Expression Profiling of microRNAs hsa-222-3p, hsa-let-7b-5p, hsa-let-7f-5p and their Putative Targets HMGA1 and CDKN1B Genes in Canine Mammary Tumor

Cancer is unregulated growth of cells that can spread to other part of body via blood circulation and lymphatic system. Mutations in genetic material can alter cell physiology, ultimately resulting in tumor. Like human cancers, dogs have relatively high incidence of cancers, relatively large body size and responses to cytotoxic and other therapeutics. Small noncoding RNA having length of 22 base pair (bp) are called micro-RNA (miRNA), that are processed by Dicer from precursors with a characteristic hairpin secondary structure. miRNAs can regulate gene expression at the post-transcriptional level by binding to the 3′ untranslated region (UTR) of target mRNAs. In this study dog mammary tumor samples were collected to investigate the expression level of miRNAs hsa-222-3p, hsa-let-7b-5p and hsa-let-7f-5p and effect of their expression on the target genes ( HMGA1 and CDKN1B ) of these miRNAs. Qiagen miScript Primer Assay based expression analyses revealed the over-expression of all three miRNAs in most of the studied canine mammary tumor samples compared to normal mammary tissue. Furthermore, this over-expression of tested miRNAs, down regulated their target genes in tumor samples compared to normal samples.


M icroRNA (miRNA) was first discovered in
Caenorhabditis elegans in 1993. It is also found in most eukaryotes, chief among them are humans (Perron and Provost, 2008). miRNAs are around 22 nucleotides long and perform as regulatory non coding RNAs. Discovery of RNA interference (RNAi) drastically increased the number of studies on miRNAs which was further sped up by the search for small endogenous RNAs of a similar type in various species (Fire et al., 1998;Treiber et al., 2019). miRNAs also play a major role in the regulation of post transcriptional gene expression via a mechanism of complimentary sequences and repression of target RNAs. These studies have established the importance of miRNAs in development, physiology and disease (Perron and Provost, 2008;Croce, 2009) The miRNA let-7 (MIRLET7) is a family that controls growing effectiveness and diversity. Generally, the loss of let-7 is seen as a major contributor in oncogenesis through increase of target oncogenes and stemness factors, its targets include cell signaling pathways, the cell cycle and cell variation. It is characterized as a tumor suppressor. It was suggested that let-7 family take part in metastasis. Let-7a was seen to influence down regulation of CCR7 by targeting its 3' UTR that resulted in the down regulation of breast cancer cells capacity for invasion and migration. Other studies confirmed similar results as let-7a acted as a tumor suppressor in zebrafish embryo models via regulating the expression of RAS and HMGA2 oncogenes. Furthermore, decreased let-7a levels were related to increased RAS levels in lung squamous Carcinoma O n l i n e F i r s t A r t i c l e (Johnson et al., 2005;Cunningham et al., 2010). However, in certain albeit rare cases the Let-7 are known to be oncogenic in nature showing increasing route, attack, chemoresistance and also increased genes expression that are related with the development and metastasis of cancer. This makes this family a major potential target as a diagnostic and prognostic marker and even a candidate for cancer therapy (Chirshev et al., 2019). hsa-let-7b-5p and hsa-let-7f-5p are two such miRNAs that are part of the let-7 family which are seen to be fatal for humans as they are major contributors in cancer pathogenesis. hsa-mir 222-3p was seen to be overexpressed in patients with carcinoid lung tumors like hsa-let-7b-5p (Di Fazio et al., 2017).
Over the decades, many similarities have been identified between human and canine mammary tumors at molecular level. These similarities include incidence of tumor, age of onset and course of disease. From a clinical perspective tumor size, stage and lymph node invasion are seen to be mostly identical (Queiroga et al., 2011). As is the case in human females, the most commonly occurring spontaneous malignancy in female canine is mammary neoplasia (Tavasoly et al., 2013), and premalignant lesions are prevalent in canine mammary glands (Antuofermo et al., 2007).
In this study, our aim was to study the expression pattern of three microRNAs, hsa-222-3p, hsa-let-7b-5p, hsa-let-7f-5p, in canine mammary tumors and further to investigate the effect of their expression on two of their putative target genes (HMGA1 and CDKN1B).

Sample collection
Dog mammary tumor and normal tissues samples (10 each) were collected from University of Veterinary and Animal Sciences (UVAS) Pet Center, after informed consent of the pet owners, and were preserved in absolute ethanol and 10% formalin solution for expression and histopathological analyses, respectively.

Histopathological examination
Histopathological examination was performed on formalin-fixed paraffin embedded (FFPE) cancerous tissues. Formalin-filled (10%) sample collection tubes were used to preserve the neoplastic tissues after grossing and isolation of core tumorous masses. The tissues were used for hematoxylin and eosin (H and E) staining to confirm the malignancy, grading and staging as described earlier (Manzoor et al, 2017).

Total RNA isolation and quantification
Total RNA was extracted from tumor and normal tissue samples by using RNeasy tissue mini kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Quantification of RNA was conducted by Nano Drop 2000 (Thermo Fisher Scientific, Pittsburg, PA, USA).

Statistical analysis
Student t-test was applied on RNU6B-normalized relative expression data (cancer vs normal) on each of the target miRNAs and similarly on GAPDH-normalized relative expression data on both target genes as well. P value <0.05 was considered as significant.

Histopathological examination
Histopathological analyses of representative dog mammary tumors showed varied population of cells with enormous nucleus and prominent mitotic characteristic (Fig. 1a). Numerous key regions were marked by using different magnifications, indicating the presence of fibroblasts and collagen fibers as shown in Figure 1.  Overall, all three types of miRNA show upregulation in most of the studied dog mammary tumor samples (Fig. 4).
Comparative expression analysis of subjected miRNAs in dog normal (DNS) and mammary tumor (DMT)  samples is shown in Figure 5. hsa-let-7b-5p has overall more expression in mammary tumor tissues as well

O n l i n e F i r s t A r t i c l e
as supported by the statistical hypothesis testing with significant p-value but with caveat emptor of the small sampled populations and of not very strongly association p-value. Similarly, Table I shows the expression (fold change) of different miRNAs in analyzed mammary tumor samples and their expression intensity. hsa-let-7b-5p has highest expression in canine mammary tumor (DMT) samples.

Expression of HMGA1 and CDKN 1B in dog mammary tumors
Expression of HMGA1 and CDKN1B genes was analyzed in dog normal (DNS) and mammary tumor (DMT) samples. The expression of HMGA1 gene was found down-regulated in all tumor samples (DMT) and strongly supported by the significant p-value of 0.000004 (Fig.  6A). Similarly, CDKN1B gene expression was also down-regulated in all tumor samples (DMT) and very strongly supported by significant p-value of 6.8×10 -08 (Fig. 6B).   GraphPad Prism software was used for combined representation of dog normal (DNS) and mammary tumor tissue (DMT) samples for better understanding of data displayed in Figure 6C. Table II represent the down-regulation values and intensity of down-regulation in miRNA target genes.

DISCUSSION
One of the most common disease in dogs is cancer although some breeds of dogs have high risk of cancer types and one of them is mammary tumor (Manzoor et al., 2019). In dog and human population mammary tumor occurs spontaneously (Egenvall et al., 2005). As both human and dog share similar environment hence epidemiology and progression of cancer is similar in both of them. Human and dog mammary tumor initiate from epithelial tissue and in both species are hormonedependent (Misdorp, 1999). To understand the human breast cancer naturally-occurring canine mammary tumor plays central role because both species have same etiology, histopathologic division and disease pathogenesis (Gray et al., 2020). Fish et al. (2020)  miRNAs could be used as a biomarker for the detection of canine mammary tumor (CMT). The findings of Kim et al. (2020) explain how the cross-species oncogenic similarities help to comprehend pathogenesis mechanisms of breast cancer progression as well as give an insight for exact diagnostics and therapeutics of breast cancer in domestic dogs. Aim of the present study was to understand the role of hsa-miR-222-3p, hsa-let-7b-5p and hsa-let-7f-5p miRNAs in canine mammary tumor and to discover it as potential biomarkers for the early diagnosis of mammary tumor in future by confirming it with next generation sequencing (NGS) and many other advanced techniques. To this end, expression of hsa-mir-222-3p, hsa-let-7b-5p and hsa-let-7f-5p miRNAs and their targeted genes (HMGA 1 and CDKN 1B) were analyzed for tumor positive samples which was confirmed by histopathological studied, all miRNAs showed up-regulation and their target genes were down-regulate in tumorous samples. These results are in comparison with a human lungs cancer study which reported the significant overexpression of hsa-mir-222-3p, and hsa-let-7f-5p and downregulation of hsa-let-7b-5p in majority of studied tumor samples, resulting in significant overexpression or stable level of CDKN 1B in majority of the samples and stable level or downregulation of HMGA2 gene (Di Fazio et al., 2017). Similarly, another study by Wang and Zhai (2020) validate our results describing the mir-222-3p/p27kip1 axis. They found that high intensity focused ultrasound (HIFU) treatment downregulated the mir-222-3p resulting in overexpression of p27kip1 and consequently apoptosis was activated. Whereas, overexpression of mir-222-3p restored cell proliferation and deactivated apoptosis, which was overturned by overexpression of p27kip1 in breast cancer cells.
The results of the study are correlated to our unpublished results in which expression of same miRNAs as well as their target genes were analyzed in humans (manuscript in preparation). In this study, all these miRNAs were found up regulated and their target genes were found downregulated in breast tumor samples compared to normal tissue samples.
hsa-let-7b-5p and hsa-let-7f-5p miRNAs have higher expression in mammary tumor which was calculated by comparing it with its reference gene RNU 6B and found fold change. Up-regulation of miRNAs have been observed in both subjected miRNAs in dog mammary tumor. We also checked its target gene response in both tumorous and normal tissues samples; in case of tumorous samples HMGA 1 gene was down-regulate in all positive samples as mentioned in Figure 6, whereas in case of normal tissues the expression of these subjected miRNAs suppressed and their target gene is upregulating and perform its functioning.
We also studied expression of hsa-miR-222-3p miRNA in dog mammary tumor, expression of this miRNA is also up-regulated in tumorous samples like Let-7 family and its target gene CDKN 1B is suppressed in all cases whereas in normal samples expression of this miRNA is suppressed and its targeted gene is up-regulated.

CONCLUSIONS
In this study, dog mammary tumor samples were collected to investigate the expression level of hsa-222-3p, hsa-let-7b-5p and hsa-let-7f-5p miRNAs and effect of their expression on the target genes (HMGA1 and CDKN1B) of these miRNAs. Qiagen miScript Primer Assay based expression analyses revealed the overexpression of all three miRNAs in canine mammary tumor samples compared to normal mammary tissue samples. Furthermore, this over-expression of tested miRNAs, down regulated their target genes in in most of the studied tumor tumor samples compared to normal samples. This study might be helpful to develop biomarkers for diagnosis of dog mammary tumor by using different advance techniques including NGS.

O n l i n e F i r s t A r t i c l e
Expression of miRNAs and their Target Genes in Canine Mammary Tumor