-MuT (TOP2A M), respectively. HEK-293T cells were pre-transfected with miR30c-2-3p-mimics and pmirGLO-TOP2A-WT, pmirGLO-TOP2A-MuT, or empty pmirGLO vectors; firefly and Renilla luciferase activity was detected 24 h after transfection using a GloMax 20/20 luminometer and a dual-luciferase reporter kit from Promega (Promega). The internal reference was the Renilla luciferase activity.

Each well of a 96-well plate received 3,000 cells overall. After transfection, an aliquot (10 μL) of CCK-8 reagent (Beyotime) was added to each well at 0 h, 24 h, 48 h, and 72 h. At 450 nm, the absorbance was measured 1 h after CCK-8 supplementation [19].

At 24 h after transfection, 1,000 cells per well in each group were inoculated into 12-well plates. Two weeks later, cells were anchored with 4% polymerized formaldehyde, washed with PBS, and stained with 0.1% crystal violet. For absorbance detection, cells were pretreated with dimethyl sulfoxide, and the absorbance of the supernatant was measured at 570 nm [19].

Apoptosis was detected 48 h after cell transfection using the Annexin V/propidium iodide double staining kit (BestBio) with a flow cytometry system (CyFlow Cube8, Sysmex).

For data representation, the mean and standard deviation are used. Data were examined using the software GraphPad Prism 8 (GraphPad Software, Inc.). Measurement data conforming to normal distribution were analyzed by Student t test or one-way analysis of variance (ANOVA). Measurement data that do not conform to the normal distribution used the rank sum test. P < 0.05 was considered to indicate a statistically significant difference.

When we examined the expression of miR-30c-2-3p in RCC tissues from the TCGA database, we discovered that it was considerably lower in 241 RCC tissues than it was in 70 healthy renal tissues (P < 0.0001; Figure 1A). miR-30c-2-3p expression was inversely correlated with the overall 5-year survival rate of RCC patients (Figure 1B). Furthermore, we investigated the expression of miR-30c-2-3p in 3 RCC cell lines to confirm these findings and found that it was less abundant in those RCC cell lines than in HK-2 cells (Figure 1C).

Figure 1.

To investigate the role of miR-30c-2-3p in RCC cells, miR-30c-2-3p mimics were synthesized and successfully transfected into 786-O, Caki-1, and ACHN cells. qRT-PCR results indicated that transfection of the miR-30c-2-3p mimics dramatically increased the expression of miR-30c-2-3p (Figure 2A). We established using the CCK-8 test and colony formation assay that miR-30c-2-3p mimics reduced RCC cell growth (Figures 2B,C). Flow cytometry experiments proved that miR-30c-2-3p mimics could increase RCC cell apoptosis (Figure 2D). Western blotting analysis revealed that miR-30c-2-3p mimics upregulate the expression of Fas, FasL, caspase 8, and caspase 3 in 786-O, Caki-1, and ACHN cells (Figure 2E). The above results indicate that miR-30c-2-3p increases apoptosis and suppresses cell growth in RCC.

Figure 2.

To determine the role of miR-30c-2-3p in the regulation of RCC development, the potential binding sites of miR-30c-2-3p on TOP2A were predicted using the TargetScan database (Figure 3A). Then, we synthesized a luciferase reporter vector of TOP2A. The sequences of wild-type (TOP2A W) and mutant (TOP2A M) genes are shown as indicated (Figure 3B). Dual-luciferase reporter assay revealed that overexpression of miR-30c-2-3p significantly reduced the luciferase activity of TOP2A W but had no influence on the luciferase activity of TOP2A M (Figure 3C). Additionally, miR-30c-2-3p mimics decreased TOP2A expression in RCC cell lines 786-O, Caki-1, and ACHN (Figures 3D,E). These results suggest that miR-30c-2-3p may control the expression of TOP2A during RCC formation.

Figure 3.

TCGA data analysis showed that, compared with normal kidney tissue (n=72), the expression of TOP2A was increased in RCC tissue (n=534); at the same time, we also analyzed the tissue data of 72 pairs of RCC patients and obtained consistent results (Figure 4A). Additionally, TOP2A expression was inversely proportional to the 5-year overall survival rate of patients (Figure 4B). Using qRT-PCR, Western blotting, and immunohistochemical staining, we verifed that TOP2A expression was elevated in RCC cell lines and in renal cancer tissue compared with human renal tubular epithelial cells and normal renal tissue, respectively (Figures 4C–E). Taken together, these data suggest that increased TOP2A expression is positively correlated with RCC occurrence.

Figure 4.

We used RNA interference technology to reduce TOP2A expression in 786-O, Caki-1, and ACHN cells in order to ascertain TOP2A's role in RCC cells. The results of qRT-PCR and Western blotting showed that after transfection with shTOP2A-1 and shTOP2A-2, the mRNA and protein expressions of TOP2A in 786-O, Caki-1, and ACHN cells decreased significantly (P < 0.01; Figures 5A,B). Down-regulation of TOP2A drastically reduced the proliferation ability of RCC cells and promoted cell apoptosis, according to CCK-8, clone creation assays, and cell apoptosis experiments (Figures 5C–E). In addition, Western blotting results showed that reducing TOP2A increased the expression of Fas, FasL, caspase 8, and caspase 3 (Figure 5F), further suggesting that TOP2A acts as an oncogene by regulating RCC cell proliferation.

Figure 5.

Figure 6.

In order to further clarify the regulatory relationship between miR-30c-2-3p and TOP2A in RCC cells, miR-30c-2-3p inhibitor was transfected into the 786-O, Caki-1, and ACHN cells. As Figure S1 shows, miR-30c-2-3p inhibitor effectively inhibited miR-30c-2-3p expression and upregulated TOP2A expression. Transfecting of shTOP2A, miR-30c-2-3p inhibitor, and shTOP2A plus miR-30c-2-3p inhibitor, respectively, into 786-O, Caki-1, and ACHN cells allowed researchers to determine whether miR-30c-2-3p targets TOP2A to prevent RCC cell proliferation and trigger cell death.

Figure S1.

qRT-PCR, Western blotting, CCK-8 assay, clone formation, and flow cytometry results demonstrated that miR-30c-2-3p inhibitor increased TOP2A protein expression, proliferation, and clonal formation, while blocking cell apoptosis. In contrast, shTOP2A induced the opposite effect. Cotransfection of shTOP2A with miR-30c-2-3p inhibitor reversed the effects of the miR-30c-2-3p inhibitor on TOP2A expression, cell viability, and apoptosis (Figures 6A–E). Based on the Western blotting analysis, shTOP2A increased Fas, FasL, caspase 8, and caspase 3 expression, whereas it had no significant influence on Bcl-2 and caspase 9 expression. However, the miR-30c-2-3p inhibitor decreased Fas, FasL, caspase 8, and caspase 3 expression but had no effect on Bcl-2 and caspase 9 expression. Transfection of shTOP2A plus miR-30c-2-3p inhibitor reversed miR-30c-2-3p inhibitor-induced changes in the expression of Fas, FasL, caspase 8, and caspase 3 (Figure 6F). These results suggest that miR-30c-2-3p inhibits RCC cell proliferation and promotes apoptosis by regulating TOP2A expression and, subsequently, by triggering Fas/FasL/caspase 8/caspase 3.