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Circadian Oscillation of Natural Antisense Transcripts Related to Human Core Clock Genes

Background: Circadian clocks are autonomous intracellular oscillators that synchronize metabolic and physiological processes with the external signals. So, misalignment of environmental and endogenous circadian rhythms leads to disruption of biological activities in living organisms. Noncoding transcripts including antisense RNAs are an important component of the molecular clocks. Commonly, the antisense transcripts are involved in the regulation of gene expression. PER2AS and CRY1AS are the only known Natural Antisense Transcripts (NAT) among the core clock genes, which overlap with the PER2 and CRY1 genes, respectively. In this study, we hypothesized that PER2AS and CRY1AS like the other clock genes, exhibit the oscillatory behavior in a 24-hour period and affect the expression of PER2 and CRY1. Methods: First, the A549 cell line was cultured under standard conditions. After horse serum shock, RNA extraction and cDNA synthesis was performed; then the expression fluctuations of PER2AS, CRY1AS, PER2, and CRY1 were measured with Real-time PCR. Results: Our result showed that PER2AS and CRY1AS had similar oscillation patterns with their sense strand during 24-hour period. Conclusions: Therefore, we suggested that PER2AS and CRY1AS transcripts probably by preventing the interaction of miRNAs with PER2 and CRY1 mRNAs, influence the expression of them, positively.


Introduction
The majority of living organisms are equipped with a time measuring system that is known as a circadian clock. The suprachiasmatic nucleus (SCN) as a master regulator of the mammalian circadian clock is responsible for synchronizing internal biological rhythms with day/night periods and controlling the peripheral clocks in the other tissues (1)(2)(3)(4). The dysregulation of circadian rhythms is closely involved in the development of different human metabolic diseases. Furthermore, clock genes play a key role in the cell cycle and cancer-related processes like cell proliferation, apoptosis, and DNA damage response; thus, improper expression of them are associated with various cancers (5)(6)(7)(8)(9).
From a molecular viewpoint, the circadian clock is composed of multiple interconnected transcriptional/translational feedback loops (TTFL). the primary feedback loop contains the core clock genes (CCG) including BMAL1, CLOCK, Period (PER1/2/3) and Cryptochrome (CRY1/2) family genes (8,10). BMAL1-CLOCK heterodimer in a positive feedback loop, activate the expression of PER and CRY genes. Consequently, PER and CRY proteins dimerize in the cytosol and after translocation to the nucleus, interfere with the BMAL1-CLOCK transcriptional activity. Eventually, by reducing the expression of PER and CRY genes, the inhibitory effect of them is eliminated from the BMAL1-CLOCK complex (6,11,12). Natural antisense transcripts (NAT) are single-stranded noncoding RNAs (ncRNAs) that are transcribed from the opposite strand of coding or non-coding genes. They can be cis or trans-acting and generally play a critical role in the regulation of their target gene expression, positively or negatively (13,14). Dysfunction of some antisense RNA (asRNAs) can lead to several diseases such as diabetes, cancers, and neurological disorders (15)(16)(17)(18). The effect of antisense transcripts on mRNA expression is based on the cellular location of sense and antisense RNA interaction. Antisense transcripts that remain in the nucleus, inhibit the translation of mRNAs, but in some cases that the sense-antisense RNA duplex form in the cytoplasm, asRNAs can increase the stability and expression of the sense strand. This is done by competing with common miRNAs and covering their binding site within target mRNAs (19). More than 70% of the human genome generates antisense transcripts. As mentioned, some of them are considered as main regulators of gene expression; thus, their disruption can cause various diseases. For instance, MACC1-AS1 is an antisense RNA that sponges the tumor suppressor miRNAs (miR-384 and miR-145-3p) related to pleiotrophin and c-Myc proto-oncogenes; hence, the upregulation of MACC1-AS1 leads to increased cell proliferation and invasion (20)(21).
So far, two cis-acting natural antisense transcripts related to the human core clock genes called PER2AS and CRY1AS have been identified that originated from PER2 and CRY1 genes, respectively. Recent studies in the mouse liver indicated that mPer2as and mPer2 transcripts have antiphasic oscillatory patterns. Furthermore, it became clear that mPer2as is one of the clock controlling factors (22).
We hypothesized that antisense transcripts present in the core loop of human circadian clock may be involved in circadian clock control. Therefore, we evaluated the expression of PER2AS and CRY1AS compared to PER2 and CRY1 genes to find out the possible interaction between antisense and parental gene expression.

Materials and Methods
Cell culture A549 cell line was obtained from the Pasteur Institute of Iran and was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and L-glutamine (PSG). Then, cells were incubated in humidified atmosphere at 37 °C with 5% CO2.

Clock genes synchronization using horse serum shock method
In order to clock genes stimulation, approximately 2×10 5 cells/well were seeded in a 24-well plate in triplicate. After three days the cells became confluent and at this time they were exposed with serum-rich media, DMEM supplemented with 50% horse serum. After 2 hours' incubation at 37 °C, this medium was exchanged with pre-warmed serum free DMEM plus PSG. Finally, every 2 hours' cells were washed twice with ice-cold PBS and harvested in 200 µl/well Trizol (Gibco, Life technologies, Carlsbad, CA, U.S.A) at 13 time points and during a 24 hours' period (23).

RNA extraction and cDNA synthesis
Total cellular RNA was isolated by Trizol reagent (Gibco, Life technologies, Carlsbad, CA, USA) according to the manufacturer's guidance. The purity of RNA was measured at 260/280 nm with a UV spectrophotometer and the extracted RNA was stored at -70 °C. Then, 1 µg of total RNA was reverse transcribed using the cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). For this purpose, according to the manufacturer's specification, 1 µg of RNA was initially treated with DNase I enzyme, then 1 µl of random hexamer primer plus RNase-free water (up to 12 µl) was added to the mixture and placed at 65 °C for 5 min. In the next step, 5X Reaction Buffer (4 μl), RiboLock RNase Inhibitor (1 μl), 10 mM dNTP Mix (2 μl) and reverse transcriptase (M-MLV) enzyme (1 μl) were added, respectively (total volume 20 µl). The mixture was incubated for 5 min at 25 °C followed by 60 min at 42 °C and 5 min at 70 °C.

Quantitative Real-time PCR
To evaluate the expression of PER2, PER2AS, CRY1, CRY1AS, and PSMB2 genes, qRT-PCR was performed in a volume of 20 μl using 10 μl SYBR Green 2X master mix (Yekta Tajhiz Azma, Tehran, Iran), 1 μl cDNA, and 0.5 μl of each primer. The Proteasome 20S Subunit Beta 2 (PSMB2) as a reference gene was used for normalization. The PCR was carried out with the following protocol: 1 cycle at 95 °C for 30 sec, followed by 40 cycles at 95 °C for 5 sec, 60 °C for 30 sec and 72 °C for 40 sec. Table 1 shows the list of primer sequences that were applied in this study. The PCR products were checked on 2% agarose gel with 100 bp DNA ladder.

Statistical analysis
The specificity of primers was controlled by NCBI blastn tool. Relative level of gene expression was obtained from the comparative ΔΔCT Method (CT Method). Charts were drawn using the SAS JMP Statistical Discovery software (version 13.0.0). Crosscorrelation function was calculated by R software (version 3.3.2). The data of common miRNAs between the sense and antisense transcripts were collected from the miRDB database and presented in Table 2.

PER2AS and CRY1AS follow similar oscillatory pattern with target mRNA
Evaluation of PER2AS and CRY1AS expression along with PER2 and CRY1 genes after horse serum shock treatment over 24 hours, showed that PER2AS and CRY1AS have a circadian oscillatory pattern and this oscillation is similar to the expression pattern of their sense strand. Indeed, although the expression level of them varies at different time points but display similar oscillatory behavior with PER2 and CRY1 genes (Fig. 1). in addition, it seems that the circadian expression pattern of PER2 and CRY1 genes be in harmony with each other.

PER2AS and CRY1AS are expressed coincidently with PER2 and CRY1
Cross-correlation analysis revealed that the expression of both antisense transcripts coincides with their target mRNA without time lag. the highest correlation coefficient for PER2AS-PER2 and CRY1AS-CRY1 transcript pairs were 0.97 and 0.93, respectively. Also, the strongest correlation of CRY1 and PER2 genes was observed at lag= 0 (r= 0.94), which means, their expression was correlated to each other (Fig. 2).

Discussion
Today, it has been revealed that some antisense transcripts play a pivotal role in cellular and molecular processes (24,25). Based on this findings, antisense transcripts of the circadian clock are probably involved in its regulation. So, in this study, we assessed the expression oscillation of NATs along with their ancestral genes in the clock. Our results showed that PER2AS and CRY1AS as the sole antisense transcripts in the molecular clock genes, have a rhythmic expression pattern within 24 hours and the circadian fluctuation of them is similar to PER2 and CRY1 mRNAs, respectively. According to ccf analysis, the strongest positive correlation between the expression of these sense and antisense transcripts was observed at lag= 0. In fact, they oscillate synchronously but independently of each other. Recently, a study demonstrated that the mouse Per2as transcript also exhibits circadian rhythmic expression, except that its fluctuation is antiphasic with the mPer2 mRNA. However, it was reported that the mPer2 level enhanced dramatically following the overexpression of mPer2as (22). Although the majority of asRNAs interfere with the expression of their target genes, the PER2AS-PER2 and CRY1AS-CRY1 pairs are expressed in coordination with each other. Therefore, it can be suggested that PER2AS and CRY1AS regulate the expression of PER2 and CRY1 by masking the miRNAs binding site within PER2 and CRY1 mRNAs.
In conclusion, PER2AS and CRY1AS may have a regulatory function in the clock network and further studies are needed to be performed about their biological and pathological significance.