LncRNA HEIH/miR-4500/IGF2BP1/c-Myc Feedback Loop Accelerates Bladder Cancer Cell Growth and Stemness

INTRODUCTION

Bladder cancer (BCa) is the most common malignant urinary tumor [1]. The incidence of BCa has gradually increased in recent years [2]. Although great improvements have been made in therapeutic strategies, including surgery, chemotherapy and immunotherapy, the prognosis of patients with BCa is still unsatisfactory [3]. A great number of researches have deepened our understanding of the pathogenesis of BCa, but the molecular mechanisms underlying this malignant disease remain poorly understood [4]. Hence, it is of great necessity to explore the molecular mechanism governing BCa and explore potential targets for BCa treatment.

Cancer stem cells (CSCs), also named as tumor-initiating cells, are a class of tumor cells with self-renewal and limitless replication capacities so as to induce the carcinogenesis of multiple cancers, including BCa [5, 6]. Thus, assessment of the molecular mechanisms underlying CSCs is required.

With advanced RNA sequencing techniques, over 90% of DNA sequences can be transcribed into RNAs, but only less than 2% of these transcripts have protein-encoding capacity, while the majority of transcripts are noncoding RNAs (ncRNAs) [7]. LncRNAs are a class of RNA molecules which possess a length of exceeding 200 nucleotides [8]. LncRNAs involve in the progression of cancers by regulating gene expression at epigenetic, transcriptional and post-transcriptional levels [9]. The mushroom growth of RNA genomics has emphasized the potential of lncRNAs in a variety of human cancers, and BCa is no exception [10]. Remarkably, lncRNAs serve as oncogenes or tumor repressors to regulate BCa cell proliferation, migration and invasion [11, 12].

Previous studies have suggested that HEIH participates in the progression of various cancers [13, 14]. For instance, HEIH functions as an oncogene in breast cancer [15]. HEIH accelerates cell migration and invasion in cholangiocarcinoma via regulating the miR-98-5p/HECTD4 axis [16]. HEIH contributes to ovarian cancer cell proliferation by regulating the miR-3619-5p/CTTNBP2 axis [17]. Nonetheless, the role of HEIH in BCa remains largely obscure.

According to GEPIA analysis, HEIH was markedly over-expressed in BCa tissues versus normal tissues. Given that, we wondered whether up-regulation of HEIH is implicated in BCa development. At the same time, the molecular mechanism by which HEIH elicits its function in BCa cells was also investigated in this study.

MATERIALS AND METHODS

RESULTS

Expression and function of HEIH in BCa

To identify potential lncRNAs associated with the pathogenesis of BCa, we firstly adopted the GEPIA analysis and discovered that lncRNA HEIH was highly expressed in BCa tissue samples versus normal tissues (P < 0.05; Fig. 1A). Then, we also detected relatively higher expression of HEIH in BCa cell lines compared with human normal bladder epithelial cell line SV-HUC-1 (Fig. 1B). To discuss the effect of HEIH on BCa tumorigenesis, T24 and J82 cells which exhibited the highest expression of HEIH were transfected with shRNAs against HEIH (sh-HEIH#1/2; Fig. 1C). The results followed by colony formation and EdU experiments showed that HEIH deficiency significantly inhibited BCa cell proliferation (Fig. 1D-1E). Besides, flow cytometry analysis and JC-1 assay demonstrated that the percentage of apoptotic cells was increased by HEIH depletion (Fig. 1F-1G). By performing transwell assays, we examined the influence of HEIH silence on the migratory and invasive capacities of BCa cells. The number of migrated and invaded cells in HEIH-silenced group was lower than the control group (Fig. 1H-1I). Collectively, these data showed the upregulation of HEIH expression in BCa cell lines and manifested that the deficiency of HEIH hampers cell proliferation, migration and invasion but promotes cell apoptosis in BCa.

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Fig. 1

HEIH silence inhibits BCa cell proliferation, migration and invasion. (A) HEIH expression in BCa tissue samples (n = 411) and normal samples (n = 19) from TCGA database (P < 0.05). (B) HEIH expression in BCa cell lines was examined by RT-qPCR. (C) The interference efficiency of sh-HEIH was detected by RT-qPCR. (D-E) Colony formation and EdU experiments were performed to test BCa cell proliferation after HEIH silence. (F-G) Flow cytometry analysis and JC-1 experiment were conducted to estimate BCa cell apoptosis after HEIH knockdown. (H-I) Transwell migration and invasion assays were carried out to evaluate BCa cell migration and invasion after the ablation of HEIH. ^*P < 0.05, ^**P < 0.01.

HEIH contributes to BCa cell stemness via up-regulating c-Myc

Since CSC properties exert important roles in the progression of human cancers [5], we assessed the effects of HEIH on CSC characteristics. Sphere formation assay illustrated HEIH inhibition reduced the size of sphere and sphere formation efficiency (Fig. 2A). It is well known that Nanog, OCT4, KLF4, SOX2 and c-Myc are major regulators involved in cancer cell stemness. Next, we investigated whether HEIH contributes to BCa cell stemness by regulating some of these CSC-associated genes. Through western blot, we found that HEIH knockdown only decreased the protein level of c-Myc, while barely affected the level of other proteins (Fig. 2B). RT-qPCR analysis further validated that HEIH positively regulates c-Myc expression in terms of mRNA level (Fig. 2C). Further, the analysis of GEPIA database displayed the correlation between Myc and HEIH in bladder urothelial carcinoma (BLCA) (Fig. 2D). Collectively, HEIH facilitates BCa cell stemness via up-regulating c-Myc.

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Fig. 2

HEIH contributes to BCa cell stemness via up-regulating c-Myc. (A) Sphere formation assay was performed to evaluate cell stemness in HEIH-silenced BCa cells. (B) Western blot was used to analyze the protein levels of Nanog, OCT4, KLF4, SOX2 and c-Myc in HEIH-inhibited BCa cells. (C) RT-qPCR was used to measure c-Myc mRNA level in HEIH-silenced BCa cells. (D) The correlation of HEIH and MYC expression in BLCA based on GEPIA analysis. ^**P < 0.01.

HEIH functions as a miR-4500 sponge in BCa cells

In addition to its biological role, we explored the regulatory mechanism of HEIH in BCa. Firstly, the distribution of HEIH was determined by FISH and nucleus/cytoplasm fractionation assays. The analysis of subcellular localization revealed that HEIH was mainly located in the cytoplasm instead of the nucleus of BCa cells (Fig. 3A-3B). Given that cytoplasmic lncRNAs affect post-transcriptional regulations such as mRNA stability and translational regulation, largely through the competing endogenous RNA (ceRNA) regulation mechanism of adsorbed miRNA [18], HEIH was likely to have the potential of post-transcriptional regulation in BCa. Next, we performed Ago2-RIP assay to verify this hypothesis since the RNA-binding protein (RBP) Ago2 is a key component of the RNA-induced silencing complex (RISC) and exerts vital functions in global alterations of miRNA levels and activity in the cytoplasm [19]. Ago2-RIP assay indicated that HEIH was highly enriched in Ago2-containing beads (Fig. 3C), verifying the potential of HEIH in ceRNA regulatory network. Thus, we conjectured that HEIH might regulate c-Myc via sponging some specific miRNAs. However, no mutual miRNA was observed to combine with HEIH and c-Myc (Fig. 3D). Hence, the starBase (https://starbase.sysu.edu.cn) database analysis was carried out to predict HEIH-interacting miRNAs. We discovered 13 miRNAs containing the putative binding sites with HEIH under the threshold of CLIP Data > = 3. To certify the binding between candidate miRNAs and HEIH, RNA pull down assays were implemented and showed that only miR-4500 was pulled down by biotinylated HEIH (Bio-HEIH) probe among candidate miRNAs (Fig. 3E). RT-qPCR analysis further demonstrated that miR-4500 was low-expressed in BCa cell lines compared with SV-HUC-1 cell line (Fig. 3F). To explore the function of miR-4500 in BCa progression, we overexpressed miR-4500 in T24 and J82 cells (Fig. S1A), and found that enforced expression of miR-4500 impeded cell proliferation, migration, invasion and stemness while promoted cell apoptosis in BCa (Fig. S1B-S1H). Moreover, we found that HEIH was pulled down by biotin-labeled miR-4500 wild-type (Bio-miR-4500-WT) instead of mutant-type (Bio-miR-4500-Mut; Fig. 3G). The binding sites of HEIH and miR-4500 were disclosed in Fig. 3H, and luciferase reporter assays further showed that the luciferase activity of HEIH wild-type (HEIH-WT) was repressed by miR-4500 mimics, whereas that of HEIH mutant-type (HEIH-Mut) was barely influenced (Fig. 3I). Altogether, HEIH acts as a miR-4500 sponge in BCa cells, and miR-4500 plays a suppressive role in BCa.

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Fig. 3

HEIH sponges miR-4500 in BCa cells. (A-B) Cellular position of HEIH was certified by FISH and subcellular fractionation assays. (C) RIP experiment detected the existence of HEIH in Ago2-RISC. (D) Venn diagram illustrated no shared miRNAs bound to HEIH and c-Myc. (E) RNA pull down assay detected the combination between HEIH and candidate miRNAs. (F) Expression of miR-4500 in BCa cells was detected via RT-qPCR. (G) RNA pull down experiment tested the abundance of HEIH in biotinylated miR-4500-WT or miR-4500-Mut. (H) The binding sites of miR-4500 and HEIH. (I) Luciferase activity of HEIH-WT and HEIH-Mut in BCa cells after miR-4500 overexpression was detected. ^*P < 0.05, ^**P < 0.01.

HEIH sponges miR-4500 to regulate IGF2BP1

With the help of bioinformatics, 9 mRNAs were predicted to be targeted by miR-4500 (Fig. 4A). Through RT-qPCR analysis, we found that only IGF2BP1 was markedly down-regulated in miR-4500-overexpressed BCa cells (Fig. 4B). Besides, we found that IGF2BP1 was expressed at a higher level in BCa cell lines versus SV-HUC-1 cell line (Fig. 4C). RIP assays showed that HEIH, miR-4500 and IGF2BP1 were abundant in Ago2 precipitates (Fig. 4D), further validating the interaction of these three RNAs. Besides, RNA pull down assays uncovered that IGF2BP1 was preferentially abundant in Bio-miR-4500-WT group while Bio-miR-4500-Mut group had no marked change versus control (Fig. 4E). Moreover, we discovered the binding sites of IGF2BP1 3’UTR and miR-4500 (Fig. 4F). Luciferase reporter assay indicated that miR-4500 elevation markedly declined the luciferase activities of IGF2BP1 3’UTR-WT while IGF2BP1 3’UTR-Mut had no marked change in luciferase activities (Fig. 4G). In addition, we found that HEIH deficiency obviously reduced IGF2BP1 mRNA and protein levels while this was abrogated after simultaneous miR-4500 inhibition (Fig. 4H). Meanwhile, we found that IGF2BP1 mRNA and c-Myc protein levels were increased by miR-4500 inhibition alone (Fig. 4H). Taken together, HEIH sponges miR-4500 to upregulate IGF2BP1 expression in BCa cells.

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Fig. 4

HEIH sponges miR-4500 to regulate IGF2BP1. (A) Venn diagram showed 9 putative target mRNAs of miR-4500 predicted by microT and RNA22 algorithms. (B) RT-qPCR was used to analyze the expression of 9 mRNAs in miR-4500-overexpressed BCa cells. (C) RT-qPCR and western blot were applied to measure IGF2BP1 expression in BCa cell lines and SV-HUC-1 cell line. (D) RIP assays were performed to detect the enrichment of HEIH, miR-4500 and IGF2BP1 in Ago2-bound precipitates. (E) RNA pull down assays were conducted to detect the abundance of IGF2BP1 in Bio-miR-4500-WT group and Bio-miR-4500-Mut group. (F) Binding sites of IGF2BP1 3’UTR and miR-4500 and mutated sequence of IGF2BP1 3’UTR-Mut. (G) Luciferase activity of IGF2BP1 3’UTR-WT and IGF2BP1 3’UTR-Mut was detected in BCa cells co-transfected with miR-4500 mimics. (H) IGF2BP1 mRNA and protein levels in BCa cells transfected with sh-NC, sh-HEIH#1, sh-HEIH#1 + miR-4500 inhibitor, miR-4500 inhibitor or NC inhibitor using RT-qPCR and western blot. ^*P < 0.05, ^**P < 0.01.

HEIH regulates c-Myc expression via targeting the miR-4500/IGF2BP1 axis

Previous literatures have indicated that IGF2BP1 can bind to and stabilize c-Myc mRNA [20]. Thus, we hypothesized that HEIH might regulate c-Myc expression via targeting the miR-4500/IGF2BP1 axis. Herein, we first detected the binding between IGF2BP1 and c-Myc in BCa (Fig. 5A). Then, we silenced IGF2BP1 expression in BCa cells (Fig. 5B), and found that knockdown of IGF2BP1 significantly repressed c-Myc expression at mRNA and protein levels (Fig. 5C). Moreover, after treatment with Actinomycin D, the stability of c-Myc mRNA was restrained when HEIH or IGF2BP1 was down-regulated (Fig. 5D). In addition, to determine whether HEIH promotes BCa cell growth and stemness via regulating c-Myc, we overexpressed c-Myc expression in BCa cells (Fig. S2A), and then severally transfected sh-NC, sh-HEIH#1, sh-HEIH#1 + pcDNA3.1/c-Myc into cells to perform rescue experiments. It was found that overexpression of c-Myc offset the inhibited proliferation of HEIH-silenced cells (Fig. S2B-S2C). Besides, the enhanced apoptosis induced by HEIH depletion was countervailed by c-Myc overexpression (Fig. S2D-2E). Moreover, we observed that the reduced migration, invasion and sphere-forming ability caused by HEIH deficiency were counteracted by synchronous c-Myc elevation (Fig. S2F-S2H). To sum up, HEIH regulates c-Myc expression via targeting the miR-4500/IGF2BP1 axis.

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Fig. 5

HEIH regulates c-Myc expression via targeting the miR-4500/IGF2BP1 axis. (A) ChIP assays were performed to detect the combination between c-Myc and IGF2BP1 in BCa cells. (B) Silencing efficiency of IGF2BP1 in BCa cells was verified by RT-qPCR and western blot. (C) RT-qPCR and western blot were used to detect c-Myc mRNA and protein levels in IGF2BP1-silenced BCa cells. (D) Stability of c-Myc mRNA was detected in IGF2BP1-silenced or HEIH-depleted BCa cells after Actinomycin D treatment. ^**P < 0.01.

c-Myc transcriptionally activates HEIH expression

We further explored the upstream mechanism of HEIH in BCa cells. With the help of UCSC (https://genome.ucsc.edu/) and JASPAR (https://jaspar.genereg.net/) databases, we discovered that c-Myc was a transcription factor that binds to the promoter region of HEIH (Fig. 6A-6B). Subsequently, c-Myc was knocked down in BCa cells and the knockdown efficiency of sh-c-Myc was verified by RT-qPCR and western blot analyses (Fig. 6C). We found that HEIH expression was restrained in c-Myc-depleted BCa cells (Fig. 6D). Further, ChIP assay showed that HEIH promoter was highly expressed in the complexes precipitated by anti-c-Myc versus control anti-IgG (Fig. 6E), suggesting the direct binding of c-Myc and HEIH promoter. Similarly, luciferase reporter assay showed that c-Myc suppression significantly weakened the luciferase activity of HEIH promoter-WT instead of that of HEIH promoter-Mut (Fig. 6F). In conclusion, c-Myc activates HEIH expression at the transcriptional level.

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Fig. 6

c-Myc transcriptionally activates HEIH expression. (A-B) UCSC and JASPAR databases predicted the c-Myc-binding sites (–171∼–160) in the sequence of HEIH promoter region. (C) Silencing efficiency of c-Myc in BCa cells was validated using RT-qPCR and western blot. (D) HEIH expression in c-Myc-depleted BCa cells was detected by RT-qPCR. (E) Combination between HEIH promoter and c-Myc in BCa cells was examined by ChIP assay. DNA bands were visualized using agarose gel electrophoresis (left panel). Quantification of HEIH promoter by RT-qPCR after ChIP was shown (right panel). (F) Luciferase activity of HEIH promoter-WT and HEIH promoter-Mut was detected in BCa cells after c-Myc deficiency. ^**P < 0.01.

DISCUSSION

Our study, for the first time, demonstrated that HEIH was highly expressed in BCa cell lines and clarified its role in BCa. Functionally, depleted HEIH inhibits BCa cell proliferation, migration, invasion while elevates cell apoptosis, suggesting that HEIH might exert an oncogenic role in BCa progression. This is in line with findings reported in previous studies that HEIH functions as a tumor facilitator in a variety of human cancers. For example, HEIH enhances cell proliferation and suppresses apoptosis in triple-negative breast cancer [21]. HEIH promotes liver cancer cell growth and metastasis [22]. HEIH facilitates the tumorigenesis of colorectal cancer [23].

Our study also revealed that HEIH induces BCa cell stemness via up-regulating c-Myc. CSCs play a vital role in the progression of malignant tumors, including BCa. Notably, c-Myc is one of the most frequently activated oncogenes in the development of human cancers, including BCa [24–26]. Besides, c-Myc has been considered as a crucial modulator of stem cell biology [27]. Mounting evidences have shown that c-Myc can regulate the stemness of various cancer cells [28, 29]. Thus, targeting c-Myc may be a promising strategy for cancer therapy.

A further finding of our study was that HEIH acts as a ceRNA of miR-4500 in BCa cells. This ceRNA mechanism was in line with previous studies on HEIH regulation in cancers. For instance, HEIH has been reported to suppress hepatocellular carcinoma cell growth by interacting with miR-199a-3p [30]. Lei Jiang et al. have pointed that HEIH contributes to gastric carcinoma cell proliferation, migration and invasion by targeting miR-214-3p [31]. Moreover, miR-4500 has been reported to exert a suppressive function in multiple human cancers. MiR-4500 hinders papillary thyroid cancer progression via modulating PLXNC1 [32]. MiR-4500 represses non-small cell lung cancer progression by affecting STAT3 [33]. MiR-4500 impedes breast cancer cell migration and invasion via targeting RRM2 [34]. Consistently, miR-4500 was proved to be poorly expressed in BCa cells, and we found that overexpression of miR-4500 inhibits BCa progression. Moreover, our study demonstrated that HEIH sponges miR-4500 to up-regulate IGF2BP1 expression.

IGF2BP1 as a RBP has been reported to regulate the stability of mRNAs, such as PEG10 [35], ELF3 [36] and SOX2 [37]. Of note, IGF2BP1 is well-known to maintain the stability of c-Myc mRNA [38]. Herein, our study further uncovered that HEIH up-regulates c-Myc via targeting the miR-4500/IGF2BP1 axis in BCa cells. Additionally, mounting studies have identified that c-Myc can activate multiple lncRNAs, such as NEAT1 [39], LINC01123 [40] and FGF13-AS1 [41] via transcriptional regulation. Herein, c-Myc was verified to act as a transcriptional factor to activate HEIH expression in BCa.

Moreover, the current study validated the promoting effect of HEIH on the tumorgenesis and metastasis of BCa through in vivo experiments. However, this study lacked the clinicopathological analysis. In the further exploration, we will investigate the correlation between HEIH expression and clinicopathological features of BCa patients.

In conclusion, our study revealed that HEIH, highly expressed in BCa cell lines, functions as an oncogene by facilitating cell proliferation, migration and invasion as well as stemness. Our findings demonstrated that HEIH expression and c-Myc activation forms a positive feedback loop to control BCa progression by targeting miR-4500 to up-regulate IGF2BP1 expression, which might offer novel insight into BCa treatment.