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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

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Front. Med.    2016, Vol. 10 Issue (3) : 278-285    https://doi.org/10.1007/s11684-016-0453-z
RESEARCH ARTICLE
iTRAQ-based quantitative analysis of cancer-derived secretory proteome reveals TPM2 as a potential diagnostic biomarker of colorectal cancer
Yiming Ma1,Ting Xiao1,Quan Xu2,Xinxin Shao2,Hongying Wang1,*()
1. State Key Laboratory of Molecular Oncology
2. Department of Gastrointestinal Cancer Surgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
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Abstract

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. We aimed to find novel molecules as potential biomarkers for the early diagnosis of CRC. A serum-free conditioned medium was successfully collected from three pairs of CRC tissue and adjacent normal tissue. iTRAQ-based quantitative proteomic analysis was applied to compare the differences in secretome between primary CRC mucosa and adjacent normal mucosa. A total of 145 kinds of proteins were identified. Of these proteins, 29 were significantly different between CRC and normal tissue. Tropomyosin 2 β (TPM2) exhibited the most significant differences; as such, this protein was selected for further validation. Quantitative real-time PCR indicated that the mRNA expression of TPM2 significantly decreased in the CRC tissue compared with the paired adjacent normal tissue. Immunohistochemical analysis also confirmed that TPM2 was barely detected at protein levels in the CRC tissue. In summary, this study revealed potential molecules for future biomarker applications and provided an efficient approach for the differential analysis of cancer-associated secretome. TPM2 may be valuable for the early diagnosis of CRC.
Keywords iTRAQ      secretome      colorectal cancer      TPM2     
Corresponding Author(s): Hongying Wang   
Just Accepted Date: 19 May 2016   Online First Date: 12 June 2016    Issue Date: 30 August 2016
 Cite this article:   
Yiming Ma,Ting Xiao,Quan Xu, et al. iTRAQ-based quantitative analysis of cancer-derived secretory proteome reveals TPM2 as a potential diagnostic biomarker of colorectal cancer[J]. Front. Med., 2016, 10(3): 278-285.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0453-z
https://academic.hep.com.cn/fmd/EN/Y2016/V10/I3/278
Patient No. Age (year) Gender Tumor location Differentiation grade TNM stage
1 53 F Rectum Medium T2N0M0
2 64 M Rectum Medium T3N1M0
3 76 M Rectum Medium T4N2M0
Tab.1  Description of human colorectal cancer samples used in secretome analysis
Fig.1  The conditional media from serum-free primary culture of CRC sample were separated by 10% SDS-PAGE and stained with Coomassie. N: adjacent normal tissue; T: tumor tissue.
Accession No. Gene name Gene symbol MW(Da) Change fold
IPI00910262 Periostin isoform 4 POSTN 83 850 9
IPI00220709 Tropomyosin β chain isoform 2 TPM2 32 989.8 -6.68
IPI00178352 Filamin-C FLNC 291 022 -6.02
IPI00550363 Transgelin TAGLN2 22 391 -4.86
IPI00024095 Annexin A3 ANXA3 36 375 4.03
IPI00337335 Myosin-14 MYH14 228 002 3.66
NA Unnamed protein product NA NA 3.32
IPI00010154 Human rab GDI GDI1 50 583 3.2
IPI00329801 Annexin A5 ANXA5 35 937 3.1
IPI00217966 L-lactate dehydrogenase A chain LDHA 36 689 3.07
NA Unnamed protein product NA NA 2.97
IPI00299301 Desmuslin SYNM 172 768 -2.97
IPI00554788 Cytokeratin 18 KRT18 48 058 2.55
IPI00218733 Cu/Zn-superoxide dismutase SOD1 15 936 2.43
IPI00169383 Phosphoglycerate kinase 1 PGK1 44 615 2.38
IPI00019502 Myosin-9 MYH9 226 532 2.31
IPI00014516 Caldesmon CALD1 93 250 -2.23
IPI00418471 Vimentin VIM 53 652 2.1
IPI00020501 Myosin heavy chain MHY11 227 339 -2.08
IPI00442073 Cysteine and glycine-rich protein 1 CSRP1 20 567 -2.07
IPI00797270 Triosephosphate isomerase TPI1 26 669 2.06
IPI00289334 Actin binding protein homolog ABP-278 FLNB 278 164 1.96
IPI00014898 Plectin PLEC 531 791 1.92
IPI00455315 Annexin A2 isoform 2 ANXA2 40 411 1.85
IPI00940464 Aminoacylase-1 ACY1 45 885 1.84
IPI00218918 Annexin A1 ANXA1 38 714 1.84
NA Unnamed protein product NA NA -1.8
IPI00646773 Gelsolin isoform b GSN 80 641 -1.55
IPI00011201 Mitochondrial malate dehydrogenase 2 ME2 65 444 1.52
Tab.2  Differentially expressed proteins identified in CM from CRC tissue and adjacent normal tissue
Fig.2  Gene ontology analysis of the proteins identified in the conditional medium. (A) The cellular component distribution of identified proteins. (B) The major biological processes enriched by DAVID. Negative log10 of P value is shown on the x-axis.
Fig.3  Validation of TPM2 expression in CRC and adjacent normal tissue by q-PCR and immunohistochemical staining. (A) qPCR analysis showed that the expression of TPM2 significantly decreased in CRC tissue. (B) qPCR analysis revealed that the expression of TPM2 significantly decreased in early-stage CRC tissue, especially T1, T2, and T3 stages. (C) Immunohistochemical staining of TPM2 in CRC tissue and adjacent normal tissue. TPM2 is mainly expressed by stromal cells, not epithelial cells, and the staining of TPM2 is rarely observed in CRC tissue. NC, negative control. (D) Statistical analysis of the immunostaining scores of the immunohistochemically stained TPM2 in 37 CRC samples. **, P<0.01; ***, P<0.001
n Negative Positive P
Gender 0.8064a
Female 13 11 2
Male 24 21 3
Age (year) 0.045b
>60 28 26 2
<60 9 6 3
Position <0.0001ac
Normal mucosa 37 4 33
Tumor 37 32 5
Primary tumor (T) stage 0.3067a
T1?T2 9 9 0
T3?T4 28 23 5
Lymph node (N) metastasis 0.1681a
N0 18 17 1
Nx 19 15 4
Distant metastasis (M) 0.5654a
M0 35 30 5
M1 2 2 0
Tab.3  Relationships between the TPM2 expression in primary CRC and clinical pathological factors
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