Posttranslational Modifications of Rev-Erbα Protein and Abnormal Inflammatory Response in Gastric Cancer

We reported that Rev-erbα, a transcriptional repressor, is reduced in human gastric cancer and that it inhibits glycolysis in cultured gastric cancer cells. However, it is unclear whether Rev-erbα undergoes posttranslational modifications in gastric cancer. Here, we determined levels of Rev-erbα and its posttranslational modifications including phosphorylation, SUMOylation, and ubiquitination in N-methyl-N-nitrosourea (MNU)/Helicobacter pylori (H. pylori)-induced gastric cancer in mice and in cultured human gastric cancer cells. Administration of MNU plus H. pylori infection successfully induced gastric tumor in C57BL/6J mice. MNU/H. pylori decreased the levels of Rev-erbα in gastric tumor tissues of mice accompanied by an increase in the level of lactic acid. Rev-erbα protein SUMOylation and ubiquitination modifications were significantly increased, whereas phosphorylation was unchanged, in gastric cancer cells line BGC-823 and MNU/H. pylori-induced mouse gastric cancer tissues. Using human gastric cancer tissues, we found that Rev-erbα was specifically reduced in mucosal epithelial cells in gastric tissue. Cytokine levels were increased in MNU/H. pylori-exposed mice compared with control mice. Similarly, the levels of IL-6 IL-10, TNF-α, and VEGF were higher in the BGC-823 cell line compared with GES-1 cells. IL-6 and IL-1 incubation did not affect Rev-erbα levels in BGC-823 cells. Furthermore, Rev-erbα was recruited on the promoters of these cytokine genes, which suppressed their expression. Conclusively, Rev-erbα SUMOylation and subsequent ubiquitination may contribute to its protein reduction, which leads to increased glycolysis and abnormal inflammatory responses during the development of gastric cancer. Targeting Rev-erbα and its SUMOylation represents promising approaches for prevention and management of gastric cancer.


Introduction
Although gastric cancer is a common malignant tumor in humans, its pathological mechanism is not fully understood. Te occurrence of gastric cancer is considered a result of multiple biological, genetic, and environmental factors, and multiple stages. Helicobacter pylori (H. pylori) infection is the most important factor. After its infection, H. pylori causes an infammatory response in the gastric mucosa, which induces the host to produce a variety of cytokines that alter the microenvironment including the physiology and immune status of the stomach. Tis can lead to cancerous transformation and the unlimited growth of gastric mucosal epithelial cells [1][2][3]. With the development of metabolomics, recent research has advanced our understanding of the relationship between metabolic regulation and cancer. Extensive research has demonstrated that metabolic reprogramming is a hallmark of cancer and is intricately linked to oncogenesis and cancer immune escape. Te concentration of lactic acid is consistently increased in the urine and/or tissue samples of gastric cancer patients, whereas glucose is considerably depleted compared with healthy individuals. Tese high lactate levels might be attributed to the special metabolism of most cancer cells because tumor cells consume a large amount of glucose for glycolysis even under the condition of sufcient oxygen (Warburg efect) [4]. Tis glycolytic switch was reported to be associated with oncogenic transformation and molecular signal transduction [5].
Rev-erbα is a nuclear receptor and critical component of the molecular clock that drives the daily rhythms of metabolism. Rev-erb family members participate in pathological processes, including sleep disorders, diabetes, atherosclerosis, Alzheimer's disease, and other diseases, by regulating the biological clock, infammatory/immune responses, and lipid metabolism. A study revealed that Reverbα KO mice had a greater infammatory response to cigarette smoke, including increased neutrophil lung infux and proinfammatory cytokine release compared with wildtype mice [6]. Stimulation of Rev-erbα activity by SR9009 greatly diminished ventilator-induced lung injury, infammatory cell infltration, and the production of the proinfammatory cytokine TNF-α [7]. Rev-erbα is critical for the regulation of infammation-and metabolism-related gene transcription. We have examined the relationship between Rev-erbα and tumors [8][9][10][11][12][13]. Infammation is usually related to the occurrence and development of cancer. Te inducement of chronic infammation increases the risk of cancer or promotes cancer progression, including H. pylori infection [14]. Infammatory cytokines IL-6 is highly upregulated in many cancers and is considered to be one of the most important cytokine families in tumorigenesis and metastasis [15]. Infammatory cytokines TNF-α can trigger the frst step of tumor transformation, act as an autocrine growth factor for tumor cells, and play a major role in metastasis [16]. Previous studies showed that the decreased activity of Rev-erbα or Rev-erbα knockout promoted the production of TNF-α and IL-6 in rodent lungs [6,7]. Knockdown of Rev-erbα is efective at modulating the production of IL-6 [17]. Te Rev-erbα agonist SR9011 stimulated the expression of the anti-infammatory cytokine IL-10 [18]. Upregulation of VEGF expression during gastric infammation may be related to the development of gastric cancer [19]. We previously reported that Rev-erbα is reduced in human gastric cancer [13] and that it inhibits glycolysis in cultured gastric cancer cells [20]. Rev-erbα can undergo various protein modifcations including phosphorylation which afects its stability. Te current study investigated whether Rev-erbα reduction is associated with its posttranslational modifcations, including phosphorylation, SUMOylation, and ubiquitination in gastric cancer. Infammation and lactate levels were also measured during the development of gastric cancer.

Patients and Samples.
Six fresh gastric cancer tissue pairs (tumor and adjacent normal tissues) were obtained by surgical resection at a similar time to avoid circadian changes from the First Afliated Hospital of Anhui Medical University and immediately stored at -80°C. Tumoradjacent tissues were obtained from as far away as 2 cm from the gastric tumor. All cases were diagnosed by histopathology. Te tissue wax blocks and sections of gastric cancer were obtained from the Department of Pathology of the First Afliated Hospital of Anhui Medical University and confrmed via histopathological diagnosis by a pathologist. Characteristics are shown in Table 1. Informed consent was obtained from each enrolled patient.

2.2.
Animals. C57BL/6J mice (both males and females) used in the present research were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). In total, 60 4-week-old male C57BL/6J mice were housed in cages with a 12/12-hour light/dark cycle and maintained at 23°C under specifc pathogen-free (SPF) conditions. Mice were divided into the following three groups: Group 1, control (n � 10). Group 2, Nmethylnitrosourea (MNU) (Sigma Chemical Co., St. Louis, MO, USA) + H. pylori (ATCC, Manassas, VA, 6 months, n � 25). Group 3, MNU + H. pylori (12 months, n � 25). MNU was dissolved in distilled water at a concentration with 200 ppm and placed in a light-shielding bottle as drinking water for mice. Mice in the MNU groups were fed drinking water containing 200 ppm MNU twice a week for 10 weeks. After completion of MNU treatment, mice in the MNU + H. pylori groups were inoculated orogastrically with 5 × 10 9 colony-forming units/mL of H. pylori (ATCC 49179), fve times every alternate day. Mice were sacrifced by CO 2 asphyxiation at 6 and 12 months after inoculation at similar times to avoid circadian changes. All the experiments performed in this study were approved by the Ethics Committee of Te First Afliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Western Blot.
Cell pellets or mouse gastric tissues were washed with phosphate-bufered saline (PBS) and homogenized with an appropriate amount of RIPA lysis bufer containing protease inhibitor cocktail and PMSF. Te processed tissues were incubated in ice for 30 min. Te supernatants were used as sample for experiment after centrifugation at 4°C with 12,000 ×g for 20 minutes. Te BCA method was used to measure the protein concentration. Samples were separated by 10% SDS-PAGE and transferred to the PVDF membrane. Te membranes were blocked with 5% fat-free milk in PBS containing 0.1% Tween 20 for 2 hours at room temperature, followed by overnight incubation with the primary antibody (Anti-Rev-erbα antibody: Santa Cruz, #sc-393215, 1 : 1,000) at 4°C. Next, the secondary antibody was incubated for 1 hour at room temperature, and the protein bands were detected using the ECL reaction solution.

Measurement of Lactate Concentrations.
Te cells as well as gastric tumor tissues of the MNU/H. pylori treatment groups and the corresponding tissues in the normal groups were collected under an empty stomach condition for the measurement of lactate concentrations using a lactate assay kit (BioVision, Milpitas, CA) in accordance with the manufacturer's instructions. Serum lactate was also measured in human subjects.

Hematoxylin and Eosin
Staining. Gastric tissues were fxed them with 4% neutral bufered paraformaldehyde. Tese fxed lungs were embedded in parafn, sectioned into 5 μm sections using a rotary microtome, and stained with hematoxylin and eosin.

2.7.
Immunofuorescence. Formalin-fxed parafn-embedded tissues were cut int 4 μm sections which were deparafnized with xylene and rehydrated through a graded series of alcohols. Te tissue sections were placed in a repair box flled with citric acid antigen retrieval solution, and antigen retrieval was performed in a microwave oven. Te slides were washed 3 times with PBS after natural cooling, 5 min each time and then, blocking was done with 3% BSA in PBS bufer for 30 min. Tissues were incubated with primary antibodies against Rev-erbα (ab174309, Abcam) and Tf1 (ab92377, Abcam) at 1 : 200 dilutions overnight at 4°C in humidifed chambers followed by incubation with the corresponding secondary antibody for 60 min at room temperature in the dark. Te slides were mounted with resin after dropping an appropriate amount of an antifuorescence quencher on the tissue. Fluorescence was detected with fuorescence microscopy. Fluorescence was detected by using a LEICA TCS NT laser confocal microscope.

Immunoprecipitation (IP).
Cells and experimental gastric cancer tissues were lysed in IP lysis bufer containing 50 mM HEPES (pH7.4), 100 mM NaCl, 5 mM MgCl 2 , 0.5% NP-40, 10% glycerol, 1 mM NaF, 1 mM Na 3 VO 4 , and 1 × protease inhibitor cocktail for 20 min in ice. Te cell lysate was centrifuged at 13,000 ×g at 4°C for 20 min. An appropriate amount of supernatant was taken for determination of protein concentration and prepared for input samples. Lysate containing about 400 μg-2 mg of total protein and equal volume of precooled immunoprecipitation bufer containing an appropriate proportion of protease inhibitors and antibody-coupled agarose beads were added, respectively. Te abovementioned mixture was incubated for overnight at 4°C. Te precipitated complex was collected by centrifugation at 3000 rpm for 1 min and then washed with TBST three times. Te immune complex was dissociated from the beads for Western blot.

Chromatin Immunoprecipitation (ChIP) Assay.
Cells were fxed with 1% formaldehyde and terminated with 2.5 mM glycine. Te scraped cells were sonicated for lysis in PBS with sodium thiosulfate. Te lysates were divided into three aliquots, one of which was a positive control and received no treatment and one of which was a negative control, which was incubated with target protein.
One-third of the cell lysate was served as the test group and was incubated with antibody against Rev-erbα (1 μg) and Protein G PLUS-Agarose. After removal of RNA and protein, DNA was extracted with phenol-chloroform, respectively. Next, the degree of enrichment on gene promoters was detected using real-time quantitative PCR.

Statistical
Analysis. SPSS 20.0 software was using for statistical analysis. Te results were subjected to one-way ANOVA and parametric t-testing and were expressed as the mean ± SEM. Statistical signifcance was accepted at a level of P value <0.05.

MNU/H. pylori Induces Gastric Tumors in Mice.
Gastric tumors were examined macroscopically and microscopically by a pathologist in a blind manner. Overall, Table 2 shows that 29.2% of mice developed gastric tumors after MNU/H. pylori treatment for 6 months, whereas 86.4% of mice developed gastric tumors after being fed MNU/ H. pylori for 12 months. Tese results demonstrate that MNU/H. pylori induced gastric cancer in mice.

Levels of Rev-Erbα Protein Are Specifcally Decreased in Gastric Mucosal Epithelial Cells in Clinical Gastric Cancer
Tissues. To determine whether Rev-erbα was decreased in human gastric cancer tissues, Rev-erbα and a gastric mucosal epithelial cell specifc marker (Tf1) were detected in human gastric cancer tissues and the corresponding adjacent tissues by immunofuorescence staining. First, we confrmed that gastric cancer tissues with moderately diferentiated adenocarcinoma through H&E staining ( Figure 1). Compared with the corresponding adjacent tissues, the fuorescence intensity of Rev-erbα protein in human gastric cancer tissues was signifcantly lower. Te fuorescence intensity of Rev-erbα protein was also reduced in Tf1-positive cells determined by co-localization analysis (Figures 1(b) and 1(c)), while IgG control showed no signals (Figure 1(d)). Tese results suggest that Rev-erbα protein is specifcally reduced in gastric mucosal epithelial cells in clinical human gastric cancer tissues.

Levels of Rev-Erbα Are Decreased in Gastric Tissues, and Lactic Acid Levels Are Increased in Mice with MNU/H. pylori-Induced Gastric Cancer and in Human Gastric Cancer Tissues.
To determine whether the formation of gastric tumors induced by MNU/H. pylori was associated with Rev-erbα, we measured Rev-erbα protein levels in experimental gastric tissues. Compared with the control group, signifcantly reduced levels of the Rev-erbα protein were observed in the gastric tissues of the MNU/H. pylori groups in a time-dependent manner (Figures 2(a) and 2(b)). Furthermore, the levels of lactic acid in the stomach tissues of the MNU/ H. pylori treatment group were signifcantly higher than those in the control group (Figure 2(c)). Additionally, lactic acid was increased in human gastric cancer tissues compared to that in adjacent normal gastric tissues (Table 1). Tese results suggest that the formation of gastric tumors is associated with a decrease in Rev-erbα levels and an increase in glycolysis.

Rev-Erbα Phosphorylation Levels Are Unchanged in BGC-823 Cell Lines or in MNU/H. pylori-Induced Moues
Gastric Tumor Tissues. We examined changes in Rev-erbα protein phosphorylation in human gastric cancer cell line (BGC-823) and in gastric cancer tissues from MNU/ H. pylori-exposed mice to determine whether it is associated with the decreased expression of Rev-erbα protein. Compared with normal human gastric mucosal epithelial cells (GES-1), there was no signifcant change in phosphorylation on the Ser55/59 or Tr275 amino acid residues of Rev-erbα protein in BGC-823 (Figures 3(a)  and 3(c)). Similarly, in MNU/H. pylori-induced mouse gastric cancer tissues, phosphorylation on Ser55/59 or Tr275 amino acid residues of Rev-erbα protein were not signifcantly changed compared with controls (Figures 3(b) and 3(d)). Tese results suggest that Reverbα phosphorylation levels is unchanged in BGC-823 cell lines or in MNU/H. pylori-induced moues gastric cancer tissues.

SUMO Modifcation of Rev-Erbα Is Signifcantly Increased in BGC-823 and MNU/H. pylori-Induced Mouse Gastric
Cancer Tissues. Te levels of Rev-erbα SUMOylation in BGC-823 cell lines and in MNU/H. pylori-induced moues gastric cancer tissues were detected by immunoprecipitation. As shown in Figures 4(a) and 4(c), compared with normal human gastric mucosal epithelial cells (GES-1), interactions between Rev-erbα protein and ubiquitin were signifcantly increased in the BGC-823 cell line. Furthermore, interactions between Rev-erbα protein and SUMO1 were increased in the BGC-823 cell line compared to GES-1. Similarly, both ubiquitination and SUMOylation of Reverbα were increased in MNU/H. pylori-exposed mouse gastric tumor tissues (Figures 4(b) and 4(d)). However, the interaction between Rev-erbα protein and SUMO2 was unchanged in the BGC-823 cell line or in MNU/H. pyloriinduced mouse gastric cancer tissues. Tese data suggest that Rev-erbα SUMOylation and subsequent ubiquitination may contribute to its degradation in gastric cancer tissues. We quantifed IL-6 and TNF-α levels in serum samples from mice to determine any signifcant diferences in cytokine concentrations between the normal and experimental groups. Te method was also used to compare GES-1 cells and the BGC-823 cell line. We found that a signifcant increase in IL-6 and TNF-α concentration was observed in serum samples from MNU/H. pylori-exposed mice compared to control mice ( Figure 5(a)). Levels of IL-6, IL-10, TNF-α, and VEGF were higher in the BGC-823 cell lines compared with those of GES-1 cells (Figure 5(b)). Likewise, the levels of these  Journal of Oncology 5 cytokines were increased in gastric cancer tissues compared to those in adjacent normal tissues (Table 1). Tese results suggest that infammatory responses are increased in gastric cancer, which is associated with reduced Rev-erbα.

IL-6 and IL-1 Treatments Do Not Afect Rev-Erbα Protein but Increase Lactic Acid Levels in Cultured Human Gastric
Cancer Cells. BGC-823 cells were treated with IL-6 or IL-1 (5 and 10 ng/ml) for 24 h. Rev-erbα levels were measured by Western blot. As shown in Figure 6, IL-6 and IL-1 treatments did not afect Rev-erbα protein levels ( Figure 6(a)). Interestingly, the levels of lactic acid were signifcantly increased in these cells treated with IL-6 and IL-1 (Figures 6(b) and 6(c)). Tis suggests that infammation may not cause Rev-erbα reduction but increases glycolysis in human gastric cancer cells.

Rev-Erbα Is Recruited on the Promoter of Cytokine Genes, Which Represses Teir Expression.
Since IL-1 and IL-6 did not afect Rev-erbα protein levels, we wanted to evaluate whether Rev-erbα inhibits cytokine gene expression. First, we deleted Rev-erbα encoding gene Nr1d1 and determined expression of cytokine genes. As shown in Figures 7(a) and 7(b), Nr1d1 gene expression was signifcantly reduced in Nr1d1 KO cells. Nr1d1 gene deletion remarkedly increased the expression of IL-6, TNF-α, VEGF, and IL-10 levels in BGC-823 cells. Furthermore, Rev-erbα protein was recruited on the promoter of IL-6, TNF-α, VEGF, and IL-10 genes (Figure 7(c)). Tese results suggest that Rev-erbα is recruited on the promoter of cytokine genes, which represses their expression.

Discussion
Rev-erb is a nuclear receptor and transcriptional repressor, and the two family members Rev-erbα and Reverbβ are encoded by the Nr1d1 and Nr1d2 genes, respectively. Rev-erbs participate in pathological processes including sleep disorders, diabetes, fatty liver, atherosclerosis, Alzheimer's disease, and abnormal bone  Figure 2: MNU/H. pylori decreases the levels of Rev-erbα in gastric cancer tissues of mice accompanied by an increase in the level of lactic acid. After C57BL/6J mice were exposed to MNU (200 ppm) for 10 weeks, they were inoculated with H. pylori (5 × 10 9 CFU/time, 5 times in total). After 6 or 12 months (m) of MNU exposure, the levels of Rev-erbα protein (a), (b), and lactic acid (c) in the gastric tissues of mice were measured. Mean ± SEM, N � 8. * P < 0.05, * * * P < 0.001 vs corresponding controls (Ctr); † † P < 0.01, † † † P < 0.001 vs MNU/H. pylori (6 m). 6 Journal of Oncology resorption/remodeling by regulating the biological clock, infammatory/immune responses, and lipid metabolism [22][23][24]. Data from a study by Sulli et al. showed that Rev-erb agonists reduced the survival of brain cancer, leukemia, breast cancer, rectal cancer, and melanoma cell lines [10]. Additionally, the activation of Rev-erbα/β inhibited the growth of mouse glioblastoma [10,11]. We recently reported that Rev-erbα is reduced in human gastric cancer tissues with an increased TMN stage. Furthermore, the low expression of Rev-erbα is associated with poor prognosis in gastric cancer patients [13]. Rev-erbα was also reduced in MNU/H. pylori-induced gastric cancer tissues. Specifcally, Rev-erbα was decreased in gastric mucosal epithelial cells in gastric tissues, suggesting epithelial cell diferentiation and tumorigenesis. Te role of Rev-erbα in the development of gastric cancer will be demonstrated using KO mice in future. Further study is warranted to determine the mechanisms of reduced Nr1d1 gene expression in gastric cancer [12,13].  Rev-erbα can undergo various protein modifcations through ubiquitination/proteasome-dependent degradation pathways that afect its stability. For example, phosphorylation of serine (Ser) residues 55 and 59 of Rev-erbα protein increased its stability, whereas phosphorylation of threonine residue (Tr) 275 reduced its stability [25,26]. Additionally, phosphorylation of N-terminal regions of Rev-erbα regulates its intracellular localization and signal pathway [26,27]. On the basis of these fndings, we tested whether Rev-erbα was phosphorylated in human gastric cancer cell lines and MNU/H. pylori-induced mouse gastric cancer tissues. Tere were no signifcant changes in the levels of Rev-erbα protein phosphorylation (Ser55/59 and Tr275) in human gastric cancer cell lines and in mouse gastric cancer induced by MNU/H. pylori, suggesting the decrease of Rev-erbα protein in gastric cancer tissues may not be related to its phosphorylation. Despite this, the ubiquitination of Rev-erbα protein was signifcantly increased in human gastric cancer cell lines and mouse gastric cancer tissues. Tese results suggest that the Rev-erbα protein might undergo other Journal of Oncology modifcations in gastric cancer tissues that cause it to bind to ubiquitin, which leads to its proteasome-dependent degradation.
In HEK293 cells, Rev-erbα protein can undergo SUMO modifcation under the stimulation of infammatory factors, leading to its ubiquitination and proteasome-dependent degradation [28]. SUMO is a ubiquitin-like protein with four family members: SUMO1, SUMO2, SUMO3, and SUMO4. SUMO1-SUMO3 are expressed in all tissues, whereas SUMO4 is expressed specifcally in organs. Te SUMO modifcation covalently binds SUMO to the amino acid residues of the target protein by activating enzyme E1, combining enzyme E2 (Ubc9) and ligase E3. It is a dynamic and reversible process, and deSUMOylation is mediated by SUMO-specifc protease family members. We, for the frst time, found that SUMO modifcation of Rev-erbα protein was signifcantly increased in human gastric cancer cell lines and mouse gastric cancer tissues. Tis may be related to the marked increase in SUMO1 expression in human gastric cancer tissues [29]. Further studies using proteasome inhibitors, ubc9, and SUMO1 transfection in gastric cancer cells would further understand whether Rev-erbα SUMOylation causes its protein degradation.
Current research of Rev-erbs in metabolism has mainly focused on lipid metabolism. For example, when the Rev-erbα gene is knocked out in mice, the expression of apolipoprotein CIII in the liver and serum is increased, and the levels of very low-density lipoprotein and triglycerides are signifcantly increased [30]. Additionally, the expressions of key genes involved in fatty acid metabolism (CD36, Fabp3, and Fabp4) were decreased in cells containing Rev-erbs mutants [31]. In terms of glucose metabolism, Rev-erbα inhibits gluconeogenesis. When heme binds to Rev-erbα in hepatocytes, it enhances its activity and inhibits the gene expression of a key enzyme (phosphoenolpyruvate carboxykinase, PEPCK) required for gluconeogenesis [32]. We reported that Rev-erbα reduction causes gastric cancer cell proliferation by upregulating glycolysis and pentose phosphate pathway (PPP) [20]. Te lactate was increased in serum of MNU/H. pylori-exposed mice, which may be due to reduced Rev-erbα. H. pylori has the ability to utilize glucose for metabolism through a glucokinase activity and enzymes of the PPP and glycolysis pathways [33,34]. Interestingly, the anti-H. pylori activity was observed when treated with high levels of exogenous lactate [35]. Tus, it is possible that H. pylori increases lactate production for gastric cancer cell proliferation but in return reduces its bacterial activity. Whether Reverbα inhibits the proliferation of gastric cancer cells in vivo and the growth of gastric cancer remains unclear.
Infammatory responses play key roles in cancer development, including tumor occurrence, promotion, progression, and metastasis. Cytokines are considered to be important mediators linking infammation to gastric cancer [36]. Our data suggested signifcantly increased levels of IL-6, IL-10, TNF-α, and VEGF in serum samples from the experimental mice compared with normal mice. Furthermore, the levels of IL-6, IL-10, TNF-α, and VEGF in the gastric cancer cell line were higher than those in normal human gastric mucosal epithelial cells. Research studies have showed that the decreased activity of Rev-erbα or Rev-erbα knockout promoted the production of IL-6 and TNF-α in rodent lungs [6,7]. Tis is corroborated by our fndings showing increased expression of these genes in Nr1d1 KO cells. Tese fndings suggest that increased infammatory response is associated with reduced Rev-erbα during the development of gastric cancer. It is noted that H. pylori induces infammation [37,38]. Further studies are required to dissect the role of H. pylori and reduced Rev-erbα in modulating infammatory responses in gastric cancer.
is the case in our fndings showing increased lactate by IL-1 and IL-6 incubation. We speculate that increased concentrations of cytokines may promote glycolysis pathway in patients with gastric cancer.

Conclusion
In summary, this is the frst study to show that the SUMO modifcation of Rev-erbα protein is observed in gastric  cancer tissues, which is associated with protein degradation. Rev-erbα reduction causes the expression of cytokine genes due to reduced recruitment on their promoters. Increased release of cytokines augments glycolysis, which is seen in gastric cancer (Figure 7(d)). Targeting Rev-erbα or its SUMO modifcation may represent promising approaches for prevention and management of gastric cancer.

Data Availability
Te data used to support the fndings of our present research are available from the corresponding authors upon request.

Additional Points
Contribution to the feld. Although gastric cancer is a common malignant tumor in humans, its pathological mechanism is poorly understood. We reported that Reverbα protein is decreased in human gastric cancer, which is associated with poor diferentiation, TMN stages, and poor prognosis. However, it is unclear whether Rev-erbα protein undergoes posttranslational modifcations leading to its degradation in gastric cancer. Here, we found that Rev-erbα SUMOylation and subsequent ubiquitination were increased in cultured gastric cancer cells and in N-methyl-N-nitrosourea/Helicobacter pylori-induced mouse gastric tumor tissues. Tis was associated with increased glycolysis and abnormal infammatory responses. Terefore, targeting Reverbα and blocking SUMOylation-mediated protein degradation represent a promising approach for prevention and management of gastric cancer.

Ethical Approval
Te study's protocols were approved by the Ethics Committee of Anhui Medical University.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Conception and design was done by ZW. Data acquisition and analysis was done by XC, KC, YW, RJ, JW, and DL. Data interpretation was done by HZ and ZW. XC KC, and YW drafted the manuscript. KC and ZW revised the manuscript. Chen Ke, Cheng Xiaowen, and Wan Yufeng contributed equally to this work.