SciELO - Scientific Electronic Library Online

 
vol.52 issue8MicroRNA-383-5p inhibits the progression of gastric carcinoma via targeting HDAC9 expressionAssociation between BDNF G196A (Val66Met) polymorphism and cognitive impairment in patients with Parkinson's disease: a meta-analysis author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Brazilian Journal of Medical and Biological Research

Print version ISSN 0100-879XOn-line version ISSN 1414-431X

Braz J Med Biol Res vol.52 no.8 Ribeirão Preto  2019  Epub July 29, 2019

http://dx.doi.org/10.1590/1414-431x20198522 

Research Article

Gene expression profiling reveals upregulated FUT1 and MYBPC1 in children with pancreaticobiliary maljunction

1Department of Radiology, Children's Hospital of Soochow University, Suzhou, China

2Clinical Laboratory, the 3rd Hospital of Yulin, Yulin, China

3Department of Pediatric Surgery, Children's Hospital of Soochow University, Suzhou, China

ABSTRACT

Pancreaticobiliary maljunction (PBM) is associated with high risk of epithelial atypical growth and malignant transformation of the bile duct or gallbladder. However, overall changes in genetic expression have not been examined in children with PBM. Genome-wide expression was analyzed using peripheral blood samples from 10 children with PBM and 15 pediatric controls. Differentially expressed genes (DEGs) were identified using microarray. Bioinformatics analysis was conducted using Gene Ontology and KEGG analyses. The top 5 in the up-regulated genes in PBM were verified with qRT-PCR. Receiver operator characteristic curve analysis was conducted to evaluate the predictive accuracy of selected genes for PBM. The microarray experiments identified a total of 876 DEGs in PBM, among which 530 were up-regulated and the remaining 346 were down-regulated. Verification of the top 5 up-regulated genes (TYMS, MYBPC1, FUT1, XAGE2, and GREB1L) by qRT-PCR confirmed the up-regulation of MYBPC1 and FUT1. Receiver operating characteristic curve analysis suggested that FUT1 and MYBPC1 up-regulation could be used to predict PBM, with the area under the curve of 0.873 (95%CI=0.735−1.000) and 0.960 (95%CI=0.891−1.000), respectively. FUT1 and MYBPC1 were up-regulated in children with PBM, and could be used as potential biomarkers for PBM.

Key words: Pediatric; Pancreaticobiliary maljunction; Gene expression

Introduction

Pancreaticobiliary maljunction (PBM) is a congenital anomaly characterized by the junction of the pancreatic and biliary ducts outside the duodenal wall (1,2). Due to the lack of control of pancreaticobiliary junction by the sphincter of Oddi, reciprocal reflux of bile and pancreatic juice occurs. Reflux of pancreatic juice into the bile duct damages the bile duct wall and could lead to bile duct dilatation (3,4). PBM is often accompanied by pancreatitis, cholestasis, stone formation, epithelial hyperplasia, and malignant transformation in the biliary tract (5 67). Once a diagnosis of PBM is established, prophylactic surgery (mostly in the form of cyst excision and Roux-en-Y hepaticojejunostomy) is recommended to prevent carcinogenesis (8,9).

In a large case series report by Tashiro et al. (10), 11% of the PBM patients had cancer in the biliary system, 2/3 in the gallbladder and the remaining 1/3 in the bile duct. Such a trend is more pronounced in PBM without choledochal cysts. Kaneko et al. (11) reported association of certain genes with the development of gallbladder cancer in PBM. In the current study, we examined whether gene expression profile could be used for early detection of PBM itself. Specifically, we conducted genome-wide transcriptional profiling using blood-derived mRNA followed by real-time PCR verification in 10 children with PBM versus 15 control cases.

Material and Methods

Study subjects

The study protocol was approved by the Institutional Review Board of the Children's Hospital of Soochow University (No. 20160106011), in compliance with the Declaration of Helsinki. There was no ethical/legal conflict involved in the study. Written informed consent was obtained from the legal guardians of all subjects.

RNA extraction

Peripheral blood mononuclear cells were isolated, and then stored at –80°C before RNA extraction using TRIZOL (Invitrogen, USA).

Microarray analysis

Microarray experiments were conducted by Shanghai KangChen Biotech (China; http: //http:/www.kangchen.com.cn) with Agilent Human 4x44K Gene Expression Microarray chips with >44,000 probes using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol.

Real-time reverse transcription-polymerase chain reaction assay

Real-time quantitative PCR was used to determine the level of mRNAs for the TYMS, MYBPC1, FUT1, XAGE2, and GREB1L genes using a SYBR Green PCR Kit (Applied Biosystems, USA). The sequence of primers is listed in Table 1. The relative expression of gene transcript was calculated using the 2−ΔΔCt method, and was normalized to β-actin mRNA.

Table 1. Upstream and downstream primer sequences. 

Gene name Primer sequences Annealing temperature (°C) Product length (bp)
β-actin (H) F: 5′ GTGGCCGAGGACTTTGATTG 3′R: 5′ CCTGTAACAACGCATCTCATATT 3′ 60 73
MYBPC1 F: 5′ GACTGGACCCTTGTCGAAACT 3′R: 5′ TCTTCACCAACTTTCACTGTTCC 3′ 60 119
FUT1 F: 5′ GACATTGGCTAAGCCTTGA 3′R: 5′ AAGATCAGGCTACTTCAGAAAG 3′ 60 53
GREB1L F: 5′ GGACAAGCGATTTCTACCA 3′R: 5′ TCTTTCTCCACAGCCGATA 3′ 60 82
XAGE2 F: 5′ TAGGCCAAGAAGAAGTTTACAG 3′R: 5′ CATCAGTGGGTTCAAGCATAG 3′ 60 62
TYMS F: 5′ GTGCATTTCAATCCCACG 3′R: 5′ GACGAATGCAGAACACTTCT 3′ 60 216

H: human.

Statistical analysis

SPSS 20.0 software (IBM, USA) was used for statistical analysis. Continuous variables are reported as means±SD, and were analyzed using the rank sum test. Receiver operator characteristic (ROC) curve analysis was conducted and area under the ROC curve (AUC) was used to evaluate the predictive accuracy of selected genes for PBM. P<0.05 was considered statistically significant.

Results

Demographic and clinical data

A total of 10 pediatric PBM cases (age: 65.2±36.6 months) and 15 controls (age: 74.1±38.8 months) were included in the study. Demographic and clinical data are summarized in Table 2.

Table 2. Demographic and clinical characteristics of pediatric pancreaticobiliary maljunction. 

Variables Todani types I (n=7) Todani types IV (n=3)
Abdominal pain 6 3
Jaundice 4 1
Mass 1 0
Fever 2 1
Vomiting 4 1
Gender (male) 2 0
Age (months) 22−105 60−85
Pathological findings
Cyst wall hyperplasia 7 3
Gallbladder wall congestion 7 3

Differential gene expression

The microarray experiment identified a total of 876 differentially expressed genes (DEGs; cutoff at 2-fold): 530 were up-regulated and the remaining 346 were down-regulated in subjects with PBM. The heat maps showed distinct gene expression profile in PBM compared with the normal control (Figure 1). Homogeneity within the 2 groups was fair.

Figure 1. Hierarchical clustering showing mRNA expression profile between the two groups and homogeneity within each group (blue: pancreaticobiliary maljunction (PBM); red: control). Red and green represent up-regulated and down-regulated genes in the PBM group. 

GO and pathway analysis

The results of the Gene Ontology (GO) analysis for up-regulated genes is shown in Figure 2. We also mapped the 876 DEGs to the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways to further identify target mRNAs and their common cellular processes. The pathways most common to the up-regulated genes are shown in Figure 3. Major pathways targeted by the up-regulated genes included those implicated in metabolism, receptor interaction, adhesion, and cancer pathway.

Figure 2. Analysis of the significant Gene Ontology terms (molecular function, cell component, and biological process analyses) for up-regulated genes. 

Figure 3. KEGG pathway analysis. The first ten pathways that exhibited significant differences (differentially expressed (DE) genes) between the 2 groups (P<0.005) are listed (up-regulated mRNAs). Seventeen genes known to be related to cancer were observed in pancreaticobiliary maljunction. 

qRT-PCR validation

Up-regulation of TYMS, MYBPC1, FUT1, XAGE2, and GREB1L, as suggested by the microarray experiments, were verified by qRT-PCR. Up-regulation of MYBPC1 and FUT1 were successfully validated by qRT-PCR (P<0.01). qRT-PCR failed to validate the preliminary findings with TYMS, XAGE2, and GREB1L (Figure 4). The AUC under ROC curve was 0.960 and 0.873 for MYBPC1 and FUT1, respectively (Figure 5).

Figure 4. qRT-PCR validation of 5 selected differentially expressed genes in pancreaticobiliary maljunction (PBM). Each spot represents the gene expression value (corrected by β-actin housekeeping gene (ΔΔCt)) of an individual patient (n=10). Midline represents the mean. **P<0.001 (rank sum test). CTRL: control. 

Figure 5. Receiver operating characteristic curve of MYBPC1 and FUT1 as biomarkers for pancreaticobiliary maljunction (PBM). Area under the curve = 0.960 and 0.873, respectively. 

Discussion

Previous studies using a genome-wide approach identified altered gene expressions in the biliary tract of children with choledochal cyst/PBM. For example, Kaneko et al. examined genome-wide expression in gallbladder epithelia obtained from 6 children with PBM versus 4 pediatric controls using microarray analysis followed by RT-PCR verification (11). They found several dysregulated genes that may contribute to the pathophysiology of PBM. Wong et al. (12) conducted exome-sequencing in 31 pedigrees of congenital bile duct dilatation of the bile-duct cases and found association of six genes carrying damaging de novo variants with human developmental disorders involving epithelial and four linked with cholangio- and hepatocellular carcinomas.

In the present study, a group of 13 up-regulated genes with varying functional importance in the PBM group was identified. The top 5 on this list (TYMS, MYBPC1, FUT1, XAGE2, and GREB1L) were examined using RT-PCR. The RT-PCR experiments confirmed the up-regulation of MYBPC1 and FUT1, but not TYMS, XAGE2, and GREB1L.

FUT1 has been shown to be over-expressed in both human and rat colon cancers (13,14). A previous study by Zhang et al. (15) reported that suppressing the expression of FUT1/4 by RNAi technology reduces the synthesis of LeY and inhibits cancer growth. The current study showed up-regulation of FUT1 in PBM patients. Whether such an increase reflects the propensity of carcinogenesis requires further study.

TYMS is located on chromosome 18p11.32, and is composed of six introns with sizes ranging from 507 to 6271 bp and seven exons with sizes ranging from 72 to 250 bp (16 1718). TYMS encodes for thymidylate synthase (TS), an enzyme indispensable for DNA synthesis and repair (19). TS level is under strict control and can be modified by genetic variations in the TYMS gene. Decreased TS activity decreases folate level and DNA repair capability, and consequently promotes carcinogenesis (20,21). Burdelski et al. reported that high TYMS expression is significantly associated with unfavorable tumor phenotypes, rapid tumor cell proliferation, and early recurrence of prostate cancer (22). In the present study, higher TYMS expression in PBM patients was found with microarray, but not confirmed with RT-PCR. Such a discrepancy may be due to the relatively small sample size, and remains to be verified in future studies.

MYBPC1 encodes a member of the myosin binding protein-C family. Geist and Kontrogianni-Konstantopoulos (23) reported that MYBPC1 and its isoforms interact with thick and thin filaments to regulate the cycling of actomyosin cross bridges. A previous study from this research group found higher expression of phosphorylated myosin regulatory light chain in the common bile duct in pediatric PBM (24), suggesting that MYBPC1 up-regulation is a consequence of hypertrophy and fibrosis in common bile duct of PBM. MYBPC1 up-regulation identified in PBM patients in the current study suggests that MYBPC1 up-regulation could be used to predict pediatric PBM.

The current study has several limitations. First, the sample size is relatively small. Second, only five DEGs identified with microarray experiments were validated with RT-PCR. Third, only the top 5 up-regulated genes were validated by RT-PCR. Future studies with larger sample sizes and in-depth verification of more candidate genes using more definitive methods such as western blot are needed. Also, the relationship between genetic profiles and damage to the bile duct epithelium and eventual carcinogenesis requires further investigation.

In conclusion, FUT1 and MYBPC1 are up-regulated in children with PBM, suggesting myosin dysfunction. More detailed genetic profiling and functional analysis are warranted.

Acknowledgments

This work was supported by Jiangsu Province Health and Family Planning Projects (H201519 and H201520), Jiangsu Province Social Development Program - Standardized Diagnosis and Treatment of Key Disease in Clinic (BE2015643), and Science and Technology Development Project of Suzhou (SS201868).

References

1. Kamisawa T, Ando H, Suyama M, Shimada M, Morine Y, Shimada H, et al. Japanese clinical practice guidelines for pancreaticobiliary maljunction. J Gastroenterol 2012; 47: 731–759, doi: 10.1007/s00535-012-0611-2. [ Links ]

2. Kamisawa T, Ando H, Shimada M, Hamada Y, Itoi T, Takayashiki T, et al. Recent advances and problems in the management of pancreaticobiliary maljunction: feedback from the guidelines committee. J Hepatobiliary Pancreat Sci 2014; 21: 87–92, doi: 10.1002/jhbp.8. [ Links ]

3. Singham J, Yoshida EM, Scudamore CH. Choledochal cysts: part 1 of 3: classification and pathogenesis. Can J Surg 2009; 52: 434–440. [ Links ]

4. Dabbas N, Davenport M. Congenital choledochal malformation: not just a problem for children. Ann R Coll Surg Engl 2009; 91: 100–105, doi: 10.1308/003588409X391947. [ Links ]

5. Tsuchiya R, Harada N, Ito T, Furukawa M, Yoshihiro I. Malignant tumors in choledochal cyst. Ann Surg 1997; 186: 22–28, doi: 10.1097/00000658-197707000-00004. [ Links ]

6. Takuma K, Kamisawa T, Hara S, Tabata T, Kuruma S, Chiba K, et al. Etiology of recurrent acute pancreatitis with special emphasis on pancreaticobiliary malformation. Adv Med Sci 2012; 57: 244–250, doi: 10.2478/v10039-012-0041-7. [ Links ]

7. Csendes A, Kruse A, Funch-Jensen P, Oster MJ, Ornsholt J, Amdrup E. Pressure measurements in the biliary and pancreatic duct systems in controls and in patients with gallstones, previous cholecystectomy, or common bile duct stones. Gastroenterology 1979; 77: 1203–1210, doi: 10.1016/0016-5085(79)90158-6. [ Links ]

8. Ishibashi H, Shimada M, Kamisawa T, Fujii H, Hamada Y, Kubota M, et al. Japanese clinical practice guidelines for congenital biliary dilatation. J Hepatobiliary Pancreat Sci 2017; 24: 1–16, doi: 10.1002/jhbp.415. [ Links ]

9. Qiao G1, Li L, Li S, Tang S, Wang B, Xi H, et al. Laparoscopic cyst excision and Roux-Y hepaticojejunostomy for children with choledochal cysts in China: a multicenter study. Surg Endosc 2015; 29: 140–144, doi: 10.1007/s00464-014-3667-7. [ Links ]

10. Tashiro S, Imaizumi T, Ohkawa H, Okada A, Katoh T, Kawaharada Y, et al. Pancreaticobiliary maljunction: retrospective and nationwide survey in Japan. J Hepatobiliary Pancreat Surg 2003; 10: 345–351, doi: 10.1007/s00534-002-0741-7. [ Links ]

11. Kaneko K, Ito Y, Ono Y, Tainaka T, Tsuchiya H, Shimoyama Y, et al. Gene expression profiling reveals upregulated UCA1 and BMF in gallbladder epithelia of children with pancreaticobiliary maljunction. J Pediatr Gastroenterol Nutr 2011; 52: 744–750, doi: 10.1097/MPG.0b013e318214bd30. [ Links ]

12. Wong JK, Campbell D, Ngo ND, Yeung F, Cheng G, Tang CS, et al. Genetic study of congenital bile-duct dilatation identifies de novo and inherited variants in functionally related genes. BMC Med Genomics 2016; 9: 75, doi: 10.1186/s12920-016-0236-z. [ Links ]

13. Sun J, Thurin J, Cooper HS, Wang P, Mackiewicz M, Steplewski Z, et al. Elevated expression of H type GDP-L-fucose: beta-Dgalactoside alpha-2-L-fucosyltransferase is associated with human colon adnocarcinoma progression. Proc Natl Acad Sci USA 1995; 92: 5724–5728, doi: 10.1073/pnas.92.12.5724. [ Links ]

14. Cordel S, Goupille C, Hallouin F, Meflah K, Le Pendu J. Role for alpha1,2-fucosyltransferase and histo-blood group antigen H type 2 in resistance of rat colon carcinoma cells to 5-fluorouracil. Int J Cancer 2000; 85: 142–8, doi: 10.1002/(SICI)1097-0215(20000101)85:1<142::AID-IJC24>3.0.CO;2-K. [ Links ]

15. Zhang Z, Sun P, Liu J, Fu L, Yan J, Liu Y, et al. Suppression of FUT1/FUT4 expression by siRNA inhibits tumor growth. Biochim Biophys Acta 2008; 1783: 287–296, doi: 10.1016/j.bbamcr.2007.10.007. [ Links ]

16. Nazki FH, Masood A, Banday MA, Bhat A, Ganai BA. Thymidylate synthase enhancer region polymorphism not related to susceptibility to acute lymphoblastic leukemia in the Kashmir population. Genet Mol Res 2012; 11: 906–917, doi: 10.4238/2012.April.10.6. [ Links ]

17. Kaneda S, Nalbantoglu J, Takeishi K, Shimizu K, Gotoh O, et al. Structural and functional analysis of the human thymidylate synthase gene. J Biol Chem 1990; 265: 20277–20284. [ Links ]

18. Ghosh S, Hossain MZ, Borges M, Goggins MG, Ingersoll RG, Eshleman JR, et al. Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies. PLoS One 2012; 7: e34426, doi: 10.1371/journal.pone.0034426. [ Links ]

19. Welsh SJ, Titley J, Brunton L, Valenti M, Monaghan P, Jackman AL, et al. Comparison of thymidylate synthase (TS) protein up-regulation after exposure to TS inhibitors in normal and tumor cell lines and tissues. Clin Cancer Res 2000; 6: 2538–2546. [ Links ]

20. Lima A, Azevedo R, Sousa H, Seabra V, Medeiros R. Current approaches for TYMS polymorphisms and their importance in molecular epidemiology and pharmacogenetics. Pharmacogenomics 2013; 14: 1337–1351, doi: 10.2217/pgs.13.118. [ Links ]

21. Shi Q, Zhang Z, Neumann AS, Li G, Spitz MR, Wei Q. Case-control analysis of thymidylate synthase polymorphisms and risk of lung cancer. Carcinogenesis 2005; 26: 649–656, doi: 10.1093/carcin/bgh351. [ Links ]

22. Burdelski C, Strauss C, Tsourlakis MC, Kluth M, Hube-Magg C, Melling N, et al. Overexpression of thymidylate synthase (TYMS) is associated with aggressive tumor features and early PSA recurrence in prostate cancer. Oncotarget 2015; 6: 8377–8387, doi: 10.18632/oncotarget.3107. [ Links ]

23. Geist J, Kontrogianni-Konstantopoulos A. MYBPC1, an emerging myopathic gene: what we know and what we need to learn. Front Physiol 2016; 7: 410, doi: 10.3389/fphys.2016.00410. [ Links ]

24. Guo WL, Zhang Q, Wang J, Jin MF. Higher expression of phosphorylated myosin regulatory light chain in the common bile duct in pancreaticobiliary maljunction accompanied by bile duct dilatation in children: a post-mortem observational study. Pediatr Surg Int 2013; 29: 293–298, doi: 10.1007/s00383-012-3225-0. [ Links ]

Received: January 26, 2019; Accepted: May 29, 2019

Correspondence: Jian Wang: <wangjiansuzhou@163.com>

*These authors contributed equally to this study.

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.