Meta-analysis of the cell cycle related C 12 orf 48

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene. BIOCELL 2013, 37(1): 11-16 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
The cell cycle is the conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division.The main function of the cell cycle is to accurately duplicate the entire genome and segregate a copy of each chromosome precisely into two daughter cells.Proper regulation of this process is crucial to the growth and development of all organisms.Therefore, understanding of the regulation of cell cycle is important to gain knowledge of the molecular mechanisms that control DNA replication and accurate segregation of chromosomes to daughter cells which are characteristically aberrant in cancer cells (Ganem et al., 2007).
The regulation of cell division relies on two major mechanisms which are phosphorylation and transcription (Delcuve et al., 2008).However, transcriptional regulation is poorly studied compared to phosphorylation dependent regulation of cell division (Delcuve et al., 2008).Understanding the transcriptional regulation of cell division requires identification of the novel genes that are involved in this process.Since the inventory of division related genes is still incomplete, we have undertaken a co-expression analysis to reveal novel genes that are involved in these processes.Previously, we have reported that Fam83d is a novel mitosis related gene and is differentially expressed in cancer by using in silico approaches (Varisli, 2012).In this study, we performed a similar analysis for C12orf48 and we identified this molecule as a novel division related gene.
C12orf48, encodes a PARP-1 interacting protein (Piao et al., 2011).Although the molecular function of this protein isn't well understood it was shown that it may be involved in recombination process at replication forks and may have proliferation related roles (Moldovan et al., 2012).In this study, we report that C12orf48 is co-expressed with mitosis related genes such as AurkA, AurkB, Plk-1, Plk-4, Cdc20, Cdk1, Nek2, Top2A and CENP family members which are well known genes that have crucial roles not only in different stages of mitosis but also in equal segregation of chromosomes to daughter cells.We also found that, some of the genes that co-expressed with C12orf48 are involved in other cell cycle related roles such as DNA replication, in concordance with molecular functions of PARP-1.Moreover, our results also show that this gene is differentially expressed in various cancers in concordance with the functions of above-mentioned co-expression partners.
Differentially expressed genes are candidates for diagnosis of cancer and are prognostic markers.Therefore, this article suggests that C12orf48 is a strong candidate for prognostic and diagnostic approaches in cancer and should be further investigated.

Meta-analysis of C12orf48
To gain insight into the function of C12orf48, coexpression analysis was performed from Oncomine database (http://oncomine.org)as described previously (Varisli, 2012;Wilson, 2008;Wilson and Giguere, 2008) with minor modifications.Threshold was adjusted as p-value <1E-4, fold change; 2 and gene rank; top 1%.8 different array fulfilled these criteria (Table 1) and the top 200 co-expressed genes were extracted and filtered to give one representative gene per study (removing

C12orf48 and cancer relationship
Oncomine cancer microarray database was used to study gene expression of C12orf48 in various tumor types and their normal control tissues.The gene transcriptome data only from the same study generated with the same methodology were used.All gene expression data were log transformed, median centered per array, and standard deviation was normalized to one per array (Rhodes et al., 2004).Student's t test was used for differential expression analysis, and only studies with P value less than 1E-4 and fold change more than two and gene rank 5% were considered, as described previously (Varisli, 2012).

C12orf48 is co-expressed with genes that are involved in mitosis
Using the Oncomine cancer microarray database C12orf48 was searched for co-expressing genes.After meta-analysis, 133 genes were found co-expressed in four or more studies (Table 2).DAVID was used to perform GO Term Enrichment analysis to obtain characteristics of the set of significant genes from our metaanalyses.This analysis provides a list of gene functions, which are over-represented in a gene set.Analysis of the 133 C12orf48 co-expressed genes with the DAVID functional annotation tool (GOTERM BP FAT) resulted 186 GO-categories (cut-off p-value < 0.1, count > 2, Fold enrichment > 1.5) (data not shown).To receive a more comprehensive and structured view of the annotation terms a DAVID clustering analysis under high stringency conditions was performed resulting in 20 annotation clusters matching the statistical criteria (p < 0.0001, count > 20, and fold enrichment > 2.0) (Table 3).Subsequently, the aforementioned DAVID annotation tool was used for identification of putative KEGG pathways associated with C12orf48 co-expressed genes.Consequently, four pathways which associated with cell cycle and related signaling pathways were significantly enriched with C12orf48 co-expressed genes (p < 0.0001, count > 5 and fold enrichment > 1.5) (Table 4).In addition, DAVID was also used for prediction of putative diseases that are linked with C12orf48 co-expressed genes using Genetic Association Database.The results revealed that breast and colorectal cancers showed significant enrichment of these genes (p<0.05, fold enrichment > 1.5) (Table 5).

C12orf48 is differentially expressed in various cancers
We investigated the expression of C12orf48 in cancer using publicly available gene expression data using Oncomine (Table 6).C12orf48 has been found up-regu-lated in various tumors including breast cancer compared to normal breast (Richardson et al., 2006), in colorectal cancer compared to normal colon or rectum in two independent studies (Sabates-Bellver et al., 2007;Skrzypczak et al., 2010), in hepatocellular carcinoma compared to normal liver (Wurmbach et al., 2007), in lung cancer compared to normal lung (Hou et al., 2010), in nasopharyngeal carcinoma compared to normal nasopharynx (Sengupta et al., 2006), in vulvar intraepithelial neoplasia compared to normal vulva (Santegoets et al., 2007) and in various sarcomas compared to their control normal tissues (Detwiller et al., 2005).

Discussion
The cell division is a complicated cellular process involving extensive functional and structural organiza-  Count, Gene counts involved in that cellular process; %, % ratio of the genes involved in that process; Fold, fold enhancement; FDR, false discovery ratio tions in a sequence of highly orchestrated events.The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during cell cycle.Therefore, the expression of many regulator genes changes during different phases of the cell cycle.Although the genes involved in the interphase are well studied, most of the genes which are involved in mitotic events are still unidentified.Therefore, various experimental or in silico approaches have been used to identify the novel mitosis related genes.Recently, we reported Fam83d as a novel mitosis related gene (Varisli, 2012) when we performed a co-expression and gene ontology analysis to find unidentified mitotic genes.
C12orf48, encodes a PARP-1 interacting protein (Piao et al., 2011).PARP-1 is a multifunctional protein that is involved in DNA repair, maintenance of genomic stability, replication, transcription, telomere dynamics and apoptosis (Hassa and Hottiger, 2008).This mol-ecule is normally distributed in whole chromatin in the nucleus (Rouleau et al., 2004).Furthermore, it was shown that PARP-1 also associates with mammalian centrosomes in a cell-cycle dependent manner and interacts with the CENP family members and other mitotic spindle checkpoint proteins (Perdoni et al., 2009;Saxena et al., 2002), thus suggesting that PARP-1 might regulate their function in controlling chromosome segregation and mitosis.The activity of PARP-1 is primarily dependent on its poly(ADP-ribosyl)ation activity.Therefore, the molecules involved in regulation of this activity are probably also involved in all PARP-1 mediated processes.In concordance with this hypothesis we suggested that C12orf48 is probably involved in all PARP-1 dependent mechanisms as this molecule positively regulates the poly(ADP-ribosyl)ation activity of PARP-1 (Piao et al., 2011).In co-expression analysis we have seen that C12orf48 is co-expressed with cell cycle and replication related genes, in concordance with the cellular functions of PARP-1.Moreover, our results have shown that C12orf48 is also co-expressed with important mitotic genes, of which many are found in association with centrosome.Since most of the cell cycle and mitosis related genes are also involved in carcinogenesis, we searched expressional changes of C12orf48 in cancers.In concordance with this hypothesis our results revealed that C12orf48 is differentially expressed in various cancers which have a direct link with centrosome and mitotic abnormalities such as breast (Lingle et al., 1998), lung (Jung et al., 2007) and colon (Nakajima et al., 2004) cancers.Breast cancer is a good model to study the relationship between cancer and mitotic abnormalities since aneuploidy is common.et al., 2011).Interestingly, our results revealed that most of these genes are co-expressed with C12orf48.Therefore, we suggest that C12orf48 may be involved in equal segregation of chromosomes during cell division and may have cell cycle dependent roles like its co-expression partners.
Taken together, we performed a meta-analysis for C12orf48 using in silico approaches.Our results have revealed that this molecule may be important for cell cycle and mitotic events and also in carcinogenesis.Therefore, further investigation of the results presented in this study by experimental approaches may increase our understanding of cell cycle, mitosis and carcinogenesis.

TABLE 1 .
The arrays that were used for co-expression analysis

TABLE 4 .
Pathway based enrichment of C12orf48 co-expressed genes.
Count, Gene counts involved in that pathway; %, % ratio of the genes involved in that pathway; Fold, fold enhancement; FDR, false discovery ratio

TABLE 5 .
Disease based enrichment of C12orf48 co-expressed genes.
Count.Gene counts involved in that disease; %. % ratio of the genes involved in that disease; Fold.fold enhancement; FDR.false discovery ratio

TABLE 6 .
Over-expression of C12orf48 in various cancer types compared to their normal counterparts.Bièche et al., reported that the expression of 49 known mitotic genes was deregulated in breast tumors compared to normal breast tissues (Bieche