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Original Article BTG3 Gene Alterations in Colorectal Carcinoma

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Original Article BTG3 Gene Alterations in Colorectal Carcinoma Int J Clin Exp Pathol 2017;10(3):3445-3450 www.ijcep.com /ISSN:1936-2625/IJCEP0046336 Original Article BTG3 gene alterations in colorectal carcinoma Zhang Cao1, Min Cui2, Peng Ma3, Hong Li1, Qian Zhang1, Ning Yu1 Departments of 1Pathology, 3Cell Biology, Binzhou Medical University, Yantai, Shandong, China; 2Department of Pediatrics, Binzhou City People’s Hospital, Binzhou, Shandong, China Received December 11, 2016; Accepted December 27, 2016; Epub March 1, 2017; Published March 15, 2017 Abstract: Aims: To investigate whether the BTG3 gene is involved in colorectal carcinogenesis. Methods and results: we have examined the genetic alterations, including somatic mutations and promoter hypermethylation of the BTG3 gene in 78 sporadic colorectal carcinomas. Results revealed that no mutation was detected in the coding region of the BTG3 gene and promoter hypermethylation was detected in 5 colorectal carcinoma samples. The expression of BTG3 proteins was also examined via immunohistochemistry in 78 colorectal carcinomas. There was loss or reduced expression of BTG3 in the 43 (55.1%) of the 78 colorectal carcinomas. Statistically, altered expression of BTG3 was not associated with clinicopathological parameters, including tumor differentiation, location, and lymph node metastasis (P>0.05). Conclusion: Our results suggest that genetic, epigenetic, and protein expression pattern alterations of the BTG3 gene might play a minor role in the development or progression of colorectal carcinomas. Keywords: BTG3, colorectal carcinoma, mutation, methylation, immunohistochemistry Introduction anti-proliferative BTG (B-cell translocation ge- ne)/Tob (Transducer of ErbB2) gene family, is Colorectal carcinoma (CRC) is the third leading localized at chromosome 21q11.2-q21.1 and cause of cancer death worldwide with a dismal found to associate with and inhibit Src tyrosine outcome [1]. A rising incidence and mortality kinase through its C-terminal domain in PC12 from CRC have recently been observed in cells [6, 8]. Recently, BTG3 was identified as a China. However, little is known about the molec- transcriptional target of p53 and its anti-prolif- ular genetic events in the development and pro- erative action was clarified through inhibition of gression of CRC [2]. Allelotype studies have E2F1 [7]. The transcription factor E2F1 controls shown that specific regions of the genome are the cell cycle by activating genes important for frequently deleted in many tumor types. In G1/S progression: these include the genes for addition, frequent loss of heterozygosity (LOH) cyclin E, PCNA, DNA polymerase a, Cdc6, dihy- at certain chromosomal regions is the hallmark drofolate reductase (DHFR) and others [8, 9]. BTG3 binds to and inhibits E2F1 through an of the presence of a tumor suppressor gene. N-terminal domain including the conserved box Therefore, the identification of consistent areas A [9]. However, no genetic alterations of the of chromosomal deletion in the DNA of tumors BTG3 gene have been reported on human spo- may point to the regions harboring a tumor sup- radic colorectal carcinoma. Therefore, we pressor gene that is associated with carcino- hypothesized that BTG3 might function as a genesis of a specific tissue type. Allelotype tumor suppressor gene in the development studies have reported frequent LOH on 21q in and/or progression of colorectal carcinoma. several human cancers, including lung, breast, oral cancer [3-7]. In order to determine whether genetic and epi- genetic alterations and expression patterns of Src nonreceptor tyrosine kinase plays an impor- the BTG3 gene are involved in the development tant role in multiple signaling pathways that or progression of CRC, somatic mutations, regulate several cellular functions including hypermethylation and protein expression of the proliferation, differentiation and transforma- BTG3 gene were analyzed in colorectal carci- tion [7]. Interestingly, BTG3, a member of the nomas. Analysis of the BTG3 in colorectal carcinoma GAACTTAT-3’ (sense primer) and 5’-AACACAA- TCAAAAACGAAG-3’ (antisense primer). Num- bering of the DNA of the BTG3 gene was done with respect to the ATG start codon according to the genomic sequence of Genbank, acces- sion no. NM006806. Each polymerase chain Figure 1. Methylation-specific PCR results of the reaction (PCR) was performed under standard BTG3 gene in colorectal carcinoma tissues and cor- conditions in a 10 μl reaction mixture contain- responding non-cancerous tissue. The tumor tissue from case no. 65 showed both the methylated and ing 20 ng of template DNA, 0.5 μM of each unmethylated PCR products, whereas the corre- primer, 0.2 μM of each deoxynucleotide tri- sponding non-neoplastic DNA demonstrated only the phosphate, 1.5 mM MgCl2, 0.4 unit of Taq poly- unmethylated PCR product. Case no. 17 showed un- merase, 0.5 μCi of [32P]dCTP (Amersham, methylated and methylated DNA in both correspond- Buckinghamshire, UK), and 1 μl of 10× buffer. ing non-neoplastic and cancer tissues. (N, non-neo- plastic DNA, T, tumor DNA. U, unmethylated DNA, M, The reaction mixture was denatured for 12 min methylated DNA). at 94°C and incubated for 35 cycles (denatur- ing for 40 s at 94°C, annealing for 40 s at 52-55°C, and extension for 40 s at 72°C). The Materials and methods final extension was continued for 5 min at 72°C. After amplification, the PCR products Tissue samples were denatured for 5 min at 95°C at a 1:1 dilu- tion of sample buffer containing 98% for- A total of 78 CRC samples were examined. mamide/5 mmol/L NaOH and were loaded Informed consent was obtained from all onto a single-strand conformation polymor- patients. No patient had a family history. Their phism (SSCP) gel (Mutation Detection Enhan- ages ranged from 35 to 85 years, with an aver- cement, FMC BioProducts, Rockland, ME, USA) age of 52 years. The male to female ratio was with 10% glycerol. After electrophoresis, the 47 to 31. Two pathologists independently gels were transferred to 3 MM Whatman paper reviewed one 6 μm section stained with and dried; autoradiography was performed Hematoxylin & Eosin. Approval was obtained using Kodak X-OMAT film (Eastman Kodak, from the institutional review board at the Rochester, NY, USA). For the detection of muta- Binzhou Medical University of Shandong. tions, DNAs showing mobility shifts were cut DNA extraction out from the dried gel and reamplified for 30 cycles using the same primer sets. Sequenc- Malignant cells were selectively obtained from ing of the PCR products was carried out using the hematoxylin and eosin-stained slides using cyclic sequencing kits (Perkin-Elmer, Foster a laser microdissection device. The genomic City, CA, USA) according to the manufacturer’s DNA was prepared using a procedure described recommendations. previously [10]. Methylation-specific PCR (MSP) Mutational analysis The methylation status of the promoter region Genomic DNAs from the cancer cells and cor- of the BTG3 gene was determined using sodi- responding non-cancerous tissue were ampli- um bisulfite treatment of the DNA followed by fied with four sets of primers covering the entire MSP, as described in the literature with slight coding region (4 exons) of the BTG3 gene. The modification [11, 12]. Briefly, DNA was incubat- primers used were the following: for exon 1: ed in 0.2 M NaOH at 42°C for 30 min in a total 5’-AATGAAGAATGAAATTGCTG-3’ (sense primer) volume of 50 μl. After adding 350 μl of 3.6 M and 5’-TGCTTAATCCAATCTAAT-3’ (antisense sodium bisulfite containing 1 mM hydroquinone primer). For exon 2: 5’-GGCAAGTATGAATAGAT- at pH 5.0, the samples were incubated for 16 h ATTAC-3’ (sense primer) and 5’-ATCAGTTCA at 55°C in the dark. The modified DNA was GCCTCTAAC-3 (antisense primer). For exon 3: recovered with 5 μl glassmilk (BIO 101, Irvine, 5’-AATTTTATTCTT AGGTATGGAG-3’ (sense prim- CA, USA) and 800 μl of 6 M NaCl. The glassmilk er) and 5’-TTTTCATGGTTACCTGG-3’ (antisense catching the modified DNA was washed three primer), and for exon 4: 5’-CCCTACAGATTCA- times with 70% ethanol at room temperature, 3446 Int J Clin Exp Pathol 2017;10(3):3445-3450 Analysis of the BTG3 in colorectal carcinoma Figure 2. BTG3 expression in colorectal cancers. Colorectal cancers exhibited cytoplasmic positive staining for BTG3 (A). Loss of BTG3 expression in colorectal cancers (B). (Original magnification, ×200). treated with 0.3 M NaOH/90% ethanol, and naissance TSA indirect kit (NEN Life Science, washed twice with 90% ethanol. The DNA was Boston, MA, USA), which included streptavidin- finally eluted from the dried pellet with 30 μl 1 peroxidase and biotinylated tyramide, was mM Tris-HCl (pH 8.0) for 15 min at 55°C; 4 μl of used. After rinsing with PBS, the slides were the bisulfite-modified DNA was subjected to treated with 1% H2O2 in PBS for 20 min at room MSP using two sets of primers for the methyl- temperature. After washing with TNT buffer (0.1 ated and unmethylated BTG3. The primers mol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl and used were the following: for methylation primer, 0.05% Tween 20) for 20 min, the slides were 5’-TTGGATTTTTTTTGGGTTTTATTTAC-3’ (sense treated with TNB buffer (0.1 mol/L Tris-HCl, pH primer) and 5’-ATAACGTCATCCTAAAACCTCGTC- 7.4, 0.15 mol/L NaCl and 0.5% blocking 3’ (antisense primer); for unmethylation primer: reagent). The sections were incubated over- 5’-TGGATTTTTTTTGGGTTTTATTTAT-3’ (sense pr- night at 4°C with BTG3 antibodies (1/100; imer) and 5’-TAACATCATCCTAAAACCTCATC-3’ Santa Cruz Biotechnology, Santa Cruz, CA, (antisense primer). The PCR was performed in USA). Detection was carried out using biotinyl- a total volume of 30 μl, containing 5 μl of the ated goat anti-rabbit antibody (Sigma, St. Louis, template DNA, 0.5 μM of each primer, 0.2 μM MO, USA), followed by incubation with a peroxi- dase-linked avidin-biotin complex.
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