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Over-Expression of TNNI3K Is Associated with Early-Stage Carcinogenesis of Cholangiocarcinoma

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Over-Expression of TNNI3K Is Associated with Early-Stage Carcinogenesis of Cholangiocarcinoma Received: 7 August 2018 | Accepted: 5 October 2018 DOI: 10.1002/mc.22925 RESEARCH ARTICLE Over-expression of TNNI3K is associated with early-stage carcinogenesis of cholangiocarcinoma Chun-Nan Yeh1 | Ming-Huang Chen2 | Yu-Chan Chang3 | Ren-Chin Wu4 | Lee-Cheng Tsao5 | Shang-Yu Wang1 | Chi-Tung Cheng1,6 | Kun-Chun Chiang7 | Tsung-Wen Chen1 | Michael Hsiao3 | Wen-Hui Weng5 1 Department of Surgery, Liver Research Center, Chang Gung Memorial Hospital, Cholangiocarcinoma (CCA) is a devastating disease with very poor prognosis due to late Chang Gung University, Taoyuan, Taiwan diagnosis and resistance to traditional chemotherapies and radiotherapies. Herein, 2 Division of Hematology and Oncology, Department of Medicine, Taipei Veterans thioacetamide (TAA)-induced rat CCA model and CGCCA cell line were used; we aim to General Hospital, Taipei, Taiwan study the cytogenetic features during tumoral development of CCA and uncover the 3 Genomics Research Center, Academia Sinica, mystery regarding carcinogenesis of CCA. The Array comparative genomic hybridiza- Taipei, Taiwan tion analysis, in silico method, gene knockdown, Western blot, cell count proliferation 4 Department of Pathology, Chang Gung Memorial Hospital, Chang Gung University, assay, clonogenecity assay, and IHC staining were applied in this study. Array Taoyuan, Taiwan comparative genomic hybridization analysis was performed on all different TAA- 5 Department of Chemical Engineering and − Biotechnology and Graduate Institute of induced phases of rat tissues to reveal the certain pattern, +2q45, +Xq22, 12p12, Biotechnology, National Taipei University of have been identified for the tumor early stage, where involve the gene TNNI3K.In Technology, Taipei, Taiwan addition, 16 genes and 3 loci were associated with rapid tumor progression; JAK-STAT 6 Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan signaling pathway was highly correlated to late stage of CCA. In silico database was 7 Department of Surgery, Chang Gung used to observe TNNI3K was highly express at tumor part compared with normal Memorial Hospital, Kee-lung, Taiwan adjacent tissue in CCA patients from TCGA dataset. Furthermore, the growth of Correspondence TNNI3K-knockdown SNU308 and HuCCT1 cells decreased when compared with cells Chun-Nan Yeh, Department of Surgery, Chang Gung Memorial Hospital, Chang Gung transfected with an empty vector cell demonstrated by proliferation and colonoge- University, No.5, Fusing St., Kwei-Shan, necity assay. Besides, over expression of TNNI3K was especially confirmed on human Taoyuan County 333, Taiwan, Republic of China. CCA tumors and compared with the intrahepatic duct stone bile duct tissues and Email: [email protected] normal bile duct tissues (P < 0.001). Our findings might uncover the mystery regarding Wen-Hui Weng, Department of Chemical Engineering and Biotechnology and Graduate carcinogenesis of CCA, and provide the potential genetic mechanism to the clinicians Institute of Biotechnology, National Taipei some ideas for the patients’ treatment. University of Technology, No. 1, Sec. 3, Zhongxiao E. Rd., Taipei 106, Taiwan, KEYWORDS Republic of China. Email: [email protected] array comparative genomic hybridization, carcinogenesis, cholangiocarcinoma, chromosomal alterations 1 | INTRODUCTION diagnostic and treatment challenge; neither the primary tumor nor metastases can be fully resected or successfully treated with Being one of the deadliest cancers, cholangiocarcinoma (CCA) comes radiotherapy or chemotherapy.2–5 with a very low survival rate—more than 90 percent of patients die The value of present tumor markers for the diagnosis of CCA within 5 years, and the majority of patients have a survival of less than remains controversial. So far the most common used markers are 12 months after diagnosis.1,2 CCA commonly presents an arduous carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen Molecular Carcinogenesis. 2018;1–9. wileyonlinelibrary.com/journal/mc © 2018 Wiley Periodicals, Inc. | 1 2 | YEH ET AL. (CEA). The combined use of CEA and CA 19-9 was reported to be able 2.2 | Genomic DNA isolation to improve the diagnosis of CCA.6 However, CA 19-9 only identifies All tissue samples and CGCCA cells were extracted using the illustra patients with advanced and unresectable CCA.7–10 Tissue and Cells GenomicPrep Mini Spin Kit (GE Healthcare, Although the genetic association of great complexity in CCA Buckinghamshire HP7 9NA, UK) according to the manufacturer's have been discovered from time to time, including specific genetic instructions. In brief, the samples were treated with proteinase K and mutations (eg, KRAS, IDH1 and IDH2,andp53) might play an lysis buffer type 1 for an hour at 56°C, then followed with RNase A for important role in CCA, and might be correlated with a more 15 min at room temperature. After applied with lysis buffer type 4 and aggressive phenotype; the epigenetic silencing, aberrant signaling centrifugation, the samples were washed by wash buffer type 6 and pathway activation (eg, interleukin (IL)-6/signal transducer and centrifugation again then resuspended in ddH2O. Genomic DNA activator of transcription 3 [STAT3], tyrosine kinase receptor-related quantity and quality was determined by 0.8% agarose gel electropho- pathways) and molecular subclasses with unique alterations (eg, resis and a NanoDrop spectrophotometer (ND-1000, Thermo proliferation and inflammation subclasses) have been mentioned as Scientific, Wilmington, DE). The size of DNA bands should be greater well.11–13 However, there were no consistent trends of tumorigenic than 10 kb; the optical density of gDNA should have an A260/A280 findings of CCA so far. greater than 1.8 and A260/A230 greater than 1.9 then were used for In our previous studies, we have successfully built a TAA- array-based comparative genomic hybridization. induced CCA rat model and established a rat CCA cell line, CGCCA. We have also revealed the molecular features of late stage CCA.14 Based on it, in order to seek markers for early diagnosis, as well as 2.3 | Array comparative genomic hybridization the therapeutic targets, array comparative genomic hybridization (aCGH) (aCGH) was used in current study. Herein, we compared the cytogenetic alterations among the different stages of TAA-induced A genome-wide analysis of rat tissue samples and CGCCA cells were CCA (an animal model recapitulating the multi-stage progression of performed by using Rat CGH 385 K Whole-Genome Tiling Array chips, CCA) in quest of initial genetic alterations, which might be which contained 385000 probes with an average spatial resolution of ∼ responsible for tumor initiation as well as gene copy number 5.3 kb (Roche NimbleGen, Inc, Madison, WI). The protocol was variations to reveal the possible tumorigenesis of CCA. Furthermore, conducted according to the instruction of NimbleGen arrays user's a panel of normal part of bile duct tissue from hepatocellular guide, CGH and CNV Arrays with slightly modification. In brief, the equal carcinoma (HCC), hepatolithiasis inflammatory hepatic bile duct amounts of gDNA (0.5 µg) were labeled with Cy5 and Cy3 on the control (IHD) tissues, and intrahepatic cholangiocarcinoma (CCA) were also DNA and tumor DNA, respectively. The labeled DNAs were then collected and provided for further confirmation. quantified using the NanoDrop spectrophotometer (ND-1000, Thermo Scientific), after mixed well the 6 µg of each labeled control and tumor DNA, the probes were hybridized onto microarray chip of Rat CGH | 2 MATERIALS AND METHODS 385 K Whole-Genome Tiling Array, and incubated for 16-20 h at 42°C in a humid chamber. The dual-color digital images were captured by MS | 2.1 TAA-induced CCA rat tissue samples 200 Microarray Scanner (Roche NimbleGen, Inc) in combination with preparation the MS 200 Data Collection Software (Roche NimbleGen) then the The TAA-induced rat CCA was induced as previously reported and images were analyzed with the NimbleScan v2.6 Software (Roche followed the animal experiment guideline of Chang Gung Memorial NimbleGen) then carried out using SignalMap v1.9 (Roche NimbleGen). Hospital, Chang Gung University, Taoyuan, Taiwan with approval The Cy5/Cy3 ratios of probes were converted into log2 ratios. The code: IACUC: 201012140914; in which, the TAA-induced CCA threshold values above or below log2 ratio of 0.2 were considered as tissues were derived from seventeen Male Sprague-Dawley (SD) gains or losses, respectively. The significant targets were further rats, and CGCCA cell line developed by ourselves and published at searched by Rat Genome Database (http://rgd.mcw.edu/). previous study15 were used for current experiments. In brief, the CCA tumor were induced by feeding TAA 300 mg/L in rats drinking 2.4 | In silico study water every day up to the time they were sacrificed; during the period, we regularly collected the TAA-induced CCA tissues from The clinical information and genomic matrix file of The Cancer the time points from the 4th week (n =3),8thweek(n =3),12th Genome Atlas (TCGA) database were download form the USCS week (n = 3), 16th week till 25th week (n = 3), the CGCCA cell line cancer browser website (https://genome-cancer.ucsc.edu/proj/site/ was established at 25th week of induced CCA rat cells. The normal hgHeatmap/).The expression level of N/T pairs and groups were bile ducts were harvest from five SD rats feeding with normal performed using Prism software, respectively. Statistical analyses drinking water, after extraction the DNAs were then
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