Investigation of Clock Gene Variations in Nannospalax Cytotypes

– Blind mole rats ( Nannospalax ) are subterranean mammals noted for their longevity and cancer resistance. It is known that these animals' vestigial eyes, particularly the Harder gland around the eyes, may generate considerable amounts of melatonin. Furthermore, the melatonin production mechanism in the circadian rhythm cycle of blind mole rats is regarded to be different from that of other living beings. The melatonin and Clock genes are hypothesized to be linked to the formation, development, and spread of cancer, but the researchers still cannot explain their extraordinary cancer resistance. In this study, we hypothesized that the melatonin production mechanism in the circadian rhythm cycle of blind mole rats, which have been shown in the literature to be cancer-resistant, may differ from that of other living species due to the difference in their amino acid variations. Differences in the DNA of the Clock genes (Cry1 and Per1) involved in melatonin biosynthesis in blind mole rats were studied compared to other model species (Spalax galili, Mus musculus, Heterocephalus glaber, Rattus norvegicus, and Homo sapiens). As a result, while no variations were found in the Cry 1 gene; only p.G7R variation was found in the Per1 gene. The SNAP2 software had demonstrated that the human analogs of this variation harmful effects. It was suggested that more or full exon sites, more samples and genes should be studied to observe more variants in Nannospalax species. Thereby, the cancer resistance of blind mole rats may be explained better by these variations and the functions of protein domains where these variants are located in.


I. INTRODUCTION
Nannospalax is a member of Spalacidae family, which is one of the most extreme cases of rodents adopting subterranean ecological niche [1], [2]. The blind mole rats, which have both behavioral and physiological adaptations to underground life, dig underground tunnels and rarely rise above ground. Their eyes are protected by a thick layer of skin and dark fur [3]. The physical features of their vestigial eye are mostly aberrant, however, there is still a distinct retinal layer remaining. Although photic information is unlikely to reach retina, it appears that the eye of the blind mole rats can perceive photoperiod. The blind mole rat's eye does not have an evoked visual potential; yet the retina may still be light sensitive and functionally implicated in the photoperiodic response [4]. It has been demonstrated that these animals' vestigial eyes, particularly Harder gland around the eye, can synthesize substantial amounts of melatonin and 5methoxy tryptophol [5]. Melatonin synthesis is controlled by the circadian cycle of living organisms. Light is one of the primary physical elements that has a direct impact on the melatonin synthesis [6]. The biological clock and consequently the 24-hour circadian rhythm are highly connected in the production of the melatonin. Melatonin production peaks at night and then reduces precipitously when exposed to light throughout the day [7]. The regulation of the circadian rhythm of melatonin has been emphasized by the persistence of the cycle in animals kept in the dark [8].
Some details, such as melatonin secretion and circadian rhythm in the blind mole rat species, are still unknown. The key information that researchers are interested in this regard are how this underground species, which has no vision, might adapt to variations in the daily rhythm of light. According to some researchers, this is because blind mole rats, which lack the ability to distinguish visual images, have a complex photo-sensing system and subcutaneous atrophic eyes that respond rhythmically to light [9], [10].
Previous studies showed that disruption of melatonin production or Clock gene expression could promote inflammation or cancer development which could be caused by a variety of disorders (including Alzheimer's disease and liver disease) [11]. Multiple studies have indicated that melatonin has oncostatic effects on many kinds of cancers [12]. The melatonin has been reported to lower the incidence of experimentally induced malignancies [13], [14], [15] and considerably inhibit the growth of some human breast tumors [16].
Any disruption in the circadian rhythm, which controls biological processes in all organisms, causes an abnormal function in the expression of genes responsible for this process (Clock), including rats, and has related to a variety of cancers, including some hormone-dependent and hormone-independent cancers. Tumor development refers to a number of complex processes. Understanding the diurnal patterns of the nucleus clock genes and their clock-controlled genes, on the other hand, is critical for determining the molecular process behind the cancer phenotype. Because the melatonin production and secretion occur in a rhythmic manner during the light-dark cycle, understanding this information can aid in the implementation of a more realistic strategy for both diagnosis and treatment, along with the progression of the disease [17].
On the other hand, spontaneous tumors were not discovered among thousands of individuals (Israeli mole rats) in cancer research conducted on mole rats over a long period of time (50 years). Animals are extremely resistant to chemically induced cancers [18].
In general, studies have shown a lot of evidence associating the circadian rhythm mechanism and circadian rhythm-related clock genes with cancer development and progression, but more research is required in this area. The results show that there is a relationship between melatonin and clock genes and the development of cancer. The entire mechanism is connected to the 24-hour circadian rhythm cycle.
In this study, variants of Clock genes (Cry 1 and Per1), which are involved in the biosynthesis of melatonin and play a role in the regulation of many biological functions such as sleep, circadian rhythm, adrenal gland and thyroid gland functions, reproduction, aging, the antioxidant system, and immunity, were investigated on blind mole rats. Additionally, this study presented DNA-based information for the Nannospalax species (Nannospalax xanthodon, Nannospalax ehrenbergi and Nannospalax leucodon) living in Turkey. We hypothesize that the melatonin biosynthesis mechanism in the circadian rhythm cycle of blind mole rats, which are known to be cancer-resistant according to the literature, may be different from that of other living creatures. As a result, the differences in the DNA of the Clock genes (Cry1 and Per1) involved in melatonin biosynthesis in blind mole rats were researched in comparison to other living model organisms (Spalax galili, Mus musculus, Heterocephalus glaber, Rattus norvegicus, and Homo sapiens).

A. Sampling
Ten different cytotypes (two samples from each cytotype) belonging to 3 different blind mole rat species (2n = 36, 48, 52, 54, 56 for N. xanthodon; 2n = 48, 52, 54, 56 for N. ehrenbergi; 2n = 56 for N. leucodon) distributed in Turkey were used in the study. For each gene, a total of 20 samples were studied. The liver, kidney, and muscle tissues of mole rats obtained from our previous studies were used for extracting DNA.

B. DNA Isolation and Polymerase Chain Reaction
CTAB (Cetyltrimethylammonium bromide) DNA isolation method was used previously described by [19]. Following conditions were used for PCR cycle: a 5-minute initial denaturation at 95°C; 40 cycles of denaturation (4 minutes at 94°C), annealing (1 minute at 58°C), and extension (1 minute at 72°C); and a 5-minute final extension at 72°C.
The PCR amplification was carried out in 25 µl reactions with 5 µl template DNA, 5U Taq DNA polymerase (Thermo Scientific TM ), 1X Taq Buffer, 10 mM dNTP, 25 mM MgCl2 (Thermo Scientific TM ), and 10 pmol/l of each primer. The PCR products were separated on a 1% agarose gel in Tris-acetate-EDTA buffer (TAE) and stained with ethidium bromide.

C. Bioinformatic Analysis
The sequence results were cleaned in the Bioedit software. The similarities between the sequences evaluated in the BLAST database and the sequence in the database were investigated, and the differentiated bases were identified. First, the variations in the amino acid sequences of Nannospalax galili and Homo sapiens, Mus musculus, Rattus norvegicus, and Heterocephalus glaber were determined using the ENSEMBL database [20].
After the variations in Cry1 and Per1 genes of humans were researched, the pathogenic features of these variants were investigated using a software program such as SNAP2. SNAP2 software was used to quantify the effect of single amino acid sequence variations on protein function in the human Cry1 and Per1 genes. The SNAP2 software was used to estimate the functional effects of single amino acid sequence variations [21].

III. RESULTS
The Bioedit sequence alignment editor was used to analyze and align the DNA sequence data. The NCBI-Blast database was used to screen all obtained DNA sequence results. Fig. 1 represents that Sequence alignment of Cry1 gene in the Bioedit software and Fig. 2 shows the alignment of the Per1 gene sequence with the reference to the Per1 gene in the GenBank Blast database.

B. In-Silico Analysis of Oncogenic Pathogenic Effects in Detected Variants
In Cry1 protein, since no alterations were detected in any species, no pathogenic effects were analyzed for humans in the SNAP2 software. On the other hand, the SNAP2 software was utilized to evaluate if human variations of Per1 proteins are pathogenic. According to the SNAP2 in-silico results, p.G7R, variant in human Per1 protein were found in 65 score, and the estimated pathogenic effect values were positive (Fig. 9). The evolution of the eye is physiologically influenced by light signals in the eye, which are the primary cause of the circadian rhythm. In this scenario, the question of potential function loss comes to mind. The functionality of blind mole rats' eyes, which have been shielded from light for millions of years, is demonstrated by their protection of the eye function [23]. The Spalax circadian clock functions similarly to other rodents via a group of clock genes [24]. Here, the genes in blind mole rats were compared with human and other model organisms to determine whether any variation in the Clock gene confers cancer resistance in this evolutionary history of blind mole rats.
The lack of variation in the Cry1 gene of the three Nannospalax species may be a coincidence, or it may be caused by the absence of variation in the sequenced exon in the study.
On the other hand, in the Per1 gene, only one variation (p.G7R) was observed compared to the reference genome Nannospalax galili. On the other hand, one variation (p.G7R) was determined compared to the Homo sapiens. These changes were not situated on any domain. But it showed a harmful amino acid change in humans, which suggested that the variation was either effective or pathogenic.
V. CONCLUSION In this DNA sequence-based study of two Clock genes Cry1 and Per1 involved in melatonin biosynthesis, although the relationship between eye evolution and cancer resistance in blind mole rats could not be clarified, one variation was found in Per1 gene in Nannospalax species compared to the Nannospalax galili and Homo sapiens.
In conclusion, the differences seen in these organisms offer no explanation for why blind mole rats are more cancer-resistant than the other species. For more information it is recommended to increase the number of samples and expand the study with more genes and whole exon regions in future studies.