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Abstract

BACKGROUND:

Coiled-coil domain containing protein-124 (Ccdc124) is a putative mRNA-binding factor associated with cell division, and ribosome biology. Previous reports mentioned an up-regulation of CCDC124 gene in cancer, and listed its mRNA in a molecular prognostic signature in breast cancer.

OBJECTIVES:

Establishing RNA-binding characteristics of Ccdc124 for a better molecular functional characterization, and carrying-out retrospective studies in order to evaluate its aberrant expression in human cancer samples from various tissue origins.

METHODS:

Bioinformatics calculations followed by RIP and RNA-seq experiments were performed to investigate mRNA targets of Ccdc124. Quantitative studies on arrays of cDNAs from different cancers and IHC assays on tissue arrays were used to assess CCDC124 expression levels in cancers.

RESULTS:

Ccdc124 was characterized as an RNA-binding protein (RBP) interacting with various mRNAs. CCDC124 mRNA levels were high in tumors, with a particular up-regulation in cancers from esophagus, adrenal gland, endometrium, liver, ovary, thyroid, and urinary bladder. IHC assays indicated strong Ccdc124 positivity in endometrial (95.4%), urinary bladder (68.4%), and ovarian cancers (86.8%).

CONCLUSION:

Ccdc124 is a cytokinesis related RBP interacting with various mRNAs. CCDC124 mRNA over-expression and an accompanied increase in Ccdc124 protein accumulation was reported in cancers, indicating this RBP as a novel cancer cell marker.

Keywords

Cancer biomarkers coiled-coil domain-containing protein-124 RNA-binding proteins gynecological cancers ovarian cancer urinary bladder cancer

1. Introduction

Cancer is a complex multifactorial disease typically characterized by uncontrolled cellular proliferation and metastatic spreading throughout the body. Worldwide, endometrial and ovarian cancers account for $\sim$ 7.9% and $\sim$ 3.6% of new cancer cases in women, and urinary bladder cancer accounts for $\sim$ 3.1% of cancers in both sex; all three types of proliferative diseases affecting annually more than 1.4 million people [1]. Research on novel biomarkers of cancers originating in these tissues is essential both for discovering accurate early detection methods, and for having a better understanding on molecular mechanisms of the disease, which could provide novel opportunities to develop personalized and more efficient treatment strategies.

Identification of cancer-conducive alterations in cellular molecular interactions has long been a subject of intensive study; and a number of such interactions have been shown to involve RNA-binding proteins (RBP) [2, 3]. RBPs assemble ribonucleoprotein (RNP) complexes with RNA, and they mediate virtually every step of the RNA metabolism [4]. Causal mutations and mis-expression of RBPs affect most stages of RNA lifecycle, including RNA splicing (e.g. SRSF2) [5], 3’ end processing (e.g. CPEB1) [6, 7], editing (e.g. ADAR1) [8], stability (e.g. ZFP36) [9], translation (e.g. eIF4E) [10], and biogenesis of small RNAs such as miRNAs (e.g. AGO2, LIN28) [11]. Alterations of RNA metabolism due to RBP dysfunction can cause global changes in the transcriptome and proteome of the cell, and can affect carcinogenesis related molecular mechanisms involved in cell growth, proliferation, invasion and apoptosis [12, 13, 14, 15, 16, 17, 18].

Coiled-coil-domain-containing protein 124 (Ccdc124) was first discovered in our past studies as a new centrosome and midbody localized protein that regulate cytokinetic abscission during progression of mitosis in cultured human cell models [19]. Our previous RNA-interference experiments have indicated that knocking-down Ccdc124 expression in HeLa cells leads to aberrant cytokinesis, as characterized by time-lapse imaging monitored formation of multinucleated cells [19]. Importantly, Ccdc124 is encoded by a gene that is broadly conserved in eukaryotes, and predictions based on in silico analysis of functional domains in Ccdc124 by the web-based NCBI/UniProt software platform indicated it as a putative RBP (https://www.uniprot.org/uniprot/Q96CT7). Indeed, in a study termed as “interactome capture” where covalent UV crosslinking of RBPs to RNA, followed by a mass-spectrometry based unbiased and comprehensive approach to define the mRNA interactome of proliferating HeLa cells, also identifies Ccdc124 in a list of 860 new RBPs [20] (supplement. Table S1). Furthermore, Lso2 (late-annotated short open reading frame-2) which is the orthologue of Ccdc124 in a lower eukaryote organism Saccharomyces cerevisiae, was shown to localize in the ribosome and interact also with the ribosomal 25S RNA in a region near the A site that overlaps the GTPase activation center [21]. Thus, these previous analysis and studies on RNA-binding characteristics of Ccdc124 have indicated a broad role for Ccdc124/Lso2 in cellular physiology and RNA metabolism, including functions in translation during starvation recovery [19, 21].

A comprehensive data mining based study by Kechavarzi and Janga analyzing comparative expression patterns of RBPs in healthy human tissues and in nine human cancers (brain, breast, colon, kidney, liver, lung-adeno, lung-squamous, prostate, and thyroid cancers) by using RNA-seq data available from the Human BodyMap (HBM) and the Cancer Genome Atlas (TCGA) [https://tcga-data.nci.nih.gov/tcga], have revealed that CCDC124 transcription was strongly upregulated in cancer tissues [22]. Furthermore, a second study on supervised network analysis on gene expression profiles in breast tumor samples experimentally identified CCDC124 mRNA among a set of transcripts displaying a prognostic signature in MYB-positive infiltrating ductal carcinoma of the breast [23].

In the present study, we initially carried out in silico analysis on Ccdc124 to predict localization of its putative RBDs, and for initial characterization of possible RNA-interaction parameters for this protein. Then, we transfected HeLa cells with a vector expressing eGFP tagged version of CCDC124 under CMV-promoter, and subsequently we used RNA-immunoprecipitation (RIP) followed by RNA-sequencing methods for experimental validations of interactions between Ccdc124 and mRNAs. Furthermore, we investigated the versatility of Ccdc124 as a cancer cell marker, initially by examining CCDC124 expression change in human cancer tissues and normal tissues using a TissueScan array (OriGene, USA) spotted with cDNAs derived from 18 different types of cancer/normal tissue origins ( $n=$ 381; adrenal glands, breasts, cervix, colon, endometrium, esophagus, kidneys, liver, lungs, lymphoid tissue, ovaries, pancreas, prostate, stomach, testes, thyroid gland, urinary bladder, and uterus). Then, we carried-out a narrowed immunohistochemistry analysis using a selected subset of human cancer tissue protein arrays (US Biomax, USA) containing a normal/tumor paired samples from ovarian ( $n=$ 38), endometrial ( $n=$ 43) and urinary bladder ( $n=$ 38) cancer tissue samples. Our results have shown that both Ccdc124 protein and mRNA expression levels were significantly high in cancer samples, identifying this novel RBP as a biomarker in cancers from ovary, endometrium, and urinary bladder tissue origins.

2. Materials and methods

2.1 Tissue cDNA arrays

TissueScan Cancer Survey cDNA Array (OriGene Technologies Inc., Rockville, MD, USA, Catalog no. CSRT-303) was used as described in manufacturers protocols in order to screen expression level of Ccdc124 in samples of indicated human cancer types. This cDNA array contained human samples from 381 patients, and included both cancerous tissues and normal samples from healthy tissues.

To investigate the mRNA expression level of Ccdc124 in ovarian cancers, a distinct cDNA array containing 48 samples (OriGene Technologies Inc., Rockville, USA, cat. no. HORT 303) covering 6 normal cDNA samples, and cancer cDNAs from 9 different stages (IA, 2-IB, 3-IC, 3-IIB, 1-IIC, 1-II, 3-IIIA, 6-IIIB, 12-IIIC, 1-III,1-IV) was used, as previously mentioned.

2.2 Ccdc124 expression analysis by real-time quantitative PCR (qPCR)

Real-time qPCRs were performed with Syber Green using Stratagene Mx3000P. Each reaction was performed at least in triplicate in 25 $\mu$ L volume containing 1X SYBR (Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. K0253) and 0.4 mM of each primer and ddH ${}_{2}$ 0. The primers used in qPCR assays for CCDC124 gene expression were 5’GACACAGCCGAGAAAGCCAA3’(fwd) and 5’CGGAGCCACTCCTTCTTGAG3’(rev), The primers used for internal control GAPDH gene amplification were 5’GGCTGAGAACGGGAAGCTTGTCAT3’ (fwd) and 5’CAGCCTTCTCCATGGTGGTGAAGA3’ (rev). qPCR analysis was completed using MxPro Software. Ct (treshold cycle) values normalized to GAPDH and CCDC124 expression levels were calculated according to 2( $-\Delta\Delta$ Ct) value. The cycling conditions were as follows: 95 ${}^{\circ}$ C for 10 sec., followed by 35 cycles of 95 ${}^{\circ}$ C for 15 sec. for denaturation, 55 ${}^{\circ}$ C for 1 minute for annealing and 72 ${}^{\circ}$ C for 30 sec for extension. The reactions were terminated with a final cycle of 95 ${}^{\circ}$ C for 1 minute, 55 ${}^{\circ}$ C for 30 sec and 95 ${}^{\circ}$ C for 30 sec. PCR reaction specificity was confirmed by DNA melting curve analysis. When needed, samples were analyzed by electrophoresis using 1.5% agarose gels in TAE buffer.

2.3 Fluorescence microscopy

Fluorescence microscopy pictures were taken at 10x magnification with FITC filter using Nikon Eclipse T $i$ . Images were processed using NIS Elements Software.

Figure 1.

Bioinformatics calculations by catRAPID algorithm predicts Ccdc124 as an RNA-binding protein. A) Prediction of interaction propensity of Ccdc124 protein by catRAPID analysis. The graphic indicates classical (0.57), putative (0.35) and non-classical (0.28) RBP classes, and the overall prediction score of Ccdc124 as 0.65. B) RNA-binding propensity profile of Ccdc124 protein showing putative RNA-binding domains and their RNA-interaction propensity. C) The algorithm identifies peaks corresponding to regions between residues 74-124, 54-67, and 152-181 of Ccdc124.

2.4 Immunohistochemistry (IHC)

The expression level of CCDC124 in ovarian, endometrium and urinary bladder cancer tissue samples was analyzed by using human cancer tissue microarrays (US Biomax Inc., Rockville, MD; cat. no. OV485, BL 1002a and UT501a). To prevent tissue detachment during antigen retrieval, slides were baked for 1 h at 60 ${}^{\circ}$ C. The tissue arrays were de-paraffinized and rehydrated using Clearify Clearing Agent (StatLab). For antigen retrieval, the samples were incubated in pH 6.1 sodium citrate buffer EnVision FLEX Target Retrieval Solution (Low pH) (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA) for 30 minutes at 97 ${}^{\circ}$ C. After antigen retrieval, the slides were washed in Tris-buffered saline solution with Tween 20 (Dako Omnis Wash Buffer) (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA) for 40 minutes. For IHC analysis, human cancer tissue samples were stained with Ccdc124 antibody (Bioss Antibodies, Woburn, USA, cat. no. bs-8083R) diluted 1:150 in 30 minutes at room temperature and blocked by using EnVision FLEX Peroxidase-Blocking Reagent (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA) for 3 minutes after washing. Then samples were washed with Dako Omnis Wash Buffer (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA) for 2 minutes. To provide signal amplification of Ccdc124 antibody EnVision FLEX $+$ Rabbit Linker was used. Slides were incubated with EnVision FLEX/HRP which contains dextran coupled with peroxidase molecules and goat secondary antibody cocktails for 20 minutes after washing with Dako Omnis Wash Buffer. Washing step was repeated with ddH ${}_{2}$ O and Dako Omnis Wash Buffer, then samples were incubated with EnVFLEX Substrate Working Solution for 5 minutes. Then, slides were washed with distilled water and wash buffer Hematoxylin (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA) for 8 minutes. Finally, slides were washed with ddH ${}_{2}$ O and once again with Dako Omnis Wash Buffer. All steps were performed with Dako Link Omnis. Immunoreactivity was described by the percentage of positive tumor cells (percent positivity) and by the staining intensity (weak, moderate, strong) were evaluated by double blind IHC evaluation methods. The intensity was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage of positivity was scored as: 5%–25% $=$ 1, 26%–50% $=$ 2, 51%–75% $=$ 3, 76%–100% $=$ 4. Samples with less than 5% staining or had no areas of positive staining were regarded as negative. IHC value was calculated by multiplying the intensity score (0–3) by percent of positivity score (0–4) and was classified as follows: 0 $=$ negative; 1–4 $=$ low; 5–8 $=$ moderate, 9–12 $=$ high, as previously described [24].

2.5 Statistical analysis

Statistical analysis was performed using GraphPad Prism software version 6.1 (GraphPad Software, San Diego, California, USA). The results were presented as data obtained in three different independent experiments. Cancer samples data was analyzed with Mann-Whitney test ( $p<$ 0.05) and distribution of Ccdc124 expression levels of different cancer stages, grades, ages were analyzed with Kruskal-Wallis test ( $p<$ 0.05). qPCR data which shows RNA targets of Ccdc124 was analyzed with Sidak’s multiple comparison test ( $p<$ 0.05).

3. Results

3.1 In silico analysis of Ccdc124 to predict its possible RNA binding potential

NCBI database search indicated a possible RNA binding function for Ccdc124 (https://www.uniprot. org/uniprot/Q96CT7). Moreover, previous studies suggested possible interactions between Ccdc124 and various RNA species [20, 21]. In order to characterize RNA binding potential of Ccdc124, we first predicted possible locations of its putative RNA-binding domain(s), and its RNA binding propensity by using the web based catRAPID algorithm developed for large-scale calculations of protein-RNA interactions [25]. Indeed, catRAPID-signature server platform (http://service.tartaglialab.com/update_submission/272 860/59d238fdcb) predicted that Ccdc124 is an RNA-binding protein with an overall propensity score of $\sim$ 0.65 on a scale from zero to one, where an interaction propensity value $>$ 0.5 indicates a protein of the classical RNA-binding class (Fig. 1A). Analysis of the primary amino acid sequence of Ccdc124 protein indicated one possible major interaction propensity region between residues 74–124 to interact with RNA targets (Figs 1B and C). Ccdc124 also contains two other regions that are close to propensity threshold value ( $>$ 0.5) between residues 54–67 and 152–181 with possibilities of RNA interaction (Fig. 1B and C). Protein sequence analysis using profile hidden Markov Models through European Bioinformatics Institute (EBI) HMMER software platform indicated Ccdc124 as a largely disordered protein (https://www.ebi.ac.uk/Tools/hmmer/search/), a result that is also confirmed by protein disorder region prediction platform IUPRED2 (https://iupred2a.elte.hu). It is known that most RBPs contain intrinsically disordered regions (IDR), and some interact with target RNAs via these regions [26, 27, 28]. Therefore, presence of IDRs in Ccdc124 was taken as an additional evidence for its RNA-interaction potential. Subsequently, in order to predict potential RNA targets of Ccdc124, once again we carried-out in silico analysis by catRAPID-omics platform, but this time for large-scale calculations of Ccdc124 interactions with human coding RNA library [25]. We observed that together with coding RNA species, the predicted interactions list also included a substantial number of RNAs belonging to pseudogenes, mRNAs with retained introns, or anti-sense RNAs (Supplement Table S1).

3.2 Characterization of Ccdc124 as an mRNA binding protein

A reliable technique for identifying targets of a RBP is RNA-immunoprecipitation (RIP) followed by the identification of protein bound RNAs by RNA-sequencing (RIP-seq) [29, 30]. We used a human cervix cancer cell model, HeLa, and transfected the cells with vectors containing either GFP-linked version of CCDC124 under control of cytomegalovirus (CMV) promoter (CMV-CCDC124-GFP), or related empty CMV-GFP vector as control (Fig. S1). Then, we used either anti-Ccdc124 antibody or anti-GFP antibody coated sephadex beads in order to precipitate Ccdc124-RNA complexes from lysates prepared by using untransfected HeLa cells (controls), or cells transfected either with pEGFP-N2 or pCMV-CCDC124-GFP vectors (Fig. S1). As a confirmatory approach, RIPs were performed by using both anti-Ccdc124 antibody and anti-GFP antibody covered beads in two different sets of experiments on lysates obtained from CMV-CCDC124-GFP transfected HeLa cells. Same immunopurification protocols were also followed with lysates obtained from untransfected cells, or cells transfected with CMV-GFP vector, as controls. Following RIP experiments, immunoprecipitated Ccdc124-bound RNA samples were analyzed by RNA-sequencing methods (Suppl. Materials and Methods and $\geqslant$ Table S2). Then, we used CatRAPID-strength algorithm [25] for in silico testing of interaction possibilities, and by comparing data obtained in these two subsequent steps we shortened our list to 27 different mRNAs as targets of Ccdc124 (Supplement Table S3). Interactions between Ccdc124 and potential target mRNAs with highest in silico predicted propensity values were experimentally tested by RIP/RNA-sequencing methods, ;nd it was observed that Ccdc124 indeed interacts specifically with mRNAs (Fig. S2).

3.3 Up-regulation of Ccdc124 mRNA in cancers of different tissue origins

Besides playing important roles in cellular homeostasis by controlling gene expression at the post-transcriptional level, RBPs are also involved in various pathways related with RNA-splicing, translation, cellular stress response, and innate intracellular immunity [31, 32, 33]. Mutations mapping various RBPs and alterations in expression levels of corresponding transcripts were mostly associated with degenerative diseases, but also with cancer [34, 35, 36]. Previously, two different in silico data-mining studies focusing on levels of RBP encoding transcripts in cancers, and on MYB transcription factor dependent gene signatures in breast cancers have indicated that CCDC124 mRNA expression could be significantly upregulated in different types of cancers [22, 23]. Therefore, after proving RNA binding capacity of Ccdc124, we aimed to study in detail the expression level of CCDC124 gene in cancer tissue samples. For this purpose, we took advantage of a ready-made human cancer and normal tissue cDNA arrays (TissueScan, Origene, USA) containing materials from 381 tumor/normal paired human samples coming from 18 different types of cancers in an array ready for qPCR analysis (Supplement Table S4). Thus, we examined CCDC124 mRNA levels in normal/cancer tissue pairs coming from adrenal gland, breast, esophagus, kidney, liver, lung, lymphoid tissue, ovary, pancreas, prostate, stomach, testis, thyroid gland, urinary bladder, uterus, and thyroid gland tissues, as described in Materials and Methods. Our data indicated that as compared to healthy tissue pairs, CCDC124 mRNA expression level was significantly high (from $\sim$ 2.9 to 7-fold increase) in cancers originated from esophagus ( $\sim$ 2.9 fold), adrenal gland ( $\sim$ 2.9 fold), endometrium ( $\sim$ 3.6 fold), liver ( $\sim$ 3.4 fold), ovary ( $\sim$ 3.8 fold), thyroid gland ( $\sim$ 5.9 fold), and urinary bladder ( $\sim$ 7 fold) tissues, as compared to normal pairs (Figs 2 and 3A). In this list, urinary bladder, thyroid, ovarian, and endometrium stand out as cancer tissues with highest ( $\sim$ 3.6 to 7 fold) increases in CCDC124 mRNA levels (Fig. 2, inner box).

Figure 2.

Expression of CCDC124 mRNA in human normal or cancer tissue samples. Transcript profiling was performed by using a ready-made cancer tissue qPCR array, as described in Methods. cDNA concentrations were normalized to $\beta$ -actin expression. CCDC124 mRNA values were integrated to the graph relative to the average values coming from the sample tissue with the lowest level of CCDC124 expression (normal prostate). Inner table indicated summary of fold change differences of indicated cancer types, normalized by Ccdc124/ $\beta$ -actin values relative to normal tissue pair averages of each cancer type.

Figure 3.

Comparisons of Ccdc124 mRNA expression levels in a selected set of cancer types. A) Comparative analysis of expression of Ccdc124 in normal and cancer tissues originating from endometrium, esophagus, urinary bladder, thyroid and liver. Asterisks indicate statistically significant differences (Mann Whitney test * $p<$ 0.05). B) Comparisons of Ccdc124 expressions in different types of tumors according to stages of cancer. Early stage indicates Stage 0 and I, late stage indicates II, III and IV stage of the disease. Kruskal-Wallis test was used for a statistical analysis of gene specific expression (Ccdc124) at stages of cancer.

Figure 4.

Analysis of Ccdc124 mRNA expression in adrenal gland and ovarian cancers. A) Graphical expressions of fold changes in Ccdc124 mRNA levels in adrenal gland and ovarian cancers. Asterisks indicated statistically significant differences (Mann Whitney test * $p<$ 0.05, $n=$ 5 normal, $n=$ 10 adrenal cancer samples). B) Graphical expressions of fold changes in Ccdc124 mRNA levels in ovarian cancer samples. Asterisks indicated statistically significant differences (Mann Whitney test ** $p<$ 0.01 $n=$ 8 normal, $n=$ 57 ovary cancer samples). C) Comparison of Ccdc124 expressions at various stages of ovary cancer samples. Early stage, indicate stages IA, IB, IC, and late stage indicate stages II, IIB, IIIA, IIIB, IIIC, IV of the disease. D) Tumor grade dependent Ccdc124 expression in ovarian cancer samples with FIGO staging system. Asterisks indicated statistically significant difference (multiple comparisons of Kruskal-Wallis test * $p<$ 0.05 and ** $p<$ 0.01. E) Ccdc124 expressions in different histopathological types of ovarian cancer expressed as fold change in mRNA levels (Kruskal-Wallis test * $p<$ 0.05 and *** $p<$ 0.001).

Interestingly, we observed that the CCDC124 mRNA was particularly high at the early stages of endometrium, esophagus and urinary bladder cancers, while it was high at relatively later stages of thyroid and liver cancers (Fig. 3B). Significantly, we detected high CCDC124 mRNA level in cancers originated from adrenal gland and ovary tissue, independent of tumor stage (Fig. 4A–C). Yet, when ovarian cancer samples were compared based on FIGO tumor grading system [37], CCDC124 mRNA expression was more pronounced in Grades 2 and 3 samples which were histologically classified as endometrioid, papillary serous and serous, as compared to normal tissue pairs (Fig. 4D and E).

Table 1

Correlations between Ccdc124 protein immunohistochemistry staining and clinicopathological data associated with endometrium cancer samples in TMA

		Endometrium cancer (Ccdc124 Staububg)
			IHC value of endometrium cancer
		$N$	Negative	Low	Moderate	High	Chi-square, df	$P$ value
Coiled coil	Age
domain	$\leqslant$ 55	20	50%	38%	47%	67%
containing	$>$ 55	23	50%	63%	53%	33%	1.517, 3	0.6783
protein 124	Grade
(Ccdc124)	Grade 1	26	100%	63%	67%	33%
	Grade 2	7	0%	13%	17%	33%
	Grade 3	9	0%	25%	17%	33%	3.938, 6	0.6851
	Stage
	Stage IB	23	0%	41%	76%	50%
	Stage IC	14	0%	47%	18%	50%
	Stage II-III	4	100%	12%	6%	0%	14.77, 6	0,0222*
	Pathology diagnosis
	Clear cell adenocarcinoma	2	0%	6%	6%	0%
	Endometrioid adenocarcinoma	38	50%	88%	94%	100%
	Serous adenocarcinoma	2	50%	6%	0%	0%	10.76, 6	0.0961

Figure 5.

Immunohistochemical analysis of Ccdc124 protein using specific antibodies on paraffin embedded endometrial carcinoma and normal tissue sections in ready-made tissue arrays. A) IHC stained sections from normal tissue (magnifications, $\times$ 10 and $\times$ 50) (IHC value $=$ 1). B) IHC stained sections from endometrioid carcinoma tissue (magnifications, $\times$ 10 and $\times$ 50) (IHC value $=$ 12). Arrows indicate the positively stained cells. (Scale bar $=$ 100 $\mu$ m.)

Figure 6.

Immunohistochemical analysis of Ccdc124 protein using specific antibodies on paraffin embedded endometrial cancer and normal tissue ready-made protein array samples. Immunohistochemical analysis of Ccdc124 protein in normal endometrium and endometrial cancer microarray tissue samples. A) IHC staining of human endometrial cancer tissues at $\times$ 10 magnification. B) IHC staining of human endometrial cancer tissues at $\times$ 50 magnification. Arrows indicate the positively stained cells. C) Ccdc124 protein expression levels in normal/cancerous endometrial tissue samples as analyzed by Mann Whitney test (* $p<$ 0.05). D) Analysis of samples according to grade of the disease (Kruskal-Wallis test, ** $p<$ 0.001). E) Analysis of samples according to stage of the disease (Kruskal-Wallis test, * $p<$ 0.05 and ** $p<$ 0.01). F) Analysis of samples by age-correlated Ccdc124 protein expression levels in endometrial cancer samples (Kruskal-Wallis test, ** $p<$ 0.001). Asterisks indicate statistically significant differences.

3.4 Increased Ccdc124 protein expression in bladder, ovarian and endometrial cancers

After identifying increased CCDC124 mRNA levels in cancers originated from urinary bladder, thyroid gland, ovary, endometrium, liver, adrenal gland, and esophagus tissues, we decided to examine by immunohistochemistry (IHC) methods the possible increase of Ccdc124 protein in this subgroup of cancers. For this purpose, we used ready-made tissue microarrays (TMA) obtained from commercial sources (US Biomax, USA). For IHC analysis; human endometrial, ovarian, and urinary bladder cancer TMAs were stained with anti-Ccdc124 antibodies (;ioss, USA) suitable for use on paraffin embedded tissue sections. Then, we evaluated tissue-specific Ccdc124 positivity, as it is done routinely by the presence of dark-colored areas in histopathological examination (Supplement Fig. S3). The results showed that regardless of the origin of cancer, Ccdc124 had broadly cytoplasmic reactivity and major epithelial distribution (for example, Figs 5 and 7). In normal endometrial TMA samples, we detected low staining pattern of Ccdc124 antibody in endometrial gland cells and no reactivity in endometrial stromal cells (Fig. 5A). However, in endometrial cancer TMA samples ( $n=$ 43) we observed that anti-Ccdc124 antibody staining was present in both glandular and stromal cells (Figs 5B and 6A). We also detected Ccdc124 positivity at different levels in 95.4% of cancer samples (37.2% low; 44.2% moderate, and 14% high), as compared to relatively low (4.6%) negative IHC value, indicating that the expression level of Ccdc124 protein in endometrial cancers was significantly higher than normal tissue samples ( $p\leqslant$ 0.01) (Fig. 6A–E). We also found a correlation between Ccdc124 expression level and grades of tumor cells, with a significant increase particularly in grades 2 and 3 tumors ( $p\leqslant$ 0.05; Fig. 6D). The frequency of positivity was significantly related with the stage of disease, especially in premenopausal patients (Fig. 6E and F). Therefore, these IHC findings confirmed our previous qPCR data on increased CCDC124 mRNA detections in endometrial cancer, and it also indicated that Ccdc124 protein level was significantly increased rather in early stages (i.e., IB and IC) of the disease (Fig. 6E). Results presented in Table 1 summarize the clinicopathological data of all analyzed endometrial cancer samples.

Regarding Ccdc124 expression in normal urinary bladder TMA samples, we detected a relatively low staining pattern in transitional epithelium and glomerulus, but not in neighboring smooth muscle tissue (Fig. 7A). However, IHC analysis of Ccdc124 presence in urinary bladder cancer samples indicated 44.7% low, 15.8% moderate, 7.9% strong staining patterns ( $n=$ 38), as compared to no staining in 31.6% of cases (Fig. 7B). Statistical analysis of IHC values indicated that there is no significant difference in Ccdc124 expression regarding gender and age of the patient. Regarding stage dependent changes, expression was relatively high in tumors at stage II, but this was not persistent at stage III ( $p<$ 0.05) (Fig. 7C, upper right). The clinicopathological data related with all analyzed urinary bladder cancer TMA samples were presented in Table 2.

Table 2
Correlations between Ccdc124 protein immunohistochemistry staining and clinicopathological data associated with urinary bladder cancer samples in TMA

		Urinary bladder cancer (Ccdc124 staining)
			IHC value of urinary bladder cancer
		$N$	Negative	Low	Moderate	High	Chi-square, df	$P$ value
Colled coil	Age
domain	$\leqslant$ 55	13	42%	18%	67%	33%
containing	$>$ 55	25	58%	82%	33%	67%	5.178, 3	0.1592
protein 124	Gender
(Ccdc124)	Female	8	33%	12%	33%	0%
	Male	30	67%	88%	67%	100%	3.316, 3	0.3455
	Stages
	Stage I	14	33%	53%	17%	0%
	Stage II	13	25%	24%	50%	100%
	Stage III	10	33%	24%	33%	0%
	Stage IV	1	8%	0%	0%	0%	11.44, 9	0.2465
	Pathology diagnosis
	Urothelial carcinoma	34	92%	82%	100%	100%
	Urothelial carcinoma with squamous	4	8%	18%	0%	0%	2.035, 3	0.5651
	differentiation

Figure 7.

Immunohistochemical analysis of Ccdc124 protein levels in normal and urinary bladder cancer tissue microarray samples. A) IHC staining of human normal urinary bladder tissue at $\times$ 10 and x100 magnifications. (IHC value $=$ 1). B) IHC staining of human urinary bladder cancer tissue at $\times$ 10 and $\times$ 100 magnifications. Arrows indicate the positively stained cells. (Scale bar $=$ 100 $\mu$ m; IHC value $=$ 6). C) Analysis of urinary bladder cancer samples according to stage (upper right), age (below left), and gender distribution (below right) as established with Mann Whitney test and Kruskal-Wallis test ( $p<$ 0.05). Asterisks indicate statistically significant differences.

Subsequently, we carried-out similar analysis in ovary cancer TMA samples. In this analysis, we first noticed that Ccdc124 protein was predominantly detectable in the cytoplasm of oocytes in normal ovary tissue sections, but not in the stromal cells of ovaries (Fig. 8A). Relatively low IHC reactivity was detected in ovarian cancer samples ( $n=$ 38) when compared with cancers originated from endometrium and urinary bladder cancer (please compare Figs 8 and 9A; with Figs 5 and 7). Yet, even though it was not observable in healthy tissue controls by IHC methods, an increased level of Ccdc124 expression ( $p<$ 0.01) was detected in 86.8% of ovarian cancer samples (%76.3 low and 10.5% moderate reactivity), as compared to no detection in 13.2% of samples (Fig. 9). Our data indicated that Ccdc124 expression was significantly increased ( $p<$ 0.01) in early stages of ovarian cancer, in grades 1 and 3 tumors ( $p<$ 0.05) (Fig. 9E), and in ovarian samples obtained from patients have serous adenocarcinoma or serous papillary carcinoma ( $p<$ 0.05) (Fig. 9F). The clinicopathological data related with all analyzed ovarian cancer samples were presented in Table 3.

Table 3

Correlations between Ccdc124 protein immunohistochemistry staining and clinicopathological data associated with ovarian cancer samples in TMA

		Ovarium cancer (Ccdc124 staining)
			IHC value of ovarian cancer
		$N$	Negative	Low	Moderate	High	Chi-square, df	$P$ value
Colled coil	Age
domain	$\leqslant$ 55	25	60%	66%	75%	0%
containing	$>$ 55	13	40%	34%	25%	0%	0.2262, 2	0.8931
protein	Grade
124 (Ccdc124)	Grade 1	8	0%	30%	33%	0%
	Grade 2–3	22	100%	70%	67%	0%	1.690, 2	0.4296
	Stage
	Stage I	33	80%	86%	100%	0%
	Stage III	5	20%	14%	0%	0%	0.8211, 2	0.6633
	Pathology diagnosis
	Dysgerminoma	1	0%	3%	0%	0%
	Endodermal sinus carcinoma	2	0%	7%	0%	0%
	Endometrioid adenocarcinoma	2	0%	7%	0%	0%
	Immature teratoma	1	20%	0%	0%	0%
	Mucinous papillary adenocarcinoma	2	0%	3%	25%	0%
	Serous adenocarcinoma	13	60%	28%	50%	0%
	Serous papillary adenocarcinoma	14	20%	45%	0%	0%
	Undifferentiated carcinoma with	1	0%	3%	0%	0%
	neuroendocrine differentiation
	Clear cell carcinoma	2	0%	3%	25%	0%	19.27, 16	0.2550

Figure 8.

Immunohistochemical analysis of cell types expressing Ccdc124 protein in normal and ovarian carcinoma tissues. A) anti-Ccdc124 Ab stained sections in normal ovary tissue (magnification, $\times$ 10 and $\times$ 50) (IHC value $=$ 1). B) anti-Ccdc124 Ab stained sections in ovarian carcinoma tissue (magnification, $\times$ 10 and $\times$ 50) (IHC value $=$ 8). Arrows indicate positively stained cells. (Scale bar $=$ 100 $\mu$ m.)

Figure 9.

Immunohistochemical analysis of Ccdc124 protein levels in normal and ovarian cancer tissue microarray samples. A) IHC staining of human ovarian mucinous papillary adenocarcinoma tissue at $\times$ 10 magnification. B) IHC staining of human ovarian mucinous papillary adenocarcinoma tissue at $\times$ 50 magnification. Arrows indicate positive staining of epithelial cells in tumor samples. C) Comparisons of Ccdc124 protein expression levels in normal ovary and ovarian cancer tissue samples statistically analyzed by Mann Whitney test (** $p<$ 0.01). D) Analysis of Ccdc124 protein expression levels by stage of disease (Kruskal-Wallis test, ** $p<$ 0.01). E) Analysis of Ccdc124 protein expression levels by grade of cancer (Kruskal-Wallis test, * $p<$ 0.05). F) Analysis of Ccdc124 protein expression levels by histological type of cancer samples (Kruskal-Wallis test, * $p<$ 0.05). Asterisks indicate statistically significant differences.

4. Discussion

In the present study, firstly we have addressed RNA-binding propensity of Ccdc124 by in silico analysis using CatRapid software platform. This analysis indicated possible intrinsic disorder regions in Ccdc124, together with one major and two minor predicted RNA binding domains (Fig. 1). Subsequent experimental approaches testing RNA binding capacity of Ccdc124 by using RIP, followed by RNA-sequencing methods have not only proven RNA binding ability of Ccdc124, but in the meantime allowed us to identify its potential mRNA targets. By these RNA-sequencing based experimental approaches, we found 27 potential mRNA targets which were physically interacted with Ccdc124 (Supplement Table S3). Interestingly, preliminary predictions on cellular functions of these 27 factors based on STRING interaction network database analysis [38] indicated that at least five of those RBPs might take part in positive regulation of Ras superfamily of small GTP-binding proteins known to be involved in formation and progression of cancers (Supplement Table S3).

Subsequently, by using qPCR methods, we tested in cervix cancer originated HeLa cells the validity of two of the highest propensity value mRNA interactions with Ccdc124, and we obtained results confirming interactions between those candidate mRNA molecules and our protein of interest (Fig. S2). These results have proven unequivocally that Ccdc124 is an RNA binding protein targeting mRNA in addition to rRNA, as the latter was previously shown to interact with the yeast orthologue of Ccdc124 [21, 39].

Recent studies indicated clear roles of RNA-binding proteins in oogenesis by regulating translations of select groups of mRNA [40, 41, 42]. Interestingly, in this study, we observed a pronounced Ccdc124 staining in oocyte follicles in normal tissues, which could indicate a functional role of Ccdc124 in these follicles. As reported previously by Telkoparan et al., Ccdc124 was diffusely distributed in cytoplasm, as expected in cells at interphase [19]. This may also indicate a similar distribution pattern of its target mRNAs. Moreover, our data showed that Ccdc124 protein had a rather epithelial staining pattern, as compared to stromal cells, indicating a primary function for this protein in epithelial cells.

One of our major results in the present study is up-regulation of Ccdc124 expression both at mRNA and protein levels in ovarian and endometrial cancers. Previously, in two independent data mining RNA-seq data, and by high-throughput studies, up-regulation of Ccdc124 was mentioned in cancers of different tissue origins (brain, breast, colon, kidney, liver, lung-adeno, lung-squamous, prostate, and thyroid cancers), but a detailed analysis on this expression was absent [22, 23]. We have now extended these studies, and addressed expression levels of CCDC124 mRNA and the encoded protein in cDNA and tissue arrays corresponding to a large number of human cancer samples with grade and stage specific classifications. In this analysis, we confirmed previously obtained results, and we better characterized cancer tissue specific over-expression of CCDC124 (Fig. 2). Significantly, at the mRNA level, we detected approximately from 2.4 to 7-fold increase in the expression of Ccdc124 in urinary bladder, thyroid gland, ovary, endometrium, liver, adrenal gland, and esophagus cancers (Figs 2–4). This result indicated the promising potential of Ccdc124 as a novel cancer biomarker.

We also observed a very significant increase in CCDC124 mRNA expression in urinary bladder cancer samples by qPCR method (Figs 2 and 3A). Yet, in contrast to results obtained in ovarian and endometrium cancers, in bladder cancers Ccdc124 protein expression levels were relatively mild (compare Fig. 2 inset with Fig. 7). This discrepancy could be related with post-transcriptional regulation mechanisms operating differentially in tumors of different tissue origins [41, 42].

In endometrial cancer samples, we noticed a $\sim$ 3,6-fold higher expression of CCDC124 mRNA as compared to normal tissue samples (Fig. 2). This result was persistent in IHC assays on protein levels. Regarding grade specific analysis of endometrial tumors, our data showed that even though a tendency of increase was detectable in Ccdc124 protein levels, statistical analysis indicated similar levels of Ccdc124 protein expressions independent of the grade (Fig. 6D).

Our data indicated nearly 4-fold higher expression of CCDC124 mRNA in ovarian cancers, particularly in papillary serous and serous ovarian carcinoma as compared to normal tissue controls (Figs 2 and 4). This result is also confirmed by IHC staining of tissue-arrays by anti-Ccdc124 antibodies (Fig. 9). Furthermore, we detected a grade dependent increase in Ccdc124 expression at relatively later stages of tumor progression, both by qPCR and by IHC methods (grade-2 and grade-3; Figs 4 and 9).

In this study, we have not assessed CCDC124 mRNA or protein expression levels in tumors in comparison to widely used proliferation markers and/or prognostic factors, such as for instance Ki-67 [43]. Yet, interestingly, similar to Ccdc124, the proliferation marker/prognostic factor Ki-67 is also upregulated in early stage ovarian cancers [44], in urinary bladder cancers [45], and in endometrial cancers [46]. Further studies are required to carry-out a comparative mRNA, protein expression, and immunohistochemical analysis of these two seemingly independent markers, in order to establish whether there exist possible relationships between their expression levels and clinicopathological features, such as degree of malignancy, recurrence, or survival.

Ccdc124 was previously shown to be a factor with a sub-cellular localization at centrosomes and midbody, and it was associated with the separation of dividing daughter cells at the end of mitosis [19]. Aberrant expression of proteins localized at centrosomes and cytokinetic midbody were known to be associated with centrosome disfunctions, aberrations in chromosome segregation, and aneuploidy [47]. Therefore, it is plausible to propose a similar cellular outcome for Ccdc124 over-expression. Indeed, this might also indicate a possible early contribution of aberrant Ccdc124 expression to carcinogenesis, as imbalanced chromosome distribution was previously shown to contribute not only to heterogeneity and drug resistance of cancer cells, but also to DNA damage and to the activation of DNA damage response involving p53 pathway [48, 49].

We have carried-out Ccdc124 mRNA expression and immunohistochemistry-driven evaluation of protein expression analysis in tissue sample sets coming from different but clinically comparable cohorts of patients at similar age intervals. Thus, mRNA and protein expression analysis data were obtained form comparable sample sets, but not from the same patients individually. This was a necessary compromise in order to access to relatively large numbers of samples (381 tumor/normal paired human cDNA samples from 18 different cancer types, and tissue arrays from 119 tumor/normal paired samples) obtained from different sources, and to carry-out a comprehensive study in a reasonable time-period. As clinically followed patient groups were not included to our study, one limitation of this work was the absence of series of tumor samples of different stages coming from patients for an individual grade-per-grade analysis of Ccdc124 expression changes in tumors of different tissue origins. Future studies will elaborate individual stage-specific changes in Ccdc124 expression in detail, and they will define its contribution to tumor type and stage specific molecular profiles.

5. Conclusion

In conclusion, our experimental assays clearly indicated that as predicted by computational methods, Ccdc124 is an RBP that interacts with select mRNA species in HeLa cells, a widely used cervix cancer cell model (Fig. 1 and Figs S1 and S2). Besides, we demonstrated that CCDC124 mRNA was over-expressed between approximately three to seven folds in human endometrial, ovarian, urinary bladder, thyroid gland, liver, and esophagus, adrenal gland cancer tissues (Fig. 2). In subsequent tests on human cancer tissue arrays by IHC methods, we have shown that this up-regulation of CCDC124 mRNA was also accompanied by a pronounced Ccdc124 protein accumulation, particularly in endometrial and ovarian cancers, and to a lesser degree in urinary bladder cancer samples. Because of significantly high levels of expression, CCDC124 mRNA, and the corresponding protein (Ccdc124) could easily be detected by IHC methods, qualifying it as a strong biomarker protein at least for ovarian, endometrial, and urinary bladder cancers. Future molecular studies on cellular functions of Ccdc124 could lead to a better understanding of roles of RBPs in cancer cell biology and provide novel detection and treatment strategies in fight against this disease.

Author contributions

Conception: Ö.A., P.T.A., U.H.T.

Interpretation and analysis of data: Ö.A., N.K.S., P.T.A., U.H.T.

Preparation of the manuscript: Ö.A., N.K. S., U.H.T.

Revision for important intellectual content: Ö.A., N.K.S., P.T.A., U.H.T.

Supervision: U.H.T.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-200802.

sj-docx-1-cbm-10.3233_CBM-200802.docx - Supplemental material

Supplemental material, sj-docx-1-cbm-10.3233_CBM-200802.docx

sj-xlsx-1-cbm-10.3233_CBM-200802.xlsx - Supplemental material

Supplemental material, sj-xlsx-1-cbm-10.3233_CBM-200802.xlsx

sj-xlsx-2-cbm-10.3233_CBM-200802.xlsx - Supplemental material

Supplemental material, sj-xlsx-2-cbm-10.3233_CBM-200802.xlsx

sj-xlsx-3-cbm-10.3233_CBM-200802.xlsx - Supplemental material

Supplemental material, sj-xlsx-3-cbm-10.3233_CBM-200802.xlsx

sj-xlsx-4-cbm-10.3233_CBM-200802.xlsx - Supplemental material

Supplemental material, sj-xlsx-4-cbm-10.3233_CBM-200802.xlsx

Footnotes

Acknowledgments

The present study was funded by grants from Turkish Scientific and Technological Research Council (TUBITAK) KBAG 114-Z-349 to U.H.T., and by Ministry of Development 2014-K-120430 to Gebze Technical University Centre Research Laboratory (GTU-MAR).

Conflict of interest

Authors declare no conflict of interest.

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Coiled-coil domain-containing protein-124 (Ccdc124) is a novel RNA binding factor up-regulated in endometrial,ovarian,and urinary bladder cancers

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Tissue cDNA arrays

2.2 Ccdc124 expression analysis by real-time quantitative PCR (qPCR)

2.3 Fluorescence microscopy

2.5 Statistical analysis

3. Results

3.1 In silico analysis of Ccdc124 to predict its possible RNA binding potential

3.2 Characterization of Ccdc124 as an mRNA binding protein

3.3 Up-regulation of Ccdc124 mRNA in cancers of different tissue origins

Table 2 Correlations between Ccdc124 protein immunohistochemistry staining and clinicopathological data associated with urinary bladder cancer samples in TMA

5. Conclusion

Author contributions

Supplementary data

sj-docx-1-cbm-10.3233_CBM-200802.docx - Supplemental material

sj-xlsx-1-cbm-10.3233_CBM-200802.xlsx - Supplemental material

sj-xlsx-2-cbm-10.3233_CBM-200802.xlsx - Supplemental material

sj-xlsx-3-cbm-10.3233_CBM-200802.xlsx - Supplemental material

sj-xlsx-4-cbm-10.3233_CBM-200802.xlsx - Supplemental material

Footnotes

Acknowledgments

Conflict of interest

References

Table 2
Correlations between Ccdc124 protein immunohistochemistry staining and clinicopathological data associated with urinary bladder cancer samples in TMA