MicroRNA-34a Attenuates Metastasis and Chemoresistance of Bladder Cancer Cells by Targeting the TCF1/LEF1 Axis Open Access

Bladder cancer ranks as the fourth and tenth leading cause of cancer-related deaths in males and females, respectively [1], and the incidence of bladder cancer continues to rise year by year [2]. The prognosis for patients with bladder cancer rests on factors such as histological subtype, tumor size, lymph node metastasis, and distant metastasis [3, 4]. Although epirubicin is commonly used to treat bladder cancer, resistance to the drug greatly inhibits its long-term curative effect [5] and the molecular mechanisms involved in chemoresistance of bladder cancer cells are unclear. It is therefore critical that effective molecular targets that could regulate bladder cancer invasion and chemoresistance are identified for the successful development of targeted therapies.

MicroRNA (miRNA) is a short, non-coding RNA (approximately 22 nucleotides in length) that binds to the 3′-untranslated region (3′-UTR) of mRNA and regulates gene expression by controlling mRNA translation or degradation [6, 7]. MiRNAs are involved in various biological processes. In conjunction with their target genes, miRNAs play a critical role in tumor proliferation, differentiation, metastasis, and chemosensitivity [8] and miR-34a in particular functions as a tumor suppressor and is downregulated in many human cancers [9]. Furthermore, reduced expression of miR-34a is associated with chemotherapy resistance in a variety of cancers [10-13]. One of the mechanisms is the regulatory function of miR-34a in the p53 signaling pathway. miR-34a is considerably induced by p53 and its upregulation induces apoptosis, cell-cycle arrest and senescence and alters cancer cell chemosensitivity by directly targeting multiple genes in the p53 signaling pathway, such as Sirt-1, CDK6, E2F3 and Bcl2 [14, 15]. In bladder cancer, miR-34a chemosensitizes bladder cancer cells during chemotherapy, regardless of the p53-Rb pathway status [12]. However, the regulatory mechanism of miR-34a in epirubicin-based bladder cancer chemotherapy is not clear and requires further investigation.

In this study, a series of in vitro and in vivo tests were performed, resulting in the discovery of consistently lower expression of miR-34a in BC tissues and epirubicin-resistant BIU87 cells. Overexpression of miR-34a significantly increased the sensitivity of BC cells to epirubicin treatment, as evidenced by greater numbers of apoptotic cells. Moreover, miR-34a overexpression in BC xenograft tumors after miR-34a mimic transfection was found to contribute to the development of sensitivity to epirubicin treatment. Finally, our data indicate that miR-34a may confer chemosensitivity to epirubicin by reducing the levels of its target TCF1 and LEF1.

All animal handling and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the Third Military Medical University, China (approval no. SYXK20070002).

The miR-34a expression profiles in BC tissues were obtained from the TCGA database (https://tcga-data.nci.nih.gov and https://genome-cancer.ucsc.edu). The prognostic value of miR-34a was analyzed using the TCGA portal. We used TCGA samples from patients at different stages of the disease to compare the tissues in our analysis of miR-34a levels. Overall survival was determined by Kaplan–Meier analysis for patients with high or low levels of miR-34a.

Human bladder cancer cell lines BIU87 and BIU87/ADR were purchased from Shanghai Meixuan Biological Technology Company (Shanghai, China) and cultured in RPMI 1640 media with 10% fetal bovine serum, penicillin, and streptomycin (Gibco, Carlsbad, CA, USA). The cell lines were maintained at 37°C and 5% CO₂ in a humidified incubator. EPI was purchased from Cayman Chemical (Ann Arbor, MI, USA). Cancer cell lines (BIU87 and BIU87/ADR) were seeded at a density of 3 x 10⁵ per well in 6-well plates, incubated overnight, and subsequently treated with EPI for 48 h. After induction, the cells were harvested for analysis.

The miR-34a mimic and miR-34a inhibitor were obtained from RiboBio (Guangzhou, China). Primary antibodies against GAPDH and TCF1, LEF1, E-cadherin, Bcl2, cyclin D1, CDK6, and c-MYC protein were obtained from Cell Signaling Technology (Danvers, MA, USA) and the primary antibodies against delta2 catenin were obtained from Abcam (Cambridge, UK)

Total RNA samples were extracted from the harvested cells using the RNAi (Dojindo Molecular Technologies, Kumanoto, Japan) according to the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was conducted using the method described by Duan et al [16].. For the detection of miRNA-34a, ADAM17, JAG1, Notch1, c-MY, cyclin D1, CDK6, and E-cadherin, 1 µg of total RNA per sample was converted to cDNA using a Reverse SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). GAPDH was used as the endogenous control for mRNA. For the detection of mRNA, the cDNA products were synthesized using a miScript Reverse Transcription Kit (Qiagen). The primers specific to miR-34a or the endogenous control U6 were purchased from Qiagen. The qRT-PCR was performed using a miScript SYBR Green PCR Kit (Qiagen). All reactions were performed in triplicate on a Bio-Rad C1000 thermal cycler (CFX-96 real-time PCR detection system; Bio-Rad, Hercules, CA, USA). The fold change of miRNA to mRNA expression was calculated according to the 2^-ΔΔct method. All primer sequences are provided in Table 1.

In the examination of transient transfection, miRNA mimics and inhibitors were transfected into BIU87 and BIU87/ADR cells using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. The miR-34a mimic and inhibitor, as well as the negative control, were obtained from RiboBio. All final concentrations of the miR-34a mimic and inhibitor and the negative control in the transfection system were 200 nM. After a period of 48 h, the cells were collected for subsequent flow cytometry, western blotting, and qRT-PCR.

Cell proliferation was evaluated using a Cell-Counting Kit 8 (CCK8; Dojindo Molecular Technologies), following the manufacturer’s instructions. BIU87 or BIU87/ADR cells (5000 per well) were cultured overnight in 96-well plates, and 10 μL of CCK-8 solution was subsequently added to each well at the indicated time points after transfection. Cells were incubated for a further 2 h at 37°C in a 5% CO₂ incubator. The absorbance was measured at 450 nm, using a Multiscan FC Microplate Photometer (Thermo Fisher Scientific, Rochester, NY, USA).

Transfected bladder cancer cells were plated in 6-well plates (500 cells/well) and cultured in RPMI-1640 medium for 14 days. Proliferating colonies were stained with crystal violet and colonies consisting of ≥ 50 cells were counted and photographed for statistical analysis. The colonies were observed using an inverted phase contrast microscope. All procedures were performed in triplicate.

Cells were analyzed in a RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). A complete protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA) was added to the lysis buffer before use. Protein concentration was determined using a Bio-Rad DC protein assay, and 30 µg of total protein from cell lysates was subjected to SDS-PAGE and subsequently transferred to nitrocellulose membrane. The membrane was then blocked in 5% non-fat milk in PBS overnight and incubated with primary antibody TCF1, LEF1, cleaved-caspase 3, Bcl2, CDK6, cyclin D1, c-myc, and E-cadherin (all from Cell Signaling Technology), delta2 catenin (Abcam), or β-actin (Cell Signaling Technology). After washing for 30 min, secondary goat anti-mouse IgG (Vector Co., Burlingame, CA, USA) was applied to the nitrocellulose membrane in TBS-Tween for 1 h and washed again. The proteins of interest were detected using a Chemiluminescent HRP Antibody Detection Kit (Denville Scientific, South Plainfield, NJ, USA). The protein signals were captured using an electrochemiluminescent system (PerkinElmer Life Science, Boston, MA, USA).

Cells were lysed and fixed in ice-cold 70% ethanol for 24 h at 4°C. Thereafter, propidium iodide (PI) was added and the cells were placed in the dark for 30 min. Cells (1 x 10⁶) were fixed using 70% ethanol at -20°C, incubated with propidium iodide (Invitrogen) for 30 min at 37°C, and then analyzed using fluorescence-activated cell sorting (FACS) flow cytometry (BD Biosciences, California, New Jersey, USA). Data analysis was performed using FlowJo software (version 9.3.2).

The extent of apoptosis was determined by flow cytometry using an Annexin V/PI staining kit (BD Biosciences), according to the manufacturer’s instructions. In brief, after collection, the cells were resuspended in 200 μL binding buffer, and samples were subsequently incubated with 2 μL annexin V-APC and 2 μL 7-AAD in the dark for 15 min at room temperature (RT). They were then analyzed using flow cytometry. Data analysis was performed using FlowJo software (version 9.3.2).

A wound healing assay was used to assess the extent of cell migration and invasion. When cells reached a 90-95% confluence, a wound was generated by scratching the surface of the plates with a pipette tip. After removal of the floating cells, the adherent cells were incubated for 24 h until the wound healed. The gaps between the different time points were normalized one by one, immediately after scratching.

The transwell cell invasion assay was performed using a Cell Migration Kit with polycarbonate membrane inserts (8-μm pore size; Millipore, Billerica, MA, USA). Cells were serum-starved overnight in DMEM prior to the experiment. The lower chambers were filled with 1 mL of conditioned media. Cells (4 x 10⁴) were re-suspended in 200 μL of DMEM and added to the upper chamber, before incubation at 37°C for 24 h to allow cell migration through the membrane. Thereafter, the cells were fixed and stained using 0.05% crystal violet. The numbers of cells that migrated from the upper to the lower chamber were counted from five random views in each experiment and each assay was performed three times.

In brief, 3′ UTR segments of TCF1 and LEF1 were predicted to interact with miR-34a (https://www.targetscan.org). Luciferase reporter genes were successfully constructed using molecular cloning technology. The target sequence was inserted into a pGL3-Basic Vector (Promega Wisconsin, USA) to obtain pGL3-TCF1-3′-UTR and pGL3-LEF1-3′-UTR containing the miR-34a binding sequences (TCF1-3′-UTR and LEF1-3′-UTR sequences). The miR-34a modified BIU87/ADR cells were seeded at a density of 3 x 10⁴ cells per well in 24-well plates for 24 h. When the cells reached 60-70% confluence, wild-type and mutant plasmids were co-transfected with miR-34a mimics or negative control (NC). Lipofectamine 3000 (Invitrogen) was used to optimize transfection. Cells were collected at 48 h post-transfection. Luciferase activities were subsequently measured using the dual-luciferase reporter gene assay kit (Promega), according to the manufacturer’s instructions. Luciferase activities were normalized to Renilla luciferase activity. Statistically significant differences between the treatment and control groups were determined using Student’s t-test.

The miR-34a mimic cells and their vector control cells were used to generate animal models. All animal handling and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the Third Military Medical University, China (approval no. SYXK20070002). All efforts were made to mitigate animal suffering and to minimize the number of animals used. Four-week old BALB/c nude mice were used in this study and maintained at room temperature (25 ± 2 °C) under a standard 12h/12h light-dark cycle in a specific, pathogen-free experimental animal room and fed a sterilized diet. For the subcutaneous tumor growth assay, 2 x 10⁶ of the indicated cells in 0.1 mL of PBS were subcutaneously injected into 6-week-old female mice (8 mice per group). When the tumors reached a size of approximately 100 mm3, the mice were started on a treatment of either PBS or EPI (6 mg/kg body weight). The treatment was administered every third day. After 3 weeks, the mice were sacrificed and tumor weights were measured. For lung and liver metastasis experiments, BC lung and liver metastatic models were generated by an injection of BC cells through the tail vein. Cells (5 x 10⁵) were suspended in 0.1 mL of PBS and injected into the lateral tail veins of 6-week-old female nude mice (8 mice per week). Three weeks after the injections, the mice were started on a treatment of either PBS or EPI (6 mg/kg body weight). Six weeks after the injections, all mice were sacrificed, and the lung and liver surface tumor foci were counted.

Tumors excised from nude mice were fixed in 4% buffered paraformaldehyde for 24 h at room temperature, dehydrated through a graded series of ethanol solutions, embedded in paraffin wax, and cut into sections of 4 μm. Sections were subsequently stained with H&E and observed under a light microscope.

Statistical analysis was performed using SPSS software (version 19.0). All data are expressed as means ± SD, unless otherwise stated. Comparisons between two groups were performed using a two-tailed unpaired t-test, while comparisons of three or more groups were performed using one-way analysis of variance (ANOVA). Differences between tumor volumes were determined using a two-way ANOVA. Overall survival analysis was performed using the Kaplan-Meier method. Statistical significance was set at p< 0.05.

First, we compared miR-34a expression levels at different stages of bladder cancer in the TCGA dataset (Fig. 1A). Notably, miR-34a expression was lower in the late stages of the disease than in the early stages, indicating that miR-34a is a tumor suppressor in bladder cancer. In addition, the Kaplan-Meier survival analysis demonstrated that the BC patient group with a lower level of miR-34a had a lower survival rate than the group with a high level of miR-34a (Fig. 1B). Further investigations into the impact of EPI chemotherapy on the expression of miR-34a in BC cells were subsequently conducted, using BIU87 and BIU87/ADR cells, and qRT-PCR analysis showed that miR-34a expression was significantly lower in BIU87/ADR cells than in BIU87 cells (Fig. 1C). Together, these results suggest that reduced expression of miR-34a indicates a poor prognosis and may be involved in the stimulation of BC metastasis and chemoresistance.

As indicated in Fig. 1D, qRT-PCR analysis confirmed a high level of miR-34a expression after transfection. To determine the effect of miR-34a on the proliferation of BC cell lines, BIU87 and BIU87/ADR cells were transfected with miR-34a inhibitor and miR-34a mimics, respectively. The proliferation of BIU87 cells 48 h after transfection was significantly lower than that of the miRNA negative control (Fig. 2A and 2B). The effect of miR-34a was also tested via colony formation, another type of proliferation assay (Fig. 2C, D, E, and F). Here, the inhibition of miR-34a significantly promoted colony formation in BIU87 cells, whereas overexpression of miR-34a significantly decreased colony formation in BIU87/ADR cells. Morphological analysis of the cells showed similar changes in both BIU87 and BIU/ADR cells, characterized by cell volume and concomitant shrinking (Fig. 2G, H, I, and J). Additionally, an apoptosis assay was conducted using flow cytometry to determine whether the effects of miR-34a on cell proliferation were related to apoptosis. BIU87 cells were first transfected with a miR-34a inhibitor for a period of 24 h and treated with EPI for another 24 h. The results showed that ectopic miR-34a inhibition decreased EPI-induced cellular apoptosis (Fig. 3A and B). Similar results were obtained from BIU87/ADR (Fig. 3C and D). Next, BIU87 cells were first transfected with miR-34a inhibitor for 24 h and treated with EPI for another 24 h to determine whether the effects of miR-34a on cell proliferation were related to the cell cycle. The results showed that ectopic miR-34a inhibition induced G1/G0 arrest, which indicated cell cycle arrest (Fig. 3E, F, G, and H). Collectively, the results show that cells with miR-34a downregulation are clearly resistant to EPI treatment in BC cells.

The following information should be considered when investigating whether the reduced expression of miR-34a was involved in BC metastasis. As shown in Fig. 3, in the in vitro wound healing and the transwell assays, the inhibition of miR-34a can promote significant cell invasion of BIU87 cells (Fig. 3I, K, M, and O), while overexpression of miR-34a reduce significant cell invasion of BIU87/ADR cells (Fig. 3J, L, N, and P). These results clearly demonstrate that cells with downregulated miR-34a are stimulated by BC metastasis.

To investigate the association between miR-34a and TCF1/LEF1 expression, the expression of TCF1/LEF1 was examined in relation to miR-34a overexpression and inhibition in BIU87/ADR and BIU87 cells at both mRNA and protein levels, respectively (Fig. 4A, B, and C). Furthermore, to investigate the underlying mechanism of miR-34a and TCF1/LEF1 on bladder cancer cell metastasis and chemoresistance, miRNA target prediction algorithms (http://targetscan.org and http://microrna.org) were used to screen miR-34a target genes, with TCF1 and LEF1 identified as tentative targets of miR-34a (Fig. 4D and E). Notably, previous studies have shown that the Wnt/β-Catenin pathway correlates closely with bladder cancer progression, metastasis, and chemoresistance [17-19]. To determine whether the regulation of TCF1-luciferase and LEF1-luciferase expression is dependent on the binding of their complementary 3′-UTR sequences to the miR-34a seed sequence, a 3-nt mutation was inserted into the TCF1 3′UTR and LEF1 3′UTR. The results revealed that the overexpression of miR-34a significantly repressed the luciferase activity associated with the wild-type 3′UTR, while the 3′UTR mutation completely abrogated the effect of miR-34a overexpression on luciferase activity in BIU87 cells (Fig. 4F and G). Cumulatively, these findings suggest that miR-34a negatively regulates the expression of TCF1/LEF1 axis by directly targeting the 3′UTR sequence.

A rescue experiment was performed to test whether miR-34a negatively regulates TCF1/LEF1 downstream signal or protein expression through direct targeting of the TCF1/LEF1 axis. The Wnt/ β-Catenin signaling pathways protein, including cleaved-caspase 3 (Fig. 5A), Bcl2 (Fig. 5A, B, and C), CDK6 (Fig. 5A, D, and E), cyclin D1 (Fig. 5A), C-myc (Fig. 5A, F, and G), E-cadherin (Fig. 5A, H, and J), and delta2 catenin (Fig. 5A, J, and K), were tested by qPCR or western blot. Our results suggest that the restoration of miR-34a can upregulate cleaved-caspse 3 and anti-metastatic protein E-cadherin and downregulate Bcl2, CDK6, cyclin D1, c-myc, and delta 2 catenin expression in BIU87/ADR cells, while overexpression of TCF1/LEF1 can rescue the properties of bladder cancer cells and lead to metastasis. The cumulative data confirms that the restoration of miR-34a and inhibition of TCF1/LEF1 reduces the properties and chemoresistance of bladder cancer cells.

BIU87/ADR cells were examined to test the effects of miR-34a overexpression on the chemosensitivity of BIU87 cells. A consistently lower tumor volume was observed in tumors with miR-34a overexpression than in those with empty vectors under EPI administration, particularly on day 21 after the initiation of EPI treatment (Fig. 6A and B). At the end of the experimental period, the mean wet weights of the tumors were significantly lower in the miR-34a transfected group than in the empty vector control group (p< 0.05; Fig. 6C). Further tests for the involvement of miR-34a in metastasis and EPI resistance in vivo involved injections into athymic nude mice with the cell lines grown as xenograft tumors. The nude mice were sacrificed 6 weeks later, and surface tumor nodules were counted on the lung and liver metastasis models. The results confirm that the overexpression of miR-34a inhibits BIU87/ADR cell lung and liver metastasis (Fig. 6D, E, F, and G). Therefore, higher expression of miR-34a is also associated with metastasis and EPl chemosensitivity in vivo.

EPI is a chemotherapeutic agent commonly used to treat multiple malignancies, including bladder cancer, but there are currently no known markers for either response or resistance to treatment. The development of new molecules for bladder cancer screening and treatment is therefore urgently required [20]. Molecular profiling of miRNAs may assist in the development of companion diagnostics in the clinic to identify subgroups of patients most likely to develop metastasis or respond to chemotherapy [21, 22]. By directly targeting protein-coding genes, miRNAs are able to inhibit the genes necessary for signaling pathways or drug-induced apoptosis [22]. In recent years, microRNAs have attracted increasing attention, with multiple miRNAs—including miR-195 [23], miR-124 [24], miR-1-3p [25], miR-218[26], and miR-222-3p [27]—highlighted as critical components in the control of proliferation, metastasis, and drug resistance in bladder cancer [28, 29]. The present study examined the influence of EPI on miR-34a expression and its target genes by using in vitro/in vivo assays and bladder cancer cells. Notably, our data implies that a miR-34a mimic is a potential therapeutic agent for treating chemoresistant bladder cancer.

miR-34a is downregulated in many types of cancer and acts as a critical tumor suppressor by targeting multiple oncogenes that modulate cancer-related processes, such as cell proliferation, apoptosis, metastasis, and chemoresistance [30]. Moreover, previous studies have indicated that miR-34a inhibits migration and invasion via Notch1 and CD44 and induces chemosensitivity to cisplatin treatment, regardless of the p53-Rb pathway in bladder cancer [12, 31, 32]. In the present study, BIU87/ADR was obtained from BIU87 through treatment with EPI. Thereafter, a series of in vitro and in vivo studies provided evidence that the reduced expression of miR-34a contributes significantly to lung and liver metastasis and chemoresistance through the inhibition of apoptosis and Wnt/ β-Catenin signaling pathways in bladder cancer. Accumulating evidence shows that the induction of apoptosis and activation of Wnt/ β-Catenin signaling pathways dramatically suppress tumor growth, metastasis, and chemoresistance, confirming that miR-34a can regulate apoptosis through Wnt/β-catenin signaling pathways. Indeed, some studies have revealed that miR-34a alters apoptosis via direct BCL2 targeting [33, 34]. Our findings clearly indicate that the overexpression of miR-34a can significantly enhance EPI-induced apoptosis and Wnt/β-Catenin signaling pathway inhibition. Furthermore, bladder cancer cell metastasis is dramatically inhibited in vitro and in vivo, indicating that restoration of miR-34a may be a useful strategy for the treatment of metastasis and chemoresistance in bladder cancer patients.

TCF1 and LEF1 are two important interactions of activated Wnt/β-catenin in nuclei. Activation of the canonical Wnt/β-catenin signaling pathway is frequently involved in the development of tumors, include colorectal cancer, malignant melanoma, and several other tumor types [35]. Indeed, our study confirmed the association between miR-34a and TCF1/LEF1 in the treatment of bladder cancer by qRT-PCR and western blot analysis. Moreover, the dual-luciferase reporter gene experiments conducted in this study show that miR-34a directly targets the 3′UTR of TCF1 and LEF1.

In summary, this study combined clinical and experimental research to determine the role and clinical significance of miR-34a in the progression of bladder cancer. The miR-34a-mediated regulation of TCF1/LEF1 is involved in bladder cancer metastasis and chemoresistance; however, the restoration of miR-34a and inhibition of TCF1/LEF1 was found to reduce the properties and chemoresistance of bladder cancer cells both in vivo and in vitro. In addition, miR-34a is one of the master tumor suppressor miRNAs, and the miR-34a/TCF1/LEF1 axis plays a crucial role in the survival of bladder cancer cells. Thus, modulation of the miR-34a and TCF1/LEF1 axis may serve as a novel therapeutic strategy for drug-resistant BC by increasing chemosensitivity and shifting BC therapy from conventional methods to targeted therapies.

LL, JX, and ZY conceived and designed the experiments; XL, LL, and YW performed the experiments; LL, ZF, QW, YH, and XY analyzed the data; QW and XH contributed reagents, materials, and analysis tools; and LL and XL wrote the paper.

This work was supported partly by the National Natural Science Foundation of China (81470989) and partly by key clinical research projects of the Second Affiliated Hospital, Third Military Medical University (2014YLC28 and 2015YLC06). We thank Dr. Qingjian Wu for constructive comments and editing the manuscript. We are grateful to the authors and publications that were not cited in this paper due to space restrictions.

The authors declare to have no conflict of interests.