Ejaz et al. Applied Cancer Research (2017) 37:35 Applied Cancer Research DOI 10.1186/s41241-017-0042-8 RESEARCH ARTICLE Open Access Down regulation of KLK7 expression in breast tissues and identification of a novel spliced KLK7 mRNA Samina Ejaz1, Faiz-ul-Hassan Nasim2*, Muhammad Ashraf2 and Gulzar Ahmad3 Abstract Background: Alternative splicing commonly occurs in cancer cells and many cancer specific splice variants have been reported as potential candidate biomarkers of the disease. We have studied human tissue Kallikrein 7 (KLK7) mRNA expression profile in breast cancer patients of our region. KLK7 is member of a multi-gene family consisting of 15 members (KLK1-KLK15). Methods: We optimized touch down nested PCR method for the amplification of KLK7 isoforms/variants. Various bioinformatics tools were used for sequence analysis, identification of splicing pattern and prediction of encoded proteins. Results: We observed an unusual splicing event consisting of exon 3 (E3) truncation at 3′ end (by 124 nucleotides), exon 4 (E4) exclusion and exon 5 (E5) truncation at 5′ end (by 33 nucleotide) in 2 normal breast tissues, one obtained from invasive ductal carcinoma grade II patient and other collected from mammary dysplasia patient. Moreover, 3 other KLK7 mRNAs (KF963190, KF963191, and KF963193) expressed in breast cancer were noticed to exhibit single nucleotide polymorphism (SNPs). Bioinformatic analysis revealed that the alternatively spliced mRNA (KF963192) will potentially encode a truncated and non-functional protein. Similarly although encoded proteins have considerable homology with normal hK7 protein, SNPs seem to cause great variations in pIs, structures and molecular weights of encoded proteins. Conclusions: There is need to further explore the impact of the unique splicing event, SNPs and characterize these population specific mutations and their possible role in the pathogenesis of breast cancer. Keywords: Alternative splicing, Biomarker, Breast cancer, KLK7, Splice variants Background Human tissue Kallikreins (KLKs) is a multigene family Alternative splicing is a way to produce functionally consisting of 15 members which encode serine prote- diverse proteins from a single gene [1, 2]. It occurs in a ases. All KLKs are known to produce alternative tran- variety of biological processes including developmental, scripts through the use of alternative, transcriptional physiological and pathogenic pathways [3]. According to start site, polyadenylation and splicing. Browsing of the one estimate at least 15% of the genetic diseases are the Genbank database revealed that KLKs have 82 reported outcome of defective pre–mRNA splicing and alternative transcripts and 49 (~ 27% of 82) are the outcome of splicing is a common phenomenon occurring in cancer alternative splicing. Some of the splice variants are cells. Various cancer–specific splice variants have been tissue, developmental stage, stimulus or disease specific. proposed as potential biomarkers of cancer [4–6]. Few KLK splice variants help in cancer diagnosis and prognosis [7–9] and can be used as potential biomarkers of cancer [10, 11]. Future work in this area will be particularly helpful to find more useful diagnostic and * Correspondence: [email protected] 2Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, prognostic biomarkers [12]. Pakistan Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ejaz et al. Applied Cancer Research (2017) 37:35 Page 2 of 13 KLK7 is a member of KLKs family and is known to Mean age of patients was 47.32 ± 12.16 years (range encode human stratum corneum chymotryptic enzyme 24–75 years) and majority of patients were suffering (hSCCE of PRSS6) and expressed in a wide range of from infiltrating ductal carcinoma (53.20%). Total 118 tissues [13–15]. According to GenBank, a total of 11 dis- breast tissue samples, 63 diseased and 55 normal tinct transcripts have been reported for KLK7 (Table 1 samples were subjected to analysis. Normal tissues were and Fig. 1) and only three can be categorized as alterna- carefully removed by a surgeon from regions away from tively spliced transcripts/isoforms. KLK7 mRNA expres- area of cancerization. However, in case of 8 patients due sion level has been quantified by RT-PCR in breast to the involvement of whole breast it was not possible to cancer tissues and reported to be a better prognostic obtain normal tissue. Following the instructions of man- marker for the unfavorable prognosis of breast carcin- ufacturers tissue samples submerged in RNAlaterR were oma. KLK7 mRNA was significantly lowered in either transported from hospital to laboratory, incubated over- stage I or stage II breast cancer patients and higher level night at 2-8 °C followed by the removal of tissues from of KLK7 mRNA was found to be associated with better RNAlaterR and storage at -70 °C. prognosis [16, 17]. Breast cancer is the second leading cause of death Extraction and quantification of RNA worldwide [18]. In Asia Pakistan has the highest preva- Tissue samples were processed for the extraction of total lence of breast cancer. On the average about 40,000 RNA using Norgen Biotek corporation ‘total RNA purifi- women die per year due to this disease [19, 20] and one cation kit’ (Catalogue No. 17200). RNA was quantified out of every nine women in Pakistan is at the risk of de- by measuring absorbance (A) at 260 nm and purity of veloping breast cancer [21]. Contrary to western females, RNA was checked by measuring the ratio of A260/280. Pakistani females suffer from the breast cancer at an RNA samples having A260/280 ratio between 1.8–2.1 early age having a higher risk of metastatic cancers and were further subjected to PCR amplifications. are presented with large lesions [22]. Considering the importance of the issue and associ- cDNA synthesis ation of KLK7 mRNAs with breast cancer we hypothe- cDNA was synthesized by reverse transcription using M- sized that based on genetic differences there can be MLV (Moloney Murine Leukemia Virus) reverse tran- certain new splice variants expressed in breast cancer scriptase (Invitrogen, catalogue no. 28025–013). Reverse (BC) patients of our region. The project was therefore transcription was carried out in a reaction mixture of initiated to evaluate the potential of KLK7 isoforms/vari- 20 μl. Initially RNA solution containing 3 μg RNA, ants as biomarkers of breast cancer in this region. This 1.5 μlof20μM poly dT primer (1.5 μM), 1 μlof10mM study is the first to report KLK7 isoforms/variants in dNTPs mix (0.5 mM) were mixed in 0.2 ml PCR tube patients of southern Punjab, Pakistan. placed on ice. Total volume was adjusted to 12.5 μl with nucleases free H2O, if required. Mixture was heated at Methods 65 °C for 5 min and quickly chilled on ice. After cooling The study was approved by appropriate institutional the contents of tube were collected by brief centrifuga- technical committees including the departmental Ethics tion. It was followed by addition of 6.5 μl RT mix and Research Committee and by the Advanced Studies containing 4 μl of 5× first strand buffer (supplied by and Research Board (ASRB) of the Islamia University of Invitrogen with M-MLV reverse transcriptase), 2 μl 0.1 Bahawalpur, Pakistan. Written Consent to participate in MDTT (10 mM) and 0.5 μl nucleases free H2O. the study was taken on a prescribed form from all Contents of tubes were gently mixed by pipetting and patients included in the study. The experimental work incubated at 37 °C for 2 min. Finally 1 μl (200 units) M- involving humans was in complete compliance with the MLV reverse transcriptase (M-MLV RT) was added to Helsinki Declaration. the reaction mixture, contents were gently mixed by Table 1 Already known KLK7 alternative transcripts Sr. no. Constitutively spliced mRNAs Constitutively spliced Alternatively spliced mRNAs Alternatively spliced mRNAs having SNP mRNAs having SNP 1 AY601109.1 NM_005046.2 AY646152.1 AF411215.1 2 NM_139277.1 BC032005.1 AK295313.1 ———— 3 ———— AF411214.1 ———— ———— 4 ———— AK289660.1 ———— ———— 5 ———— L33404.1 ———— ———— 6 ———— DQ018784.1 ———— ———— Ejaz et al. Applied Cancer Research (2017) 37:35 Page 3 of 13 Fig. 1 Alternative transcripts of KLK7. Sequences of transcripts were retrieved from GenBank, aligned again KLK7 gene sequence to deduce KLK7 mRNA’s architecture. All transcripts are drawn in the 5′ -3′ direction pipetting up and down and incubated at 37 °C for In this technique annealing temperature is initially kept 50 min. After 50 min of incubation at 37 °C M-MLV RT above the melting temperature of primers being used for was inactivated by heating at 70 °C for 15 min and the amplification and then over the course of successive cycles reaction mixture was stored at either 4 °C (for short transitions are progressively made to a lower annealing term storage) or -70 °C for long term storage. temperature. TD PCR is better employed to amplify tran- scripts which are difficult to amplify. Moreover, TD-PCR is Designing/selection of primers for PCR amplification helpful for rapid optimization of PCR conditions avoiding Primers for PCR amplification of KLK7 and β-actin lengthy experimentation for optimization [23]. (used as internal control) cDNAs were selected from a In nested PCR 2 sets of primers are used and PCR list of possible suitable primers predicted by the primer amplification is done in two successive runs of PCR.