Gene expression analysis of potential genes and pathways involved in the pathogenic mechanisms of parvovirus B19 in human colorectal cancer

In order to investigate the pathogenic mechanisms of parvovirus B19 in human colorectal cancer, plasmids containing the VP1 or VP2 viral capsid proteins or the NS1 non-structural proteins of parvovirus B19 were constructed and transfected into primary human colorectal epithelial cells and LoVo cells. Differential gene expression was detected using a human genome expression array. Functional gene annotation analyses were performed using Database for Annotation, Visualization and Integrated Discovery v6.7 software. Gene ontology (GO) analyses revealed that VP1-related functions included the immune response, immune system process, defense response and the response to stimulus, while NS1-associated functions were found to include organelle fission, nuclear division, mitosis, the M-phase of the mitotic cell cycle, the mitotic cell cycle, M-phase, cell cycle phase, cell cycle process and cell division. Pathway expression analysis revealed that VP1-associated pathways included cell adhesion molecules, antigen processing and presentation, cytokines and the inflammatory response. Moreover, NS1-associated pathways included the cell cycle, pathways in cancer, colorectal cancer, the wnt signaling pathway and focal adhesion. Among the differential genes detected in the present study, 12 genes were found to participate in general cancer pathways and six genes were observed to participate in colorectal cancer pathways. NS1 is a key molecule in the pathogenic mechanism of parvovirus B19 in colorectal cancer. Several GO categories, pathways and genes were selected and may be the key targets through which parvovirus B19 participates in colorectal cancer pathogenesis.


Introduction
Colorectal cancer is the third most common type of cancer and the second most frequent cause of cancer mortality in numerous industrialized countries (1). The majority of tumors arise sporadically with no clear cause or genetic predisposition. Several risk factors have been considered as causes of colorectal cancer, but little has been confirmed. Viruses are among the few known causes of cancer and contribute to various malignancies worldwide (2). Previous studies on viral etiology in colon cancer have reported contradictory findings (3,4).
Parvovirus B19 (B19) is a non-enveloped virus with a linear, single-stranded DNA genome. The B19 viral genome encodes three proteins: The non-structural protein, NS1, and two viral capsid proteins, VP1 and VP2 (5). In our previous study, significantly higher levels of B19 nucleic acids and proteins were found in neoplastic colon tissues (6). This finding indicates that an association may exist between B19 infection and the development of colon neoplasia.
Infection with parvovirus B19 is a global concern. The infection rate is similar in the United States, Europe and Asia, with ~50% of 15-year-old adolescents and >60% of adults being seropositive (5). A previous study has shown that B19 infection may contribute to the pathogenesis of acute lymphoblastic and myeloblastic leukemia (7). However, few studies have investigated B19 in solid tumors or the mechanisms or regulatory proteins that could be involved. Therefore, it is important to establish whether B19 contributes to the pathogenesis of colorectal cancer and its underlying mechanism.
The present study aimed to investigate the pathogenic mechanisms underlying B19 in colon carcinoma by analyzing differential gene expression and biological functions, through assessing the changes in primary human colorectal epithelial cells (HCECs) and LoVo cells following transfection with plasmids containing VP1, VP2 and NS1.
Fluorescence microscopy. The expression of enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) in the transfected HCECs and LoVo cells was observed using a fluorescence microscope (TE2000-U, Nikon Corporation, Tokyo, Japan) equipped with a fluorescence filter. Digital images of the cells were captured using a spot camera system (Nikon Corporation).
Microarray hybridization and data analysis. Microarray hybridization was performed by Shanghai Biochip Co., Ltd., (Shanghai, China) using an Agilent SurePrint G3 Human GE 8x60k microarray (Agilent Technologies, Santa Clara, CA, USA) that targeted 27,958 Entrez Gene RNAs and 7,419 long non-coding RNAs (reference). In brief, total RNAs from the transfected cells were extracted and purified using the Qiagen RNeasy ® Mini kit (Qiagen, Hilden, Germany). Total RNA was amplified using the Low Input Quick Amp Labeling kit, One-Color (Agilent Technologies). For hybridization, each slide was hybridized with 1.65 µg Cy-3 labeled complementary RNA using the Gene Expression Hybridization kit (Agilent Technologies) in a Hybridization Oven (Agilent Technologies) according to the manufacturer's instructions. Subsequent to 17 h of hybridization, the slides were washed in staining dishes (Thermo Fisher Scientific, Waltham, MA, USA) with Gene Expression Wash Buffer (Agilent Technologies) according to the manufacturer's instructions. The slides were scanned at a 3-µm resolution using the green dye channel in an Agilent Microarray Scanner (Agilent Technologies). The data were read using Feature Extraction Software 10.7 (Agilent Technologies), and were normalized using Quantile Algorithm, Gene Spring 11.0 software (Agilent Technologies).
The data from three replicates were averaged. Genes were defined as differentially expressed if the intensity ratio (Cy5) was found to increase or decrease >2-fold and if the intensity ratio (Cy5) showed the same direction of change (upregulated or downregulated) in all three experimental repeats. Gene ontology (GO) and pathway analyses were performed using Database for Annotation, Visualization and Integrated Discovery v6.7 software (10,11).
Quantitative (q)PCR analysis. qPCR analysis using SYBR ® Green (Invitrogen Life Technologies) was performed in order to verify the results of the microarray analysis. Total RNA was extracted from the transfected cells. The RNA was reverse transcribed using Murine Leukemia Virus reverse transcriptase (Promega Corp., Madison, WI, USA). The expression of the 12 genes that were identified as being associated with apoptosis in the microarray analysis was determined using qPCR analysis with SYBR-Green I (Invitrogen Life Technologies). GAPDH was used as an internal control and distilled water was used as a negative control. The amplification reaction consisted of 10X PCR buffer, 1.25 units of JumpStart™ Taq (Sigma-Aldrich), 10 pmol forward and reverse primers, 0.2 µmol dNTP, 100 ng template and 0.2X SYBR-Green I (Amresco Inc., Solon, OH, USA) in a final volume of 50 µl. The reactions were performed using the StepOne TM Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA, USA). The mRNA expression of the 12 genes was normalized with GAPDH using the 2 -∆∆Ct method (12). The primer sequences used for GAPDH and the 12 genes were retrieved from PrimerBank (http://pga.mgh.harvard.edu/primerbank/).
Differential gene analysis. Using the human genome expression microarray, differential gene expression was detected in the HCECs and LoVo cells transfected with pReceiver-M03-VP1, pReceiver-M33-VP2 or pReceiver-M16-NS1 compared with those transfected with pReceiver-M03, pReceiver-M33 or pReceiver-M16, respectively. The number of upregulated and downregulated genes (P<0.05; false discovery rate <0.05; fold-change >2.0) are shown in Table I. The top five differential genes in the six groups are shown in Table II. The fold change of the differentially-expressed genes associated with colorectal cancer are shown in Table III.
GO analysis. The differentially-expressed genes were classified into different functional categories based on GO analysis for biological process, molecular function and cellular components. The primary GO categories for the upregulated genes in the HCECs and LoVo cells transfected with pReceiver-M03-VP1 included immune response, immune system process, defense response and response to stimulus, and for the downregulated genes was primarily cellular amino acid metabolic process. The predominant GO categories for the upregulated genes in the HCECs and LoVo cells transfected with pReceiver-M16-NS1 included organelle fission, nuclear division, mitosis, the M-phase of the mitotic cell cycle, the mitotic cell cycle, M-phase, cell cycle phase, cell cycle process and cell division (Table IV).
Pathway analysis. Significant pathways for the upregulated and downregulated differentially-expressed genes are shown in Table V. No pathways or specified pathways were found among the upregulated genes in the HCECs following Table III. Fold-change of differentially-expressed genes associated with colorectal cancer.

Confirmation of microarray results using qPCR analysis.
To verify the microarray analysis data, the expression of the 12 differentially-expressed genes selected using microarray analysis was confirmed by qPCR analysis in the different groups. Consistent results were observed with regard to the nine genes in the microarray and qPCR analysis data (Table VI).

Discussion
Despite our current understanding of the genetic alterations associated with the progression of colon cancer, the specific etiology of colorectal cancer has yet to be elucidated. Epidemiological studies have indicated that environmental factors and host immunological characteristics may contribute to the initiation and progression of colon cancer. Infectious agents, primarily viral infection, are acquired through the environment and have the potential to alter numerous regulatory processes, which may result in the development of colorectal cancer. Our previous study showed that B19 infection may cause colon carcinoma (6). However, little is known regarding the pathogenic mechanisms responsible for B19-induced tumorigenesis. B19 was discovered in 1974 and is the only Parvoviridae family member that is known to be pathogenic in humans. The genome of B19 has two large open reading frames encoding a single non-structural protein, NS1, and two capsid proteins, VP1 and VP2, which form an icosahedral capsid (5). The contribution of these viral proteins to B19 infectivity have yet to be experimentally demonstrated due to problems with in vitro culture and the lack of an infectious clone. Due to the difficulty in culturing B19 in vitro, little experimental evidence exists regarding the known and putative roles of B19 viral proteins in infectivity. In the present study, plasmids containing VP1, VP2 and NS1 were constructed and transfected into cultured HCECs and LoVo cells. Through the analysis of differentially-expressed genes and their functional enrichment, the present study aimed to identify potential targets to enable further investigation of the function of B19 in colon carcinoma, rather than to identify specific signaling pathways or molecules leading to colon carcinoma in which B19 participated.
Current understanding of the B19 viral proteins is primarily based on studies of other parvoviruses. The B19 NS protein is a multifunctional protein, for which sequence analysis has revealed that, in addition to transregulation of the p6 promoter (13,14), NS contains motifs for nucleoside triphosphate (NTP) binding and hydrolysis (15) associated with helicase activity, thus indicating a role for the protein in B19 DNA replication. A previous study has also indicated that the NTP-binding motifs of NS play roles in the induction erythroid lineage cell apoptosis during B19 infection (16). VP2 is the major capsid protein, comprising 95% of the capsid and 58-kDa in size (17). Previous studies in insect cells have reported that VP2 can self-assemble into virus-like particles (17) and that it is capable of binding directly to blood group P antigen, which is the cellular receptor of B19 (18). VP1 is the minor capsid protein, which has an identical amino acid sequence to VP2, plus an extra 227 amino acids termed the VP1-unique region (VP1u)) at the amino terminus (19). Previous studies have shown that the VP1u, which is found on the exterior of the capsid, contains the primary neutralizing epitopes of B19 (20)(21)(22). Furthermore, a conserved phospholipase A2 motif has been identified in the VP1u of members of the Parvoviridae family, including B19 (23,24). Two small 7.5-and 11-kDa proteins, are encoded by the small abundant mRNA of B19 (25)(26)(27) and are unique among those parvoviruses that have so far been characterized. A number of proline-rich motifs are contained within the 11-kDa protein and are conserved to the Src homology 3 binding domain of eukaryotic proteins (28); however, the function of the 7.5-and 11-kDa proteins in B19 replication and/or pathogenesis has yet to be elucidated. In the present study, plasmids containing VP1, VP2 and NS1 were constructed for transfection into cultured HCECs and LoVo cells. Hundreds of differentially-expressed genes were identified in the HCECs and LoVo cells following VP1, VP2 and NS1 protein expression in different ontological pathways and functional GO groups. GO analyses revealed that the significant VP1-related ontology categories included that of immune response, immune system process, defense response and response to stimulus, while significant NS1-related ontology categories included organelle fission, nuclear division, mitosis, M-phase of the mitotic cell cycle, mitotic cell cycle, M-phase, cell cycle phase, cell cycle process and cell division. Pathway expression analysis identified that VP1-related pathways included cell adhesion molecules, antigen processing and presentation, cytokines and inflammatory response. Pathway expression analysis identified that NS1-related pathways included cell cycle, pathways in cancer, colorectal cancer, the wnt signaling pathway and focal adhesion. The functional GO categories and pathways associated with VP1 and NS1 that were identified in the present study were consistent with the functions of VP1 and NS1 reported previously (6,9,13,14,(16)(17)(18)(19)(20)(21)(22)(23)(28)(29)(30). This indicates that NS1 has a significant role in the pathogenesis of B19 in colorectal carcinoma.