Microsatellite Instability and MMR Genes Abnormalities in Canine Mammary Gland Tumors

Early diagnosis of mammary gland tumors is a challenging task in animals, especially in unspayed dogs. Hence, this study investigated the role of microsatellite instability (MSI), MMR gene mRNA transcript levels and SNPs of MMR genes in canine mammary gland tumors (CMT). A total of 77 microsatellite (MS) markers in 23 primary CMT were selected from four breeds of dogs. The results revealed that 11 out of 77 MS markers were unstable and showed MSI in all the tumors (at least at one locus), while the other markers were stable. Compared to the other markers, the ABC9TETRA, MEPIA, 9A5, SCNA11 and FJL25 markers showed higher frequencies of instability. All CMT demonstrated MSI, with eight tumors presenting MSI-H. The RT-qPCR results revealed significant upregulation of the mRNA levels of cMSH3, cMLH1, and cPMSI, but downregulation of cMSH2 compared to the levels in the control group. Moreover, single nucleotide polymorphisms (SNPs) were observed in the cMSH2 gene in four exons, i.e., 2, 6, 15, and 16. In conclusion, MSI, overexpression of MMR genes and SNPs in the MMR gene are associated with CMT and could be served as diagnostic biomarkers for CMT in the future.


Introduction
Genomic instability, a hallmark of cancer, is generally characterized by DNA mismatch repair (MMR) defect, that leads to microsatellite instability (MSI). Therefore, cell MSI plays a critical role in the genesis of mammary gland tumors. Maintenance of genomic stability ensures the inheritance of a complete copy of genetic material in the daughter cells. Moreover, during replication, cells may develop multiple forms of mutations in several genes, such as chromosomal rearrangements, as well as a gain or a loss of part(s) of or the entire chromosome [1].
Repetitive sequences of 1-6 nucleotide base pairs in DNA are known as microsatellites. In addition, alterations in microsatellites are an important form of genomic instability, referred to as MSI. These tandem repeat-sequences are dispersed across the genomes of eukaryotes, usually in noncoding regions. Inactivation of the MMR system results in mutations, particularly, highly repetitive sequences. Additionally, the distribution of microsatellites throughout the genome leads to MSI [2,3].
More than 2000 identified canine microsatellite (MS) markers have been identified and considered useful genetic markers for genetic mapping [4]. MSI most likely occurs during the replication of genetic material, and any errors introduced during this process result in the addition or deletion of a base pair. These genomic changes may cause abnormalities in cell division and hence lead to an imbalance between cell growth and death or ultimately cause cancer. In addition, the MMR system

Ethical Statement
Tumor samples were collected with the consent of the owners and according to the recommendations published in the Guide for the Care and Use of Laboratory Animals of Jiangsu province (SYXK2017-0027). The Committee on the Ethics of Animal Experiments of Jiangsu province approved the protocol (NJAU-20171019, 10 October 2017).

Tumor Sample Collection
Twenty-three CMT from 23 female dogs of four different breeds were provided by the Teaching Hospital of Nanjing Agricultural University. The age of the patients ranged from 5 to 15 years (9 years mean age). Tumors were collected from the female dogs by mastectomy, followed by confirmation through histopathological evaluation. The adjacent normal mammary glands were also excised during the surgical procedure. The tumor tissues and adjacent normal mammary glands were divided into two parts, one part of which was fixed in 10% formalin solution for histopathological assessment and the other was frozen at −80 • C for DNA and RNA extraction.

Tumor Histopathological Assessment
The tumor tissues samples as well as the normal tissue samples were removed from each dog and fixed into 10% formalin solution. Then, these samples were embedded in paraffin wax via a routine process. All sections were stained with hematoxylin and eosin (H&E) and histopathologically examined using an optical microscope. Mammary tumors were classified according to the classification proposed by Goldschmidt et al. [13].

DNA Extraction and PCR Amplification
DNA was isolated from the frozen samples (tumor and adjacent normal mammary glands) using commercially available kits (Invitrogen USA) according to the manufacturer's instructions. The quality of the DNA was assessed on 0.8% (w/v) agarose gel by electrophoresis and was quantified with a spectrophotometer. Isolated DNA was diluted (80 ng/L) and stored at −80 • C to determine MSI.

Microsatellite Instability (MSI)
Seventy-seven microsatellite markers in the canine linkage map were used to screen the 23 samples taken from four different dog breeds for MSI evaluation [14][15][16]. The eleven unstable markers are shown in Table 1. The PCR product of each marker was denatured at 95 • C for 5 min with gel loading dye, and immediately put on ice for 5 min prior to loading. Approximately 3 µL vol of the PCR products were separated on 10% polypropylene polyamide gels (1 mm thick). The gels were subsequently stained with AgNO 3 . The bands of each locus were counted and evaluated. The MSI-H group of tumors was defined as having MSI in ≥30-40% of the loci, whereas MSI-L group exhibited MSI in <30% of the loci [17].

Expression Profiling by RT-qPCR Assay
The total RNA was isolated from tumor and adjacent normal mammary gland tissue (50 mg) with TRIZOL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Approximately 250 ng/µL of the total RNA template was used to synthesize the first-strand cDNA with Prime Script RT Master Mix Perfect Real Time (Takara Co., Otsu, Japan) according to the manufacturer's instructions. The abundance of RNA transcripts of different genes was estimated using quantitative real-time PCR (RT-qPCR) for gene-specific primers. The primers for cMSH2, cMSH3, cMLH1, cPMS1, and GAPDH (as a housekeeping gene) were designated by primer premier 5.0 software, the information is available in GenBank database NCBI reference sequence (XM_538482.5, XM_022417321.1, XM_534219.6, XM_536002.6 and NM_001003142.2 respectively) ( Table 2). The RT-qPCR mixture (20 µL) contained cDNA (2 µL) and the reverse and forward primers (0.4 µM) in the SYBR Green master mix. ABI 7300 Fast Real-time PCR System (Applied Biosystem, USA) was used for amplification, programmed at 95 • C for 15 s of initial denaturation, and annealed for 40 cycles at 95 • C for 5 s, followed by 60 • C for 31 s of primer extension. The GAPDH housekeeping gene of the dog was used as an internal Control. The relative value to gene expression was computed on the basis of

Detection of Exon Mutations of the cMSH2 Gene
The sixteen pairs of primers were designed from the related sequence information available in the GenBank database (NC_006592.3) using Primer Premier 5.0 software to amplify the 16 exons of the cMSH2 gene. The amplification conditions were the same as mentioned above, except for the annealing temperature, which varied according to each primer used ( Table 3). The targeted amplicons from the PCR product of each primer were retrieved from the agarose gel, purified, and cloned into the pGEM-T easy plasmid vector (Promega, Madison, WI, USA). The cloned exons were sequenced on an ABI PRISM 377 capillary sequencer using vector-and exon-specific primers. Table 3. Primers used to amplify exon of cMSH2 gene.

Statistical Analysis
One-way analysis of variance (ANOVA) was performed using Predictive Analytics Software 18.0. In addition, Duncan's multiple-range test was used, with differences considered significant at p < 0.05.

Histopathological Assessment
A total of 23 mammary gland tumors were observed. According to Goldschmidt et al. [13], these tumors were classified as benign (4/23, 17.

Microsatellite Instability (MSI) Screening
Changes in MS markers in the CMT samples compared with the DNA of normal mammary tissue samples were recognized as alterations in the electrophoretic migration or loss of major band(s). Among 77 MS markers, MSI existed in 11, whereas 66 markers showed stability. In all the tumor-affected patients, MSI was identified at one or more loci. The highest incidence of MSI was observed in the tumor from dog no. 19, which exhibited MSI at seven loci (7/11, 63.6%), whereas only one MSI locus was observed in each of dogs no. 1, 4, 6, 7, 8, 11, 12, 16 and 22. According to this criterion, canine mammary gland tumors from nine dogs (dog no. 2, 5, 13, 14, 15, 17, 19, 21 and 23) presented MSI-H, and the rest of the canine mammary gland tumors demonstrated MSI-L in this study (Table 4). In addition, MSI frequency for each microsatellite marker was presented in (Table 5). Among the markers, the ABC9TETRA marker showed the highest instability frequency (10/23, 43.4%) in the tumor samples. In addition, MEPIA exhibited MSI in eight tumors (8/23, 34.8%), and 9A5, SCNA11 and FJL25 exhibited MSI in seven tumors (7/23, 30.4%).

Mismatch Repair-Related Gene Expression
The RT-qPCR results revealed that mRNA expression of the MMR system genes significantly increased in CMT (Figure 4). Compared to those in the normal tissues, the genes cMLH1 (p < 0.0043), cPMS1 (p < 0.046) and cMSH3 (p < 0.026) in the tumor tissues were significantly upregulated, but cMSH2 (p < 0.016) was downregulated.

Mismatch Repair-Related Gene Expression
The RT-qPCR results revealed that mRNA expression of the MMR system genes significantly increased in CMT (Figure 4). Compared to those in the normal tissues, the genes cMLH1 (p < 0.0043), cPMS1 (p < 0.046) and cMSH3 (p < 0.026) in the tumor tissues were significantly upregulated, but cMSH2 (p < 0.016) was downregulated.

The Extent of Polymorphism in cMSH2 Gene
The sequence of cMSH2 in the dog genome retrieved from the gene bank database of NCBI revealed a 3152 bp gene sequence consisting of 16 exons. Hence, exon-specific primers were designed and amplified from the CMT and normal DNA templates using PCR. The resulting amplicons were separated on 10% nondenaturing polyacrylamide gels. These PCR amplicons were then sequenced to determine the presence of polymorphism among the samples.
The PCR amplicons obtained from the DNA templates of the tumor and normal tissues of the 23 female dogs used in this study were sequenced to identify the nature of the changes or polymorphisms. The amplified products of the 16 exons were sequenced (

The Extent of Polymorphism in cMSH2 Gene
The sequence of cMSH2 in the dog genome retrieved from the gene bank database of NCBI revealed a 3152 bp gene sequence consisting of 16 exons. Hence, exon-specific primers were designed and amplified from the CMT and normal DNA templates using PCR. The resulting amplicons were separated on 10% nondenaturing polyacrylamide gels. These PCR amplicons were then sequenced to determine the presence of polymorphism among the samples.
The PCR amplicons obtained from the DNA templates of the tumor and normal tissues of the 23 female dogs used in this study were sequenced to identify the nature of the changes or polymorphisms. The amplified products of the 16 exons were sequenced ( female dogs used in this study were sequenced to identify the nature of the changes or polymorphisms. The amplified products of the 16 exons were sequenced ( Figure 5). Single nucleotide polymorphisms were observed in exons number 2, 6, 15 and 16 of cMSH2 in the sequences of all the normal and the tumor dog samples. The genotypes of exon 2 were TT (5/23, 21.7%), TC (17/23, 73.9%) and CC (1/23, 4.3%). The genotypes of exon 6 were GG (9/23, 39.1%), GA (11/23, 47.8%) and AA (3/23, 13.0%). The genotypes of exon 15 were GG (16/23, 69.6%), GA (5/23, 21.7%) and AA (2/23, 8.7%

Discussion
Dogs, primarily unspayed female dogs, are frequently victims of mammary neoplasia. Lack of effective diagnostic tools during the earlier stages of carcinogenesis is a serious concern, as this disease is unresponsive to treatments at later stages [18]. To enhance the effectiveness of CMT treatment, it is essential to diagnose it in its early stages. Hence, this study hypothesized that the successful diagnosis could be made possible by employing the biomarkers that could distinguish the tumorous glands from the healthy ones. The dog marker genome map has been comprehensively developed during recent years. To date, more than 2000 microsatellite markers have been reported, and many of them have been assigned to linkage groups and specific chromosomes [4]. Some studies have reported a few biomarkers for the diagnosis and prognosis of CMT [19,20]. However, there is a lack of rapid, sensitive and specific biomarkers, which are required for the diagnosis of carcinogenesis in earlier stages.
In this study, a panel of 77 microsatellite markers was used to check for MSI, among which 11 markers showed instability and 66 markers showed uniform amplification. These results were in accordance with an earlier published study that evaluated 35 canine mammary tumors, among which 13 (37%) had stable genotypes, and 22 (63%) exhibited aberrations in 1 or 2 MS and 4 tumors (11%) demonstrated high instability, with aberrations in 29% to 61% of MS [15]. MSI was detected at one or more loci in all the tumor-affected dogs in this study. In particular, the highest frequency of MSI was observed for the tumor from dog no. 19, which had seven loci (7/11, 63.6%) affected displaying MSI-H.
Moreover, among the markers, ABC9TETRA showed the highest frequency of instability (10/23, 43.4%). In addition, MSI was also evident in MEPIA in eight tumors (8/23, 26%). Furthermore, FLJ32685, SCNA11, and 9A5 exhibited MSI in seven tumors (7/23, 30.4%). The results of this study were comparable to those of an earlier published report related to human HPNCC disease, which used 21 patients and analyzed 78 tumor samples for MSI by microsatellite markers. They classified 26.9% tumors as high MSI (MSI-H), 11 (14.1%) as low instability (MSI-L) and 46 patients (59%) as medium instability (MS-S) [21]. Similarly, the findings of the present study are in agreement with those of Ando et al. [22], who showed a higher frequency of MSI-L in human breast tumors. The results of this study were also in accordance with another study, which demonstrated MSI-L in grade III ductal and lobular breast cancers [23]. Furthermore, Yee et al. [24] also observed a high frequency of MSI-H in human breast cancers.
The RT-qPCR-based findings showed upregulation of some MMR genes (cMSH3, cMLH1, cPMSI), but downregulation of cMSH2, compared to the levels in the normal group. The expression of cMSH2 has been associated with tumor development in studies on gastric cancers [25], glioblastomas [26], salivary gland tumors [27], malignant melanomas [28], ovarian carcinomas [29], urothelial carcinomas [30], and endometrial carcinomas [31]. In addition, higher expression of MMR genes could be acquired by a genetic change, followed by an alteration in gene expression, thereby

Discussion
Dogs, primarily unspayed female dogs, are frequently victims of mammary neoplasia. Lack of effective diagnostic tools during the earlier stages of carcinogenesis is a serious concern, as this disease is unresponsive to treatments at later stages [18]. To enhance the effectiveness of CMT treatment, it is essential to diagnose it in its early stages. Hence, this study hypothesized that the successful diagnosis could be made possible by employing the biomarkers that could distinguish the tumorous glands from the healthy ones. The dog marker genome map has been comprehensively developed during recent years. To date, more than 2000 microsatellite markers have been reported, and many of them have been assigned to linkage groups and specific chromosomes [4]. Some studies have reported a few biomarkers for the diagnosis and prognosis of CMT [19,20]. However, there is a lack of rapid, sensitive and specific biomarkers, which are required for the diagnosis of carcinogenesis in earlier stages.
In this study, a panel of 77 microsatellite markers was used to check for MSI, among which 11 markers showed instability and 66 markers showed uniform amplification. These results were in accordance with an earlier published study that evaluated 35 canine mammary tumors, among which 13 (37%) had stable genotypes, and 22 (63%) exhibited aberrations in 1 or 2 MS and 4 tumors (11%) demonstrated high instability, with aberrations in 29% to 61% of MS [15]. MSI was detected at one or more loci in all the tumor-affected dogs in this study. In particular, the highest frequency of MSI was observed for the tumor from dog no. 19, which had seven loci (7/11, 63.6%) affected displaying MSI-H.
In addition, MSI was also evident in MEPIA in eight tumors (8/23, 26%). Furthermore, FLJ32685, SCNA11, and 9A5 exhibited MSI in seven tumors (7/23, 30.4%). The results of this study were comparable to those of an earlier published report related to human HPNCC disease, which used 21 patients and analyzed 78 tumor samples for MSI by microsatellite markers. They classified 26.9% tumors as high MSI (MSI-H), 11 (14.1%) as low instability (MSI-L) and 46 patients (59%) as medium instability (MS-S) [21]. Similarly, the findings of the present study are in agreement with those of Ando et al. [22], who showed a higher frequency of MSI-L in human breast tumors. The results of this study were also in accordance with another study, which demonstrated MSI-L in grade III ductal and lobular breast cancers [23]. Furthermore, Yee et al. [24] also observed a high frequency of MSI-H in human breast cancers.
The RT-qPCR-based findings showed upregulation of some MMR genes (cMSH3, cMLH1, cPMSI), but downregulation of cMSH2, compared to the levels in the normal group. The expression of cMSH2 has been associated with tumor development in studies on gastric cancers [25], glioblastomas [26], salivary gland tumors [27], malignant melanomas [28], ovarian carcinomas [29], urothelial carcinomas [30], and endometrial carcinomas [31]. In addition, higher expression of MMR genes could be acquired by a genetic change, followed by an alteration in gene expression, thereby leading to an increased level of MMR proteins with impaired functions [32]. Since tumor cells acquire comprehensive genetic changes, increased levels of MMR gene expression could hence be associated with a cellular adaptation aimed at repairing the DNA lesions [33]. Moreover, the overexpression of MMR genes in the cancerous cells could represent the response to the fast-growing number of replication errors in a tissue with an increased rate of cell divisions [34]. Furthermore, germline mutations in DNA MMR genes, particularly in MLH1 and cMSH2 genes, were associated with tumor development in humans in earlier studies [35,36]. Due to the observation of lower mRNA expression of the cMSH2 gene, the present study further investigated its role in the genesis of CMT. The results of the present study confirmed polymorphisms in the cMSH2 gene in exons 2, 6, 15 and 16.
In conclusion, all CMT demonstrated MSI using the eleven microsatellite markers selected in this study, with eight tumors presenting MSI-H MMR gene abnormalities, such as overexpression of cMSH3, cMLH1, and cPMSI and SNPs in the cMSH2 gene, were also found in these tumors. MSI may be related to MMR genes abnormalities and may be used as diagnostic tools for the CMT in the future.