LncRNA MIR497HG suppresses bladder cancer progression through disrupting the crosstalk between Hippo/Yap and TGF-β/Smad signaling


 Objectives

A subclass of long non-coding RNAs (lncRNAs), categorized as miRNA-host gene lncRNAs (lnc-miRHGs), is processed to produce miRNAs and involve in cancer progression. This work aimed to investigate the influence and the molecular mechanisms of lnc-miRHGs MIR497HG in bladder cancer (BCa).
Materials and methods

The miR-497 and miR-195 were derived from MIR497HG. Cell proliferation, migration and invasion assays were used to measure the function of MIR497HG, miR-497 and miR-195 in BCa. Bioinformatics, RT-qPCR, western blot, luciferase reporter assay, ChIP, and so on, were used to reveal the upstream and downstream mechanisms of MIR497HG in BCa.
Results

We identified that lnc-miRHG MIR497HG and two harbored miRNAs, miR-497 and miR-195, were downregulated in BCa by analyzing TCGA and our dataset. MIR497HG overexpression inhibited BCa cell proliferation, migration and invasion in vitro. MiR-497/miR-195 inhibitor rescued significantly the inhibiton effects of overexpression of MIR497HG in BCa. Mechanistically, miR-497 and miR-195 coordinately suppressed multiple key components in Hippo/Yap and TGF-β signaling, and particularly attenuated the interaction between Yap and Smad3. In addition, E2F4 was proved to be critical for silencing MIR497HG transcription in BCa cells.
Conclusions

We propose for the first time that MIR497HG suppressed BCa progression and its upstream and downstream mechanisms. Blocking the pathological process may be a potential strategy for the treatment of BCa.


Introduction
Bladder cancer (BCa) is one of the most prevalent epithelial malignancy worldwide [1]. Most of diagnosed patients have non-muscle-invasive bladder cancer (NMIBC) con ned to the mucosa or lamina propria and approximately 25% have muscle-invasive (MIBC) that invade the detrusor muscle [2,3]. MIBC is more aggressive and have a worse prognosis [2,4]. Existing therapies for MIBC have not changed mortality rates over the past years [4]. Genome and transcriptome pro ling studies have revealed considerable differences in the molecular and genetic features of bladder cancer cells, such as mutations, copy number, and gene epigenetic alterations, determining tumor heterogeneity and therapeutic resistance [2,5,6]. However, non-genetic or epigenetic mechanisms in BCa remain elusive.
Furthermore, miR-195 and miR-497 expression are repressed and they function as tumor suppressors in various types of human cancer including breast cancer [17], lung cancer [18], colorectal cancer [19] and hepatocellular carcinoma [20]. However, the impact of these lncRNAs or derived miRNAs in BCa progression are largely unknown.
Many signaling pathways such as JAK-STAT, NF-KB, mTOR and MAPK have been reported to affect the survival of BCa [21]. Evolutionarily conserved Hippo pathway has been shown to paly crtical roles during BCa progression and tumorigenesis [22]. The Hippo pathway regulates cell proliferation and migration via the transcriptional coactivator Yes-associated protein (YAP) [23]. YAP is highly expressed and acts as oncogenes in BCa [24,25]. Transforming growth factor beta (TGF-β) complex, a serine/threonine kinase complex, binds to TGF-β receptors and further activates different downstream substrates and regulatory proteins, mainly the SMAD, inducing transcriptions of different target genes involving in cell proliferation, differentiation, and so on [26]. Besides, activation of the TGF-β/Smad signaling pathway often correlates with the malignancy of lots of human cancer, including BCa [27].
Here we report that MIR497HG plays anti-tumorigenic roles in BCa. We found that miR-497, miR-195 and their host gene MIR497HG were down-expressed in BCa tissues and cell lines. MiR-497 and miR-195 synergistically targeted YAP and SMAD3 and their downstream genes, and decreased the formation of a YAP-SMAD complex [28]. We also identi ed that E2F4 is a critical transcriptional suppressor of MIR497HG. Our ndings uncover an ncRNAs-mediated epigenetic mechanism to block the crosstalk between Hippo/Yap and TGFβ/Smad signaling. It may contribute to search for effective personalized targeted therapies to treat BCa.

BCa specimens
All BCa samples were collected from the Peking University Shenzhen Hospital and Shenzhen University Nanshan Hospital. The study was approved by the Ethics Committee of Shenzhen University Nanshan Hospital with written informed consent obtained from all patients. The pathological status of the specimens was provided by the board-certi ed pathologist.

Western blot
Protein extracts were prepared in RIPA Lysis Buffer (#P0013B; Beyotime), supplemented with PMSF, protease inhibitor and phosphatase inhibitor cocktail. Protein concentrations were determinated using Bicinchoninic Acid Kit (Sigma-Aldrich) according to the manufacturer's protocol. Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 hour with 5% non-fat milk in TBST (Trisbuffered saline containing 0.1% Tween 20) and incubated overnight at 4 °C with primary antibodies and required secondary antibodies conjugated to horseradish peroxidase and developed by chemiluminescent substrates.

RT-qPCR and ChIP
Total RNA was extracted using TRIzol™ Reagent (Invitrogen) and puri ed using RNeasy Mini Columns (Qiagen), according to the manufacturer's protocol. For mRNA and lncRNA MIR497HG detection, cDNA was generated using SureScript™ First-Strand cDNA Synthesis Kit (genecopoeia). Quantitative PCR was then performed using the SYBR Green qPCR MasterMix (Takara). To analyze miR-195 and miR-497 expression, cDNA was synthesized by a mir-X miRNA First-Strand Synthesis Kit (Takara,Dalian, China). MiRNA expression were used mir-X miRNA qRT-PCR SYBR Kit (TOYOBO, Osaka, Japan) according to the manufacturer's instructions, with U6 as the control.

Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) assays were performed according to manufacturer's instructions using Cell Signaling Immunoprecipitation Kit (#9002). Brie y, 5637 cells were xed paraformaldehyde and lysed in Buffer A supplemented with DTT, Protease Inhibitor Cocktail (PIC), and PMSF, and incubated on ice for 10 min. Nuclei were pelleted and resuspended in Buffer B + DTT. DNA was digested with 0.5 ul Micrococcal Nuclease. Nuclei were again pelleted, resuspended in ChIP buffer + PIC and PMSF, and incubated at 4 °C. The lysates were clari ed by centrifugation and ChIP was carried out by incubating the sample with Rabbit IgG or E2F4 antibody (Abcam), followed by immobilization on protein A/G-agarose beads (Life Technologies). The chromatin was eluted from Antibody/Protein G Agarose Beads, crosslinks were reversed, and DNA was puri ed using spin columns. The qPCR was performed using MIR497HG promoter or GAPDH primers. Ct values were normalized to input DNA. The detailed primer sequences for RT-qPCR and ChIP assays are listed in supplementary Table S1. 2.6 Cell Counting Kit-8 (CCK-8) assay Cells were cultured in 96-well plates at a concentration of 3 × 10 3 cells per well. After treatment, 10 µl CCK-8 reagent (Dojindo, Kumamoto, Kyushu, Japan) was added to each well to react for 0.5 h. The absorbance was measured at 450 nm using a microplate reader.

Colony formation assay
1000 cells/well were plated onto six-well plates, which were incubated at 37℃ and 5% CO2 until colonies were formed. After 10-15 days, colonies were xed using 0.05% crystal violet in 4% paraformaldehyde and counted using Image J program.

Cell migration assay
Cells were seeded in six-well plate (5 × 10 5 /per well) and incubated at 37 °C in a humidi ed incubator containing 5% CO2 to get 100% con uence before transfection. A clear line was created by scratching with a sterile 200 µl pipette tip. Images were taken from each well quickly. After 24 hours, pictures were taken again with the help of a digital camera system. Migration distance was calculated at the time of 0 and 24 h. Experiments were performed at least three times.

Cell invasion assay
Cells were transfected with siRNAs/plasmids for 48 h and then trypsinized and resuspended in serumfree medium. A total of 1 × 10 5 cells were then added to the upper chambers of the Transwell inserts (Millicell; Merck KGaA) and allowed to migrate toward the bottom of the chambers. After 24 h, the remaining cells in the upper chamber were removed, and cells on the underside were xed in 4% paraformaldehyde for 30 min at room temperature, stained with 0.1% crystal violet for 30 min at room temperature and captured using an Olympus type light microscope sz30. Quanti cation of the migrated cells was performed by counting cell numbers.
To establish stable cell lines, the concentrated lentivirus were directly added into cancer cells and incubated at 37 °C for 48 hours before they were washed out PBS. Finally, cells were selected with 2.5 mg/mL puromycin for 4 days. For luciferase reporter assay, the 3' UTR fragments of YAP1, SMAD3,CCND1 and BIRC5 containing the wild-type or mutant miR-497 ~ 195 cluster putative target sites were directly synthesized from GeneCreate (Wuhan, China), and cloned downstream of the Renilla luciferase cassette in psiCHECK-2 (Promega);The CTGF-luc plasmids were generated as described previously [29]. The promoter fragments of human MIR497HG were directly synthesized from GeneCreate (Wuhan, China), and cloned into pGL3-Basic vector (Promega). A site-directed mutagenesis kit (Thermo Fisher Scienti c) was used to mutate the miR-497 ~ 195 cluster or E2F4 binding sites of these vectors.
The primer sequences used for the site-directed mutagenesis are provided in supplementary Table S1. All sequences were con rmed by sequencing.
The miRNA inhibitors and mimics of miR-ctrl, miR-195 and miR-497 were obtained from Ribobio (Guangzhou, China). The indicated cancer cells were transfected with 50 nM of mimic or inhibitor of miRctrl, miR-195 and miR-497 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The expression levels of miR-195 and miR-497 were quanti ed after 48 h transfection.

Luciferase reporter assay
For 3' UTR luciferase reporter assays, miR-497 or miR-195 mimic or control mimic (Ambion) and indicated psiCHECK-2-3' UTR wild-type or mutant plasmids were co-transfected into T24 or 5637 cells cultured in 24-well plates using lipofectamine 3000 (Thermo Fisher Scienti c). Renilla and re y luciferase activities were measured with the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's manual.
For luciferase reporter assay, in order to measure promoter activities, the MIR497HG promoter fragment was inserted into pGL3-basic plasmid, named pGL3-MIR497HG. Then pcDNA3.1-E2F4 expression plasmid or empty vector control and pGL3-MIR497HG or pGL3-MIR497HG-mut were co-transfected into T24 or 5637 cells cultured in 24-well plates using lipofectamine 3000 (Thermo Fisher Scienti c). The re y and Renilla luciferase activity was measured after 24-48 h with the dual-luciferase reporter assay system (Promega). Fire y luciferase activity was normalized to Renilla activity.

Statistical analysis
Statistical analysis was performed by the SPSS 20 (SPSS Inc., Chicago, IL, USA). Two-tailed unpaired or paired Student's t test, ANOVA (Dunnett's or LSD post hoc test) and Pearson correlation coe cients were used according to the type of experiment. Kaplan-Meier method was used to estimate survival curves, which were compared using the Log-rank (Mantel-Cox) test. The statistical signi cance between data sets was expressed as P values, and P < 0.05 was considered signi cant; * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

MIR497HG and MIR497HG-derived miR-497 and miR-195 were downregulated in BCa
MIR497HG is the host gene of the miR-497 ~ 195 cluster on chromosome 17 (Fig. 1A). We rst examined the expression of MIR497HG, miR-497 and miR-195. The qRT-PCR analysis con rmed the downregulation of endogenous MIR497HG, miR-497 and miR-195 expression in BCa tissues compared with normal adjacent tissues (Fig. 1B-1D). Furthermore, we analyzed The Cancer Genome Atlas (TCGA) bladder urothelial carcinoma (BLCA) RNA sequencing data and revealed that the expression levels of miR-497 and miR-195 were inhibited in various stage of BCa ( Fig. 1E and 1F); and these data also showed that MIR497HG was downregulated in BCa (Fig. 1G). Similarly, the expression levels of MIR497HG, miR-195 and miR-497 were repressed in different BCa cell lines including 5637, T24, UMUC-3, SW780, and TCCSUP. Thus, MIR497HG, miR-497 and miR-195 were downregulated in both human BCa tissues and cell lines (Fig. 1H). These data suggest that MIR497HG, miR-497 and miR-195 may involve in the progression of BCa.

MIR47HG suppressed BCa cell growth, migration and invasion in vitro
To explore the biological functions of MIR497HG, MIR497HG was overexpressed after transfection with MIR497HG overexpression plasmid. Cell Counting Kit-8 assays and colony formation assays showed that MIR497HG overexpression signi cantly inhibited cell proliferation in both 5637 and T24 cells ( Fig, 2A-2C). BCa cells transfected with MIR497HG presented signi cantly reduced scratch wound healing (Fig. 2D-2E) and lower invasion abilities (Fig. 2F, G) compared with vector control.

MiR-497 and miR-195 mediate MIR497HG-induced inhibition of BCa progression
Inspiring that a major role of miRNA-host gene lncRNAs (lnc-miRHGs) is depend on their derived miRNAs [12,13,30,31], we test whether the phenotypes associated with MIR497HG are mediated by miR-497 and miR-195 in BCa cell lines. miR-497 and miR-195 inhibitor were transiently transfected into MIR497HG overexpressing 5637 and T24 cell lines. As shown in Fig. 3, miR-497 or miR-195 inhibitor partially reversed the inhibition of cell proliferation (Fig. 3A, B), migration (Fig. 3C, D), and invasion (Fig. 3E, F) induced by MIR497HG overexpression. Collectively, these data suggested that miR-497 and miR-195 are indispensable in the biological function of MIR497HG in BCa.

MiR-497 and miR-195 directly suppressed multiple key components in Hippo/Yap and TGF-β signaling
To explore the molecular pathways of miR-195 and miR-497 in BCa cells, we performed in silico analyses using mirPath v.3 and TargetScan. Based on the KEGG pathway enrichment analysis, 14 pathways were enriched in miR-195/497 cluster putative targets ( Fig. 4A and supplementary Table S2). Then, we focused on genes predicted as miR-195/497 cluster targets and involved in hippo signaling and TGF-β pathway.

MiR-497 and miR-195 coordinately attenuated Yap and Smad3 dependent transcriptional activity
Previous studies suggest that YAP-Smad3 interaction is essential for the crosstalk between hippo signaling and TGF-β pathway [34][35][36]. To examine whether miR-497 ~ 195 clusters affect the formation of YAP-Smad complex, we preformed immunoprecipitations to detect YAP-Smad3 interaction in 5637 cell lines. Our results showed that the interaction between YAP and Smad3 was inhibited/enhanced via miR-497 or miR-195 mimic/inhibitor treatment, respectively (Fig. 5A-5B). Interaction between Yap and Smad3 is required to positively regulate transcriptional activity of their common downstream genes including connective tissue growth factor (CTGF), a critically oncogenic target [34]. Next, CTGF luciferase assays showed that miR-497 ~ 195 clusters blocked CTGF promoter transcriptional activity mediated by Yap and Smad3 (Fig. 5C). CTGF protein expression was decreased with miR-497 or miR-195 overexpression (Fig. 5D). These data suggest that miR-497 and miR-195 synergistically repress Yap and Smad3 mediated transcriptional activity of CTGF.

E2F4 transcriptionally repressed MIR497HG expression in BCa cells
To determine the mechanism of downregulation of miR-497 ~ 195 clusters in BCa, we analyzed the host gene MIR497HG promoter sequence using MethPrimer and JASPAR. Firstly, no CpG island exists in the promoter region of MIR497HG predicted by MethPrimer. It suggested that MIR497HG expression might not repressed by promoter methylation. Then we identi ed conserved DNA binding sites for fork head/winged helix transcription factors. Among these transcription factors, we focused on E2F transcription factor 4 (E2F4), which was negatively correlated with MIR497HG expression (Fig. 6A) and had relatively high scores (supplementary Table S3). Therefore, we individually knocked down E2F4 or E2F6 (another high score transcription factor) in 5637 cell line using siRNA and analyzed the effect of these knockdowns on MIR497HG expression. Silencing of E2F4 signi cantly promoted MIR497HG expression (Fig. 6B), except knock down of E2F6 (supplementary Fig. S2). It inspired us to determine whether the transcription factor E2F4 directly targets MIR497HG. Thus, we rst utilized the Ensembl and JASPAR to identify forkhead/winged helix motif (GGCGGGAA) in the MIR497HG 1.5 kb promoter region and found four potential binding sites ( Fig. 6C and 6D). Next, real-time PCR after ChIP con rmed that E2F4 was signi cantly enriched at the MIR497HG promoter (Fig. 6E). We further con rmed that E2F4 directly regulates MIR497HG expression using luciferase reporter assays. E2F4 overexpression signi cantly decreased the activity of the MIR497HG promoter (Fig. 6F). Sequential mutations and deletions of four binding sites revealed that forkhead/winged helix-binding site "-189~-179" was the major site for E2F4 repressing MIR100HG transcriptional activity (Fig. 6G-I). Altogether, these data clearly demonstrate that E2F4 inhibited MIR497HG transcription by directly binding to its promoter in BCa cells.
The schematic diagram of mechanism of lncRNA MIR497HG was shown in Fig. 7. E2F4 bound to the promoter of MIR497HG to inhibit the expression of MIR497HG. miR-497 and miR-195 were derived from MIR497HG and suppressed the crosstalk of YAP and SMAD3 which were key components in Hippo/Yap and TGF-β/Smad signaling. Then, the downstream of YAP/SMAD3 complex, oncogenic CTGF, was restrained to curb cell proliferation of BCa.

Discussion
MIR497HG is a host gene of the miR-497 and miR-195 embedded in its rst intron. MIR497HG was signi cantly downregulated and might serve as a potential diagnostic marker in BCa [15]. MiR-495 and miR-195 were also reduced in BCa and inhibited cancer cell progression [16,37,38]. Consistent with these ndings, we con rmed that concomitant low expression of MIR497HG, miR-497 and miR-195 occurred in BCa, and that miR-497 and miR-195 coordinately play critical anti-oncogenic roles in BCa cells. In vitro, we found that overexpression of MIR497HG signi cantly inhibits the viability and proliferation of BCa cells. Mechanistically, integrated in silico analyses and luciferase reporter assays revealed that miR-195/497 cluster directly targeted YAP, SMAD3, BIRC5 and CCND1, the key components in the Hippo and TGF-β pathway. Moreover, we found that miR-195/497 cluster decreased the formation of regulatory transcription complex containing YAP and Smad3, while decreasing their common target genes, such as the gene-encoding connective tissue growth factor (CTGF) [28].
Although we focused on the targets genes in the Hippo and TGF-β pathway, it is likely that other genes or pathways are changed in bladder cancer cells as a result of miR-195/497 cluster mediated posttranscriptional gene silencing. For example, miR-195/497 cluster maintained Notch activity and HIF-1α protein expression by targeting FBXW7 in endothelial cells [39]. However, we observed no similar changes of HIF-1α and FBXW7 expression after miR-195/497 mimic treatment in BCa cells (data not shown). The reasons for the disparities remain unclear. One possibility is that miRNA perform distinct genetic programs in the different cells and microenvironments in which the cells reside.
Numerous studies points toward an important role for E2F4 with reports of transcriptional stimulatory or repressive effects of E2F4 in different cell type or tissue context [40][41][42][43]. It is possible that E2F4 serves as transcriptional activator or repressor upon binding distinct transcriptional cofactors. E2F4 drives genes transcription by interacting with acetyltransferase GCN5 and the essential cofactors TRRAP [44]. Conversely, E2F4 silence target genes relying on its interaction with the "pocket protein", such as retinoblastoma (Rb), p107, and p130 that recruit DNMTs [40,45]. Our data showed that E2F4 repress MIR497HG transcriptional activity, while inhibiting its embedded miR-497 and miR-195 expression. Further studies are needed to investigate whether a novel corepressor that interacts with E2F4 exists.
In conclusion, E2F4 suppressed the expression of MIR497HG, and MIR497HG suppresses bladder cancer progression through disrupting the crosstalk between Hippo/Yap and TGF-β/Smad signaling by reducing expression of four key genes involved in the these two pathways and YAP-Smad3 interaction. Our ndings may open up avenues for developing effective therapeutic strategies to treat BCa.

Declarations
Ethics approval and consent to participate The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the Ethics Committee of Shenzhen University Nanshan Hospital. Written informed consent was obtained from individual or guardian participants.

Consent for publication
All authors agree to publish.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
All authors declare no competing interests.       Chromatin immunoprecipitation was performed in 5637 cells with either IgG or E2F4 and subsequently subjected to qPCR analysis with the indicated primers. Student's t test, *p < 0.05 and ***p < 0.001. (F) Hif-1a promoter activity was determined in 5637 with E2F4 overexpression. One-way ANOVA with Dunnett's post-test, *p < 0.05 and **p < 0.01. (G) A schematic representation of deletion or mutation constructs spanning the −1,500 to +200 region of the MIR497HG promoter. (H-I) The luciferase vector pGL3 driven by either wild-type, deletion or mutant MIR497 promoter was transfected in 5637 cells, and luciferase activity was measured. n = 3 independent experiments. *P < 0.05, **P < 0.01 by Student's t test.

Figure 7
The schematic diagram of mechanism of lncRNA MIR497HG in BCa. E2F4 bound to the promoter of MIR497HG to act as inhibitory transcription factors to suppress the expression of MIR497HG. miR-497 and miR-195 were derived from MIR497HG and they suppressed the crosstalk of YAP and SMAD3 which were key components in Hippo/Yap and TGF-β/Smad signaling. Then, the downstream of YAP/SMAD3 complex, oncogenic CTGF, was restrained to curb cell progression of BCa.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.