Introduction
Nasopharyngeal carcinoma (NPC) is the most common cancer in Guangdong Province in China, known as "Guangdong cancer". In addition, it is also one of the common malignant tumors in Southeast Asia, which is susceptible to genetic, viral, environmental and other factors [1–3]. Because of the occult location of nasopharyngeal carcinoma, which is surrounded by important blood vessels or nerves, it is high risk and difficult to remove the tumor body by surgery. Moreover, poorly differentiated squamous cell carcinoma is the most common pathological type, accounting for about 85%—90%. Radiation can kill most of its tumor cells, which determines that the preferred treatment for nasopharyngeal carcinoma is radio-therapy [4]. Tumorigenesis is a progressive process, involving multistage reactions and accumulation of mutations, in which circadian rhythm disorder and tissue hypoxia [5] are important factors affecting its occurrence.
Circadian rhythm disorder can cause a variety of diseases, such as hypertension, depression, tumor etc. [6], and the correlation with tumor is a hot topic of current research. According to existing research, when people's circadian rhythm is disturbed, the incidence of liver cancer, breast cancer, glioblastoma and other diseases increases [7–11]. Tumor cells in these cancers disrupt the expression of circadian genes, which is thought to have the potential to promote cancer progression [10, 12, 13]. The suprachiasmatic nucleus of the hypothalamus is the central core structure that controls the circadian rhythm of mammals. When the external light information is transmitted to the brain through the retina, it responds to the circadian changes of the surrounding environment through the neurohumoral regulatory system and transmits the information to various organs and tissues of the organism [14]. Circadian rhythm genes are also called biological clock genes. With the deepening of human research, more and more biological clock genes have been found, including per, cry, clock, BMAL1, Rev ERBS, Tim and other genes [15–17]. Among them, BMAL1 gene exists as the core element of circadian rhythm system, and mutation can cause circadian rhythm disorder of organisms, thus causing the occurrence and development of various diseases. Gwon et al. found that overexpression of BMAL1 gene can inhibit the invasion and metastasis of glioma cells [18], while BMAL1 expression was significantly increased in malignant pleural mesothelioma, playing a cancer promoting role [19]. It can be seen that BMAL1 plays different roles in tumors of different tissues.
Due to the rapid growth of malignant solid tumors and insufficient supply of neovascularization, the whole tumor body and its environment are in a state of ischemia and hypoxia. The hypoxic microenvironment often makes the treatment methods of tumors ineffective, such as radiation resistance and chemotherapy resistance, which are the key factors that make the treatment of solid tumors fail [20]. Hypoxia inducible factor-1α ( hypoxia inducible factor -1α, HIF -1α) as the most important regulator of maintaining oxygen balance, its induced genes play an important role in the development and metastasis of tumors, which is the main reason for tumor cells to survive in the hypoxic microenvironment. Studies have shown that HIF-1α Genes are overexpressed in many types of tumors, such as nasopharyngeal carcinoma, colon cancer and breast cancer [21], and the poor prognosis of patients with malignant tumors is often related to HIF-1α High expression of genes.
HIF-1α and its binding partner Arnt are transcription factors containing the per Arnt sim (PAS) domain, which are structurally similar to the clock genes clock and BMAL1 [22, 23]. This structural similarity also indicates that there is a link between these two paths. Relevant domestic studies have confirmed [24]: the hypoxia response pathway is monitored by the biological clock genome in vivo, and the hypoxia signal in turn regulates the clock by slowing down the physiological cycle and suppressing the amplitude of oscillation in a dose-dependent manner. Wu [24] et al. found a highly conserved canonical E-box (CACGTG) in HIF-1α, Using chromatin immunoprecipitation combined with deep sequencing (ChIP-seq), it was confirmed that BMAL1 binds to this E-box site of human osteosarcoma U2OS cells, and the presence of E-box in HIF-1α promoter suggests that it is directly regulated by clock genes.It has also been found [25] that in mouse nucleus pulposus cells, the BMAL1 gene controls the transcriptional activity of HIF-1α, which once removed, leads to inhibition of nucleus pulposus growth. Similarly, expression of HIF-1α in zebrafish under hypoxic conditions also exhibits a periodic rhythm similar to that of clock-controlled genes [26]. However, the relationship between the clock gene BMAL1 and Hypoxia-inducible factors HIF-1α has not been reported in NPC.
Our previous study [27] found that BMAL1 is a potential tumor suppressor gene in the poorly differentiated nasopharyngeal carcinoma cell line CNE2. In order to further verify the role of BMAL1 in poorly differentiated squamous cell carcinoma, we selected another poorly differentiated nasopharyngeal carcinoma cell line HONE1 for experiments. In this study, we investigated the effects of overexpression of BMAL1 gene or knockdown of HIF-1α gene on the biological behavior and radiosensitivity of nasopharyngeal carcinoma cell line HONE1 in vitro, at the same time, the effect of BMAL1 gene on HIF-1α protein was preliminarily verified by immunoblotting experiment, which also provided some theoretical basis and experimental data for searching new targets for future therapy, in order to provide a new idea for clinical treatment of nasopharyngeal carcinoma.
Materials and methods
Cell culture
The human nasopharyngeal carcinoma cell line HONE1 was purchased from the Cancer Prevention and Control Center of Sun Yat-sen University. HONE1 cells were cultured in medium containing 10% fetal bovine serum and RPMI 1640 (GIBCO) was cultured in an incubator containing 5% CO2 and saturated humidity at 37 ℃.When the cells grow to 80%, they are digested and subcultured with trypsin.
Cell transfections
In order to further explore the effect of BMAL1 and HIF-1α on NPC cells, BMAL1 was respectively overexpressed and silenced by the lentiviral vector, at the same time HIF-1α was silenced.The overexpressed BMAL1 group was labeled as BMAL1-OE, the corresponding control group as OENC, the silenced BMAL1 group as sh-BMAL1, the corresponding control group as ShNC, while the silenced HIF-1α group as sh-HIF-1α, corresponding to negative controls as ShNC.Transfected cells were cultured, passaged, inoculated, and cultured in an incubator.Cell line establishment was determined by the Western blot method.
Western blot analysis
The transfected cells were lysed with Ripa lysate to extract the total protein, and the protein concentration was detected with BCA protein determination kit; The expression level of the target protein was detected by Western blot, and the gray level was analyzed by Image-A software.
CCK-8 assay
Take synchronized cells, conduct manual cell counting and plant them in 96 well plates. Add 200 μL cell suspensions into each well, including 2 × 103 cells. Each group of cells is provided with 3 reperforations each time. Place 96 well plates in the cell incubator for 24 h (37℃, 5% CO2), suck out the culture solution at the corresponding time points of 1d, 2d and 3d respectively, add fresh and complete culture solution again with a liquid transfer gun, the liquid volume is 100 μL, and then add 10 μL CCK-8 reagents. Avoid bubbles in the holes during operation, which will affect the final experimental results and lead to experimental errors. Continue to culture in the incubator for 1–4 h, then take out the 96 well plate, use the microplate reader to measure the absorbance value of each group of cells at 450 nm, record the results and draw the cell growth curve according to the results.
Scratch test
First, use marker pen to draw three horizontal lines on the back of the 6-hole plate with a ruler. The interval between each line is about 0.5—1 cm. The lines cross each hole of the 6-hole plate. Each line is required to be uniform and parallel.Take out each group of cells in the logarithmic phase, add about 5 × 105 cells into the 6-well plate according to the experimental requirements, shake the cells slowly in a figure of eight to make the plates evenly laid. The next day, when the cells are full, use a 20 μl gun head to compare with a ruler, and try to hang it as far as possible to the horizontal line scratch on the back. The gun head should be perpendicular to the horizontal line during the marking process, and try not to tilt it.After the scribing is completed, use PBS to wash the cells that have been scribbled, and the scratch marks of the gun head are clearly visible to the naked eye. Then add fresh medium without serum or low serum (< 2%) to continue to culture in the incubator. Take samples at the time point (0, 24 h), take photos, and calculate the cell migration rate according to the results. Cell migration rate = (0 h scratch distance—24 h scratch distance)/0 h scratch distance × 100%, the experiment was repeated 3 times in each group.
Flow cytometry
In the previous experiment of our research group, it was found that the inhibition rate of cell radiotherapy was about 50% when receiving 8Gy X ray irradiation, so the radiation dose of 8Gy was used in this experiment. Add 104 cells per milliliter of cell suspension into T25 culture bottle, put it into the incubator and continue to culture for 24 h. After observing the cell adhesion, irradiate the cell with 8Gy dose of X-ray, continue to culture in the incubator after the completion of radiotherapy, extract the cells 72 h later, add 500ul binding buffer into the cell sediment with the transfer gun, blow repeatedly and mix well, then successively add 5ul Annexin V-APC, 5ul 7aad and mix well at room temperature After about 15 min of photophobic reaction, flow cytometry was used to detect the apoptosis of cells in each group after radiotherapy.
Clone formation experiment
Take the synchronized cells, inoculate them into a 6-well plate, and take different doses of X ray for irradiation, namely 0Gy, 2Gy, 4Gy, 6Gy. Select the corresponding number of cells for inoculation according to the different radiation doses. Observe the cells after continuing to culture for 24 h. If the cells adhere to the wall and grow well, irradiate them with X ray. Continue to culture for 8–14 days after radiotherapy. During the culture period, change the solution irregularly according to the cell growth, After the formation of cell clones, the culture medium shall be sucked out with a pipette gun. After PBS washing, it shall be fixed with paraformaldehyde for 50 min. The washing process shall be gentle to avoid affecting the experimental results. After fixation, crystal violet solution shall be added for dyeing. The amount of crystal violet solution shall cover all clones. The dyeing time is about 30 min. After dyeing, the crystal violet solution in the hole shall be washed out with PBS, The colony forming rate (PE) and cell survival fraction (SF) were calculated by recording the number of colonies with more than 50 cells, and the radiobiological parameters were calculated by fitting the results of the multi target click model.
Statistical analysis
The image J software was used to detect the gray value of the bands in the immunoblotting experiment and the cell scratch experiment. The statistical results of all experimental data were analyzed using the Graphad Prism 8.0.2 statistical software. Before the analysis, the data were tested for normality, and it was considered that the data were in line with normality if P < 0.05. Independent sample t test,, paired sample t test and repeated measurement analysis of variance were used for comparison between groups. Radiobiological parameters and cell survival curve were fitted with SigmaPlot software. P < 0.05 was statistically significant.
Results
Western blot verification of BMAL1 protein expression in cell lines with high and low BMAL1 gene expression
Through gene transfer technology mediated by lentivirus vector, we constructed a high expression and interference vector of BMAL1 gene, transfected the nasopharyngeal carcinoma cell line HONE1, added purinomycin to screen for 2 weeks, and detected the expression effect of BMAL1 at the protein level by Western Blot to determine the virus infection efficiency. Western blot results showed that infection with overexpression lentivirus could increase the expression of BMALl protein in HONE1 cells of nasopharyngeal carcinoma. The expression of BMALl protein in the overexpression group of BMAL1 gene (BMAL1-OE) was 1.76 times higher than that in the overexpression control group (OENC); Infection with interference lentivirus can reduce the expression level of BMALI protein in HONE1 cells of nasopharyngeal carcinoma. The expression level of BMALI protein in the BMAL1 gene interference group (sh-BMAL1) is 0.45 times higher than that in the interference control group (ShNC), indicating that the cell lines with high and low expression of BMAL1 gene were successfully constructed (P < 0.05, Fig. 1).
Fig. 1 [Images not available. See PDF.]
The expression of BMAL1 protein detected by Western blot, *P < 0.05
Western blot verification of HIF-1α protein expression in cell lines with low expression of HIF-1α gene
Knock down HIF-1α gene in nasopharyngeal carcinoma cell HONE1 by lentivirus infection and verify it with Western blot. The results showed that the expression of HIF-1α protein in the HIF-1α knockdown group (sh-HIF-1α) was lower than that in the interference control group (ShNC), and the expression of HIF-1α protein in the HIF-1α knockdown group was 0.58 times higher than that in the interference control group, indicating that the construction of nasopharyngeal carcinoma cell line with HIF-1 knockdown was successful (P < 0.05, Fig. 2).
Fig. 2 [Images not available. See PDF.]
The expression of HIF-1α protein detected by Western blot, *P < 0.05
Effects of overexpression of BMAL1 gene or knockdown of HIF-1α gene on proliferation of nasopharyngeal carcinoma cell line HONE1
In order to verify the effect of BMAL1 and HIF-1α on the proliferation of nasopharyngeal carcinoma cells in vitro, the absorbance values of nasopharyngeal carcinoma cells at 24 h, 48 h and 72 h were continuously measured by CCK-8 test.The results showed that compared with the overexpression control group (OENC), the absorbance measurement value of the overexpression group of BMAL1 gene (BMAL1-OE) decreased (P < 0.05), and the proliferation rate of nasopharyngeal carcinoma cells in BMAL1-OE group slowed down, suggesting that the proliferation of nasopharyngeal carcinoma cells HONE1 can be inhibited by BMAL1 gene (Fig. 3).Similarly, the cell proliferation rate of HIF-1α knockdown group (sh-HIF-1α) was lower than that of its negative control group (ShNC) (P < 0.05), indicating that knockdown of HIF-1α gene can also inhibit the proliferation of nasopharyngeal carcinoma cell HONE1 (Fig. 3).
Fig. 3 [Images not available. See PDF.]
The effect of overexpression of BMAL1 or knockdown of HIF-1α on the proliferation of nasopharyngeal carcinoma cells HONE1. Compared to the control group (*P < 0.05)
Effect of overexpression of BMAL1 gene or knockdown of HIF-1α gene on migration of HONE1 cells in nasopharyngeal carcinoma
Through the cell scratch test, we found that the cell migration rate of the BMAL1 gene overexpression group (BMAL1-OE) was significantly slower than that of the overexpression control group (OENC), and the cell migration rates were (17.623 ± 2.683)% and (52.480 ± 6.670)%, respectively, indicating that overexpression of BMAL1 gene can inhibit the migration ability of nasopharyngeal carcinoma cell HONE1 (P < 0.01, Fig. 4). Similarly, the cell migration rate of the HIF-1α gene knockdown group (sh-HIF-1α) was slower than that of the interference control group (ShNC), and the cell migration rates were (52.050 ± 5.646)% and (67.107 ± 0.777)% respectively (P < 0.05, Fig. 4), suggesting that knockdown of HIF-1α gene can inhibit the migration ability of HONE1 cells in nasopharyngeal carcinoma.
Fig. 4 [Images not available. See PDF.]
The effect of overexpression of BMAL1 or knockdown of HIF-1α on migration of nasopharyngeal carcinoma cells HONE1 (× 100, *P < 0.05, **P < 0.01)
Effect of overexpression of BMALl gene or knockdown of HIF-1α gene on apoptosis after X-ray irradiation
The apoptosis rate of cells in each group after 72 h of 8Gy X-ray irradiation was analyzed. The results showed that the apoptosis rate of BMAL1 gene overexpression group (BMAL1-OE) was higher than that of the over expression control group (OENC) (P < 0.05). Similarly, the apoptosis rate of HIF-1α knockdown group (sh-HIF-1α) was also higher than that of interference control group (ShNC) (P < 0.01), suggesting that overexpression of BMALl gene or knockdown of HIF-1α gene can increase the apoptosis of cells after radiotherapy (Fig. 5).
Fig. 5 [Images not available. See PDF.]
The apoptosis rate of cells irradiated 8Gy in each group after 72 h
Effect of overexpression of BMALl gene or knockdown of HIF-1α gene on radiotherapy sensitivity of nasopharyngeal carcinoma cell line HONE1
After the four groups of cells were irradiated with different doses of X ray (0Gy, 2Gy, 4Gy, 6Gy), the cloning rate and survival fraction of cells in each group were calculated, and the cloning rate and survival fraction of BMAL1 gene overexpression group and HIF-1α gene knockdown group were calculated to be lower than those of their respective negative control groups (P < 0.05, Fig. 6, Tables 1 and 2). At the same time, the cell survival curve was fitted with a multi target click model, and the biological parameters of radiotherapy were calculated. The results showed that the D0, Dq and SF2 values of BMAL1 gene overexpression group and HIF-1α gene knockdown group were lower than those of their respective negative control groups, and the radiation sensitivity enhancement ratios (SER) were 1.381 and 1.063, respectively, which increased the radiosensitivity (P < 0.05, Fig. 7 and Table 3). Therefore, overexpression of BMAL1 gene or knockdown of HIF-1α gene has radiosensitization effect on HONE1 cells.
Fig. 6 [Images not available. See PDF.]
The effect of BMALl or HIF-1α on plating efficiency irradiated 2Gy
Table 1. Cell clony formation under different radiation doses
Grouping | Sample size | PE | |||||
|---|---|---|---|---|---|---|---|
0Gy | 2Gy | 4Gy | 6Gy | t | P** | ||
BMAL1-OE | 3 | 0.830 ± 0.016 | 0.285 ± 0.020 | 0.069 ± 0.026 | 0.009 ± 0.004 | ||
OENC | 3 | 0.845 ± 0.029 | 0.474 ± 0.030 | 0.186 ± 0.045 | 0.053 ± 0.014 | 6.033 | 0.008 |
sh-HIF-1α | 3 | 0.819 ± 0.016 | 0.398 ± 0.022 | 0.108 ± 0.011 | 0.017 ± 0.005 | ||
ShNC | 3 | 0.856 ± 0.023 | 0.504 ± 0.015 | 0.199 ± 0.028 | 0.084 ± 0.026 | 5.549 | 0.007 |
PE Clone formation rate, PE number of clones formed/number of planking cells, repeated 3 times for each group of experiments
P** indicates that the P value is less than 0.01
Table 2. Survival fraction of cells under different radiation doses
Grouping | Sample size | SF | |||||
|---|---|---|---|---|---|---|---|
0Gy | 2Gy | 4Gy | 6Gy | t | P** | ||
BMAL1-OE | 3 | 1.000 ± 0.000 | 0.345 ± 0.022 | 0.082 ± 0.030 | 0.011 ± 0.005 | ||
OENC | 3 | 1.000 ± 0.000 | 0.560 ± 0.016 | 0.219 ± 0.045 | 0.063 ± 0.018 | 7.703 | 0.007 |
sh-HIF-1α | 3 | 1.000 ± 0.000 | 0.485 ± 0.017 | 0.131 ± 0.011 | 0.030 ± 0.020 | ||
ShNC | 3 | 1.000 ± 0.000 | 0.589 ± 0.033 | 0.233 ± 0.038 | 0.098 ± 0.033 | 6.448 | 0.005 |
SF Survival score, SF Clone formation rate in the irradiation group/Clone formation rate in the non irradiation group, with each experiment repeated 3 times
P** indicates that the P value is less than 0.01
Fig. 7 [Images not available. See PDF.]
The cell survival curve fitted by the multi-target single-click model
Table 3. The relevant radiobiological parameters
Grouping | D0 | Dq | SF2 | SER |
|---|---|---|---|---|
BMAL1-OE | 1.305 | 0.717 | 0.345 | |
OENC | 1.802 | 1.308 | 0.560 | 1.381 |
sh-HIF-1α | 1.373 | 1.261 | 0.485 | 1.063 |
ShNC | 1.953 | 1.341 | 0.589 |
D0: One dose of irradiation can kill 63% of cells, D0 = 1/K, Dq = D0 * ln (N), SF2 Cell survival fraction under 2Gy irradiation, SER Radiosensitization ratio, SER Control group D0/Experimental group D0
Western blot detection of HIF-1α protein expression in nasopharyngeal carcinoma cell lines with high and low BMAL1 gene expression
In order to preliminarily explore the effect of BMAL1 gene on HIF-1α protein, the cell lines were constructed by overexpression and knockdown of BMAL1 gene respectively, and the expression of HIF-1α protein was detected by Western blot. The results showed that, compared with their respective negative control groups, the expression of HIF-1α protein in HONE1 cells of nasopharyngeal carcinoma cells in the BMAL1 gene overexpression group (BMAL1-OE) was inhibited, while the expression of HIF-1α protein in HONE1 cells of nasopharyngeal carcinoma cells in the BMAL1 gene knockdown group (sh-BMAL1) was enhanced (Fig. 8), with a statistically significant difference.
Fig. 8 [Images not available. See PDF.]
Effect of BMAL1 on HIF -1α expression in HONE1 cells(*P < 0.05)
Discussion
Almost all organisms show diurnal changes in many physiological processes, such as metabolism, behavior and gene expression. This obedience of organisms to the time niche is controlled by the biological autonomous pacemaker, which is called the biological clock. Its existence makes the biological physiology, biochemistry and behavior show a 24-h cycle rhythm, which is called the circadian rhythm [28–30]. Circadian rhythm is regulated by gene expression, which is called biological clock gene.BMAL1 gene, also known as ARNTL (aryl hydrogen receiver nuclear translator like) gene, is the core gene in the biological clock gene family and an important element of the molecular center and peripheral rhythm oscillator. BMALl gene is located in the short arm of chromosome 11 and encodes BMAL1 protein. It combines with CLOCK to form the complex CLOCK/BMALI, and then enters the nucleus to combine with specific regions of the target gene promoter. For example, it combines with the promoters of PER gene and CRY gene. The coded protein forms the complex PER/CRY, which can enter the nucleus and in turn inhibit the transcription activity of CLOCK/BMALI complex, The gene expression oscillations caused by these feedback loops have a cycle length of about 24 h, so they cause obvious circadian rhythm.At the same time, there are additional feedback loops involving RoR and REV-ERB, which regulate the biological clock rhythm by increasing and inhibiting the expression of BMAL1 respectively [31–33]. HIF-1α mainly exists in the cytoplasm, is the active subunit of HIF-1, and is the key factor of hypoxia signal pathway. When cells are in normal oxygen saturation, HIF-1α subunit can hardly be detected because of its degradation. On the contrary, when cells are under hypoxia, the synthesis of proteolytic enzymes is inhibited, so that HIF-1α subunits can survive. HIF-1α and β Subunit formation HIF-1.Active HIF-1 participates in the regulation of biological effects by regulating the transcription of multiple genes [24], such as the formation of new blood vessels, erythropoiesis, energy metabolism of three major nutrients etc. The main target genes include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor II, endothelin-1 (ET-1), platelet derived growth factor (PDGF) Glucose carrier proteins 1, 3 (GLUT-1, 3) and glycolytic enzymes. The expression of these genes can help maintain the stability of tissues and cells under hypoxia conditions, so as to adapt to hypoxia.
Hypoxia plays a key role in the pathophysiology of many diseases, including cancer, cardiovascular disease, metabolic disease, pulmonary disease, and obstructive sleep apnea [34–36]. The occurrence and sequelae of these hypoxic diseases usually show daily changes [37], which indicates that the interaction between hypoxia and circadian rhythm may have an important impact on human health. The disorder of circadian rhythm has been proved to be related to various cancers such as head and neck squamous cell carcinoma [38], colon cancer [39] etc. Hypoxia can affect the expression of circadian rhythm genes in some cancer cell lines, including hepatocellular carcinoma and renal cell carcinoma [40, 41]. In addition, serious consequences caused by acute hypoxia, such as heart attack, are also related to the defect of circadian rhythm. The study found that the risk of cardiac surgery in the afternoon was more than 50% lower than that in the morning. It is believed that under pathophysiological conditions, the clock plays a role in fine-tuning the hypoxic response.
In this study, we constructed a nasopharyngeal carcinoma cell line HONE1 with BMAL1 gene overexpression and HIF-1α gene knockdown. Through experiments, we learned the relationship between BMAL1 gene, HIF-1α gene and the malignant biological behavior of nasopharyngeal carcinoma cells, cell apoptosis after radiotherapy and radiotherapy sensitivity in vitro. At the same time, we preliminarily discussed the influence of BMAL1 gene on the expression of HIF-1α protein, providing preliminary laboratory research data for further exploring the mechanism of BMAL1 gene and HIF-1α gene in regulating the radiosensitivity related pathway of nasopharyngeal carcinoma cells in the later stage, and whether the biological clock gene BMAL1 is related to HIF pathway.
The relationship between biological clock gene BMAL1 and proliferation and migration of nasopharyngeal carcinoma cell HONE1
BMAL1 gene, as the core gene of the circadian rhythm system, plays different roles in different tumors. It can not only promote cancer [19], but also inhibit cancer [42, 43]. Tiphane et al. [44] detected the expression of BMAL1 gene in thyroid follicular carcinoma and papillary carcinoma by quantitative RT-PCR, and found that the expression of BMAL1 gene in them was higher than that in benign thyroid nodules, and BMAL1 gene was a cancer promoting gene.However, Du et al. [45] found that the expression of BMAL1 gene in colon tumor tissues is significantly lower than that in adjacent tissues. Low expression of BMAL1 gene can reduce cell apoptosis, reduce the number of cells distributed in G2/M phase, and increase the expression of cyclin B1 and cyclin E, suggesting that BMAL1 gene is a tumor suppressor gene in colon cancer.It can be seen that the relationship between BMAL1 and tumor is complex, and the mechanism of BMAL1 gene action may be changed due to different tumor microenvironments.
In the previous study, our research team found that [42], the expression of BMAL1 mRNA and BMAL1 protein in nasopharyngeal carcinoma tissues was significantly lower than that in normal tissues. The group with high BMAL1 gene expression may have a longer overall survival OS than the group with low BMAL1 gene expression, and may have a longer progression free survival PFS and a relapse free survival RFS, The trend of distant metastasis free survival (DMFS) indicates that BMAL1 gene may play a role as tumor suppressor gene in nasopharyngeal carcinoma and can inhibit the occurrence and development of nasopharyngeal carcinoma.In order to further understand how the BMAL1 gene affects the proliferation and migration of nasopharyngeal carcinoma cells in vitro, we overexpressed the BMAL1 gene in the nasopharyngeal carcinoma cell line HONE1, and carried out the CCK-8 experiment in vitro. The absorbance value of the BMAL1 gene overexpression group and the overexpression control group at 450 nm was detected by the microplate reader for three consecutive days. The cell proliferation was determined by the absorbance value, The stronger the cell proliferation ability is, the higher the absorbance value is. On the contrary, if the absorbance value is low, the lower the cell proliferation ability is.The experimental results showed that the absorbance value of the overexpression group of BMAL1 gene was lower than that of the overexpression control group, indicating that overexpression of BMAL1 gene could inhibit the proliferation of nasopharyngeal carcinoma cells. At the same time, the cell scratch experiment was also carried out. By measuring the scratch distance of 0 h and 24 h, the results showed that the cell migration rate of the overexpression group of BMAL1 gene was slower than that of the overexpression control group, suggesting that overexpression of BMAL1 gene inhibited the migration of nasopharyngeal carcinoma cells. The above two experimental results provide another strong evidence to prove that BMAL1 gene is a tumor suppressor gene in nasopharyngeal carcinoma.
The relationship between biological clock gene BMAL1 and apoptosis and radiosensitivity of nasopharyngeal carcinoma cell HONE1 after radiotherapy
The inhibition of normal apoptosis pathway is an important reason for the rapid and unlimited proliferation of tumor cells, which makes it impossible for tumor cells to die normally. Jiang et al. [46] found that BMAL1 gene can promote the apoptosis of pancreatic cancer tumor cells by promoting the expression of apoptosis promoting protein downstream of p53 and inhibiting the expression of anti apoptosis protein, and can also directly transcribe and activate p53 gene, thereby activating p53 tumor suppression signal pathway, so as to achieve the role of tumor suppression.Wei et al. [47] also found that BMAL1 gene can promote the spontaneous and orderly death of gastric cancer cells, which may be related to the inhibition of the expression of oncogenes Bcl-2 and c-Myc. These studies indicate that BMAL1 gene can inhibit the growth of tumor cells by increasing apoptosis in vitro. In order to further understand the relationship between BMAL1 gene and apoptosis of nasopharyngeal carcinoma cell HONE1 after radiotherapy, we irradiated nasopharyngeal carcinoma cells with 8Gy X ray, continued to culture for 72 h, and then detected the apoptosis rate of BMAL1 gene overexpression group and overexpression control group with flow cytometry. It was found that overexpression of BMAL1 gene can increase the apoptosis rate after radiotherapy, It shows that BMALI gene can promote the apoptosis of nasopharyngeal carcinoma cell HONE1 under 8Gy X-ray irradiation. The correlation between BMAL1 gene and apoptosis may provide new ideas and means for subsequent cancer treatment. In addition, as a malignant tumor mainly treated by radiotherapy, the overall survival rate of patients with nasopharyngeal carcinoma has also been significantly improved with the significant improvement of radiotherapy technology. However, the improvement in the efficacy of patients with advanced nasopharyngeal carcinoma has not changed much, and even some patients have the radiation resistance phenomenon of radiotherapy.Therefore, it is urgent to understand the potential mechanism of anti radiation and develop radiosensitive drugs to improve the local control and survival rate of patients with advanced nasopharyngeal carcinoma. Wang [48] found that during the period of high expression of Per2 gene, glioma cells can be blocked in G2/M phase, thus improving the radiosensitivity of glioma cells. At the same time, Gu [49] also found that the radiotherapy sensitivity of glioma stem cell U87 can also be improved when the Per2 gene is overexpressed. These research results indicate that there is a close relationship between biological clock and radiotherapy sensitivity, but there are few reports on the relationship between BMAL1 gene and radiotherapy sensitivity of nasopharyngeal carcinoma.Therefore, in this study, we conducted cloning experiments under different radiation doses (0Gy, 2Gy, 4Gy, 6Gy). The results showed that the cloning rate and survival fraction of cells in the BMAL1 gene overexpression group were lower than those in the overexpression control group. At the same time, the radiobiological parameters were calculated. The D0, Dq, SF2 values in the BMAL1 gene overexpression group were lower than those in the overexpression control group, and the radiosensitization ratio was 1.381, It is suggested that BMAL1 gene can increase the radiosensitivity of nasopharyngeal carcinoma cell HONE1 in vitro. This is consistent with the research results of animal experiments previously studied by our research group [21]. Overexpression of BMAL1 gene can reduce the growth rate of nasopharyngeal carcinoma CNE1 transplanted tumor in nude mice, and improve its radiosensitivity; Knocking down BMAL1 gene can promote the growth of transplanted tumor, leading to its radiation resistance. All the above studies indicate that BMAL1 gene may be an important tumor suppressor gene of nasopharyngeal carcinoma, which can enhance radiotherapy sensitivity.
The relationship between hypoxia inducible factor HIF-1α and proliferation and migration of nasopharyngeal carcinoma cell line HONE1
Hypoxic microenvironment of solid tumors is an important reason for their occurrence, development and metastasis, which has also been confirmed by relevant reports in nasopharyngeal carcinoma. HIF-1α is a key element in hypoxia signal pathway. Its expression can accelerate the formation of new blood vessels in tumor tissues and promote tumor cells to metastasize to the human body.Gong [50] et al., in order to compare the expression of HIF-1α protein in nasopharyngeal carcinoma and chronic rhinitis, examined tissue samples from 92 nasopharyngeal carcinoma patients and 20 chronic nasopharyngitis patients, and found that the expression of HIF-1α protein in nasopharyngeal carcinoma tissue was significantly higher than that in chronic nasopharyngitis tissue, suggesting its potential carcinogenic role in nasopharyngeal carcinoma.In order to explore the effect of HIF-1α gene on the proliferation and migration of nasopharyngeal carcinoma cells, we first found through CCK-8 experiment that the absorbance value of cells in the HIF-1α gene knockdown group was lower than that in the knockdown control group, indicating that knockdown HIF-1α can inhibit the proliferation of nasopharyngeal carcinoma cells. Then the scratch test was used to further explore the effect of HIF-1α gene knockdown on the migration potential of HONE1 cells in nasopharyngeal carcinoma. The results showed that the cell migration rate of the HIF-1α gene knockdown group was slower than that of the control group, suggesting that HIF-1α gene knockdown could reduce the migration capacity of HONE1 cells in nasopharyngeal carcinoma. The above research results show that knockdown of HIF-1α gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cells, providing strong laboratory data to prove that HIF-1α has a cancer promoting effect in nasopharyngeal carcinoma cells.
The relationship between hypoxia inducible factor HIF-1α and apoptosis and radiosensitivity of nasopharyngeal carcinoma cell HONE1 after radiotherapy
Apoptosis is an important index to evaluate radiosensitivity, and its inhibition may be one of the common reasons for radioresistance of cells. The hypoxia phenomenon in the tumor microenvironment often leads to the disorder of the physiological system of malignant tumors, so the degree of hypoxia of tumor cells also determines the therapeutic effect of radiotherapy.Research shows that HIF-1α gene can inhibit the apoptosis of tumor cells by promoting the expression of Bcl-2 [51]. Therefore, the up regulation of HIF-1α gene expression after radiotherapy can reduce the apoptosis of tumor cells and reduce the radiosensitivity of tumor cells. Moeller et al. [52] also confirmed the above point of view. They found that the activity of HIF-1α in tumor cells can be induced to increase by radiation during radiotherapy, thereby promoting the expression of downstream target genes VEGF and bFGF, making endothelial cells tolerant and less sensitive to radiation, thus affecting the efficacy of radiotherapy.In order to confirm the effect of HIF-1α gene on apoptosis and radiosensitivity of nasopharyngeal carcinoma cells after radiotherapy, we successively used flow cytometry to analyze apoptosis, cell cloning experiment to detect the rate of clone formation and cell survival fraction. After the experiment, we used SigmaPlot software to analyze the experimental data, used a multi target click model to fit the cell survival curve, and calculated the radiation sensitization ratio (SER). The results showed that 72 h after radiotherapy, the apoptosis rate of HIF-1α knockdown group was significantly higher than that of the knockdown control group, and SER was greater than 1, suggesting that interference with HIF-1α gene has radiosensitization effect. The above research shows that HIF-1α may be a oncogene in nasopharyngeal carcinoma cells, and knockdown can increase radiotherapy sensitivity.
Correlation between biological clock gene BMAL1 and HIF-1α protein
It was found that in mice, circadian rhythm regulator CRY1 is a negative regulator of HIF-1α [53]. In terms of mechanism, CRY1 interacts with the bHLH domain of HIF-1α through its tail region. CRY1 can reduce the half-life of HIF-1α and the binding of HIF-1α to the target gene promoter, which means that HIF-1α can be inhibited during the period of CRY1 overexpression. In the period of low CRY1 expression, HIF-1α activity increased, which in some cases led to cell proliferation and migration. In addition, the study also found that PER2 combines with HIF-1α to increase the activity of HIF-1α, which is contrary to the effect of CRY2 [54]. WU et al. also found that [24] BMAL1 can bind to the HIF-1α promoter in human osteosarcoma cells, and the same report has been reported in mouse nucleus pulposus cells. BMAL1 gene controls the transcriptional activity of HIF-1α. Once removed, it will lead to inhibition of the growth of nucleus pulposus cells [25].
Although these studies show that there is an association between the biological clock and the hypoxia signal pathway [55–57], there are few research reports on the relationship between the clock gene BMAL1 and the hypoxia inducible factor HIF-1α in nasopharyngeal carcinoma.Therefore, in order to preliminarily understand the effect of BMAL1 gene on HIF-1α protein in nasopharyngeal carcinoma cells, we conducted an immunoblot test. The results showed that in HONE1 cells, overexpression of BMAL1 gene can inhibit the expression of HIF-1α protein, whereas knockdown of BMAL1 gene can promote the expression of HIF-1α protein, laying a foundation for further understanding the mechanism between the two.
The inadequacies of this study
This study explored the effects of the biological clock gene BMAL1 and hypoxia inducible factor HIF-1α on the proliferation, migration and radiosensitivity of nasopharyngeal carcinoma cells. However, this study only discussed at the cell level, and further studies are needed from the molecular, tissue, animal and clinical levels in future studies to understand the specific mechanisms of BMAL1 and HIF-1α affecting the malignant biological behavior and radiosensitivity of nasopharyngeal carcinoma cells, At the same time, the interaction mechanism between BMAL1 and HIF-1α was studied to provide new ideas and basis for the treatment of nasopharyngeal carcinoma.
Acknowledgements
The authors would like to thank Clinical Research Center of Guizhou Medical University.
Authors’ contributions
YXT conceived the idea and contributed to the drafting of the manuscript. YYL and CFZ extracted the data. LNL performed the analysis. QYH contributed to the material preparation. YXL helped to edit pictures. DAZ contributed to the study conception and revision of the manuscript, and FJ approved the final version of the submit-ted manuscript. All authors read and approved the final manuscript.
Funding
This research was supported in part by grants from the National Natural Science Foundation of China under grant number 82060556, 81560437; the Department of Science and Technology, Guizhou Province, under grant number [2018] 2755; the Ordinary Colleges and Universities Youth Science and Technology Talent Growth Project, Guizhou Province, under grant number [2021] 187; The Health Commission Science and Technology Fund, Guizhou Provincial under grant number gzwkj2021-050; Guizhou Medical University 2021 National Foundation Cultivation Project [20NSP041], and the Hospital-level Science and Technology Project of Guizhou Cancer Hospital under grant number YJ2019-33.
Availability of data and materials
The author will provide all the data detailed in this article for free.
Declarations
Ethics approval and consent to participate
This article does not contain any research conducted by any author on human participants or animals.
Consent for publication
Everyone’s data included in the study was approved for publication.
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Abstract
Objective
To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α) on proliferation, migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time, whether the biological clock gene BMAL1 can affect the expression of HIF-1α protein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.
Methods
BMAL1 gene overexpression and interference lentivirus and HIF-1α gene interference lentivirus were constructed respectively, and were transfected into nasopharyngeal carcinoma cells HONE1. Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1α gene knockdown cell line, and to investigate the expression of HIF-1α protein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1α on the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses (0Gy, 2Gy, 4Gy, 6Gy) were detected by clone formation assay.
Results
The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile, HIF-1α gene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1α protein in nasopharyngeal carcinoma cell line HONE1, and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1α protein in HONE1 of nasopharyngeal carcinoma cells, while knockdown of BMAL1 gene promoted the expression of HIF-1α protein in HONE1 of nasopharyngeal carcinoma cells(P < 0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1α gene could inhibit the proliferation and migration of HONE1 cells (P < 0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h, the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group, similarly, the apoptosis rate of HIF-1α gene knockdown group was higher than that of the knockdown control group (P < 0.05).After X-ray irradiation at different doses (0Gy, 2Gy, 4Gy, 6Gy), clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1α knockdown group were lower than those of negative control group (P < 0.05).Sigmaplot analysis showed that the D0, Dq and SF2 of the BMAL1 overexpression group or HIF-1α knockdown group were lower than those of the negative control group, and the radiosensitization ratios were 1.381 and 1.063, respectively.
Conclusion
Overexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1, increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1α Gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1, increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1, overexpression of BMAL1 gene can inhibit the expression of HIF-1α protein while knockdown of BMAL1 gene can promote the expression of HIF-1α protein.
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Details
1 Guizhou Medical University, School of Clinical Medicine, Guiyang, P.R. China (GRID:grid.413458.f) (ISNI:0000 0000 9330 9891)
2 Affiliated Hospital of Guizhou Medical University, Department of Oncology, Guiyang, P.R. China (GRID:grid.452244.1); Affiliated Cancer Hospital of Guizhou Medical University, Department of Head and Neck Oncology, Guiyang, P.R. China (GRID:grid.413458.f) (ISNI:0000 0000 9330 9891)
3 Guizhou Medical University, School of Clinical Medicine, Guiyang, P.R. China (GRID:grid.413458.f) (ISNI:0000 0000 9330 9891); Affiliated Hospital of Guizhou Medical University, Department of Oncology, Guiyang, P.R. China (GRID:grid.452244.1); Affiliated Cancer Hospital of Guizhou Medical University, Department of Head and Neck Oncology, Guiyang, P.R. China (GRID:grid.413458.f) (ISNI:0000 0000 9330 9891)
4 The Affiliated Hospital of Guizhou Medical University, Clinical Research Center, Guiyang, P.R. China (GRID:grid.452244.1)