The miR-133a, TPM4 and TAp63γ Role in Myocyte Differentiation Microfilament Remodelling and Colon Cancer Progression

Abstract

MicroRNAs (miRNAs) play an essential role in the regulation of a number of physiological functions. miR-133a and other muscular miRs (myomiRs) play a key role in muscle cell growth and in some type of cancers. Here, we show that miR133a is upregulated in individuals that undertake physical exercise. We used a skeletal muscle differentiation model to dissect miR-133a's role and to identify new targets, identifying Tropomyosin-4 (TPM4). This protein is expressed during muscle differentiation, but importantly it is an essential component of microfilament cytoskeleton and stress fibres formation. The microfilament scaffold remodelling is an essential step in cell transformation and tumour progression. Using the muscle system, we obtained valuable information about the microfilament proteins, and the knowledge on these molecular players can be transferred to the cytoskeleton rearrangement observed in cancer cells. Further investigations showed a role of TPM4 in cancer physiology, specifically, we found that miR-133a downregulation leads to TPM4 upregulation in colon carcinoma (CRC), and this correlates with a lower patient survival. At molecular level, we demonstrated in myocyte differentiation that TPM4 is positively regulated by the TA isoform of the p63 transcription factor. In muscles, miR-133a generates a myogenic stimulus, reducing the differentiation by downregulating TPM4. In this system, miR-133a counteracts the differentiative TAp63 activity. Interestingly, in CRC cell lines and in patient biopsies, miR-133a is able to regulate TPM4 activity, while TAp63 is not active. The downregulation of the miR leads to TPM4 overexpression, this modifies the architecture of the cell cytoskeleton contributing to increase the invasiveness of the tumour and associating with a poor prognosis. These results add data to the interesting question about the link between physical activity, muscle physiology and protection against colorectal cancer. The two phenomena have in common the cytoskeleton remodelling, due to the TPM4 activity, that is involved in stress fibres formation.

Keywords: TAp63; TPM4; Tropomyosins; circulating miRs; colon carcinoma (CRC); miR-133a; miRNAs; physical activity.

Figure 1

Analysis of the presence of c-myomiRs from RNA extracted from plasma subjects. Levels of miR-133a (A), miR-1 (B), and miR-206 (C) were analysed by RTqPCR and presented as fold-over-control. Upregulation of miR-133a is detectable in most of the probands. The miR-206 seems not be widely regulated in this context. A mean value of expression was calculated for the presented miRs (D), showing miR-133a as the most upregulated. (n = 3, * p < 0.01 t-test, two tails, equal variance).

Figure 2

miR-133a putative target analysis. (A) The putative targets of miR-133a were analysed by means of the TargetScan-7.1 software and here are represented the consensus sequence and the conservation across different species [45]. A conserved putative binding site was retrieved in the 3′ UTR of the TPM4 transcript. (B) A 300 bp fragment, containing the putative responsive element, was cloned into the pGL3 vector, replacing part of the 3′ UTR of the luciferase gene, this will permit to control the luciferase gene if the cloned sequence contains a genuine binding site for miR-133a. (C) Assay showing the inhibition of luciferase when the vector is co-transfected with miR-133a demonstrates its interaction with the cloned responsive element (n = 6, * p < 0.01, t-test two tails, coupled). (D) miR-133a amplification from RNA extracted from plasma and from isolated exosomes. (n = 3).

Figure 3

miR-133a and TPM4 expression. miR-133a (A) and TPM4 (B) expression during myocyte differentiation was analysed by RTqPCR, values were expressed as fold over control. The ΔΔCts were calculated by using housekeeping transcript (actin and GAPDH), and all data were normalised on the proliferating cells condition (n = 3, * p < 0.01, t-test two tails, coupled). (C) Structure of TPM4 with primers amplifying different combinations of exons and of the 3′ UTR (NCBI identification number NM_001145160.1). (D) Results of the RT-PCR showing the amplification of the full-length transcript including the 3′ UTR (DNA marker: Thermo Scientific™ O’GeneRuler 1 kb Plus DNA Ladder).

Figure 4

TPM4 and differentiation marker analysis in differentiating myocytes. (A) RTqPCR analysis of the differentiation marker SOX4, direct target of miR-133a. The marker shows an inverse expression in respect to miR-133a, confirming the published data. (B,C) Additionally, LAMP1 and MAPK2 involved in myocyte differentiation show the correct expression; they are upregulated in the time course (A–C n = 3). (D) Protein analysis of TPM4, Myf5 and α-actinin, showing TPM4 and α-actinin upregulation and Myf5 downregulation (n = 6). (E) RTqPCR TAp63 analysis: the transcription factor is expressed in the early phases of differentiation (n = 3). The RTqPCR is shown in fold over control. The ΔΔCt was calculated using actin as housekeeping transcript, and all data was normalised on the proliferating cells condition (* p < 0.01, t-test two tails, coupled).

Figure 5

TPM4 and α-actinin staining in differentiating myocytes. (A,B) Confocal immunofluorescence analysis performed at day 1. In the field are present mono-nucleated cells expressing TPM4 (green) organised in filaments and differentiated myotubes with a lower and less organised expression of the protein. (C,D) Panels with separate channels showing the concomitant expression of TPM4 (green) and α-actinin (red) in the cytoplasm. Starting from the left first panel is a merged image of all channels, the second panel shows the captured image using only the 488 nm laser (FITC), the third is the capture using the 561 nm laser (TRITC), the last using the 401 nm laser (DAPI, blue). DAPI is used for nuclei staining. Bars = 10 µm.

Figure 6

TPM4 and p63 expression in differentiating myocytes. (A,B) Identification of p63 (red) positive nuclei in differentiating myocytes. Only some mono-nucleated cells (stars) are positive. (C) Enlargement of mononucleated cells shows fibrillar distribution of TPM4 (green) and perinuclear localisation of p63 (red), probably identifying the time of the transport from ER to nucleus (arrow and stars). DAPI (blue) was used for nuclei staining. Laser wavelengths as the previous figure. Bars = 10 µm.

Figure 7

p63 regulatory element on TPM4 promoter. (A) Jaspar prediction (sequence in the upper part of the panel (A)), and reads counted from RNAseq, suggest the possible presence of a p63 responsive element. Jaspar prediction is obtained by using a specific software that analyses DNA sequence with a mathematical matrix to identify putative transcription factor binding sites. Interestingly, the “in silico” analysis and the RNA-Seq return the same site in TPM4 5′ UTR. (B) Enlargement of the genomic region containing the reads from the RNA-Seq. (C) Luc-Assay using a 490 base pair region of the promoter containing the responsive element, showing that the TAp63γ isoform nicely regulates the TPM4 promoter (n = 4).

Figure 8

In vivo transactivation of TPM4 by TAp63γ, and overall-survival analysis of a CRC patient cohort. (A) TAp63γ (approx. 50 KDa) expression increases during early differentiation of myocytes, and TPM4 expression follows the same kinetic, “Prol” indicates the proliferating myocytes, “1D” indicated myocytes at 1 day of differentiation. (B) Upper panel, analysis, performed by using cBioportal, on colorectal adenocarcinoma (TCGA, PanCancer Atlas) showed higher survival for patients with low TPM4 expression (Logrank Test p-Value: 0.011; p-Value = 0.0190, q-Value = 0.0379). Lower panel, data reanalysed from Wang et al. [59], showing the inverse correlation with survival of miR-133a respect to TPM4. (C) TAp63γ is able to drive the expression of TPM4 also in H1299 tumour cells, demonstrating the presence of TAp63γ- TPM4 regulation also in cancer cells. (D) Upregulation of TPM4 protein expression by the ectopic expression of TAp63γ in caco-2 cells. (E) Upregulation of TPM4 protein expression by TAp63γ in HCT-116 cells, showing that the transcription factor is able to drive the transcription of TPM4 also in these cells. (F) RTqPCR, showing downregulation of TPM4 after inhibition (siRNA) of p63 in HCT-116 (blue) and Caco-2 (green, n = 3) cells, demonstrating that the regulatory pathway is TAp63 specific and valid also at protein level. (G) qPCRT amplification of miR-133a in CaCo-2, HCT-116 and DLD-1 (n = 3; * p < 0.05, t-test two tails, coupled). (H) mRNA TPM4 expression in CaCo-2, HCT-116 and DLD-1 (n = 3). (I) WB showing TPM4 expression in CaCo-2, HCT-116 and DLD-1 (n = 3; *** p < 0.01, t-test two tails, coupled).

Figure 9

Cell cycle analysis and TPM4 expression in CRC patient specimens. (A) Cell cycle analysis in HCT-116 CRC cells expressing TAp63γ (B) control cells. Representative experiments are shown (n = 3). (C) Vitality analysis showing that TAp63 expression does not alter the apoptotic program of these cells. (D) PCR of TAp63 in control and CRC tissue (top) TPM4 Western blot in normal and CRC tissues (Low). (E) TPM4 expression level in control/CRC tissues. (F) miR-133a expression level comparison between colon tissue and colon cancer tissue (**** p < 0.001, t-test two tails, coupled) (G,P) representative sections from two patients. (G,I) show the tumour adjacent normal area of the mucosa, in which is recognised the presence of regular glands, monolayered with a large luminal vacuole containing mucus, whose cytoplasm is TPM4 stained (green). In (H,L), a contiguous neoplastic area (adenocarcinoma) is represented, consisting of irregular glands, multi-layered, free of mucus, arranged back to back. TPM4 is present in the wall of the mucosal vessels (DAPI, blue, identify the nuclei). (M–O) Tumour adjacent normal area of mucosa, (N–P) Neoplastic area characterised by a high detection of TPM4 (green). In particular, adenocarcinoma cells recognisable by typical morphology, showing high cytoplasmic TPM4 staining (stars).

Figure 10

Cartoon explaining the mechanism of interaction between TAp63, miR133, and TPM4 in muscle and colon carcinoma.