Ionizing radiation altered the expression profiles and intracellular distribution of the circadian gene per1 in mouse brain neuronal cells

doi:10.21203/rs.3.rs-4666704/v1

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Ionizing radiation altered the expression profiles and intracellular distribution of the circadian gene per1 in mouse brain neuronal cells

https://doi.org/10.21203/rs.3.rs-4666704/v1

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Circadian rhythms are highly conserved in virtually all organisms. They regulate numerous biological functions and keep them synchronized with alterations in the external environment. Environmental factors such as light, temperature and microgravity have been shown to affect circadian rhythms, but the impact of ionizing radiation (IR) on circadian rhythm is still elusive. Here, the transcription and translation of key circadian genes, such as per, clock, cry and bmal1, were detected in mouse brain neurons after 2Gy X-ray or 2Gy carbon ion beam (CIB) irradiation and were compared with those in the unirradiated group. Moreover, the nuclear import of PER1 was detected by immunoblotting and immunofluorescence. The data showed that the expression phase of circadian genes was not significantly impacted by IR in either HT22 or BV2 cells, while the expression levels of per1 were markedly altered by both X-rays and carbon ion beams. Moreover, IR significantly promoted the nuclear import of PER1. Taken together, our findings suggest that IR, as an exogenous factor, disturbs per1 expression and promotes the nuclear import of PER1.

Biological sciences/Biophysics

Biological sciences/Cell biology

Biological sciences/Molecular biology

Ionizing radiation

Circadian rhythm

Circadian gene

Per

The circadian rhythm is a self-sustaining endogenous oscillation that acts as an internal clock adapted to the Earth’s 24 hour rotational schedule. It exists ubiquitously in nearly all organisms, from prokaryotes to mammals, and regulates many physiological and behavioral processes, keeping them synchronized with alterations in the external environment^[1]. The mammalian circadian rhythm system consists of a central pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. It receives time information from internal photosensitive retinal ganglion cells (ipRGCs) and is further transmitted through the retinohypothalamic tract (RHT) to ensure its synchronization to light/dark changes during the day^{[2, 3]}. Rhythm misalignment has been shown to increase the risk of metabolic syndrome^[4], cardiovascular disease^[5], cancer^[6–8], and even COVID-19^[9].

Previous reports indicate that a stable circadian rhythm exists not only in biological individuals but also in cells cultured in vitro^[10–12]. As the central circadian clock, the SCN receives light information through the retinohypothalamic tract embedded in the optic nerve, and the SCN transmits circadian information to peripheral clocks through the sympathetic and parasympathetic nervous systems^{[3, 13]}. Therefore, brain nerve cells are ideal experimental subjects for research on circadian rhythm^[14–17].

In individual cells, the molecular circadian rhythm is genetically controlled by conserved transcription translation feedback loops, including circadian locomotor output cycles kaput (CLOCK), brain and muscle ARNT-Like 1 (BMAL1), period (PER), and cryptochrome (CRY). BMAL1:CLOCK heterodimers bind to E-box elements containing enhancers upstream of the promoters of per and cry to induce their expression. PER accumulates in the cytoplasm and is gradually phosphorylated; then, it forms a heterodimer with CRY and translocates into the nucleus. The PER-CRY heterodimer in the nucleus inhibits the activity of BMAL1:CLOCK, consequently resulting in the inhibition of PER transcription and CRY transcription as well as the transcription of other circadian genes that compose secondary transcriptional translation feedback loops ^{[10, 18]}.

Light and temperature are the most significant zeitgebers of the body’s circadian rhythm^[19], and other periodic synchronizers, such as age^[20], mood^[21] and the schedule of ingestion/fasting^[22], also affect the circadian rhythm. Moreover, recent studies revealed that changes in abiotic environmental conditions, such as oxygen saturation^[23], chemical conditions^[24], and microgravity^[25], can disturb or entrain circadian rhythms, resulting in a greater risk of cardiovascular diseases, metabolic diseases, psychiatric disorders, neurodegenerative diseases, and even cancers^[26].

Ionizing radiation (IR), such as X-rays and carbon ion beam (CIB), can induce DNA damage and specific responses^[27]. Thus far, according to the few studies focusing on the influence of IR, the expression of circadian genes is altered both in vivo and in vitro by radiation, as is the behavioral rhythm of animals. For example, X-ray irradiation affects the molecular clockwork in the liver, pancreas and adrenal glands, leading to phase advances^[28]. X-rays (3 Gy) cause dramatic reproductive disruption in male mice, as indicated by decreased mRNA expression of clock genes in the testis, decreased sperm motility, increased daily sperm production, and reduced activity of testis enzymes^[29]. A low dose of γ-ray radiation interferes with the expression of bmal1b and clock in zebrafish, increases embryo melatonin secretion, and affects behavioral rhythm^[30]. Although the nervous system plays an important role in circadian biology^{[3, 13]}, the impact of IR on neural cells is not well understood. Moreover, as a kind of particle radiation, carbon ion beam radiation causes more serious cellular damage than photon radiation, such as X-rays and γ-rays, while differences in circadian gene expression after X-ray and carbon ion beam radiation have not been studied.

In this study, 2 Gy X-rays and CIB were used to treat HT22 mouse hippocampal neuronal cells and BV2 mouse microglia, and the mRNA and protein levels and subcellular localization of circadian genes were tested. The results showed that IR hardly shifted the expression phase of circadian genes but significantly altered the amplitude of per1 expression, especially at 12 to 24 hours after radiation. Moreover, IR significantly promoted the nuclear import of PER1. Taken together, our findings suggest that IR, as an exogenous factor, disturbs per1 expression and promotes the nuclear import of PER1.

Cell culture

The HT22 mouse hippocampal neuronal cells and BV2 mouse microglia were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures). HT22 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma‒Aldrich, Darmstadt, Hesse, Germany) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. BV2 cells were cultured in DMEM (Sigma‒Aldrich, Darmstadt, Hesse, Germany) supplemented with 10% FBS, 1% 1 M HEPES, 100 U/mL penicillin and 100 µg/mL streptomycin. The culture was maintained at 37°C and 5% CO₂ in a constant temperature incubator with saturated humidity.

Irradiation

The X-rays were generated by an X-Rad 225 generator (Precision, North Branford, CT, US) with an energy of 225 kV/13.3 mA. Carbon ion (¹²C⁶⁺) beams with an energy of 80 MeV/u were generated by the Heavy Ion Research Facility in Lanzhou (HIRFL), Institute of Modern Physics, Chinese Academy of Sciences (China), at a dose rate of 0.5 Gy/min.

Serum starvation

After 24 hours of cell plating, the HT22 cells were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 1 µM dexamethasone for 24 hours at Zeitgeber time 0 hours (ZT0). BV2 cells were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 1% 1 M HEPES and 1 µM dexamethasone for 24 hours at ZT0. All the cultures were maintained at 37°C and 5% CO₂ in a constant temperature incubator with saturated humidity.

Serum shock

After 24 hours of cell plating, the HT22 cells were cultured in DMEM supplemented with 50% horse serum, 100 U/mL penicillin and 100 µg/mL streptomycin for 2 hours at ZT0. Then, the HT22 cells were cultured in DMEM without FBS. BV2 cells were cultured in DMEM supplemented with 50% horse serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1% 1 M HEPES for 2 hours at ZT0. Then, BV2 cells were cultured in DMEM without FBS. All the cultures were maintained at 37°C and 5% CO₂ in a constant temperature incubator with saturated humidity.

Flow cytometry

The cells were fixed in 70% ethanol for 24 hours. After centrifugation, the supernatant was discarded, and 200 µL of 20 µg/mL propidium iodide (PI, Solarbio, C0080) was added to the precipitate. The cells were incubated for 30 minutes in the dark, followed by detection using a Beckman MoFloAstrios EQ flow cytometer.

qRT‒PCR

TRIzol was used to extract RNA, and cDNA was obtained by reverse transcription of the RNA according to the reverse transcription kit manual (GeneCopoeia, QP056). The cDNA used for real-time quantitative detection was SYBR Green (GeneCopoeia, QP041). The final volume was 20 µL, and a total of 40 cycles were executed. Predenaturation was implemented at 95°C for 10 min, followed by denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec, and primer template extension at 72°C for 15 sec at a heating rate of 0.5°C/6 sec between 72°C and 95°C. All data collection was completed using Bio-Rad CFX96 PCR system software (Bio-Rad Laboratories, Hercules, CA, USA). We calibrated the collected Ct values to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference and analyzed target gene expression using the 2-∆∆CT method. The primer sequences are shown in Table 1.

Table 1

Primer sequences. F, forward; R, reverse
Name of primer	Sequences
per1	F: 5’-GATGTGGGTGTCTTCTATGGC-3’
per1	R: 5’-AGGACCTCCTCTGATTCGGC-3’
cry1	F: 5’-CACTGGTTCCGAAAGGGACTC-3’
cry1	R: 5’-CTGAAGCAAAAATCGCCACCT-3’
clock	F: 5’-ATGGTGTTTACCGTAAGCTGTAG-3’
clock	R: 5’-CTCGCGTTACCAGGAAGCAT-3’
bmal1	F: 5’-ACAGTCAGATTGAAAAGAGGCG-3’
bmal1	R: 5’-GCCATCCTTAGCACGGTGAG-3’
pi3kca	F: 5’-TTATTGAACCAGTAGGCAACCG-3’
pi3kca	R: 5’-GCTATGAGGCGAGTTGAGATCC-3’
akt1	F: 5’-ATGAACGACGTAGCCATTGTG-3’
akt1	R: 5’-TTGTAGCCAATAAAGGTGCCAT-3’

Western blot

The cells were collected and lysed with RIPA lysis buffer (Beyotime, P0013) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) on ice. Equivalent concentrations of protein were separated via gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane via wet transfer, and blocked for 3 h at room temperature with 5% skim milk in PBS containing 0.1% Tween-20 (PBST). The membranes were subsequently incubated overnight with primary antibodies against PER1 (Proteintech, 13463-1-AP, 1:2000 dilution), GAPDH (Proteintech, 60004-1-IG, 1:50000 dilution), and β-actin (Proteintech, 20536-1-AP, 1:2000) at 4°C with oscillation. The blots were washed four times with PBST, and the membranes were reprobed with appropriate secondary antibodies conjugated with HRP (Epizyme, LF101/LF102, 1:5000 dilution) for 2 h at room temperature. The blots were processed with an enhanced chemiluminescence kit and exposed to the film. Gray analysis of multiple WB bands was performed using ImageJ software (National Institutes of Health, MD, USA) to quantify the relative expression of PER1 (normalized to that of GAPDH or β-actin).

Cytoplasmic and nuclear protein separation

The proteins were extracted with a Nuclear and Cytoplasmic Protein Extraction Kit (P0028) from Beyotime according to the manufacturer’s instructions. A total of 2×10⁶ cells were collected into 1.5 ml tubes and washed with PBS. After centrifugation, the cells were precipitated with 200 µL of separation reagent A (Beyotime, P0028-1) containing PMSF (Beyotime, ST506), vortexed for 5 seconds and placed on ice for 15 minutes. Then, separation reagent B (Beyotime, P0028-2) was added to the suspension, which was vortexed for 5 seconds and placed on ice for 1 minute. The cells were then centrifuged for 5 minutes at 4°C and 14000 × g, and the supernatant contained the cytoplasmic protein. Then, 50 µl of separation reagent C (Beyotime, P0028-3) containing PMSF was added to the precipitate, which was vortexed for 20 seconds and placed on ice for 2 minutes; this process was repeated for a total of 30 minutes. Subsequently, the cells were also centrifuged for 5 minutes at 4°C and 14000 × g, and the supernatant contained the nuclear protein.

Luciferase reporter transfection

The lentiviral vectors (LVCON335) and transfection-promoting reagents (REVG003) were obtained from GeneChem. Twenty-four hours after 5×10⁴ HT22 cells were plated in 12-well plates, the cells were transfected with 500 µL of complete DMEM containing 50 µL of 1×10⁸ TU/mL lentivirus and 20 µL of HiTransG P. After 16 hours of transfection, the medium was replaced with complete DMEM. Subsequently, the cells were screened with 2 µg/mL puromycin (MCE, HY-B1743A) for 48 hours to obtain stably positive cells. All the cultures were maintained at 37°C and 5% CO₂ in a constant temperature incubator with saturated humidity.

Immunofluorescence staining

The cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and then incubated with methanol on ice for 20 min. The fixed cells were permeabilized in 0.5% Triton X-100 for 10 min and blocked with 5% goat serum for 2 h at room temperature. Then, the cells were incubated with an anti-PER1 primary antibody (Santa Cruz, sc-398890, 1:100 dilution) at 4°C overnight after the blocking step. The bound antibodies were visualized using a goat anti-mouse IgG secondary antibody (Invitrogen, CA, USA; 1:1000). The cell nucleus was stained with 4′,6-diami-dino-2-phenylindole (DAPI) molecular probes (Invitrogen, OR, USA). Images were obtained using an RVL-100-G (ECHO, CA, USA) fluorescence microscope. Gray analysis of the images was performed using ImageJ software (National Institutes of Health, MD, USA) to quantify the relative expression of PER1.

Statistical analysis

All the data are presented as the arithmetical mean ± standard error. Student’s t test was used to determine the differences between the irradiated groups and the control group. Differences were considered significant at P < 0.05 (marked with *), P < 0.01 (marked with **), P < 0.001 (marked with ***), and P < 0.0001 (marked with ****).

Expression rhythm of circadian genes in HT22 and BV2 cells

The expression of circadian genes such as per1, cry1, clock and baml1 was tested in BV2 mouse microglia and HT22 mouse hippocampal neuronal cells to determine the biological rhythm in vitro. Since the circadian rhythm and cell cycle are mutually regulated in a coupled manner^[31], to avoid the influence of cell cycle regulatory factors, cell cycle synchronization was chosen to stabilize the cell rhythm prior to radiation treatment^[31]. In detail, cells were subjected to serum starvation^{[32, 33]}, dexamethasone (Dex)^[34], serum shock^[35], or a combination of both serum starvation and Dex, after which the cell cycle distribution was tested by flow cytometry (FACS).

Treatment with 1 µM Dex for 24 hours induced a significant increase in the percentage of HT22 cells in the S phase and a significant decrease in the percentage of HT22 cells in the G2/M phase but did not change the percentage of cells in the G₀/G₁ phase (Fig. 1a). As expected, the serum starvation treatment induced a marked cell cycle synchronization at the G₀/G₁ phase in HT22 cells. The combination of serum starvation plus 1 µM Dex for 24 hours increased the proportion of cells in both the G₀/G₁ phase and S phase but decreased the proportion of cells in the G₂/M phase (Fig. 1a). The BV2 cells showed a similar result. Combined treatment for 24 hours increased the proportion of BV2 cells in both the G₀/G₁ phase and S phase but decreased the proportion of cells in the G₂/M phase (Fig. 1b). Considering the effect of all treatments on both cell lines, the combined treatment was used to stabilize the cell cycle in subsequent studies, and the end time of the combined treatment was regarded as zeitgeber time 0 (ZT0).

At ZT0, ZT6, ZT12, ZT18, ZT24, and ZT30, RNA samples were collected, and the mRNA levels of per1, cry1, clock, and bmal1 were measured via RT‒qPCR. As shown in Fig. 1c and 1d, in both HT22 and BV2 cells, the mRNA levels of per1 decreased significantly in the first 6 hours after cell cycle synchronization and then climbed steadily until ZT30 (Fig. 1c and 1d). The expression of cry1 in HT22 cells increased at ZT6 but decreased rapidly from ZT6 to ZT12 and then started to increase. However, we detected a decrease in cry1 at ZT6 in BV2, and the expression of cry1 decreased in BV2 at 12 hours after cell cycle synchronization and then increased steadily until ZT30. In both HT22 and BV2 cells, the mRNA levels of clock and bmal1 increased in the first 6 hours and then peaked gradually with fluctuating decreases and increases (Fig. 1c and 1d). The amplitudes of increase were much greater at ZT30 in both HT22 and BV2 cells (Fig. 1c and 1d). In addition, a serum shock treatment using 50% horse serum^[35] was also performed to stabilize the rhythm of the HT22 cells. After 24 hours of cell plating, the HT22 cells were treated with medium containing 50% horse serum for 2 hours, after which the medium was replaced with serum-free medium for subsequent serum starvation treatment (Supplementary Fig. S1a). Treatment with serum shock for 2 hours did not significantly change the proportion of cells in the cell cycle, while subsequent serum starvation induced significant G₀/G₁ synchronization. The proportion of cells in the G₂/M phase also decreased, but the proportion of cells in the S phase did not change significantly (Supplementary Fig.. S1b). The changes in per1, cry1, clock, and bmal1 expression were similar to those in the serum starvation plus Dex combined treatment group (Supplementary Fig. S1c).

In summary, the expression levels of circadian genes varied with culture time. Although only the expression of per1 and cry1 showed stable periodic oscillations, the data verified that there were stable rhythms and oscillations in mouse neurons in vitro.

Transcriptional changes in per1 and cry1 were altered by IR

To investigate the effect of IR on circadian gene expression in mouse brain neurons, HT22 and BV2 cells were synchronized at the G₀/G₁ phase by combined treatment and irradiated with X-rays or CIB at the end of treatment at zeitgeber time 0 (ZT0). RNA samples were collected at ZT0, ZT6, ZT12, ZT18, ZT24, and ZT30, and the mRNA levels of per1 and cry1 were detected by RT‒qPCR (Fig. 2a).

As shown in Fig. 2B, the mRNA levels of per1 in HT22 cells were the same in both the control and irradiated groups. In particular, in the first 12 hours after radiation, the expression decreased and remained steady. Then, the per1 mRNA levels started to increase after ZT12, and the increase in the irradiated groups was faster than that in the control group, while a significant difference was detected only at ZT24 in the CIB-irradiated group and X-ray-irradiated group. Notably, the increase in per1 in the CIB group was much greater than that in the X-ray and control groups at ZT24 (Fig. 2b). The mRNA level of cry1 also showed the same phase of expression in the different groups: the expression level increased, peaked at ZT6, then gradually decreased and stabilized. However, there was no significant difference in the expression level within 30 hours (Fig. 2b). In irradiated BV2 cells, the expression phase of per1 and cry1 was also similar to that in the unirradiated group. A significant increase in per1 in irradiated BV2 cells was detected at ZT18; however, we failed to detect a significant difference in cry1 expression (Fig. 2c).

In addition, we confirmed the effect of IR on circadian gene expression in a serum shock system (Supplementary Fig. S1a). The results showed that the expression phase of per1 in HT22 and BV2 cells did not change but remained at a minimum at ZT6 and then increased until ZT30. However, the expression level of per1 was also significantly upregulated in HT22 and BV2 cells at ZT18 and ZT30, respectively (Supplementary Fig. S1d). This result was also similar to that under serum starvation conditions.

In summary, IR, such as X-rays and CIB, failed to impact the expression phase of circadian genes in vitro but significantly changed the gene transcription levels of per1 in neurons, especially at 18 to 24 hours after radiation.

IR influenced the protein levels of PER1

HT22 and BV2 cells were synchronized and irradiated, and total cellular proteins were subsequently extracted at the same time points to examine changes in per1 expression at the protein level via western blotting.

As shown in Fig. 3A, the expression phase of the PER1 protein in unirradiated HT22 cells was consistent with that of the PER1 mRNA (Fig. 1c and 2b). The protein and mRNA levels of PER1 decreased significantly in the first 6 hours after it increased gradually. The levels of PER1 in the irradiated HT22 cells were greater than those in the control cells, while a significant increase was detected only in the ZT18 and ZT24 CIB groups. The change in the level of PER1 in the HT22 CIB group showed a trend similar to that of the change in the mRNA level (Fig. 3a and 2b). In addition, the range of increase in the CIB group was greater than that in the X-ray group, which was also consistent with the change in mRNA levels (Fig. 3a and 2b). In BV2 cells, the change in the level of the PER1 protein in the unirradiated group was also consistent with the change in the mRNA level, as shown in Fig. 1D. PER1 protein expression in BV2 cells was increased after irradiation with 2 Gy of X-rays and CIB. However, significant upregulation only occurred at 6 hours and 12 hours in the X-ray group (Fig. 3b). The upregulation of the PER1 protein occurred in the early stage after irradiation when BV2 cells were irradiated with 2 Gy X-rays, and this change was accompanied by a 12–18-hour difference in the expression phase.

In summary, IR had a significant impact on PER1 protein expression, and the changes were consistent with those of per1 mRNA.

IR promoted the nuclear import of PER1

Since the activation and nuclear transfer of PER1 play a nonnegligible role in transcription-translational feedback loops, in consideration of the unified increase in PER1 at both the mRNA and protein levels after IR treatment, we further detected the intracellular distribution of PER1 by western blotting and immunofluorescence.

At almost all time points, the levels of PER1 in both the cytoplasm and nucleus after IR treatment were much greater than those in the control, although significant differences were not always detected. For example, the PER1 levels in HT22 cells treated with X-rays were much greater than those in control cells, and a significant increase in nuclear PER1 was detected at ZT12, ZT18 and ZT24. CIB irradiation also upregulated the expression of PER1. An increase in both the cytoplasm and nucleus occurred after ZT12, and significant changes in the cytoplasm were detected at ZT18 and ZT24 (Fig. 4a and 4b). In BV2 cells subjected to X-ray irradiation, PER1 was significantly upregulated at ZT12 in the cytoplasm and at ZT0 in the nucleus. PER1 was also upregulated in the cytoplasm and nucleus of BV2 cells treated with CIB, but the differences were not significant. In addition, it was downregulated at ZT30 in the nucleus (Fig. 4c and 4d). In summary, X-rays increased the amount of cytoplasmic and nuclear PER1 in both HT22 cells and BV2 cells. CIB also induced upregulation in the cytoplasm, while the change in nuclear PER1 after radiation was not significant in HT22 and BV2 cells.

In addition to observing changes in the levels of the PER1 protein in the cytoplasm and nucleus, we also explored the effects of radiation on the nuclear import of PER1. Considering the stable periodic oscillation of per1 expression at both the mRNA and protein levels after X-ray radiation and CIB radiation in HT22 cells, the intracellular distribution of PER1 in HT22 cells was verified by immunofluorescence. The per1 gene promoter is linked to a luciferase reporter in HT22 cells, and a stable PER1:Luc expression cell line was constructed and characterized. We observed the subcellular localization of PER1 at ZT12 and analyzed the intensity of total cellular PER1:Luc and PER1:Luc in the nucleus. The results showed that the ratio of nuclear PER1:Luc to total cellular PER1:Luc increased significantly after 2 Gy X-ray irradiation (Fig. 4e) or 2 Gy CIB irradiation (Fig. 4f). Moreover, immunofluorescence analysis with an anti-PER1 antibody showed similar results (Fig. 4g), which also proved that X-rays promote the transport of PER1 from the cytoplasm to the nucleus.

In summary, the results confirmed that IR promoted the entry of PER1 into the nucleus in HT22 cells.

Nuclear import of PER1 regulated the downstream PI3K/AKT pathway

The circadian gene per1 is a negative element in the primary transcription-translation feedback loop. Complexes containing the PER1 and CRY1 proteins inhibit the activity of CLOCK and BMAL1 after nuclear import, preventing their own continued expression and downstream genes. Therefore, the nuclear import of PER1 is an important factor in regulating the expression of downstream genes. To inhibit the nuclear import of PER1, we knocked down the levels of cry1 with siRNAs (Fig. 5b). As expected, the nuclear import of the PER-CRY heterodimer was significantly suppressed at ZT12 (Fig. 5d). Interestingly, the level of per1 increased during the early period from ZT0 to ZT12 (Fig. 5c).

To explore whether the nuclear import of the PER-CRY heterodimer can regulate the expression of PI3K/AKT in HT22 cells, the mRNA levels of pi3kca and akt1 were detected. Compared with those in the corresponding controls, the mRNA levels of pi3kca and akt1 in si-cry1-HT22 cells were significantly greater at ZT0 (Fig. 5e). Furthermore, the expression levels of the downstream genes pi3kca and akt1 decreased more greatly from ZT18 to ZT24 because of a phase shift as the cry1 expression level decreased.

Taken together, these results indicated that the inhibition of the nuclear import of the PER-CRY heterodimer promoted the continued expression and activation of the PI3K/AKT downstream pathway.

Early studies reported the effects of radiation on circadian rhythms, but most of them focused on nonionizing radiation. Weak broadband radiofrequency fields are reported to slow the circadian rhythms of German cockroaches and Drosophila, but the effect may be due to sensitivity to static magnetic fields in insects^[36]. Rats exposed to radiofrequency-electromagnetic radiation exhibit phase shifting as well as a significant increase in activity^[37], and the expression level of the circadian gene clock is affected in a site-specific way^[38]. According to the few recent studies focusing on the influence of IR, the expression of circadian genes changes both in vivo and in vitro in response to radiation, as is the behavioral rhythm of animals. Although the nervous system plays an important role in circadian biology, the impact of IR on neural cells is not well understood.

Here, 2Gy X-rays or CIB were applied to the cells to investigate the effect of ionizing radiation on circadian rhythm. We demonstrated that the upregulation of per1 occurred mainly at ZT18 in BV2 cells and at ZT24 in HT22 cells, with a 6-hour difference. The mRNA level of cry1 did not show a steady or unified change. Overall, IR changed the expression levels of per1 uniformly under various conditions. In addition, consistent with the results obtained with UVC, we also found that the protein content of PER1 and the relative fluorescence intensity of PER1 in the nucleus were significantly increased, suggesting that ionizing radiation promoted the nuclear import of PER1.

To demonstrate the nuclear entry of PER1, cytoplasmic proteins and nuclear proteins were extracted from HT22 and BV2 cells to examine the expression levels of PER1. We further tested the extent of PER1 expression in the cytoplasm and nucleus of HT22 cells. In the cytoplasm, the expression level of PER1 tended to increase during the first 6–12 hours, after which it peaked and then decreased gradually. The expression phase of the irradiated group was similar to that of the unirradiated group in both the X-ray group and the CIB group (Fig. 4A and Fig. 4C). Unlike that of the cytoplasmic protein, the change in the expression phase of the nuclear PER1 protein significantly differed from that in the HT22 cells after exposure to 2 Gy X-rays and 2 Gy CIB radiation (Fig. 4B). The change in the expression phase was greatest at ZT12. The nuclear expression of PER1 increased from ZT6 to ZT12 in the irradiated group, while it decreased in the unirradiated group. In BV2 cells, the expression phases of cytoplasmic PER1 in both the X-ray- and CIB-irradiated groups were similar to those in the unirradiated group. Cytoplasmic PER1 showed a downward trend in the first 6 hours to reach a trough and then tended to stabilize. Similar to the results in HT22 cells, we found that the expression of PER1 in the nucleus of BV2 cells increased in the irradiated group but decreased in the unirradiated group from ZT6 to ZT12. PER1 in the nucleus increased markedly from ZT6 to ZT12 in the irradiated group, especially in BV2 cells treated with 2 Gy of CIB, and decreased slightly in the unirradiated group. The time point ZT12 was chosen to verify the intracellular distribution of PER1.

Ionizing radiation is widely used in clinical practice for medical imaging, especially radiotherapy. Radiotherapy is the primary treatment for cancer and is used by more than 50% of cancer patients^[39]. Currently, the connection between abnormal circadian rhythm occurrence and tumorigenesis is acknowledged^{[7, 40, 41]}. Therefore, combined research on ionizing radiation and circadian rhythms is important for understanding tumor development. The PI3K/AKT pathway has been shown to induce cell proliferation, migration, and invasion in various tumors^{[42, 43]}. The effect of PER1 on the PI3K/AKT pathway has recently been demonstrated; PER1 overexpression significantly inhibited the PI3K/AKT pathway and tumor growth, while the opposite effects were observed in PER1-knockdown OSCC cells^[44]. However, the mechanism by which PER1 regulates the PI3K/AKT pathway has not been verified. Here, we focused on the nuclear import of PER1 to explore the role of PER1 in regulating downstream pathways. We found that si-cry1 increased the expression of per1. In contrast to previous research, a higher expression level of per1 did not inhibit but rather upregulated the PI3K/AKT pathway. We believe that this phenomenon is due to the inhibition of PER1 nuclear import, as detected by immunofluorescence. Therefore, in addition to the expression of PER1, the nuclear import of PER1 also plays an indispensable role in its regulation.

In this article, we provide evidence that IR upregulates the expression level and promotes the nuclear import of PER1. These results suggest that IR may inhibit the PI3K/AKT pathway via the circadian protein PER1, thereby influencing downstream factors. These findings also provide a new idea for cancer research, starting with the expression and nuclear transport of circadian proteins.

Acknowledgment

We are thankful for the support of the Heavy Ion Research Facility in Lanzhou (HIRFL) at the Institute of Modern Physics, Chinese Academy of Science (Lanzhou, China), for carbon ion radiation. We also want to thank the Public Technical Service Center, Institute of Modern Physics, Chinese Academy of Sciences, since part of the experimental work was carried out on the biomedical platform.

Funding

This research was funded by the National Natural Science Foundation of China (no. 12275329, Nan Ding, no. 12375355, Jinpeng He, and no. 12175289, Jufang Wang), the West Light Foundation of The Chinese Academy of Sciences (xbzglzb2022003, Nan Ding), the Science and Technology Research Project of Gansu Province (no. 145RTSA012, Jufang Wang, no. 21JR7RA108, Nan Ding, and 21JR7RA107, Yanan Zhang), the Gansu Provincial Science Fund for Distinguished Young Scholars (no. 22JR5RA942, Wei Wang, and no. 20JR5RA555, Jinpeng He), and the CuiYing Science and Technology Innovation Program of the Second Hospital of Lanzhou University (CY2023-MS-A03, Wei Wang).

Author contributions

Conceptualization: Nan Ding, Jinpeng He and Jufang Wang; formal analysis: Zhiang Shao, Nan Ding, Yuan Wang, Pei Qu, Yanan Zhang and Jinpeng He; investigation: Zhiang Shao, Yuan Wang, Pei Qu, Yanan Zhang, Jinpeng He, Dong Lu, Wenjun Wei and Junrui Hua; data curation: Dong Lu, Wenjun Wei; writing—original draft preparation: Zhiang Shao and Nan Ding; writing-review and editing: Zhiang Shao, Nan Ding, and Jufang Wang; supervision: Jufang Wang; project administration: Jufang Wang and Nan Ding; funding acquisition: Nan Ding, Jufang Wang and Wei Wang. All authors have read and agreed to the published version of the manuscript.

Availability of data and materials

All data needed to evaluate the conclusions in the paper are presented in the paper and/or the Additional files. Additional data related to this paper may be requested from the authors.

Conflicts of interest

The authors declare no conflicts of interest.

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No competing interests reported.

SupplementaryFigureS1.tif
Figure S1. Rhythm in the brain neurons of mice subjected to serum shock. a, Scheme of IR in serum shock-treated HT22 cells. b, The cell cycle distribution of HT22 cells was determined using flow cytometry 24 hafter treatment with serum shock. c, mRNA levels of circadian genes (per1, cry1, clock and bmal1) in HT22 cells after cell cycle synchronization were measured using quantitative PCR. d-e, mRNA levels of the circadian gene per1in HT22 cells (d) and BV2 cells (e) treated with 2 Gy X-ray radiation. The data are shown as the means ± s.d.s;n=3. *P<0.05 and **P<0.001 according to two-tailed Student’s t test indicatestatistical significance.

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Ionizing radiation altered the expression profiles and intracellular distribution of the circadian gene per1 in mouse brain neuronal cells

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Abstract

Figures

Introduction

Materials and methods

Cell culture

Irradiation

Serum starvation

Serum shock

Flow cytometry

qRT‒PCR

Western blot

Cytoplasmic and nuclear protein separation

Luciferase reporter transfection

Immunofluorescence staining

Statistical analysis

Results

Expression rhythm of circadian genes in HT22 and BV2 cells

IR influenced the protein levels of PER1

IR promoted the nuclear import of PER1

Nuclear import of PER1 regulated the downstream PI3K/AKT pathway

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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