A novel miR-206/hnRNPA1/PKM2 axis reshapes the Warburg effect to suppress colon cancer growth

Rong Fu a, b, 1, Peng Yang a, b, 1, Sajid Amin a, Zhuoyu Li a, c, *

Abstract

Alternative splicing of pyruvate kinase gene (PKM) results in a higher PKM2/PKM1 ratio that contributes to the Warburg effect and reversing the Warburg effect has opened novel avenues for cancer treatment. miR-206 functions as a tumor suppressor in several types of cancer. However, the effect and underlying mechanisms of miR-206 on the Warburg effect are not yet elucidated. Here, we showed that miR-206 expression was obviously decreased in CRC tissues based on LinkedOmics. A significant decrease in miR-206 expression was negatively correlated with advanced tumor stage, while inversely correlated with overall survival in CRC patients. Ectopic overexpression of miR-206 has dramatically restricted the cell proliferation, glucose consumption and lactate production in CRC cells, whereas transfection of miR206 inhibitor exhibited the opposite results. Furthermore, miR-206 overexpression induced switching from PKM2 to PKM1 via modulating alternative splicing of PKM gene. The alternative splicing factor hnRNPA1 is identified as the direct functional target of miR-206. Mechanistically, miR-206 overexpression directly targeted hnRNPA1 to suppress PKM2 expression to attenuate Warburg effect and cell proliferation of CRC. Importantly, the restoration of hnRNPA1 expression mostly abrogated the miR-206meditated Warburg effect. Collectively, these results revealed that the novel miR-206/hnRNPA1/PKM2 axis plays a pivotal role in the Warburg effect to modulate CRC progression.

Keywords: miR-206 hnRNPA1
Alternative splicing
Warburg effect
Colorectal cancer

1. Introduction

Colorectal cancer (CRC) remains the most devastating alimentary tract cancer in the world. Elevated aerobic glycolysis also known as metabolic reshuffling or Warburg effect, is now considered as a common hallmark of cancer. Tumor cells shift their metabolism from oxidative phosphorylation to aerobic glycolysis to promote cell proliferation and survival [1]. Aerobic glycolysis not only provides energy to cancer cells, but also generates a large number of building blocks to meet their requirements of rapid growth, and shapes the tumor microenvironment conducive to the survival of tumor cells [2]. Given the metabolic changes caused by oncogenes oblige cancer cells addicted to glucose, pathways involved in Warburg effect could be exploited as therapeutically actionable target and have received substantial attention [3].
Pyruvate kinase (PK) is a critical rate-limiting enzyme in glycolysis that responsible for the conversion of phosphoenolpyruvate to pyruvate and ATP. PKM2 is cancer-specific spliced isoform of PK and has been recognized as one of the key regulators in the Warburg effect [4]. The alternative splicing of mutually exclusive exons 9 and 10 in the primary transcript of PKM gene gives rise to the PKM1 with exon 9 and PKM2 with exon 10. Several splicing factors such as hnRNPA1, hnRNPA2, hnRNPI (also named as PTBP1) and SRSF3 have been reported to rewire a trend toward a higher PKM2/PKM1 ratio via binding to exon 9 flanking sequences to blockade exon 9 inclusion and foster PKM2-specific exon 10 [5]. A growing body of evidence has been highlighted an intimate link between a higher PKM2/PKM1 ratio and poor prognosis in cancer patients [1,6]. Therefore, targeted intervention of alternative splicing of PK and reinstate of cancer-specific spliced isoform PKM2 to PKM1 is of great significance in the development of antitumor therapy.
MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs with the length of about 18e24 nucleotides. By combining with 30-untranslated regions (30-UTRs) of the target genes, miRNAs lead to translation inhibition or mRNA degradation. miR-206, a well-characterized miRNA, is downregulated in various types of cancers [7]. Several studies have demonstrated that miR-206 exerted tumor suppressive functions in CRC to suppress cell proliferation, invasion and metastasis, induce apoptosis, trigger cell cycle arrest and enhance chemosensitivity [8e11]. Moreover, miR206 could be used as a biomarker for the diagnosis and prognosis of CRC [12e14]. Apparently at odds with aforementioned observations, Parasramka et al found that miR-206 was upregulated in rat colon tumor and restoration of miR-206 significantly enhanced CRC cell proliferation [15]. However, the role of miR-206 on Warburg effect has not been delineated.
Herein, we demonstrated that miR-206 is downregulated in CRC cells that dramatically attenuated the cellular viability via directly degrading its target hnRNPA1, which further skewed PKM splicing toward PKM1 expression to suppress the Warburg effect. Our findings indicate a novel role of miR-206/hnRNPA1/PKM2 axis in the Warburg effect and restoration of miR-206 could be worthy of considering as promising approach for the CRC treatment.

2. Materials and methods

2.1. Chemicals and antibodies

Antibodies against PKM2 and GFP were purchased from Abcam (Cambridge, MA, USA). hnRNPA1 antibody was provided by Proteintech (Wuhan, China). GAPDH antibody was bought from Santa Cruz Biotechnology (Carlsbad, CA, USA). PKM1 antibody was obtained from Signalway Biotechnology (Nanjing, China). The transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Glucose Assay Kit was bought from Biovision, Inc. (Mountain View, CA, USA). Lactic acid assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Seahorse XF glycolysis stress test kit (Seahorse Bioscience, USA).

2.2. Cell culture

Human colorectal cancer cells HCT-116, DLD1, SW620, HT29, SW480 cells and normal human colorectal epithelial cells FHC were obtained from the American Type Culture Collection (Manassas, VA, USA). The corresponding cell culture method was described as previously reported [16].

2.3. Quantitative real-time PCR

TRIzol Reagent (Invitrogen, MA, USA) was used to extract total RNA following the manufacturer’s protocol. miRNA expressions were quantified by qPCR using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA). mRNA expressions were quantified by qPCR using SYBR Green PCR master mix (Takara, Dalian, China). U6 and GAPDH were used as control for miRNA and mRNA normalization, respectively. The primers were shown as follows. PKM: 50-AGAAACAGCCAAAGGGGACT-30; 50-CATTCATGGCAAA GTTCACC-30. PKM1: 50-GAAGAACTTGTGCGAGCCT-30; 50-CGTCAGAACTATCAAAGCTGC-30. PKM2: 50- GCTGCCATCTACCACTTGC -3’; 50- CCAGACTTGGTGAGGACGATT-3’. hnRNPA1: 50-GCTCACGGACT GTGTGGTAA-30; 50-GGCCTTGCATTCATAGCTGC-30. hnRNPA2: 50GGAGTGGAAGAGGAGGCAAC-30; 50-CAGGTCCTCCTCCATACCCA-30. PTBP1: 50-CTCTCCGTATGCAGGAGCTG-30; 50-TCTGGGTTGAGGTT GCTGAC-30. SRSF3: 50-CACTCCGAAGTGTGTGGGTT-30; 50-AGGTGGG CCACGATTTCTAC-30. GAPDH: 50-AAGGTCGGAGTCAACGGATTT-30; 50- CCTGGAAGATGGTGATGGGATT-30. miR-206, 50-TGGAATGTAAGGAAGTG-30; 50-CAGTGCGTGTCGTGGAGT-30. U6: 50-CGCTTCGGCAG CACATATAC-30; 50-AAAATATGGAACGCTTCACGA-30.

2.4. Western blot

Western blot was conducted as previously described [17]. The cells were lysed, and equal amounts of protein lysates were separated on SDS-PAGE, electrotransferred to PVDF membrane and then incubated with primary antibodies. The specific protein was visualized with the enhanced chemiluminescence by radiographic film.

2.5. MTT assay and clonogenic assay

The indicated cells were plated into the 96-well culture plates at 5000 cells per well. After overnight, cells were transfected with miRNA control, miR-206 mimics or miR-206 inhibitor for various times. Cell survival rate was determined by MTT assay. DLD1 and SW480 cells were seeded in 60 mm culture dish at 5 104 cells per dish. After cells were transfected with miRNA control, miR-206 mimics or miR-206 inhibitor for 72 h, cells were trypsinized and 3000 single viable cells were seeded in 60 mm culture dish and incubated for 2 weeks. The colonies were stained with 0.1% crystal violet and photographed.

2.6. Plasmid construction and cell transfection

The DNA sequence of human hnRNPA1 gene was amplified from cDNA of HCT-116 cells. The primers (forward, 50-GGACTCAGATCTCGAATGTCTAAGTCAGAGTCTCCTAA-30; reverse, 50-CATGACCGGTGGATCAAATCTTCTGCCACTGCCATAG-30) were carried out to obtain the hnRNPA1 gene and then fused to pLVX-AcGFP1-N1 vector using the seamless cloning master mix following the manufacturer’s instructions (Sangon Biotech, Shanghai, China) and verified by sequencing. miR-206 mimics, miR-206 inhibitor and negative control (src-NC) were synthesized by GenePharma (Shanghai, China). DLD1 and SW480 cells were transfected with 100 nM miR-206 inhibitor or 10 mg/mL plasmid using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to our previously described method [18].

2.7. Glucose consumption and lactate production measurements

DLD1 and SW480 cells were seeded in 60 mm culture dish at a total of 5 106 per dish. After attachment, cells were transfected with src-NC miR-206 mimics or miR-206 inhibitor for indicated time. The glucose consumption and lactate production were measured using a glucose assay kit (Biovision, CA, USA) and lactic acid assay kit (Nanjing Jiancheng Bioengineering Institute, China) per the manufacturer’s recommendations, respectively.

2.8. Glycolysis stress tests

DLD1 and HCT-116 cells seeded into a XFe24-well culture plate (Seahorse Bioscience, MA, USA) at a density of 8000 cells per well and subsequently transfected with src-NC, miR-206 mimics or indicated plasmids for 72 h. The extracellular acidification rates (ECAR) was performed after automatically injection of 10 mM glucose, 1 mM oligomycin, and 50 mM 2-deoxy-glucose (2-DG). The experiments were independently performed in triplicate and the ECAR data were recorded after each injection.

2.9. Luciferase reporter assay

The 30-UTR of hnRNPA1 gene containing the seed sequence was amplified and inserted into a pGL4.23 luciferase reporter vector. The mutant 30-UTR of hnRNPA1 gene was generated by the Easy Mutagenesis System (Transgen Biotech, Beijing, China). The primers were depicted as follows. hnRNPA1 30UTR-WT: 50-AGAGGAGAGCCAGAGAAGTG-30, 50GCATAGGATGTGCCAACAAT-30.hnRNPA1 30UTR-mutant: 50-GTGGAAAGTGTAAAGCTTACGAACAAAGGGT-30, 50-CGTAAGCTTTACACTTTCCACAGAACAGA-30. HEK-293T cells were plated in a 24-well plate at 70% confluence and cotransfected with wild type or mutant 30UTR vector with srcNC or miR-206 mimics using Lipofectamine 3000 (Thermo Fisher Scientific, USA). A Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was performed to measure luciferase activity following the manufacturer’s instructions.

2.10. Statistical analysis

The statistical analyses were conducted by GraphPad Prism 5.0 software (San Diego, CA, USA). The results are presented as mean ± SD. P values < 0.05 are deemed as statistically significant.

3. Results

3.1. Decreased expression of miR-206 is negatively corelated with CRC cell viability

To explore the predictive value of miR-206 on the pathologic T stage of CRC, LinkedOmics platform was performed to retrieve the expression level of miR-206 and patient information from TCGA CRC datasets [19]. The results showed that miR-206 expression level was negatively correlated with the pathologic T stage of CRC (Fig. 1A). The results of Fig. 1B displayed that CRC patients with a lower level of miR-206 linked to the poorer overall survival. The qPCR analysis also confirmed that miR-206 expression was dramatically decreased in CRC cell lines compared to normal colonic epithelial cell FHC (Fig. 1C). These findings prompted us to focus on speculating whether miR-206 affected the proliferation of CRC cells. DLD1 and SW480 cells were transfected with miR-206 mimics or inhibitor (Fig. 1D). Furthermore, miR-206 mimics  caused a robust decrease of cell viability and colony formation, whereas miR-206 inhibitor notably promoted the viability of CRC cells (Fig.1E, F and G). Altogether, these data indicated that miR-206 was negatively correlated with CRC progression and it dramatically repressed CRC cell viability.

3.2. miR-206 shifts the balance from PKM2 to PKM1 to repress the Warburg effect in CRC cells

There was a notable decrease in glycolytic rate in CRC cells upon miR-206 mimics treatment, as evidenced by the suppression of glucose consumption and lactate production (Fig. 2A and B). By contrast, miR-206 inhibitor significantly enhanced glucose uptake and lactate production. Considering the importance of PKM alternative splicing in the Warburg effect, we further determined whether the miR-206-inhibited the Warburg effect correlate with the regulation of PKM splicing. The results from qPCR and western blot analysis displayed that miR-206 mimics attenuated the PKM2 expression and significantly promoted the level of PKM1 in CRC cells (Fig. 2C and D). While, PKM1 and PKM2 were downregulated and upregulated by miR-206 inhibitors, respectively. Previous studies have implicated that PKM1 and PKM2 isoforms are generated by mutually antagonistic alternative splicing of the PKM gene and replacing PKM2 with PKM1 significantly reduced the tumorigenicity of cancer cells [5]. As shown in Fig. 2E, there was virtually no changes in the PKM pre-mRNA expression upon miR-206 mimics or inhibitors in CRC cells, which further substantiated miR-206-reverted PKM alternative splicing. Collectively, these findings suggested that miR-206 tipping the balance of PKM splicing into PKM1 expression to interrupt the Warburg effect in CRC cells.

3.3. miR-206 directly targets hnRNPA1 to reprogram PKM alternative splicing in CRC cells

A growing number of studies have demonstrated that a small subset of splicing factors such as hnRNPA1, hnRNPA2, PTBP1 and SRSF3 modulates the alternative splicing of PKM gene to favor the PKM2 expression and counteract PKM1 expression [7]. As shown in Fig. 3A, miR-206 mimics led to a significant decrease in hnRNPA1 expression in both DLD1 and SW480 cells and had only a mild effect on PTBP1 expression in DLD1 cells, and no observable effects on hnRNPA2 and SRSF3 expressions. In contrast, miR-206 inhibitors obviously promoted hnRNPA1 expression. Western blot analysis further confirmed that miR-206 mimics significantly reduced, while miR-206 inhibitors enhanced the expression of hnRNPA1 (Fig. 3B). Moreover, three online miRNA computational algorithms including miRanda (microRNA.org), TargetScan (v7.0; targetscan. org), PicTar (pictar.org) were conducted to predict potential miRNA binding to the 30-UTR of hnRNPA1 transcript. Intriguingly, only miR-206 was conservatively target to hnRNPA1 among all three algorithms (Fig. 3C). To further decipher whether miR-206 can negatively mediate the transcriptional expression of hnRNPA1 in CRC cells, wild type and mutant 30-UTR of hnRNPA1 mRNA were generated and co-transfected with miR-206 mimics or control (src-NC) (Fig. 3D). As shown in Fig. 3E, the luciferase activity in hnRNPA1 30-UTR was notably attenuated in HEK-293T cells ectopically expressing the miR-206 mimics, while there were no as significant changes in the mutant 30-UTR. Moreover, the downregulation of PKM1 and upregulation of PKM2 caused by miR-206 was reversed by hnRNPA1 overexpression (Fig. 3F and G). Taken together, our findings indicated that miR-206 directly impaired the hnRNPA1 expression to reshape PKM splicing to favor PKM1 expression.

3.4. Attenuation of hnRNPA1 fulfils a key role in miR-206 triggering the shift of PKM isoform expression from PKM2 to PKM1

Given that, miR-206 directly targets hnRNPA1 and high expression of PKM2 promotes the Warburg effect that featured by upregulated lactate abundance and glucose uptake [20], we further ascertain the role of hnRNPA1 in the miR-206-mediated Warburg hnRNPA1 plasmid.
effect. As speculated, there was a significant decrease in glucose uptake and lactate production in CRC cells upon miR-206 mimics treatment and the inhibition effects caused by miR-206 mimics were compensated by hnRNPA1 overexpression (Fig. 4A and B). It has been reported that extracellular acidification rate (ECAR) reflects a temporal measure of glycolytic function of the test reagent [21]. Consistently, CRC cells transfected with miR-206 mimics exhibited lower ECAR and the suppressive effect of ECAR upon miR206 mimics was compromised by hnRNPA1 overexpression (Fig. 4C). In addition, MTT assay also demonstrated that miR-206 mimics resulted in a significant suppression of CRC cell viability was abrogated by hnRNPA1 overexpression (Fig. 4D). Taken together, these findings indicated that miR-206 directly targets and degrades hnRNPA1 to suppress the Warburg effect.

4. Discussion

As an RNA-binding protein, extensive research efforts have shown that hnRNPA1 is highly expressed in several tumor types including CRC. It has been reported that hnRNPA1 affects metabolic reshuffle via regulating the PKM alternative splicing, and is highly correlated with poor prognosis of tumor [7]. Although hnRNPA1 is essential for malignant transformation, it lacks druggable domain and small molecules which directly target hnRNPA1 [22]. Manipulating tumor-suppressive miRNAs expression providing a promising new therapeutic approach for cancer treatment.
Accumulating evidence has indicated that decreased expression of miR-206 (serves as a tumor suppressor) was observed in different types of cancer cells [23]. So far, the role of miR-206 in CRC remains somehow controversial. Several oncogenes such as ANXA2 [24,25], Notch3 [10], A3AR [26,27], TM4SF1 [9], and Bcl-2 [8], have been identified as potential targets of miR-206 in CRC. In one place for example, studies have shown that miR-206 directly targets ANXA2, Notch3, A3AR, TM4SF1 and Bcl-2 to attenuate tumor cell proliferation, inhibit cell migration and invasion, cause cell cycle arrest, induce apoptosis and facilitate chemosensitivity [8e10,24,26]. However, previous research has supported the oncogenic property of miR-206 via targeting Klf4 [17]. There was no available evidence demonstrating the potential targets of miR-206 involved in the PKM splicing. Based on miRNAs can effectively regulate gene network, which render them to be the potential anticancer therapeutic arsenal [28], miR-206 presumably directly inhibited these potential targets to suppress the CRC growth. Herein, we revealed that miR-206 directly targeted and degraded hnRNPA1, which subsequently skewed the PKM splicing toward PKM1 expression and resulting in suppressing the Warburg effect to attenuate CRC cell proliferation (Fig. 4E). In view of the advantages of miRNAs, such as stability, convenient synthesis and easy introduction into cells [29], miR-206 presents an opportunity for potential therapeutic strategies in CRC treatment.
Studies have implicated that hnRNPA1 is also fundamental for the regulation of gene expression via controlling the splicing of various gene transcripts. Besides PKM gene, CD44, SRSF1 and HMGCR has been identified as the known alternative splicing substrates of hnRNPA1 [30,31]. Our study revealed that miR-206 attenuated CRC cell growth via directly targeting hnRNPA1 to impair the Warburg effect. This raises an interesting question whether miR-206 affects other alternative splicing substrates of hnRNPA1? More experiments were needed to verify the feasibility.
In summary, we uncovered that miR-206 was decreased in CRC cells and it suppressed CRC cell growth by reshaping the Warburg effect through directly targeting hnRNPA1. Our findings propose a fundamental role of miR-206/hnRNPA1/PKM2 axis in the Warburg effect and open new opportunities for CRC treatment.

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