Effect of miR-29b-1* and miR-29c knockdown on cell growth of the bladder cancer cell line T24

Abstract

Objective

To investigate the role of the microRNAs miR-29b-1-5p (miR-29b-1*) and miR-29c in bladder urothelial cancer (BUC).

Methods

Levels of miR-29b-1* and miR-29c in normal urothelial cells (HU609) and BUC cells (T24) were determined via quantitative real-time reverse transcription–polymerase chain reaction. T24 cells were transfected with small interfering RNA targeting miR-29b-1* or miR-29c, and cell growth was assessed using 3-(4,5-dimehylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The predicted targets and oncogenic pathways of these microRNAs were determined using bioinformatics analysis.

Results

MiR29b-1* and miR-29c levels were higher in T24 cells than normal urothelial cells. Knockdown of miR-29b-1* or miR-29c suppressed T24 cell growth. Bioinformatic analysis showed that miR-29b-1* and miR-29c co-regulated a subset of putative target genes, about 10% of which have been experimentally validated.

Conclusion

Both miR-29b-1* and miR-29c regulate cell growth in BUC. The targets of miR-29b-1* and miR-29c may be functionally associated with proliferation, cell cycle and apoptosis.

Introduction

Bladder urothelial carcinoma (BUC) is the most common urinary tract cancer worldwide, ranking tenth amongst all malignant tumours and accounting for 90% of bladder cancers. ¹ BUC is three to four times more common among men than women and has high rates of relapse, metastasis and mortality. The prognosis is poor, with a 5-year survival rate of about 50%. ²

Alteration of genomic regions^3,4 and epigenetic modification ⁵ in urothelial cells contribute to the pathogenesis of BUC. Disease progression is driven by two mutually exclusive pathways. Noninvasive tumours frequently have mutations in fibroblast growth factor receptor 3 (FGFR3), whereas invasive tumours often display tumour protein p53 (TP53) mutations. ⁶ Progression of noninvasive to invasive tumours requires subsequent TP53 mutations that result in further cellular transformation. ⁶

MicroRNAs (miRNA) are endogenous small RNA molecules that negatively regulate gene expression at a post-transcriptional level, thereby playing pivotal roles in gene regulatory networks during both physiological conditions and disease (including cancer). ⁷ Studies investigating aberrant miRNA expression in BUC have helped to elucidate the underlying mechanisms of proliferation, invasion and metastasis.^8–11 We have previously identified 11 miRNAs that are differentially expressed between BUC and normal urothelial samples, and seven that are differentially expressed between invasive and noninvasive BUCs. ¹² Of these differentially expressed miRNAs, expression of the miR-29 family has been shown to be decreased in non-small cell lung cancer, ¹³ mantle cell lymphoma ¹⁴ and liver cancer, ¹⁵ but increased in breast cancer ¹⁶ and acute myeloid leukemia. ¹⁷ Members of the miR-29 family appear to function as either tumour suppressors or oncogenes, depending on the cellular context.

The aim of the current study was to investigate the expression of miR-29b-1-5p (miR-29b-1*) and miR-29c in a BUC cell line, and determine the effect on cell growth of miR-29b-1* and miR-29c knockdown. In addition, the predicted targets and oncogenic pathways of these miRNAs were determined using bioinformatics analysis.

Materials and methods

Cell lines

The human cell lines T24 (BUC), 293 T (embryonic kidney) and HU609 (normal bladder urothelium) (all GeneChem, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Cells were incubated at 37℃ with 5% carbon dioxide in air.

Quantitative real-time reverse transcription–polymerase chain reaction

Total RNA was extracted from T24 and HU609 cells (1 × 10⁷ cells/extraction) using Trizol (Invitrogen, Carslbad, CA, USA), according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Polymerase chain reaction was performed for miR-29b-1*, miR-29c and U6 (internal control) using a Bulge-Loop™ miRNA qRT-PCR kit (Ribo Bio, Guanzhou, China) that included miR-29b-1* and miR-29c primers. Primer sequences for U6 were: forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCCACGAATTTGCGTGTCAT-3′. The cycling conditions were: initial denaturation step at 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s, 60℃ for 20 s and 72℃ for 20 s, followed by a final extension step of 72℃ for 6 min.

Lentiviral vector construction

Small interfering RNA (siRNA) directed against miRNA-29b-1* or miRNA-29c was synthesized by Ribo Bio using the following primer sequences: miR-29b-1*-AgeI-forward, 5′-CCGGTAACACTGATTTCAAATGGTGCTATTTTTG-3′ and miR-29b-1*-EcoRI-reverse, 5′-AATTCAAAAATAGCACCATTTGAAATCAGTGTTA-3′; miR-29c-AgeI-forward, 5′-CCGGTTAACCGATTTCAAATGGTGCTATTTTTG-3′ and miR-29c-EcoRI-reverse, 5′-AATTCAAAAATAGCACCATTTGAAATCGGTTAA-3′.

The siRNAs were cloned into pGCsil-H1-CMV-GFP vector (GeneChem) via AgeI and EcoRI sites. To package the recombinant lentiviral particles, 70–80% confluent 293 T cells in 15 cm culture dishes were co-transfected with 20 µg pGCsil-H1-CMV-GFP containing siRNA, 15 µg pHelper1.0 and 10 µg pHelper2.0 plasmids (Genechem), using Lipofectamine2000 (Invitrogen). At 48 h after transfection, viral particles were harvested from the culture media by centrifugation at 8542  g for 90 min at 4℃. The viral particles were then titrated and aliquoted for long-term storage at −80℃.

T24 cells were grown to 60% confluence in 15 cm² flasks then cultured with viral particles (5 × multiplicity of infection) containing miR-29b-1* siRNA, miR-29c siRNA or empty vector (as control). Infection efficiency was assessed via fluorescence microscopy at 48 h after infection.

MTT assay

A standard 3-(4,5-dimehylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to evaluate the effect of miR-29b-1 or miR-29c knockdown on T24 cell growth. Nontransfected control cells, or cells transfected with empty vector, vector containing miR-29b-1* siRNA or vector containing miR-29c siRNA were seeded into 96-well plates (1 × 10⁴ cells/well) and cultured for 1–5 days. At each time point, 20 µl MTT solution (5 mg/ml) was added to each well and incubated at 37℃ for 4 h. The medium was removed and the formazan dye crystals were dissolved in 150 µl of dimethyl sulphoxide, with shaking at low speed for 10 min. Absorbance at 490 nm was measured in a spectrophotometer. Assays were performed in triplicate.

Bioinformatic analysis

miRNA target prediction

The miRWalk database ¹⁸ (available at http://miwalk.uni-hd.de) was used for miR-29b-1* and miR-29c target prediction. This database offers a comparative platform of 10 datasets including miRWalk, Diana-mt, miRanda, RNA22, miRDB, TargetScan, RNAhybrid, PITA, PICTAR4 and PICTAR5.

Annotation of miRNA putative targets

Validated targets were imported into the DAVID bioinformatic database (available at david.abcc.ncifcrf.gov/home.jsp) for functional annotation analysis.

Statistical analyses

Data were presented as mean ± SD and compared using one-way analysis of variance or Dunnett’s t-test. Statistical analyses were carried out using SPSS® version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values < 0.05 were considered statistically significant.

Results

To validate the findings from our miRNA profiling on BUC cell lines, we detected the expression of miR-29b-1* and miR-29c genes in bladder urothelial cancer T24 cells and normal urothelial HU609 cells using real-time PCR. In comparison to the normal urothelial HU609 cells, levels of miR-29b-1* and miR-29c were significantly higher in T24 cells than HU609 cells (3.47-fold and 4.40-fold, respectively; P < 0.05 for both comparisons). We then investigated whether miR-29b-1* and miR-29c are involved in the proliferation of T24 cells. Using lentivirus-mediated siRNA expression, we were able to reach >95% infection efficiency and effectively knockdown the level of miR-29b-1* and miR-29c (Figure 1). Next, we monitored the proliferation rate of T24 cells with miR-29b-1 or miR-29c knockdown. At 4 and 5 days after transfection, cell growth was significantly lower (lower absorbance in MTT assay) in T24 cells transfected with miR-29b-1 or miR-29c siRNA compared with nontransfected controls or cells transfected with empty vector (P < 0.01 for all comparisons; Figure 2).

Figure 1.

Lentiviral vector (pGCsil-H1-CMV-GFP)-mediated transfection of T24 cells (a human bladder urothelial cancer cell line) with small interfering RNA (siRNA) targeting microRNA (miR)-29b-1* and miR-29c. Left panels: light photomicrographs indicating T24 cell growth. Right panels: fluorescence photomicrographs indicating transfection efficiency (green fluorescent protein). Control cells were transfected with empty vector. Original magnification × 400.

Figure 2.

Cell growth (assessed by 3-(4,5-dimehylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) of the human bladder urothelial cancer cell line T24, transfected with small interfering RNA (siRNA) targeting microRNA (miR)-29b-1* or miR-29c. NC, nontransfected control cells; CON, cells transfected with empty vector; OD, optical density. **P < 0.01; one-way analysis of variance.

MicroRNA can interact with multiple mRNAs leading to inhibitory regulation in gene circuitry. We searched putative targets of miR-29b-1 or miR-29c using miRWalk database, which contains 10 predictive algorithm programs. 6063 miR-29b-1 targets and 7312 miR-29c targets were identified (Figure 3A). Of these predicted targets, 2563 were coregulated by both miR-29b-1 and miR-29c. About 200 of these were experimentally validated in the literature. Results of functional analysis using DAVID software are shown in Figure 3B.

Figure 3.

A: Putative targets of microRNA (miR)-29b-1* and miR-29c (larger circles) predicted using miRWalk. ¹⁸ The smaller circles indicate experimentally validated targets. B: The most frequent validated targets were used for pathway enrichment analysis using DAVID (david.abcc.ncifcrf.gov/home.jsp). Target genes are listed on the right, with pathways along the bottom edge. Green: gene is enriched in pathway; black: gene is not present in pathway.

Discussion

miRNAs are a class of endogenous, small (approximately 22 nucleotides), noncoding, regulatory RNAs. ¹⁹ A single miRNA can target up to 200 mRNAs, and one mRNA may be regulated by several miRNAs. ²⁰ As modulators of gene expression, miRNAs play important roles in the formation and progression of tumours. Gain- and loss-of-function studies suggest that miRNAs may have oncogenic or tumour suppressive functions in different types of cancer. ⁷ Several miRNAs have been found to be involved in the onset and progression of BUC, ¹⁰ and may be useful to assist in diagnosis, grading and prognosis.^8,9 Studies regarding BUC-specific miRNAs and their precise functions in tumorigenesis may provide novel approaches to the diagnosis and treatment of BUC.

We have previously shown that miR-29b-1* was upregulated in both invasive and non-invasive BUC, suggesting a role in tumour development. ¹² On the other hand, miR-29c is upregulated more highly in invasive than noninvasive BUC, suggesting that this miRNA may be involved in tumour invasion and metastasis. ¹² It remains to be investigated whether miR-29b-1* and miR-29c interaction, and whether members of the miR29 family are synergistic or antagonistic in BUC.

The current study found that both miR-29b-1* and miR-29c are upregulated in T24 cells compared with normal bladder urothelial cells. This suggests that higher expression of these miRs may contribute to bladder cancer development. Indeed, the miR-29 family was previously reported to act as tumour suppressors for oncogenes in several types of cancers,^13–17 depending on cellular context. The role of miR-29b-1* and/or miR-29c in bladder cancer oncogenesis is largely unknown. Therefore, we developed specific siRNAs molecules to inhibit miR-29b-1* or miR-29c expression in T24 cells using a lentiviral delivering system (Figure 1). With significant knockdown of either miR-29b-1* or miR-29c expression in T24 cells, the cell proliferation was significantly reduced at day 4 and day 5 relative to control or sham-treated cells. The findings demonstrated a causal link between miR-29s and bladder cancer. In order to understand what the putative targets may affected by either of these two microRNAs, we intensively searched 10 microRNA datasets including miRWalk, Diana-mt, miRanda, RNA22, miRDB, TargetScan, RNAhybrid, PITA, PICTAR4 and PICTAR5. The bioinformatic analysis revealed that miR-29b-1* and miR-29c co-regulated 2562 putative target genes, about 10% of which have been experimentally validated. These target genes included several transcription factors (PAX3, PAX7, HOXA10, FOXO1, E2F1, ETF7, YY1 and MYC) that may contribute to cancer development via a E2F/Myc/miR-17-92 negative feedback loop. ²¹ Targets may be functionally associated with proliferation, cell cycle and apoptosis. More interestingly, DNA-methyltransferase genes and histone deacetylase genes are the targets of miR-29 s, suggesting the epigenetic modification by miR-29 s. Further studies are required to validate these mRNA targets, as well as investigate the changes in protein levels associated with miR-29b-1* and miR-29c.

In conclusion, we have demonstrated that higher expression of either miR-29b-1* or miR-29c in bladder cancer cell line T24 and knockdown either of these miRs inhibits the cell proliferation. In silico analysis suggests multiple transcription factors or epigenetic modifying genes may be regulated by miR-29 s.