OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2014.1910 ol-07-05-1537 Articles Overexpression of collagen VI α3 in gastric cancer XIEXIAOJUN LIUXIAOSUN ZHANGQING YUJIREN Department of Gastrointestinal Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China Correspondence to: Professor Jiren Yu, Department of Gastrointestinal Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang 310003, P.R. China, E-mail: yujr0909@zju.edu.cn 5 2014 25 02 2014 7 5 1537 1543 26 06 2013 07 01 2014 Copyright © 2014, Spandidos Publications 2014 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Collagen VI is significant in the progression of numerous types of cancer. Type VI collagen consists of three α-chains and collagen VI α3 (COL6A3) encodes the α3 chain. The overexpression of COL6A3 has been demonstrated to correlate with high-grade ovarian cancer and contributes to cisplatin resistance; however, its role in human gastric cancer (GC) remains unclear. Using microarray meta-analysis, COL6A3 was observed to be frequently overexpressed in the GC tissues, furthermore, this overexpression was identified in five GC cell lines. A microarray-based co-expression network analysis was conducted and identified a total of 62 genes that were co-expressed with COL6A3, with the majority of the genes being involved in cancer-related processes, such as cell differentiation, migration and adhesion. Network analysis of these 62 genes demonstrated that fibronectin 1, a well-characterized oncogene, was located at the center of the COL6A3 co-expression network. Therefore, COL6A3 may act as an oncogene in human GC and the antagonism of COL6A3 may be an effective therapeutic treatment for GC.

 collagen VI α3 microarray meta-analysis gastric cancer Introduction

Gastric cancer (GC) is the fourth most common type of malignancy worldwide, which results in 989,600 novel cases and 738,000 fatalities annually, specifically in Asian countries (1). Recent advancements in diagnosis and treatment modalities have been made, however, the prognosis of GC patients remains poor. As current therapeutic strategies are insufficient and do not achieve complete tumor ablation, it is important to analyze the molecular mechanisms of GC and identify novel biomarkers, as well as targets for therapeutic approaches, which may improve the clinical outcome for GC patients.

Collagen VI was initially identified as an extracellular matrix protein. It forms a microfilament network and binds to extracellular matrix proteins via its functional subdomains, which is important for the organization of fibrillar collagens and adhesion to the basement membrane (2). Collagen VI has recently attracted interest due to its involvement in breast and ovarian cancers (3–5). It is composed of three distinct α-chains (α1, -2 and -3) and collagen VI α3 (COL6A3) encodes the α3 chain, which is markedly longer than the other two chains (6). In a previous study, COL6A3 was shown to be upregulated in ovarian cancer (7), and Sherman-Baust et al (5) identified that the expression of COL6A3 was correlated with cisplatin resistance in ovarian cancer cell lines. Furthermore, highly or moderately differentiated ovarian tumors expressed lower levels of COL6A3 than poorly differentiated tumors, which indicated that the expression of COL6A3 was associated with the grade of the ovarian tumor (5). A recent exon array analysis study demonstrated that an alternative long isoform of COL6A3 was expressed, almost exclusively, in cancer samples, and may potentially serve as a novel cancer biomarker (8). Currently, the majority of studies relating to the oncogenic role of this gene focus on ovarian and breast cancer, however, the expression pattern and the biological functions of COL6A3 in human GC remain unknown.

In the present study, the authors investigated whether the expression level of COL6A3 was altered in GC, and a microarray meta-analysis was performed in order to assess the functional characteristics and molecular mechanisms of COL6A3 in GC.

 Materials and methods Gene expression patterns in GC

The Oncomine database (http://www.oncomine.org) was used to examine the differences in the transcriptional profiles between GC tissues and the adjacent normal tissues (9). Only the datasets that contained cancer versus normal analysis at the mRNA expression level were selected for analysis in the present study. In total, four GeneChip datasets, consisting of 318 paired GC and non-cancerous tissues, were selected according to the criteria shown in Table I.

 Cell culture

Five human GC cell lines (AGS, HGC-27, BGC-823, SGC-7901 and MGC80-3) and one immortalized gastric cell line (GES-1) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). All cell lines were incubated in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Carlsbad, CA, USA) with 10% fetal bovine serum (SAFC Biosciences Inc., Lenexa, KS, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

 Quantitative polymerase chain reaction (qPCR) analysis

TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to extract the total RNA from whole cells, and reverse-transcription was conducted using a TaqMan® Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The DNA was amplified using an ABI® 7500 Real-Time PCR system (Applied Biosystems) and SYBR Premix Ex Taq (Takara, Kusatsu, Japan). The ΔΔCt method was used to calculate the relative RNA expression, which was normalized to GAPDH expression. PCR was performed using the following primers: forward, 5′-GAGACGCAGTGAGTGGGAAA-3′ and reverse, 5′-AGAGTCTTGTGCTGCTTGCT-3′ for COL6A3; and forward, 5′-CTCTCTGCTCCTCCTGTTCGAC-3′ and reverse, 5′-TGAGCGATGTGGCTCGGCT-3′ for GAPDH.

 Co-expression analysis

The Oncomine database co-expression analysis tool was used to conduct the co-expression analysis of the microarray datasets. Using the co-expression score, the top 150 genes of each dataset were selected. The genes that appeared in at least two of the three datasets were defined as COL6A3 co-expressed genes.

 Gene ontology (GO) and pathway enrichment analysis

GO and pathway enrichment analysis were conducted to examine COL6A3 co-expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov/). The categories, GOTERM_BP_3, GOTERM_CC_2 and GOTERM_MF_3 were selected, and the other options were set as defaults.

 Construction of the gene interaction network

The gene interaction network was constructed using a gene expression pattern scanner (GePS: http://www.genomatix.de/) as described previously (10).

 Statistical analysis

The independent Student’s t test was used to analyze the differences between two groups. Statistical analysis was performed using SPSS software version 16.0 (SPSS, Chicago, IL, USA). Data are presented as the means ± SD. P<0.05 was considered to indicate a statistically significant difference.

 Results COL6A3 is commonly overexpressed in GC

To determine the changes in the transcriptional pattern of GC cells, microarray datasets from the studies by Chen et al (11), Cho et al (12), D’Errico et al (13) and Wang et al (14) were analyzed using the Oncomine database. COL6A3 demonstrated a significant overexpression in the GC cells (P=3.98×10−15; Fig. 1A). To confirm this finding, the expression of COL6A3 in one immortalized gastric cell line (GES-1) and five GC cell lines (AGS, HGC-27, BGC-823, SGC-7901, MGC80-3) was analyzed using qPCR. The five GC cell lines exhibited ≥2.5-fold overexpression of COL6A3 compared with that of GES-1 cells (Fig. 1B).

 Genes co-expressed with COL6A3

A previous study indicated that genes which are co-expressed in different conditions may be functionally related or co-regulated (15). Therefore, a microarray co-expression analysis was conducted to identify the genes that were co-expressed with COL6A3. The dataset from the study by D’Errico et al (13) did not contain any co-expression data, therefore, the other three datasets consisting of 249 paired tissues were selected for inclusion in the co-expression analysis. Using a cut-off of the top 150 genes, which were identified by the co-expression score from each dataset, and with at least two appearances on the co-expressed list, 62 genes were identified as genes that were co-expressed with COL6A3 (Table II).

 GO and pathway enrichment analysis of COL6A3 co-expressed genes

GO and pathway enrichment analysis were conducted using the DAVID functional annotation chart tool (16) to further analyze the underlying mechanisms of COL6A3 and its co-expressed genes. In total, 36 biological process, seven cellular constituents, seven molecular function terms and six Kyoto encyclopedia of genes and genomes pathways were indicated to be significantly enriched (P<0.01; Table III). The extracellular matrix organization indicated the most marked enrichment among the GO biological process terms. The predominant function of COL6A3 has been identified to be the organization of matrix components, which supported the reliability of the present analysis. Furthermore, cell processes, such as cell differentiation, cell-substrate adhesion, regulation of cell proliferation, regulation of cell migration, cell motion and cell migration, which are considered to be cancer-related biological processes, were enriched (Fig. 2). This result indicated that COL6A3 may have been involved in the biological processes that promote the progression of GC.

 Network analysis of COL6A3

A network analysis was conducted using Genomatix GePS to construct the functional connections of COL6A3 co-expressed genes. FN1 was highlighted in this network, as it functionally associated with 50 (81.9%) COL6A3 co-expressed genes, which indicated that FN1 may act as a significant regulator in the COL6A33 regulatory network (Fig. 3).

 Discussion

COL6A3 is located on chromosome 2q37 and codes for the α-3 chain, one of the three α-chains of type VI collagen. It is hypothesized that COL6A3 accelerates cell anchoring and signaling through its interaction with integrin (17) and disruption of this gene results in muscular dystrophy (2). In addition to integrin, COL6A3 interacts with other matrix components, such as decorin, hyaluronan, heparan sulfate and NG2 proteoglycans (18). Furthermore, COL6A3 may promote neural crest cell migration and attachment, which is significant in the later stages of neural crest development (19).

Recently, COL6A3 has received increasing attention, due to its abnormal expression and the occurrence of alternative splicing in numerous types of cancer. Previous genome exon array studies have identified cancer-specific alternative splicing of exons 3, 4 and 6 of COL6A3 in colon, pancreatic, bladder and prostate cancer (8,20). Furthermore, COL6A3 was identified to be overexpressed in pancreatic (21) and ovarian cancer (7), which was associated with the poor differentiation of tumor cells (5). Although COL6A3 has been investigated in numerous other types of cancer, its biological mechanisms and expression pattern in GC remain unclear.

In the era of post-genomic medicine, microarray meta-analysis has been demonstrated to be an effective strategy for identifying gene expression changes in various types of cancer (22,23). In the present study, a microarray meta-analysis was performed to identify that COL6A3 was frequently overexpressed in hepatocellular carcinoma tissues, indicating that an increased expression of COL6A3 was associated with the carcinogenesis of GC. The underlying mechanisms that result in the increased expression of COL6A3 may relate to the transcriptional regulation of transforming growth factor (TGF)-β (24), however, this requires further investigation. To further define the biological mechanisms of COL6A3, a co-expression analysis was conducted to investigate the genes that are functionally related to, or co-regulated by, COL6A3. This identified 62 co-expression genes for COL6A3, the majority of which are involved in the processes of extracellular matrix organization such as lysyl oxidase, collagen type IV α2, TGF-β-induced and laminin γ1 (Table II). The functional network analysis of these co-expression genes was dominated by FN1, which demonstrated its predominant functional connections with other genes. FN1 is an adhesive protein of the extracellular matrix and it contains two apparently identical subunits with a range of binding sites for cell surface and extracellular ligands. It has been indicated that FN1 is involved in various aspects of cancer-related biological processes, such as cellular adhesion and migration. FN1 was identified to be overexpressed in hepatocellular, gastrointestinal, head and neck cancers (25,26), which indicated its involvement in tumorigenesis. Furthermore, Waalkes demonstrated that advanced-stage renal cancer patients exhibited increased FN1 expression when compared with patients exhibiting organ-confined diseases (27). Thus, the present study provided a mechanistic insight into the role of COL6A3 in GC.

In conclusion, the present study indicated that COL6A3 was regularly overexpressed in GC cells. A list of potential partner genes of COL6A3 was generated, the majority of which are involved in cancer-related processes, and a functional network of COL6A3 was constructed, which provided promising results to enable future studies to identify the precise role of COL6A3.

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COL6A3 was overexpressed in gastric carcinoma tissue. (A) The expression pattern of COL6A3 in four GC datasets that were obtained using the Oncomine database; whiskers, 10th and 90th percentile; box boundaries, 75th and 25th percentile; line within the box, median. *P<0.001. (B) Relative COL6A3 expression of five GC cell lines (HGC-27, MGC80-3, SGC-7901, BGC-823 and AGS) compared with the mean value of a normal GC cell line (GES-1). COL6A3; collagen VI α3; GC, gastric cancer.

Gene ontology analysis of collagen VI α3 co-expressed genes was conducted using the Database for Annotation, Visualization and Integrated Discovery functional annotation chart tool. *P<0.01 for the pathway enrichment of COL6A3 co-expressed genes compared with Homo sapiens transcriptome background.

Network construction of COL6A3 co-expressed genes. The biological interactions of COL6A3 co-expressed genes were analyzed and visualized using a gene expression pattern scanner. The category of each gene is distinguished by its shape for factors, such as kinases and transporters. The direction of the arrow demonstrates whether a gene is upstream or downstream of another gene. Dashed line, co-cited genes; solid line, genes with an expertly curated connection. Genes with no interactions are not shown.

Oncomine datasets obtained for use in the present study.

Dataset (Ref no.) Samples Data link
Chen Gastric (11) 103 gastric adenocarcinomas and 29 normal gastric mucosa samples http://genome-www.stanford.edu/gastric_cancer2/index.shtml
Cho Gastric (12) 65 gastric adenocarcinoma, 19 paired surrounding normal tissue and six gastrointestinal stromal tumor samples http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13861
D’Errico Gastric (13) 31 paired gastric carcinoma and adjacent normal gastric mucosa and seven unmatched gastric carcinoma samples http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13911
Wang Gastric (14) 12 paired gastric carcinoma and normal gastric mucosa samples and three normal gastric tissue samples http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19826

Collagen VI α3 co-expressed genes with the cut-off for selection defined as an appearance in two datasets.

Gene Gene name No. of appearances
COL6A3 Collagen type VI α3 3
COL1A2 Collagen type I α2 3
COL1A1 Collagen type I α1 3
COL12A1 Collagen type XII α1 3
THY1 Thy-1 cell surface antigen 3
THBS2 Thrombospondin 2 3
BGN Biglycan 3
CTHRC1 Collagen triple helix repeat containing 1 3
SULF1 Sulfatase 1 3
FAP Fibroblast activation protein-α 3
SFRP4 Secreted frizzled-related protein 4 3
TIMP1 Tissue inhibitor of metallopeptidase 1 3
WNT2 Wingless-type mouse mammary tumor virus integration site family member 2 3
COL11A1 Collagen type XI α1 3
BMP1 Bone morphogenetic protein 1 3
SPOCK1 Sparc/osteonectin cwcv and kazal-like domains proteoglycan (testican) 1 3
SERPINH1 Serpin peptidase inhibitor clade H (heat shock protein 47) member 1 (collagen binding protein 1) 2
CPXM1 Carboxypeptidase X (M14 family) member 1 2
INHBA Inhibin β A 2
CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 2
RAB31 Member of the RAS oncogene family 2
ANTXR1 Anthrax toxin receptor 1 2
NID2 Nidogen 2 (osteonidogen) 2
PDGFRB Platelet-derived growth factor receptor β polypeptide 2
COL4A2 Collagen type IV α2 2
COL4A1 Collagen type IV α1 2
TGFBI Transforming growth factor β-induced (68kDa) 2
PLAU Plasminogen activator urokinase 2
PRRX1 Paired related homeobox 1 2
LOX Lysyl oxidase 2
PLXDC2 Plexin domain containing 2 2
LAMC1 Laminin γ1 (formerly LAMB2) 2
OLFML2B Olfactomedin-like 2B 2
CLDN4 Claudin 4 2
FAM83D Family with sequence similarity 83, member D 2
ITGB5 Integrin β5 2
TNC Tenascin C 2
SNAI2 Snail family zinc finger 2 2
FRMD6 FERM domain containing 6 2
COL6A1 Collagen type VI α1 2
NUAK1 NUAK family, SNF1-like kinase 1 2
HSPG2 Heparan sulfate proteoglycan 2 2
NOTCH3 Notch 3 2
CD276 Cluster of differentiation 276 molecule 2
WNT5A Wingless-type mouse mammary tumor virus integration site family member 5A 2
ECM1 Extracellular matrix protein 1 2
PDPN Podoplanin 2
TNFAIP6 Tumor necrosis factor α-induced protein 6 2
ADAM12 A disintegrin and metallo-peptidase domain 12 2
GAS1 Growth arrest-specific 1 2
THBS1 Thrombospondin 1 2
COL10A1 Collagen type X α1 2
FNDC1 Fibronectin type III domain containing 1 2
SPHK1 Sphingosine kinase 1 2
MMP11 Matrix metallopeptidase 11 (stromelysin 3) 2
CST1 Cystatin SN 2
KRT80 Keratin 80 2
PMEPA1 Prostate transmembrane protein, androgen induced 1 2
SPP1 Secreted phosphoprotein 1 2
TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b 2
IGF2BP3 Insulin-like growth factor 2 mRNA binding protein 3 2
MFAP2 Microfibrillar-associated protein 2 2
EHD2 EH-domain containing 2 2

GO and pathway enrichment analysis of COL6A3 co-expressed genes.

Category Term Function Count P-value Fold enrichment FDR
GOTERM _BP_3 GO:0030198 ECM organization 11 5.88×10−12 26.86738026 8.02×10−9
GO:0048731 System development 29 2.30×10−9 3.161608227 3.13×10−6
GO:0048513 Organ development 24 2.54×10−8 3.507740409 3.47×10−5
GO:0009653 Anatomical structure morphogenesis 19 2.42×10−7 4.032045523 3.30×10−4
GO:0009888 Tissue development 14 9.88×10−7 5.347765641 1.35×10−3
GO:0022603 Regulation of anatomical structure morphogenesis 7 1.89×10−4 8.119324546 2.57×10−1
GO:0030154 Cell differentiation 17 3.01×10−4 2.637947926 4.10×10−1
GO:0051093 Negative regulation of developmental process 7 4.64×10−4 6.865374809 6.31×10−1
GO:0031589 Cell-substrate adhesion 5 5.47×10−4 12.96014632 7.43×10−1
GO:0051239 Regulation of multicellular organismal process 12 7.52×−4 3.253176537 1.02
GO:0048519 Negative regulation of biological process 17 9.44×10−4 2.383179224 1.28
GO:0050793 Regulation of developmental process 10 9.95×10−4 3.768825934 1.35
GO:0060348 Bone development 5 1.28×10−3 10.32597024 1.73
GO:0009887 Organ morphogenesis 9 1.33×10−3 4.053492573 1.80
GO:0006928 Cell motion 8 2.20×10−3 4.278212512 2.96
GO:0042127 Regulation of cell proliferation 10 2.90×10−3 3.227685742 3.88
GO:0032101 Regulation of response to external stimulus 5 3.27×10−3 7.988014715 4.36
GO:0002683 Negative regulation of immune system process 4 4.02×10−3 12.24187315 5.34
GO:0009611 Response to wounding 8 4.05×10−3 3.834247063 5.39
GO:0030334 Regulation of cell migration 5 4.06×10−3 7.515351122 5.40
GO:0016477 Cell migration 6 4.13×10−3 5.522149303 5.49
GO:0016337 Cell-cell adhesion 6 4.13×10−3 5.522149303 5.49
GO:0050865 Regulation of cell activation 5 4.60×10−3 7.257681941 6.08
GO:0008284 Positive regulation of cell proliferation 7 5.02×10−3 4.295005013 6.64
GO:0009790 Embryonic development 8 5.90×10−3 3.577730534 7.75
GO:0007566 Embryo implantation 3 5.94×10−3 25.40188679 7.80
GO:0044259 Multicellular organismal macromolecule metabolic process 3 6.33×10−3 24.58247109 8.30
GO:0040012 Regulation of locomotion 5 6.37×10−3 6.615074686 8.34
GO:0051272 Positive regulation of cell motion 4 6.39×10−3 10.36811706 8.36
GO:0040017 Positive regulation of locomotion 4 6.39×10−3 10.36811706 8.36
GO:0048870 Cell motility 6 6.46×10−3 4.964538135 8.46
GO:0051270 Regulation of cell motion 5 6.48×10−3 6.580799687 8.49
GO:0048523 Negative regulation of cellular process 14 8.98×10−3 2.142327802 11.57
GO:0050867 Positive regulation of cell activation 4 9.00×10−3 9.153833078 11.59
GO:0009792 Embryonic development ending in birth or egg hatching 6 9.13×10−3 4.563213196 11.76
GO:0032844 Regulation of homeostatic process 4 9.67×10−3 8.912942734 12.41
GOTERM_CC_3 GO:0031012 ECM 26 2.51×10−26 19.52139523 2.54×10−23
GO:0005578 Proteinaceous ECM 25 1.40×10−25 20.23702331 1.41×10−22
GO:0044420 ECM part 15 8.00×10−18 33.20947414 8.10×10−15
GO:0005581 Collagen 10 5.53×10−15 74.00968523 5.62×10−12
GO:0005604 Basement membrane 6 1.13×10−5 19.92568449 1.14×10−2
GO:0005615 Extracellular space 12 4.49×10−5 4.537820116 4.55×10−2
GO:0005886 Plasma membrane 25 3.82×10−3 1.71454791 3.80
GO:0031252 Cell leading edge 3 9.65×10−2 5.631171702 64.22
GOTERM_MF_3 GO:0019838 Growth factor binding 6 3.98×10−5 15.21982507 3.80×10−2
GO:0005518 Collagen binding 4 3.06×10−4 29.59410431 2.92×10−2
GO:0005102 Receptor binding 11 1.21×10−3 3.306790436 1.15
KEGG_PATHWAY hsa04512 ECM-receptor interaction 14 1.41×10−16 28.25 9.99×10−14
hsa04510 Focal adhesion 14 1.52×10−11 11.80597015 1.35×10−08

GO, gene ontolgy; COL6A3, collagen VI α3; FDR, false discovery rate; BP; biological process; CC, cellular constituent; ECM, extracellular matrix; MF, molecular function; KEGG, Kyoto encyclopedia of genes and genomes. P<0.01 indicated a statistically significant difference.