ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway

ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway SHUYANG PANG, YUTING SHEN, YANAN WANG, XUANNING CHU, LINGMAN MA, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3836816/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Biochimica et Biophysica Acta (BBA) - General Subjects → Version 1 posted You are reading this latest preprint version Abstract Background: Dysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity. Methods: The biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. The coimmunoprecipitation assay confirmed the direct binding between ROCK1 and c-MYC, and the luciferase reporter assay clarified the binding between c-MYC and the promoter of the PFKFB3-encoding gene. These results were verified in animal experiments. Results: ROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased Gemcitabine (GEM) sensitivity in vivo and in vitro. Conclusion: ROCK1 promotes glycolytic activity in PC cells and PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC. ROCK1 c-MYC PFKFB3 Glycolysis Pancreatic cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pancreatic cancer (PC) is a fatal disease, with a gradually increasing annual mortality rate approaching the annual incidence rate 1 . Statistics for 2020 show that the incidence of PC continues to rise, mainly in Western countries, and PC is expected to become the second leading cause of death among cancers by 2030 2 . In the early stage of PC, there are often no obvious symptoms, and it is difficult to detect. Patients tend to miss the golden treatment period and thus lose the opportunity for radical surgery. The pancreas is hidden in the abdominal cavity and close to major blood vessels, and accurate serum markers and imaging models for early diagnosis are lacking, making early detection of and screening for PC extremely difficult 3 . Additionally, PC shows significant resistance to radiotherapy and chemotherapy, resulting in a further increase in patient mortality. Surgical resection is still an effective treatment, but 80%-90% of patients have unresectable tumors, and even after successful resection, the 5-year survival rate of patients is only 10%-25% 4 . Therefore, understanding how the upstream and downstream molecular mechanisms of PC affect its occurrence and development may provide theoretical guidance for the clinical treatment of this deadly disease. The mechanisms by which Rho-associated coiled coil-containing protein kinases (ROCKs) participate in tumor progression, especially in the stages of tumorigenesis, tumor development and metastasis, remain a focus of current research 5 – 7 . It has been reported that the ROCK isomer ROCK1 is highly expressed during breast cancer metastasis, thus enhancing the aggressiveness of cancer cells, and its overexpression was found to be significantly negatively correlated with the survival rate of patients 8 , 9 . High expression of ROCKs and high kinase activity have also been detected in patients with advanced breast cancer. A trend of increased ROCK expression has also been observed in PC patients 10 – 12 . In a study of colorectal cancer, polymorphisms in ROCKs were found to be closely related to the progression of cancer 13 , 14 . High expression of ROCKs has also been found in hepatocellular carcinoma 15 . However, the mechanism by which ROCK1 plays a role in regulating tumor glucose metabolism remains unclear. The MYC family oncoproteins, particularly c-MYC, are indispensable master regulators of metabolic reprogramming in various cancer types, including pancreatic cancer 16 . Studies have shown that c-MYC can participate in the regulation of tumor cell growth, cell cycle progression, metabolism, angiogenesis and other processes. However, the action of c-MYC alone does not seem to be the decisive factor of cancer. Instead, c-MYC must exert synergistic or antagonistic effects with other oncogenes and tumor suppressor genes to produce a series of effects 17 . The phosphorylation of c-Myc at certain sites governs its activation and consequential biological functions through transcriptional activation of target genes that are necessary for cell growth, and its phosphorylation at Ser 62 is essential for its oncogenic activity 18 . Previous studies have revealed that ROCK1 play a crucial role in regulating prostate tumor growth through interaction with c-Myc 19 , 20 . Therefore, the complex role of c-MYC in pancreatic cancer needs further study. Glycolysis is a major metabolic pathway that provides energy requirements for tumor growth, leading to a high rate of glycolytic flux and a greater dependence on glucose in tumor cells 21 .The committed step in glycolysis is controlled by the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), which converts fructose 6-phosphate (F6P) to fructose 2, 6-diphosphate (F2, 6BP). PFKFB3 is a major regulator of glycolysis in rapidly proliferating cells. Its high expression and/or dysregulation in multiple types of cancer has made PFKFB3 a potential therapeutic target 22 . In this paper, we demonstrate that the expression of ROCK1 is significantly increased in PC tissues and that silencing ROCK1 can effectively inhibit the proliferation and migration of PC cells, promote their apoptosis and increase their sensitivity to gemcitabine (GEM). ROCK1 was also confirmed to promote tumor growth through and increase sensitivity to GEM in in vivo transplantation experiments. Mechanistically, ROCK1 could enhance the stability of c-MYC by increasing its phosphorylation at Ser 62 and inhibiting its degradation, further increasing the transcription and expression of the key glycolytic enzyme PFKFB3, thus promoting glycolytic activity in PC cells. The findings provide vital strategies for the treatment of PC mediated by ROCK1. Materials and methods Cell line maintenance and transfection Human PC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1, BxPC-3, SW1990) and the normal pancreatic duct epithelial cell line HPDE6-C7 were purchased from The American Type Culture Collection (American Type Culture Collection, VA, USA). These cell lines were cultured in Dulbecco’s modified Eagle’s medium with high glucose (Gibco Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies, NY, USA), 100 U/mL penicillin, and 100 g/mL streptomycin. The si-ROCK1/NC plasmid and shROCK1/NC lentiviral plasmid were synthesized by Shanghai GenePharma (Shanghai, China). PC cells with good growth status were seeded in 24-well plates or 6-well plates, transfected, and grown to approximately 70% confluence. Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA) was employed for transfection. Transfection efficiency was verified by qRT‒PCR at 24 h and by Western blotting at 48 h. Clinical samples Adjacent normal mucosa and tumor tissues were collected from PC patients with informed written consent and with the approval of local medical ethics committees of Ruijin Hospital Affiliated with Shanghai Jiao Tong University. RNA isolation and quantitative reverse transcription–polymerase chain reaction (qRT‒PCR) Total RNA of the treated group and the control group was extracted by TRIzol reagent, and the extracted RNA was then reverse transcribed into cDNA by a ChamQ SYBR qPCR Master Mix Kit (Vazyme Biotech, Jiangsu, China) and analyzed on the StepOne™ Real-Time PCR system (Thermo Fisher Scientific, MA, USA). The relative mRNA expression levels were normalized to those of GAPDH and were calculated using the comparative Cq method (2 −ΔΔCq ) 20 . Primers used for RT-PCR were as follows: 5'-AACATGCTGCTGGATAAATCTGG-3' (forward) and 5'-TGTATCACATCGTACCATGCCT-3' (reverse) for ROCK1; 5'-TTCCGTGTCCCCACTGCCAACGT-3' (forward) and 5'-CAAAGGTGGAGGAGTGGGTGTCGC' (reverse) for GAPDH; 5'-GCCCGTGAGGCAGAGGCTGC-3' (forward) and 5'-TGGTGAGGACGATTATGGCCC-3' (reverse) for PKM2; 5'-GCCATCCTGCAACACTTAGGGCTTGAG-3' (forward) and 5'-GTGAGGATGTAGCTTGTAGAGGGTCCC-3' (reverse) for HK2; 5'-ATGGCAACTCTAAAGGATCAGC-3' (forward) and 5'-CCAACCCCAACAACTGTATCT-3' (reverse) for LDHA; 5'-TCCATGTGACCATGAGGAAATG-3' (forward) and 5'-TCGGCTAGTTAGGGTACACTTC-3' (reverse) for HIF-1α; 5'-ATTGCGGTTTTCGATGCCAC-3' (forward) and 5'-GCCACAACTGTAGGGTCGT-3' (reverse) for PFKFB3; 5'-AAACACAAACTTGAACAGCTAC-3' (forward) and 5'-ATTTGAGGCAGTTTACATTATGC-3' (reverse) for c-MYC. Immunofluorescence assay (IF) Cell suspensions were inoculated in confocal dishes. When the cells were 60%-70% confluent, the medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min. Then, the cells were washed with PBS, permeabilized with 0.2% Triton X-100 (Shanghai Genebase Gene-Tech, Shanghai, China) for 30 min, washed with PBS, and further blocked with 5% BSA solution at room temperature for 30 min. The cells were incubated with the primary antibody at 4°C overnight. After rewarming, the cells were washed with PBS, a fluorescent secondary antibody (ProteinTech Group, IL, USA) was added, and the cells were incubated for 3 hours at room temperature in the dark. Nuclei were stained with DAPI staining solution (Beyotime Biotechnology, Shanghai, China) for 30 min. The cells were washed with PBS, observed under a laser confocal microscope (Olympus Corporation, Tokyo, Japan) and photographed 23 . MTT assay Cells were seeded into a 96-well plate at a density of 5×10 4 cells/mL, with 100 µL of cell suspension per well and 4 wells per group. After 6, 24, 48 and 72 h of culture, the cell density was examined under a light microscope (Nikon Corporation, Tokyo, Japan), and the cells were photographed. Then, 10 µL of MTT solution (Sigma‒Aldrich, MO, USA) was added to each well in the dark, and culture was continued for 4 h. After discarding the medium, 150 µL of DMSO was added to each well, and the plate was incubated with gentle shaking for 10 min. Then, the absorbance of each group was measured with a microplate reader (Molecular Devices, CA, UCA). The detection wavelength was 492 nm. Colony formation assay After transfection, AsPC-1 and PANC-1 cells were digested by trypsin, inoculated in a 6-well plate at a density of 600 cells/mL and incubated at 37°C for 10 days. The original medium was discarded, and the cells were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min. The fixative was discarded, and the cells were washed with PBS twice. One milliliter of 0.1% crystal violet staining solution (Beijing Solarbio Science & Technology, Beijing, Chian) was added to each well and stained for 20 min. The staining solution was discarded, and the cells were washed with PBS 3–5 times until the culture plate had no background color. The colonies formed were counted and photographed. Apoptosis assay Cells were seeded into 6-well plates at a density of 3×10 5 cells/mL. Transfection was carried out at approximately 70% confluence, and then the cells were cultured for 48 h. The cell culture medium was discarded, and the cells were digested with trypsin (without EDTA). The trypsin was discarded, and the cells were washed twice with PBS by centrifugation at 400 x g for 5 min. After the supernatant was discarded, 500 µL of binding buffer was added to each tube in the dark to resuspend the cells. Then, the cells were gently mixed with 5 µL of Annexin V-FITC, and finally, 5 µL of propidium iodide (Vazyme Biotech, Jiangsu, China) was added. The reaction was allowed to proceed in the dark for 10 minutes. The stained cells were captured by flow cytometry (Becton, Dickinson and Company, NY, USA). Transwell invasion assay Cells were suspended in serum-free basal medium at a density of 2.5×10 5 cells/mL. Two hundred microliters of the suspension was added to the upper chamber (Corning Incorporated, NY, USA), and 500 µL of medium containing serum was added to the lower chamber and cultured for 24 h. The liquid in the chamber was discarded, and the cells were washed twice with PBS. The cells were fixed with 500 µL 4% paraformaldehyde for 30 min in a 24-well plate, washed with PBS twice, and then air dried appropriately. The cells were stained with 300 µL of 0.1% crystal violet solution for 20 min and washed with PBS twice. Then, the membrane in the upper chamber was wiped with a cotton swab to remove the remaining cells, washed with PBS 3 times, and air dried at room temperature for 5 min. Cell migration was observed and photographed under a 100× microscope (Olympus, Tokyo, Japan). Glucose uptake and lactate release assays Cell suspensions were seeded into 96-well plates at a density of 5×10 4 cells/mL, with 100 µL of cell suspension per well and 3 wells per group. Cell culture medium was taken for detection after 6, 24 and 48 h of culture. The glucose concentration in the medium was measured using a glucose determination kit (Shanghai Rongsheng BIOTECH, Shanghai, China), and the lactic acid concentration was measured according to the instructions of the lactic acid test kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) 24 . Western blotting Cells were lysed with RIPA buffer, and the protein concentration was measured with a BCA kit (Beyotime Biotechnology, Shanghai, China). The primary antibodies used were as follows: rabbit anti-ROCK1, rabbit anti-β-actin, rabbit anti-GAPDH, rabbit anti-c-MYC, rabbit anti-p-c-MYC (Ser 62), and rabbit anti-PFKFB3 (1:1000; all from ZEN-BIOSCIENCE). Coimmunoprecipitation (Co-IP) assay Transfected AsPC-1 cells were cultured for 48 h, 200 µL of precooled NP40 lysate (Beyotime Biotechnology, Shanghai, China) was added to each well, and the cells were lysed for 30 min with vortexing every 10 min. The supernatant was centrifuged at 13800 x g for 10 min at 4°C. Then, 150 µL of cell supernatant was removed as the input sample. Ten microliters of protein A/G agarose beads (Selleck Chemicals, TX, USA) was added to the IgG sample and target protein sample incubated with slow rotation at 4°C for 2 h. The protein samples were recovered, and the magnetic beads were discarded. The corresponding antibody (2 µg) was added to each tube, and the tubes were incubated overnight at 4°C with slow rotation. Magnetic beads (30 µL) were added to the IgG tube and the target protein tube, and the tubes were incubated at 4°C with slow rotation for 2 h. The samples were cleaned 5 times on the magnetic rack with NP40 containing PMSF (Beyotime Biotechnology, Shanghai, China) for 2 min each time, and 50 µL of samples was reserved during the last cleaning step. Loading buffer (5×) was added to the samples and boiled for 5 min. The IgG group and target protein group were prepared as described and stored at -80°C for later use. The antibodies used were as follows: rabbit anti-ROCK1 (ZEN-BIOSCIENCE); Alexa Fluor 488 (Fcmacs Biotech, Jiangsu, China); DAPI, mouse anti-c-MYC (Beyotime Biotechnology, Shanghai, China); and rhodamine (TRITC)–conjugated goat anti-mouse IgG (H + L) (1:50). Luciferase reporter assay Cells were seeded in 24-well plates at a density of 5×10 4 cells/mL, and transfection began when the confluence was approximately 70%. The cells were divided into three groups according to the types of transfected plasmids, with three replicates per group. The first group was transfected with plasmids pcDNA3.1(+)-NC and pGL3-basic-PFKFB3 pro (-2000 to -1800). The second group was transfected with pcDNA3.1(+)-c-MYC and pGL3-basic-PFKFB3 pro (-2000 to -1800). The third group was transfected with the plasmids pcDNA3.1(+)-c-MYC and pGL3-basic-mutPFKFB3 pro (-2000 to -1800). At the same time, each group was transfected with Renilla plasmid as an internal reference. The dose ratio of the three plasmids was 1:1:1, and the total amount of plasmids per well was 0.2 µg. The samples were collected after 48 h. The luciferase activity was measured following the instructions for the luciferase reporter assay kit (Vazyme Biotech, Jiangsu, China) with a full wavelength scanner (Thermo Fisher Scientific, MA, USA) 25 . Phosphofructokinase enzymatic activity assay AsPC-1 and PANC-1 cells were seeded into 6-well plates at a density of 2×10 5 cells/mL. After adherence, the ROCK1 inhibitor GSK429286A (MCE, 10 µM) was added, and samples were taken at 12 h, 24 h and 48 h after treatment. According to the instructions of the phosphofructokinase test kit (Naniing Jiancheng Bioengineering Institute, Jiangsu, China), the cells were lysed by ultrasonication and centrifuged at 4°C and 8000 × g for 10 min, and the supernatant was collected for detection. After adding the specified amount of working solution, the samples were quickly analyzed by spectrophotometry (Thermo Fisher Scientific, MA, USA), and the OD 340 value of each sample was recorded. Xenograft assay Male BALB/c nude mice were housed in the specific pathogen-free (SPF) laminar flow cabinet in the animal experimental center, with 5 mice in a cage 26 . The feeding temperature was 22 ± 1°C, and the animals were exposed to light for 12 h/day. The food and water were sterilized in advance and available ad libitum. All experiments were conducted in accordance with the regulations of the Ethics Committee for Animal Experiments. AsPC-1 cells transduced with shNC and shROCK1 lentiviral plasmids were digested by trypsin and washed with PBS three times at 200 x g for 5 min each. The cells were suspended in normal saline, and the density was adjusted to 6×10 6 cells/mL. Each nude mouse was injected subcutaneously with 200 µL of the cell suspension, with 7 mice per group. The nude mice were weighed every four days, and the length and width of tumors were accurately measured with Vernier calipers when obvious axillary tumors appeared. The tumor volume was calculated according to the formula (length× width 2 ) / 2. Beginning on the 20th day after cell inoculation, the nude mice were intraperitoneally injected with GEM (MedChemExpress, NJ, USA) (25 mg/kg, 50 mg/kg) every Monday and Thursday. On the 44th day, all mice in the experiment were euthanized by cervical dislocation. The tumors were dissected, weighed and photographed, and the tumor inhibition rate was calculated. Some tumor tissues were soaked in 4% paraformaldehyde for immunohistochemical analysis. The antibodies used were as follows: rabbit anti-ROCK1, rabbit anti-p-c-MYC (Ser 62) (ZEN-BIOSCIENCE), and rabbit anti-PFKFB3 (Beijing Solarbio Science & Technology, Beijing, China) (1:50). Results High expression of ROCK1 is associated with poor survival in pancreatic cancer patients The expression level of ROCK1 in PC was investigated with the GEPIA database ( http://gepia.cancer-pku.cn/ ). The mRNA level of ROCK1 was abnormally high in the tissues of PC patients ( p < 0.05, Fig. 1 A). The protein expression level of ROCK1 in PC tissues and adjacent tissues was further analyzed by immunohistochemical staining of clinical samples, and the results were consistent with those of the database analysis (Fig. 1 B). Then, the GEPIA database was utilized to predict the relationship between the ROCK1 expression level and overall survival rate of patients with PC. The ROCK1 expression level was found to be closely related to the prognosis of patients. As the ROCK1 expression level increased, the overall survival rate of patients with PC decreased significantly ( p = 0.025, Fig. 1 C), indicating poor prognosis. Then, the expression of ROCK1 in six PC cell lines was analyzed by qRT‒PCR. Compared with those in HPDE6-C7 normal pancreatic duct epithelial cells, ROCK1 mRNA and protein levels were significantly higher in AsPC-1 and PANC-1 cells and lower in Capan-1, MIAPaCa-2, BxPC3 and SW1990 cells (Fig. 1 D, E). The above results indicated that the expression of ROCK1 differed among PC cell lines. Although more PC cells in the screen showed a trend of low ROCK1 expression, AsPC-1 and PANC-1 cells are derived from a primary pancreatic head tumor and an in situ pancreatic head tumor, respectively. These cells have a greater metastatic ability. Therefore, they still have research value and significance. Since the subcellular localization of the ROCK1 protein is closely related to its function, the specific localization of ROCK1 in PC cells was explored. Due to the relatively high expression levels of ROCK1 in AsPC-1 and PANC-1 cells, these two cell lines were selected for immunofluorescence staining. ROCK1 was concentrated in the cytoplasm of AsPC-1 and PANC-1 cells, and only a small amount was distributed in the nucleus (Fig. 1 F). The above results suggest that abnormally high expression of ROCK1 is negatively correlated with the overall survival rate of patients with PC. Thus, ROCK1 has obvious prognostic value and could be considered an oncogenic factor for further study. Silencing ROCK1 inhibits the proliferation and migration of pancreatic cancer cells and promotes apoptosis To confirm the hypothesis that ROCK1 plays a role as an oncogenic factor in the progression of PC, two cell lines with high expression of ROCK1, AsPC-1 and PANC-1, were selected to construct a ROCK1 silencing model. Three siRNA-ROCK1 sequences were designed and their silencing efficiency was compared, and the ROCK1-Homo-1410 plasmid (si-ROCK1-2) was found to have the best ROCK1 silencing efficiency in two PC cell lines. The qRT‒PCR results showed that the silencing efficiency in AsPC-1 and PANC-1 cells was 89.1% ( p < 0.001, Fig. 2 A) and 61.8% ( p < 0.001, Fig. 2 B), respectively. Western blotting was subsequently employed to further confirm the silencing effect. It Again, the plasmid ROCK1-Homo-1410 had the best silencing efficiency (Fig. 2 C, D); thus, this plasmid was selected to construct the ROCK1 silencing model in subsequent experiments. Then, the effect of ROCK1 silencing on the biological behavior of PC cells, including cell proliferation, apoptosis and migration, was evaluated. First, the effect of ROCK1 on the proliferation of PC cells was analyzed. After transfection with the ROCK1-Homo-1410 plasmid, the cells were cultured for 6, 24, 48 and 72 h, and proliferation was evaluated by an MTT assay. The growth activity of AsPC-1 and PANC-1 cells was significantly inhibited after culture for 72 h, with inhibition rates of up to 29.8% and 9.7%, respectively (Fig. 2 E, F), suggesting that ROCK1 silencing can effectively suppress the proliferation of these two PC cell lines, with a more obvious effect on AsPC-1 cells. The impact of ROCK1 on PC cell colony formation was evaluated by a colony formation assay. As shown in Fig. 2 G and H, after silencing ROCK1, the colony formation of AsPC-1 and PANC-1 cells was significantly inhibited, with inhibition rates of 51.3% and 15.8%, respectively. The function of ROCK1 in PC cell apoptosis was further explored by flow cytometry. The ROCK1 silencing promoted the apoptosis of AsPC-1 and PANC-1 cells, with a more obvious effect on AsPC-1 cells was more obvious. Relative to the control group, after silencing of ROCK1, the total apoptosis rate in AsPC-1 cells was increased (1.1% vs. 25.0%) (Fig. 2 I), and that of PANC-1 cells was increased (8.0% vs. 10.7%) (Fig. 2 J). A Transwell assay was employed to investigate the effect of ROCK1 on the migration of two PC cell lines. After silencing ROCK1, cells were cultured for 24 h in a Transwell chamber to evaluate cell migration. The migration of AsPC-1 and PANC-1 cells was significantly suppressed by ROCK1 silencing, with inhibition rates of 61.6% and 37.9%, respectively (Fig. 2 K). These results suggest that ROCK1 plays a positive regulatory role in the occurrence and development of PC. ROCK1 enhances glycolytic activity in pancreatic cancer cells Tumor cells utilize glycolysis as their main energy source even under sufficient oxygen, a phenomenon called the “Warburg effect”. The cytoplasm is the main site of glycolysis in tumor cells. Therefore, we hypothesized that the ROCK1 enriched in the cytoplasm was related to glycolysis in tumor cells and played a role as an oncogenic factor by promoting glycolytic activity. The effects of ROCK1 on glucose uptake and lactate release in PC cells were examined. The glucose intake and lactic acid release of AsPC-1 and PANC-1 cells were significantly reduced after ROCK1 silencing for 48 h. Relative to the control group, the glucose uptake rate of AsPC-1 cells was decreased (21.3% vs. 13.4%) after silencing of ROCK1 (Fig. 3 A). The glucose uptake rate of PANC-1 cells was also decreased relative to that in the control group (12.7% vs. 9.7%) (Fig. 3 B). After silencing ROCK1, the lactate release rate of AsPC-1 and PANC-1 cells was decreased by 31.0% and 25.8%, respectively (Fig. 3 C, D). These results indicated that ROCK1 can promote glycolytic activity in PC cells. To identify the mechanism by which ROCK1 promotes glycolysis in PC cells, changes in the expression of related genes, including PKM2, HK2, LDHA, PFKFB3 and the related transcription factors HIF-1α and c-MYC, were detected by qRT‒PCR after ROCK1 silencing. As shown in Fig. 3 E, HK2, LDHA, PFKFB3 and c-MYC were downregulated in both AsPC-1 and PANC-1 PC cells after ROCK1 silencing. Among these genes, PFKFB3 ( p < 0.001, p < 0.05) and c-MYC ( p < 0.05, p < 0.01 ) exhibited significantly downregulated mRNA expression in both PC cell lines. Further analysis using the GEPIA database showed that ROCK1 gene expression was significantly positively correlated with the mRNA expression of PFKFB3 and c-MYC (Fig. 3 F, G). The above data suggest that ROCK1 can promote glycolytic activity in PC cells by increasing the mRNA expression levels of PFKFB3, a key enzyme promoting glycolysis, and c-MYC, a transcription factor associated with glycolysis. ROCK1 directly binds to c-MYC and promotes the phosphorylation of c-MYC (Ser 62) but does not directly affect the kinase activity of PFKFB3 After the correlations of the ROCK1 mRNA level with the c-MYC and PFKFB3 mRNA levels were confirmed, the influence of ROCK1 silencing on PFKFB3 and c-MYC protein levels was further evaluated. Western blotting demonstrated that the total c-MYC protein levels are downregulated in both AsPC-1 and panc-1 cells. after silencing of ROCK1. Then, analysis of phosphorylated c-MYC (Ser 62) showed that after ROCK1 silencing, the phosphorylated c-MYC (Ser 62) protein level and the PFKFB3 protein level were significantly decreased in both PC cell lines (Fig. 4 A, B). Since ROCK1 itself acts as a kinase, it was speculated that its enzymatic activity may also affect the expression of downstream proteins. In this study, the ROCK1 inhibitor GSK429286A was applied to treat the two PC cell lines. When the GSK429286A concentration was 20 µM, the protein levels of phosphorylated c-MYC (Ser 62) and PFKFB3 were effectively decreased in both PC cell lines (Fig. 4 C, D). These results demonstrated that both the high protein expression and the increase in the enzymatic activity of ROCK1 promoted the protein expression of the key glycolytic enzyme PFKFB3 and the phosphorylation of the transcription factor c-MYC (Ser 62) in PC cells, thus promoting glycolytic activity in PC cells. Due to the kinase activity of ROCK1, we speculated that the increase in this kinase activity may enhance the enzymatic activity of the downstream enzyme PFKFB3, thus promoting tumor glycolysis. Then, the two PC cell lines were treated with 10 µM GSK429286A to evaluate the kinase activity of PFKFB3. No significant difference in intracellular PFKFB3 kinase activity was observed between AsPC-1 cells and the control group after 12 h of treatment. Although intracellular PFKFB3 kinase activity was decreased after 24 h of treatment, it was slightly increased after 48 h of treatment (Fig. 4 E). Similarly, the intracellular kinase activity of PFKFB3 was increased in PANC-1 cells after 12 h and 48 h of treatment with the ROCK1 inhibitor (Fig. 4 F). These results suggested that the decrease in ROCK1 kinase activity itself did not directly lead to the decrease in downstream PFKFB3 kinase activity. Since ROCK1 had no direct effect on the kinase activity of PFKFB3, we aimed to determine whether there was direct binding between them at the protein level. If not, does the transcription factor c-MYC act as the bridge between these proteins? The protein interaction between ROCK1 and PFKFB3 was predicted by the protein interaction database STRING ( https://cn.string-db.org/ ). The results indicated that there was no direct binding between the ROCK1 and PFKFB3 proteins. However, direct binding interactions might occur between the ROCK1 and c-MYC proteins and between the c-MYC and PFKFB3 proteins (Fig. 4 G). A co-IP assay was applied to verify these predictions in AsPC-1 cells. No direct binding between the ROCK1 and PFKFB3 proteins was observed, but a direct and strong binding interaction between the ROCK1 and c-MYC proteins was found. In addition, the results of the IF assay showed that c-MYC colocalized with ROCK1 in the cytoplasm. ROCK1 promotes glycolytic activity in pancreatic cancer cells through the c-MYC/PFKFB3 signaling axis It has been reported that phosphorylation of c-MYC (Ser 62) inhibits its degradation by the ubiquitin‒proteasome pathway, thereby increasing the stability of the c-MYC protein. Thus, it was speculated that ROCK1 can enhance the stability of c-MYC, thus promoting the transcription of downstream target genes. AsPC-1 cells were transfected with the ROCK1 silencing plasmid, cultured for 48 h, and then treated with 10 µM MG-132 (a proteasome inhibitor) for 4 h. MG132 treatment significantly increased the accumulation of c-MYC protein compared with that in the untreated siNC group, indicating that c-MYC was indeed degraded through the proteasome pathway. However, when ROCK1 was silenced, MG132 treatment still reduced the accumulation of c-MYC protein compared with that in the untreated siNC group (Fig. 5 A). Furthermore, CHX (10 mg/mL, a protein synthesis inhibitor) was used to treat AsPC-1 cells with ROCK1 silencing, and samples were collected for Western blot analysis after 2 h, 4 h and 8 h of treatment. The experimental results suggested that the c-MYC protein began to degrade significantly at the 4th hour in the control group but was obviously degraded at the 2nd hour after ROCK1 silencing, further demonstrating ROCK1’s stabilizing and protective effect on the c-MYC protein (Fig. 5 B). The above results suggest that ROCK1 binds to the c-MYC protein in the cytoplasm to promote the phosphorylation of c-MYC (Ser 62), thereby enhancing its stability and inhibiting its degradation by the proteasome pathway. The relationship between c-MYC and the downstream enzyme PFKFB3 was further explored. GEPIA database analysis showed a significant positive correlation between the expression of these two genes (Fig. 5 C). Because c-MYC can promote the transcription of its downstream genes, the possible binding site of c-MYC to the promoter region of the PFKFB3-encoding gene was further predicted by the JASPAR and NCBI databases (Relative score = 0.85568): GGGCATGTGCTC (Fig. 5 D). This relationship was verified by a luciferase reporter assay. After transfection of corresponding plasmids into HEK293T cells, the luciferase activity of the group transfected with the pGL3-basic-PFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-c-MYC plasmids was significantly increased (1.5-fold) compared with that in the control group transfected with the pGL3-basic-PFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-NC plasmids. In cells transfected with pGL3-basic-mutPFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-c-MYC, the luciferase activity was decreased significantly compared with that in the experimental group. These results indicated that c-MYC bound to the promoter of the PFKFB3-encoding gene and promoted the transcription of this gene. Further validation was carried out in two PC cell lines, AsPC-1 and PANC-1. With an increase in the quantity of pcDNA3.1(+)-c-MYC plasmid transfected, there was a dose-dependent increase in luciferase activity (Fig. 5 E). These results indicated that c-MYC can promote the transcription of the PFKFB3-encoding gene in a dose-dependent manner, thus promoting the protein expression of PFKFB3. To further explain the relationship among ROCK1, c-MYC and PFKFB3, the c-MYC inhibitor 10058-F4 was applied to treat AsPC-1 and PANC-1 cells after silencing ROCK1 or while treating them with the ROCK1 inhibitor GSK429286A. Treatment with 10058-F4 alone did not seem to effectively inhibit the protein expression of PFKFB3. However, when ROCK silencing or ROCK1 inhibitor treatment was combined with 10058-F4 treatment, the inhibitory effect on PFKFB3 was excellent, much better than the single effects (Fig. 5 F-I). Collectively, these results suggest that ROCK1 binds to the c-MYC protein in the cytoplasm and promotes the phosphorylation of c-MYC (Ser 62), thereby enhancing the stability of the c-MYC protein and inhibiting its degradation. C-MYC could bind to the promoter of the PFKFB3-encoding gene to promote its mRNA transcription and subsequent protein expression, thus promoting tumor glycolysis (Fig. 5 J). AsPC-1 tumor growth is effectively inhibited and GEM sensitivity is increased after ROCK1-silencing in nude mice To investigate whether ROCK1 also has carcinogenic activity in vivo, the silencing efficiency of shRNA was first verified by Western blotting. The protein expression of ROCK1 was significantly inhibited after transduction with the shROCK1 lentiviral plasmid, indicating successful transduction of the shROCK 1 silencing plasmid. We then investigated whether ROCK1 deficiency enhanced the sensitivity of pancreatic cancer cells to GEM. AsPC-1 cells were transduced with shROCK1 and shNC lentiviral plasmids, plated and cultured with a concentration gradient of GEM (0, 1, 2.5, 5, 7.5 µM) for 48 h. Cytotoxicity was then detected by a MTT assay. Silencing ROCK1 significantly increased the sensitivity of AsPC-1 cells to GEM (Fig. 6 A). Then, the xenograft tumors in nude mice were analyzed. AsPC-1 cells transduced with shNC and shROCK1 were inoculated into nude mice (approximately 5 weeks old) under the left axilla, and the mice were weighed every four days. On day 44, the mean tumor volume was 181.2 mm in the shROCK1 group compared to 281.9 mm 3 in the shNC group. Thus, silencing ROCK1 significantly suppressed the increase in the tumor volume in nude mice ( p < 0.05) compared with that in the shNC group (Fig. 6 B, C). These data suggest that silencing ROCK1 can effectively inhibit tumor growth in tumor-bearing mice. In vitro experiments have shown that the protein levels of ROCK1, p-c-MYC (Ser 62), and PFKFB3 are positively correlated; thus, we aimed to further validate the above results through in vivo experiments. Immunohistochemical analysis of different tumor tissues showed that the p-c-MYC (Ser 62) and PFKFB3 protein levels were also relatively low in tissue samples with low ROCK1 expression. Conversely, tumor tissues with high ROCK1 expression also exhibited increased protein levels of p-c-MYC (Ser 62) and PFKFB3 (Fig. 6 D), consistent with the results of the in vitro experiments. Then, GEM sensitivity was analyzed in vivo. According to the above subcutaneous xenograft method, beginning when subcutaneous tumors began to form in nude mice on the 20th day, GEM (25 mg/kg, 50 mg/kg) was injected intraperitoneally every Monday and Thursday. The nude mice were sacrificed after 44 days, and the xenograft tumors were removed. The tumor volume in the shROCK1 group was significantly lower than that in the shNC group, indicating that the sensitivity of xenograft tumors to GEM ( p < 0.05) was significantly increased after silencing of ROCK1 (Fig. 6 E). Discussion Recent studies have shown that ROCK dysregulation is involved in the metastasis and progression of various malignant tumors. ROCK1 shows high kinase activity and protein expression levels during breast cancer metastasis and advanced breast cancer development, and a similar phenomenon has been observed in colorectal cancer 27 – 30 . However, the relationship between ROCK1 expression and PC activity is unclear. In this study, we show that ROCK1 enhances the stability of c-MYC by promoting its phosphorylation (at Ser 62) and inhibiting its degradation by the ubiquitin‒proteasome pathway, thus promoting the transcription of the downstream key glycolytic enzyme PFKFB3 at the mRNA level and subsequently increasing its protein expression, thereby increasing glycolytic activity in PC cells. These results define a theoretical basis for finding important PC treatment strategies. Our study revealed that ROCK1 was highly expressed in PC and associated with poor prognosis in patients. The GEPIA database and immunohistochemical analyses showed that the expression of ROCK1 was abnormally high in the tissues of PC patients. High expression of ROCK1 was found to be significantly negatively correlated with the prognosis of patients, indicating that ROCK1 is a key oncogenic factor in the development of PC. The qRT‒PCR results further confirmed that ROCK1 mRNA showed significantly high expression in AsPC-1 and PANC-1 cells. The other four PC cell lines showed low expression of ROCK1, indicating that ROCK1 expression differed among cell lines, an effect that may be related to cell genotype. The genes that regulate this phenomenon may need to be further verified by gene mutation or deletion assays. The immunofluorescence results showed that ROCK1 was distributed mainly in the cytoplasm, which is the main site of glycolysis. To verify the biological function of ROCK1 in the growth of PC cells, we evaluated the effects of ROCK1 expression changes on cell proliferation, migration and apoptosis. ROCK1 silencing significantly inhibited the growth of the two tested PC cell lines and effectively suppressed colony formation. After silencing ROCK1, the colony formation of AsPC-1 and PANC-1 cells was significantly inhibited, with inhibition rates of 51.3% and 15.8%, respectively. The flow cytometry results showed that ROCK1 silencing promoted the apoptosis of AsPC-1 and PANC-1 cells. The Transwell assay indicated that silencing ROCK1 significantly inhibited the migration of PC cells, with inhibition rates of 61.6% and 37.9% in AsPC-1 and PANC-1 cells, respectively. These results suggest that ROCK1 plays a role as an oncogenic factor in the development and progression of PC. Furthermore, the mechanism by which ROCK1 promotes PC cell activity was explored. Since cancer cells utilize glycolysis as the main energy source for their rapid proliferation even under oxygen-rich conditions, the cytoplasm is the main site of glycolysis 31 , and it was speculated that ROCK1 may play a role as an oncogenic factor by affecting glycolysis in PC cells. The glucose uptake and lactic acid release assays showed that ROCK1 silencing significantly inhibited glucose uptake and lactic acid release in the two tested PC cell lines, indicating that ROCK1 can promote glycolytic activity in PC cells. The mRNA expression of glycolysis-related genes after silencing ROCK1 was further evaluated, and PFKFB3 and c-MYC mRNA expression was obviously downregulated in both tested PC cell lines. Studies have shown that cancer cells adapt to stressful conditions and continue to proliferate rapidly, in part because the actions of PFKFB3 are diverse and reversible 32 – 35 . Moreover, the c-MYC protein has been reported to be involved in the regulation of tumor cell growth, cell cycle progression, metabolism, and angiogenesis 36 , 37 . Western blot analysis showed that both silencing of ROCK1 and treatment with the ROCK1 inhibitor GSK429286A significantly reduced the protein levels of p-c-MYC (Ser 62) and PFKFB3, while ROCK1 did not directly affect the kinase activity of PFKFB3. The co-IP results showed that ROCK1 directly binds to c-MYC but not to PFKFB3. The immunofluorescence experiments also intuitively proved that ROCK1 and c-MYC were obviously colocalized in the cytoplasm. Subsequently, treatment of AsPC-1 cells with MG132 and CHX showed that ROCK1 enhanced the stability of c-MYC by promoting its phosphorylation (at Ser 62) and inhibiting its degradation. In addition, the luciferase reporter assay showed that c-MYC can promote the transcription of the PFKFB3-encoding gene in a dose-dependent manner. These results indicate that the ROCK1 protein promotes the glycolytic activity in PC cells through the c-MYC/PFKFB3 signaling axis, thereby promoting PC tumor growth. Finally, our findings further verified the role and mechanism of ROCK1 in promoting the growth of PC in vivo. By establishing a nude mouse xenograft model of AsPC-1 cells, we found that silencing ROCK1 significantly reduced tumor volume and weight in nude mice. Immunohistochemical analysis of tumor tissues showed that the p-c-MYC (Ser 62) and PFKFB3 protein levels were also relatively high in tumor tissues with high ROCK1 expression and were also low in tumor tissues with low ROCK1 expression. The above results indicated that ROCK1 promoted the growth of PC in vivo, and the positive correlations of ROCK1 expression with p-c-MYC (Ser 62) and PFKFB3 expression were consistent with the results in vitro. However, the relationships between the protein levels of ROCK1, p-c-MYC (Ser 62) and PFKFB3 were verified only by immunohistochemistry at the animal level, and the changes in glycolysis in animals were not described in detail, although this will be the focus of subsequent research. Moreover, the mechanism by which ROCK1 affects enzymatic activity in the ubiquitin‒proteasome degradation pathway to stabilize c-MYC needs to be further studied. Conclusion In conclusion, our findings demonstrate that ROCK1 acts on PC cells through the c-MYC/PFKFB3 signaling axis to enhance glycolytic activity in PC cells, thereby enhancing the growth activity of PC. Moreover, knockdown of ROCK1 can also increase the sensitivity of PC cells to GEM. This paper indicates that ROCK1 is an effective therapeutic target for PC and that its positive regulation of glycolysis may provide an effective clinical therapeutic strategy for PC. Declarations Acknowledgements Not applicable. Funding The present study was supported by the National Key Research and Development Program of China (2018YFA0902000) and the "Double First-Class" University project (CPU2022QZ09). Availability of data and materials The data and materials are available from the corresponding author on request. Authors' contributions Shuyang Pang: conceptualization, methodology and writing - original draft; Yuting Shen, Yanan Wang data curation, writing - reviewing and editing; Lingman Ma and Yiran Zhou supervision and funding acquisition. Ethics approval and consent to participate All animal experiments were performed according to the protocols approved by the Ethics Committee of China Pharmaceutical University (approval no. SYXK 2021‑0011). Patient consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References T K, LD W, T I, K T.-Pancreatic cancer. 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A Potential Oncogenic Role for PFKFB3 Overexpression in Gastric Cancer Progression. Clin Transl Gastroenterol 2021;12:e00377 Liao DJ, Dickson RB. c-Myc in breast cancer. Endocr Relat Cancer 2000;7:143–164 Dhanasekaran R, Deutzmann A, Mahauad-Fernandez WD, Hansen AS, Gouw AM, Felsher DW. The MYC oncogene-the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol 2022;19:23–36 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Biochimica et Biophysica Acta (BBA) - General Subjects → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3836816","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265511499,"identity":"324eea13-7562-49e9-907e-fa4216fb56f3","order_by":0,"name":"SHUYANG PANG","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"SHUYANG","middleName":"","lastName":"PANG","suffix":""},{"id":265511500,"identity":"7e7f30e8-1f6a-4fdf-a0cd-d64ec959db13","order_by":1,"name":"YUTING SHEN","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"YUTING","middleName":"","lastName":"SHEN","suffix":""},{"id":265511501,"identity":"aef3d427-5a56-4273-8fbb-0d3f10527cdb","order_by":2,"name":"YANAN WANG","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"YANAN","middleName":"","lastName":"WANG","suffix":""},{"id":265511502,"identity":"00a086a4-ba2c-4c11-93c8-971f96225f7b","order_by":3,"name":"XUANNING CHU","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"XUANNING","middleName":"","lastName":"CHU","suffix":""},{"id":265511503,"identity":"cb0c4a75-c930-4faf-8419-68011d930dc0","order_by":4,"name":"LINGMAN MA","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"LINGMAN","middleName":"","lastName":"MA","suffix":""},{"id":265511504,"identity":"6b581ea8-a5d9-4477-800d-0feebfbb5e25","order_by":5,"name":"YIRAN ZHOU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie2RIQ7CQBBFh5C0ZhKQiylXmATbm2C2ZmpYwgEQq8CQYHuPJughTVCFC2Cq0E2wFbQCHEtxJOxTI+Zl/uQDeDw/itQU4wiAexuDY7biaGK/UIYF1sWMBIJ++9PMVILESX6VWwXreA7hWdypspREUZwcLjolOLGxuNTuVIpBqL1yKIHVwBbGKiSnEnSKpiLJd30V7BSh9n3sqyi8wdESR6oLpttfNrhwK9MtD+9N01a5Q1b1Ojb7sHQrMNavjBq6+XM7I3lOobzf8ng8nr/mAUh4RGJ6VWRGAAAAAElFTkSuQmCC","orcid":"","institution":"Ruijin Hospital, Shanghai Jiaotong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"YIRAN","middleName":"","lastName":"ZHOU","suffix":""}],"badges":[],"createdAt":"2024-01-05 09:14:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3836816/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3836816/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.bbagen.2024.130669","type":"published","date":"2024-07-01T12:11:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49323041,"identity":"2e105b88-8388-47af-855f-1a55981de0c2","added_by":"auto","created_at":"2024-01-08 17:05:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHigh expression of ROCK1 is associated with low survival in pancreatic cancer patients. \u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e The mRNA expression of ROCK1 in pancreatic cancer tissues and pericancerous tissues predicted by GEPIA database, with red representing cancer tissues and black representing pericancerous tissues. \u003cstrong\u003eB\u003c/strong\u003e Immunohistochemical analysis of ROCK1 protein expression in pancreatic cancer clinical samples (\u003cem\u003en \u003c/em\u003e= 5, left: 40×, right: 100×). \u003cstrong\u003eC\u003c/strong\u003e Relationship between ROCK1 expression level and overall survival rate of patients analyzed by GEPIA database. \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR detection of the relative expression of ROCK1 in six pancreatic cancer cells (HPDE6-C7 group as a reference, \u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eE\u003c/strong\u003e WB detection of the relative expression of ROCK1 in five pancreatic cancer cells (HPDE6-C7 group as a reference,\u003cem\u003e n\u003c/em\u003e = 4). \u003cstrong\u003eF\u003c/strong\u003e Localization of ROCK1 in AsPC-1 and PANC-1 cells was detected by immunofluorescence assay (\u003cem\u003en \u003c/em\u003e= 3, 400×). (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/f59d24d8213f45873dc5ac83.png"},{"id":49324908,"identity":"2ed67021-2e18-442a-a555-f67752bd1a15","added_by":"auto","created_at":"2024-01-08 17:21:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":215831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSilencing ROCK1 inhibits the proliferation and migration of pancreatic cancer cells and promotes the apoptosis.\u003c/em\u003e \u003cstrong\u003eA, B\u003c/strong\u003e The silencing efficiency of ROCK1 in AsPC-1 and PANC-1 cells was detected by qRT-PCR (\u003cem\u003en \u003c/em\u003e= 3). \u003cstrong\u003eC, D\u003c/strong\u003e Western Blotting detecting the silencing effect of ROCK1 in AsPC-1 and PANC-1 cells (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eE, F \u003c/strong\u003eMTT assay to determine the effect of ROCK1 silencing on proliferation of AsPC-1 and PANC-1 cells (\u003cem\u003en \u003c/em\u003e= 4). \u003cstrong\u003eG, H\u003c/strong\u003e Colony formation assays detecting the silencing effect of ROCK1 on the ability of AsPC-1 and PANC-1 cells to form colonies (\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eI, J \u003c/strong\u003eThe apoptotic rate of AsPC-1 and PANC-1 cells was measured by an Annexin V (AV) and the propidium iodide (P) apoptosis detection kit. The percentages of apoptosis are shown. The data in the flow cytometry plots are representative images, while the data in the bar charts represent the overall data. Thus, the data in the plots and graphs do not necessarily represent each other (\u003cem\u003en\u003c/em\u003e = 3，*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001). \u003cstrong\u003eK\u003c/strong\u003e Migration of AsPC-1 and PANC-1 cells was assessed by Transwell assay. (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/1f2a99b5b8719e83088d9386.png"},{"id":49323040,"identity":"3f10dee6-faea-4d22-8c1d-8e983ba57f58","added_by":"auto","created_at":"2024-01-08 17:05:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eROCK1 enhances glycolysis activity of pancreatic cancer cells.\u003c/em\u003e \u003cstrong\u003eA, B\u003c/strong\u003e Quantitative map of glucose uptake rate in AsPC-1 and PANC-1 cells (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eC, D\u003c/strong\u003e Quantification of lactic acid release in AsPC-1 and PANC-1 cells (\u003cem\u003en \u003c/em\u003e= 3). \u003cstrong\u003eE, F\u003c/strong\u003e qRT-PCR was used to detect ROCK1 - related glycolysis genes in pancreatic cancer cells (\u003cem\u003en \u003c/em\u003e= 3). \u003cstrong\u003eG, H\u003c/strong\u003e The correlation between ROCK1 and PFKFB3 and c-MYC was analyzed by GEPIA database. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/a0323012d8bc11e796d645e2.png"},{"id":49323042,"identity":"0daff9d2-9ce3-4731-8c56-12b4e91c9c31","added_by":"auto","created_at":"2024-01-08 17:05:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":165562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eROCK1 is directly bound to c-MYC but not directly affect the kinase activity of PFKFB3. \u003c/em\u003e\u003cstrong\u003eA-D\u003c/strong\u003e Western Blotting assay was applied to detect the effects of silencing ROCK1 or ROCK1 kinase inhibitor treatment on glycolysis related proteins in AsPC-1 and PANC-1 cells (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eE, F\u003c/strong\u003e The effects of ROCK1 kinase activity on PFKFB3 kinase activity were detected by phosphofructokinase enzyme activity assay (\u003cem\u003en \u003c/em\u003e= 3). \u003cstrong\u003eG\u003c/strong\u003e The relationships among ROCK1, c-MYC and PFKFB3 proteins were analyzed by STRING database. \u003cstrong\u003eH\u003c/strong\u003e The direct binding of ROCK1 protein to c-MYC and PFKFB3 protein was verified by Co-IP assay (\u003cem\u003en \u003c/em\u003e= 3). \u003cstrong\u003eI\u003c/strong\u003e Localization of ROCK1 and c-MYC in cells was detected by immunofluorescence assay (n = 3, 400 ×). (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/3b19afd7f6974c9ee1a7a5a4.png"},{"id":49323821,"identity":"49fa0576-338c-47e1-971b-7765db616645","added_by":"auto","created_at":"2024-01-08 17:13:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eROCK1 promotes glycolysis activity of pancreatic cancer cells through c-MYC /PFKFB3 signaling axis. \u003c/em\u003e\u003cstrong\u003eA, B\u003c/strong\u003e MG132 or CHX treated AsPC-1 cells to detect the stabilizing effect of ROCK1 on c-MYC protein (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eC\u003c/strong\u003e The correlation between c-MYC and PFKFB3 was analyzed by GEPIA database. \u003cstrong\u003eD\u003c/strong\u003e JASPAR and NCBI databases predicted the binding sites of c-MYC to the promoter region of PFKFB3 encoding gene. \u003cstrong\u003eE \u003c/strong\u003eThe binding of c-MYC to the PFKFB3 gene promoter was verified by luciferase reporter assay (\u003cem\u003en\u003c/em\u003e= 4). \u003cstrong\u003eF-I\u003c/strong\u003e Effects of ROCK1 silencing or ROCK1 kinase inhibitor combined with c-MYC inhibitor on PFKFB3 protein expression in AsPC-1 and PANC-1 cells (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eJ\u003c/strong\u003e Action mechanism diagram of ROCK1, c-MYC and PFKFB3. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/eaa7feafb97cd4ab7d8b80e1.png"},{"id":49323822,"identity":"0ea96a13-b191-4902-910e-0d15de899dfd","added_by":"auto","created_at":"2024-01-08 17:13:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":182351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAsPC-1 tumor is effectively inhibited, and more sensitivity to GEM is increased in ROCK1 silence nude mice. \u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e The effect of silencing ROCK1 by shRNA lentivirus plasmid was verified by Western Blotting assay (\u003cem\u003en\u003c/em\u003e = 3). MTT assay was used to detect the cell viability of shNC/shROCK1 AsPC-1 cells treated with GEM at different concentrations for 48 h. The value of IC50 was shown as indicated. \u003cstrong\u003eB\u003c/strong\u003e The image of tumor bearing mice. \u003cstrong\u003eC \u003c/strong\u003eThe image of tumors and Quantified tumor volume \u003cem\u003e(n\u003c/em\u003e = 7). \u003cstrong\u003eD\u003c/strong\u003e Representative images of IHC measurement showed the protein levels of ROCK1, p-c-MYC (Ser62) and PFKFB3 in tumor tissues with high and low ROCK1 levels. Scale bar: 100 μm. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003eE\u003c/strong\u003e The image of tumors and Quantified tumor volume (\u003cem\u003en \u003c/em\u003e= 5).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/644606130c1dc02d77624eee.png"},{"id":61161079,"identity":"f68659d2-9fa0-4c21-8995-ce696b3228cd","added_by":"auto","created_at":"2024-07-26 12:11:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1784748,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3836816/v1/360a7f4b-37d8-47ca-9883-58d3cbd8d0a6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer (PC) is a fatal disease, with a gradually increasing annual mortality rate approaching the annual incidence rate \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Statistics for 2020 show that the incidence of PC continues to rise, mainly in Western countries, and PC is expected to become the second leading cause of death among cancers by 2030 \u003csup\u003e2\u003c/sup\u003e. In the early stage of PC, there are often no obvious symptoms, and it is difficult to detect. Patients tend to miss the golden treatment period and thus lose the opportunity for radical surgery. The pancreas is hidden in the abdominal cavity and close to major blood vessels, and accurate serum markers and imaging models for early diagnosis are lacking, making early detection of and screening for PC extremely difficult \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Additionally, PC shows significant resistance to radiotherapy and chemotherapy, resulting in a further increase in patient mortality. Surgical resection is still an effective treatment, but 80%-90% of patients have unresectable tumors, and even after successful resection, the 5-year survival rate of patients is only 10%-25% \u003csup\u003e4\u003c/sup\u003e. Therefore, understanding how the upstream and downstream molecular mechanisms of PC affect its occurrence and development may provide theoretical guidance for the clinical treatment of this deadly disease.\u003c/p\u003e \u003cp\u003eThe mechanisms by which Rho-associated coiled coil-containing protein kinases (ROCKs) participate in tumor progression, especially in the stages of tumorigenesis, tumor development and metastasis, remain a focus of current research \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It has been reported that the ROCK isomer ROCK1 is highly expressed during breast cancer metastasis, thus enhancing the aggressiveness of cancer cells, and its overexpression was found to be significantly negatively correlated with the survival rate of patients \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. High expression of ROCKs and high kinase activity have also been detected in patients with advanced breast cancer. A trend of increased ROCK expression has also been observed in PC patients \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In a study of colorectal cancer, polymorphisms in ROCKs were found to be closely related to the progression of cancer \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. High expression of ROCKs has also been found in hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the mechanism by which ROCK1 plays a role in regulating tumor glucose metabolism remains unclear.\u003c/p\u003e \u003cp\u003eThe MYC family oncoproteins, particularly c-MYC, are indispensable master regulators of metabolic reprogramming in various cancer types, including pancreatic cancer \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Studies have shown that c-MYC can participate in the regulation of tumor cell growth, cell cycle progression, metabolism, angiogenesis and other processes. However, the action of c-MYC alone does not seem to be the decisive factor of cancer. Instead, c-MYC must exert synergistic or antagonistic effects with other oncogenes and tumor suppressor genes to produce a series of effects \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The phosphorylation of c-Myc at certain sites governs its activation and consequential biological functions through transcriptional activation of target genes that are necessary for cell growth, and its phosphorylation at Ser 62 is essential for its oncogenic activity \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Previous studies have revealed that ROCK1 play a crucial role in regulating prostate tumor growth through interaction with c-Myc \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, the complex role of c-MYC in pancreatic cancer needs further study.\u003c/p\u003e \u003cp\u003eGlycolysis is a major metabolic pathway that provides energy requirements for tumor growth, leading to a high rate of glycolytic flux and a greater dependence on glucose in tumor cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.The committed step in glycolysis is controlled by the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), which converts fructose 6-phosphate (F6P) to fructose 2, 6-diphosphate (F2, 6BP). PFKFB3 is a major regulator of glycolysis in rapidly proliferating cells. Its high expression and/or dysregulation in multiple types of cancer has made PFKFB3 a potential therapeutic target \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this paper, we demonstrate that the expression of ROCK1 is significantly increased in PC tissues and that silencing ROCK1 can effectively inhibit the proliferation and migration of PC cells, promote their apoptosis and increase their sensitivity to gemcitabine (GEM). ROCK1 was also confirmed to promote tumor growth through and increase sensitivity to GEM in in vivo transplantation experiments. Mechanistically, ROCK1 could enhance the stability of c-MYC by increasing its phosphorylation at Ser 62 and inhibiting its degradation, further increasing the transcription and expression of the key glycolytic enzyme PFKFB3, thus promoting glycolytic activity in PC cells. The findings provide vital strategies for the treatment of PC mediated by ROCK1.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell line maintenance and transfection\u003c/h2\u003e \u003cp\u003eHuman PC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1, BxPC-3, SW1990) and the normal pancreatic duct epithelial cell line HPDE6-C7 were purchased from The American Type Culture Collection (American Type Culture Collection, VA, USA). These cell lines were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium with high glucose (Gibco Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies, NY, USA), 100 U/mL penicillin, and 100 g/mL streptomycin.\u003c/p\u003e \u003cp\u003eThe si-ROCK1/NC plasmid and shROCK1/NC lentiviral plasmid were synthesized by Shanghai GenePharma (Shanghai, China). PC cells with good growth status were seeded in 24-well plates or 6-well plates, transfected, and grown to approximately 70% confluence. Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA) was employed for transfection. Transfection efficiency was verified by qRT‒PCR at 24 h and by Western blotting at 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003e Adjacent normal mucosa and tumor tissues were collected from PC patients with informed written consent and with the approval of local medical ethics committees of Ruijin Hospital Affiliated with Shanghai Jiao Tong University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative reverse transcription\u0026ndash;polymerase chain reaction (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA of the treated group and the control group was extracted by TRIzol reagent, and the extracted RNA was then reverse transcribed into cDNA by a ChamQ SYBR qPCR Master Mix Kit (Vazyme Biotech, Jiangsu, China) and analyzed on the StepOne\u0026trade; Real-Time PCR system (Thermo Fisher Scientific, MA, USA). The relative mRNA expression levels were normalized to those of GAPDH and were calculated using the comparative Cq method (2\u003csup\u003e\u0026minus;ΔΔCq\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Primers used for RT-PCR were as follows: 5'-AACATGCTGCTGGATAAATCTGG-3' (forward) and 5'-TGTATCACATCGTACCATGCCT-3' (reverse) for ROCK1; 5'-TTCCGTGTCCCCACTGCCAACGT-3' (forward) and 5'-CAAAGGTGGAGGAGTGGGTGTCGC' (reverse) for GAPDH; 5'-GCCCGTGAGGCAGAGGCTGC-3' (forward) and 5'-TGGTGAGGACGATTATGGCCC-3' (reverse) for PKM2; 5'-GCCATCCTGCAACACTTAGGGCTTGAG-3' (forward) and 5'-GTGAGGATGTAGCTTGTAGAGGGTCCC-3' (reverse) for HK2; 5'-ATGGCAACTCTAAAGGATCAGC-3' (forward) and 5'-CCAACCCCAACAACTGTATCT-3' (reverse) for LDHA; 5'-TCCATGTGACCATGAGGAAATG-3' (forward) and 5'-TCGGCTAGTTAGGGTACACTTC-3' (reverse) for HIF-1α; 5'-ATTGCGGTTTTCGATGCCAC-3' (forward) and 5'-GCCACAACTGTAGGGTCGT-3' (reverse) for PFKFB3; 5'-AAACACAAACTTGAACAGCTAC-3' (forward) and 5'-ATTTGAGGCAGTTTACATTATGC-3' (reverse) for c-MYC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay (IF)\u003c/h2\u003e \u003cp\u003eCell suspensions were inoculated in confocal dishes. When the cells were 60%-70% confluent, the medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min. Then, the cells were washed with PBS, permeabilized with 0.2% Triton X-100 (Shanghai Genebase Gene-Tech, Shanghai, China) for 30 min, washed with PBS, and further blocked with 5% BSA solution at room temperature for 30 min. The cells were incubated with the primary antibody at 4\u0026deg;C overnight. After rewarming, the cells were washed with PBS, a fluorescent secondary antibody (ProteinTech Group, IL, USA) was added, and the cells were incubated for 3 hours at room temperature in the dark. Nuclei were stained with DAPI staining solution (Beyotime Biotechnology, Shanghai, China) for 30 min. The cells were washed with PBS, observed under a laser confocal microscope (Olympus Corporation, Tokyo, Japan) and photographed \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eCells were seeded into a 96-well plate at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL, with 100 \u0026micro;L of cell suspension per well and 4 wells per group. After 6, 24, 48 and 72 h of culture, the cell density was examined under a light microscope (Nikon Corporation, Tokyo, Japan), and the cells were photographed. Then, 10 \u0026micro;L of MTT solution (Sigma‒Aldrich, MO, USA) was added to each well in the dark, and culture was continued for 4 h. After discarding the medium, 150 \u0026micro;L of DMSO was added to each well, and the plate was incubated with gentle shaking for 10 min. Then, the absorbance of each group was measured with a microplate reader (Molecular Devices, CA, UCA). The detection wavelength was 492 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eAfter transfection, AsPC-1 and PANC-1 cells were digested by trypsin, inoculated in a 6-well plate at a density of 600 cells/mL and incubated at 37\u0026deg;C for 10 days. The original medium was discarded, and the cells were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min. The fixative was discarded, and the cells were washed with PBS twice. One milliliter of 0.1% crystal violet staining solution (Beijing Solarbio Science \u0026amp; Technology, Beijing, Chian) was added to each well and stained for 20 min. The staining solution was discarded, and the cells were washed with PBS 3\u0026ndash;5 times until the culture plate had no background color. The colonies formed were counted and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eCells were seeded into 6-well plates at a density of 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL. Transfection was carried out at approximately 70% confluence, and then the cells were cultured for 48 h. The cell culture medium was discarded, and the cells were digested with trypsin (without EDTA). The trypsin was discarded, and the cells were washed twice with PBS by centrifugation at 400 x g for 5 min. After the supernatant was discarded, 500 \u0026micro;L of binding buffer was added to each tube in the dark to resuspend the cells. Then, the cells were gently mixed with 5 \u0026micro;L of Annexin V-FITC, and finally, 5 \u0026micro;L of propidium iodide (Vazyme Biotech, Jiangsu, China) was added. The reaction was allowed to proceed in the dark for 10 minutes. The stained cells were captured by flow cytometry (Becton, Dickinson and Company, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTranswell invasion assay\u003c/h2\u003e \u003cp\u003eCells were suspended in serum-free basal medium at a density of 2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL. Two hundred microliters of the suspension was added to the upper chamber (Corning Incorporated, NY, USA), and 500 \u0026micro;L of medium containing serum was added to the lower chamber and cultured for 24 h. The liquid in the chamber was discarded, and the cells were washed twice with PBS. The cells were fixed with 500 \u0026micro;L 4% paraformaldehyde for 30 min in a 24-well plate, washed with PBS twice, and then air dried appropriately. The cells were stained with 300 \u0026micro;L of 0.1% crystal violet solution for 20 min and washed with PBS twice. Then, the membrane in the upper chamber was wiped with a cotton swab to remove the remaining cells, washed with PBS 3 times, and air dried at room temperature for 5 min. Cell migration was observed and photographed under a 100\u0026times; microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGlucose uptake and lactate release assays\u003c/h2\u003e \u003cp\u003eCell suspensions were seeded into 96-well plates at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL, with 100 \u0026micro;L of cell suspension per well and 3 wells per group. Cell culture medium was taken for detection after 6, 24 and 48 h of culture. The glucose concentration in the medium was measured using a glucose determination kit (Shanghai Rongsheng BIOTECH, Shanghai, China), and the lactic acid concentration was measured according to the instructions of the lactic acid test kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed with RIPA buffer, and the protein concentration was measured with a BCA kit (Beyotime Biotechnology, Shanghai, China). The primary antibodies used were as follows: rabbit anti-ROCK1, rabbit anti-β-actin, rabbit anti-GAPDH, rabbit anti-c-MYC, rabbit anti-p-c-MYC (Ser 62), and rabbit anti-PFKFB3 (1:1000; all from ZEN-BIOSCIENCE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCoimmunoprecipitation (Co-IP) assay\u003c/h2\u003e \u003cp\u003eTransfected AsPC-1 cells were cultured for 48 h, 200 \u0026micro;L of precooled NP40 lysate (Beyotime Biotechnology, Shanghai, China) was added to each well, and the cells were lysed for 30 min with vortexing every 10 min. The supernatant was centrifuged at 13800 x g for 10 min at 4\u0026deg;C. Then, 150 \u0026micro;L of cell supernatant was removed as the input sample. Ten microliters of protein A/G agarose beads (Selleck Chemicals, TX, USA) was added to the IgG sample and target protein sample incubated with slow rotation at 4\u0026deg;C for 2 h. The protein samples were recovered, and the magnetic beads were discarded. The corresponding antibody (2 \u0026micro;g) was added to each tube, and the tubes were incubated overnight at 4\u0026deg;C with slow rotation. Magnetic beads (30 \u0026micro;L) were added to the IgG tube and the target protein tube, and the tubes were incubated at 4\u0026deg;C with slow rotation for 2 h. The samples were cleaned 5 times on the magnetic rack with NP40 containing PMSF (Beyotime Biotechnology, Shanghai, China) for 2 min each time, and 50 \u0026micro;L of samples was reserved during the last cleaning step. Loading buffer (5\u0026times;) was added to the samples and boiled for 5 min. The IgG group and target protein group were prepared as described and stored at -80\u0026deg;C for later use. The antibodies used were as follows: rabbit anti-ROCK1 (ZEN-BIOSCIENCE); Alexa Fluor 488 (Fcmacs Biotech, Jiangsu, China); DAPI, mouse anti-c-MYC (Beyotime Biotechnology, Shanghai, China); and rhodamine (TRITC)\u0026ndash;conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (1:50).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL, and transfection began when the confluence was approximately 70%. The cells were divided into three groups according to the types of transfected plasmids, with three replicates per group. The first group was transfected with plasmids pcDNA3.1(+)-NC and pGL3-basic-PFKFB3 pro (-2000 to -1800). The second group was transfected with pcDNA3.1(+)-c-MYC and pGL3-basic-PFKFB3 pro (-2000 to -1800). The third group was transfected with the plasmids pcDNA3.1(+)-c-MYC and pGL3-basic-mutPFKFB3 pro (-2000 to -1800). At the same time, each group was transfected with Renilla plasmid as an internal reference. The dose ratio of the three plasmids was 1:1:1, and the total amount of plasmids per well was 0.2 \u0026micro;g. The samples were collected after 48 h. The luciferase activity was measured following the instructions for the luciferase reporter assay kit (Vazyme Biotech, Jiangsu, China) with a full wavelength scanner (Thermo Fisher Scientific, MA, USA) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhosphofructokinase enzymatic activity assay\u003c/h2\u003e \u003cp\u003eAsPC-1 and PANC-1 cells were seeded into 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL. After adherence, the ROCK1 inhibitor GSK429286A (MCE, 10 \u0026micro;M) was added, and samples were taken at 12 h, 24 h and 48 h after treatment. According to the instructions of the phosphofructokinase test kit (Naniing Jiancheng Bioengineering Institute, Jiangsu, China), the cells were lysed by ultrasonication and centrifuged at 4\u0026deg;C and 8000 \u0026times; g for 10 min, and the supernatant was collected for detection. After adding the specified amount of working solution, the samples were quickly analyzed by spectrophotometry (Thermo Fisher Scientific, MA, USA), and the OD\u003csub\u003e340\u003c/sub\u003e value of each sample was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eXenograft assay\u003c/h2\u003e \u003cp\u003eMale BALB/c nude mice were housed in the specific pathogen-free (SPF) laminar flow cabinet in the animal experimental center, with 5 mice in a cage \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The feeding temperature was 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and the animals were exposed to light for 12 h/day. The food and water were sterilized in advance and available ad libitum. All experiments were conducted in accordance with the regulations of the Ethics Committee for Animal Experiments.\u003c/p\u003e \u003cp\u003eAsPC-1 cells transduced with shNC and shROCK1 lentiviral plasmids were digested by trypsin and washed with PBS three times at 200 x g for 5 min each. The cells were suspended in normal saline, and the density was adjusted to 6\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. Each nude mouse was injected subcutaneously with 200 \u0026micro;L of the cell suspension, with 7 mice per group. The nude mice were weighed every four days, and the length and width of tumors were accurately measured with Vernier calipers when obvious axillary tumors appeared. The tumor volume was calculated according to the formula (length\u0026times; width \u003csup\u003e2\u003c/sup\u003e) / 2. Beginning on the 20th day after cell inoculation, the nude mice were intraperitoneally injected with GEM (MedChemExpress, NJ, USA) (25 mg/kg, 50 mg/kg) every Monday and Thursday. On the 44th day, all mice in the experiment were euthanized by cervical dislocation. The tumors were dissected, weighed and photographed, and the tumor inhibition rate was calculated. Some tumor tissues were soaked in 4% paraformaldehyde for immunohistochemical analysis. The antibodies used were as follows: rabbit anti-ROCK1, rabbit anti-p-c-MYC (Ser 62) (ZEN-BIOSCIENCE), and rabbit anti-PFKFB3 (Beijing Solarbio Science \u0026amp; Technology, Beijing, China) (1:50).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of ROCK1 is associated with poor survival in pancreatic cancer patients\u003c/h2\u003e \u003cp\u003eThe expression level of ROCK1 in PC was investigated with the GEPIA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The mRNA level of ROCK1 was abnormally high in the tissues of PC patients (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The protein expression level of ROCK1 in PC tissues and adjacent tissues was further analyzed by immunohistochemical staining of clinical samples, and the results were consistent with those of the database analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Then, the GEPIA database was utilized to predict the relationship between the ROCK1 expression level and overall survival rate of patients with PC. The ROCK1 expression level was found to be closely related to the prognosis of patients. As the ROCK1 expression level increased, the overall survival rate of patients with PC decreased significantly (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.025, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating poor prognosis. Then, the expression of ROCK1 in six PC cell lines was analyzed by qRT‒PCR. Compared with those in HPDE6-C7 normal pancreatic duct epithelial cells, ROCK1 mRNA and protein levels were significantly higher in AsPC-1 and PANC-1 cells and lower in Capan-1, MIAPaCa-2, BxPC3 and SW1990 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). The above results indicated that the expression of ROCK1 differed among PC cell lines. Although more PC cells in the screen showed a trend of low ROCK1 expression, AsPC-1 and PANC-1 cells are derived from a primary pancreatic head tumor and an in situ pancreatic head tumor, respectively. These cells have a greater metastatic ability. Therefore, they still have research value and significance. Since the subcellular localization of the ROCK1 protein is closely related to its function, the specific localization of ROCK1 in PC cells was explored. Due to the relatively high expression levels of ROCK1 in AsPC-1 and PANC-1 cells, these two cell lines were selected for immunofluorescence staining. ROCK1 was concentrated in the cytoplasm of AsPC-1 and PANC-1 cells, and only a small amount was distributed in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The above results suggest that abnormally high expression of ROCK1 is negatively correlated with the overall survival rate of patients with PC. Thus, ROCK1 has obvious prognostic value and could be considered an oncogenic factor for further study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSilencing ROCK1 inhibits the proliferation and migration of pancreatic cancer cells and promotes apoptosis\u003c/h2\u003e \u003cp\u003eTo confirm the hypothesis that ROCK1 plays a role as an oncogenic factor in the progression of PC, two cell lines with high expression of ROCK1, AsPC-1 and PANC-1, were selected to construct a ROCK1 silencing model. Three siRNA-ROCK1 sequences were designed and their silencing efficiency was compared, and the ROCK1-Homo-1410 plasmid (si-ROCK1-2) was found to have the best ROCK1 silencing efficiency in two PC cell lines. The qRT‒PCR results showed that the silencing efficiency in AsPC-1 and PANC-1 cells was 89.1% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and 61.8% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), respectively. Western blotting was subsequently employed to further confirm the silencing effect. It Again, the plasmid ROCK1-Homo-1410 had the best silencing efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D); thus, this plasmid was selected to construct the ROCK1 silencing model in subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, the effect of ROCK1 silencing on the biological behavior of PC cells, including cell proliferation, apoptosis and migration, was evaluated. First, the effect of ROCK1 on the proliferation of PC cells was analyzed. After transfection with the ROCK1-Homo-1410 plasmid, the cells were cultured for 6, 24, 48 and 72 h, and proliferation was evaluated by an MTT assay. The growth activity of AsPC-1 and PANC-1 cells was significantly inhibited after culture for 72 h, with inhibition rates of up to 29.8% and 9.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F), suggesting that ROCK1 silencing can effectively suppress the proliferation of these two PC cell lines, with a more obvious effect on AsPC-1 cells.\u003c/p\u003e \u003cp\u003eThe impact of ROCK1 on PC cell colony formation was evaluated by a colony formation assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H, after silencing ROCK1, the colony formation of AsPC-1 and PANC-1 cells was significantly inhibited, with inhibition rates of 51.3% and 15.8%, respectively. The function of ROCK1 in PC cell apoptosis was further explored by flow cytometry. The ROCK1 silencing promoted the apoptosis of AsPC-1 and PANC-1 cells, with a more obvious effect on AsPC-1 cells was more obvious. Relative to the control group, after silencing of ROCK1, the total apoptosis rate in AsPC-1 cells was increased (1.1% \u003cem\u003evs.\u003c/em\u003e 25.0%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), and that of PANC-1 cells was increased (8.0% \u003cem\u003evs.\u003c/em\u003e 10.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). A Transwell assay was employed to investigate the effect of ROCK1 on the migration of two PC cell lines. After silencing ROCK1, cells were cultured for 24 h in a Transwell chamber to evaluate cell migration. The migration of AsPC-1 and PANC-1 cells was significantly suppressed by ROCK1 silencing, with inhibition rates of 61.6% and 37.9%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). These results suggest that ROCK1 plays a positive regulatory role in the occurrence and development of PC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eROCK1 enhances glycolytic activity in pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eTumor cells utilize glycolysis as their main energy source even under sufficient oxygen, a phenomenon called the \u0026ldquo;Warburg effect\u0026rdquo;. The cytoplasm is the main site of glycolysis in tumor cells. Therefore, we hypothesized that the ROCK1 enriched in the cytoplasm was related to glycolysis in tumor cells and played a role as an oncogenic factor by promoting glycolytic activity. The effects of ROCK1 on glucose uptake and lactate release in PC cells were examined. The glucose intake and lactic acid release of AsPC-1 and PANC-1 cells were significantly reduced after ROCK1 silencing for 48 h. Relative to the control group, the glucose uptake rate of AsPC-1 cells was decreased (21.3% \u003cem\u003evs.\u003c/em\u003e 13.4%) after silencing of ROCK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The glucose uptake rate of PANC-1 cells was also decreased relative to that in the control group (12.7% \u003cem\u003evs.\u003c/em\u003e 9.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). After silencing ROCK1, the lactate release rate of AsPC-1 and PANC-1 cells was decreased by 31.0% and 25.8%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These results indicated that ROCK1 can promote glycolytic activity in PC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the mechanism by which ROCK1 promotes glycolysis in PC cells, changes in the expression of related genes, including PKM2, HK2, LDHA, PFKFB3 and the related transcription factors HIF-1α and c-MYC, were detected by qRT‒PCR after ROCK1 silencing. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, HK2, LDHA, PFKFB3 and c-MYC were downregulated in both AsPC-1 and PANC-1 PC cells after ROCK1 silencing. Among these genes, PFKFB3 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and c-MYC (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) exhibited significantly downregulated mRNA expression in both PC cell lines. Further analysis using the GEPIA database showed that ROCK1 gene expression was significantly positively correlated with the mRNA expression of PFKFB3 and c-MYC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). The above data suggest that ROCK1 can promote glycolytic activity in PC cells by increasing the mRNA expression levels of PFKFB3, a key enzyme promoting glycolysis, and c-MYC, a transcription factor associated with glycolysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eROCK1 directly binds to c-MYC and promotes the phosphorylation of c-MYC (Ser 62) but does not directly affect the kinase activity of PFKFB3\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter the correlations of the ROCK1 mRNA level with the c-MYC and PFKFB3 mRNA levels were confirmed, the influence of ROCK1 silencing on PFKFB3 and c-MYC protein levels was further evaluated. Western blotting demonstrated that the total c-MYC protein levels are downregulated in both AsPC-1 and panc-1 cells. after silencing of ROCK1. Then, analysis of phosphorylated c-MYC (Ser 62) showed that after ROCK1 silencing, the phosphorylated c-MYC (Ser 62) protein level and the PFKFB3 protein level were significantly decreased in both PC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Since ROCK1 itself acts as a kinase, it was speculated that its enzymatic activity may also affect the expression of downstream proteins. In this study, the ROCK1 inhibitor GSK429286A was applied to treat the two PC cell lines. When the GSK429286A concentration was 20 \u0026micro;M, the protein levels of phosphorylated c-MYC (Ser 62) and PFKFB3 were effectively decreased in both PC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). These results demonstrated that both the high protein expression and the increase in the enzymatic activity of ROCK1 promoted the protein expression of the key glycolytic enzyme PFKFB3 and the phosphorylation of the transcription factor c-MYC (Ser 62) in PC cells, thus promoting glycolytic activity in PC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the kinase activity of ROCK1, we speculated that the increase in this kinase activity may enhance the enzymatic activity of the downstream enzyme PFKFB3, thus promoting tumor glycolysis. Then, the two PC cell lines were treated with 10 \u0026micro;M GSK429286A to evaluate the kinase activity of PFKFB3. No significant difference in intracellular PFKFB3 kinase activity was observed between AsPC-1 cells and the control group after 12 h of treatment. Although intracellular PFKFB3 kinase activity was decreased after 24 h of treatment, it was slightly increased after 48 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Similarly, the intracellular kinase activity of PFKFB3 was increased in PANC-1 cells after 12 h and 48 h of treatment with the ROCK1 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results suggested that the decrease in ROCK1 kinase activity itself did not directly lead to the decrease in downstream PFKFB3 kinase activity. Since ROCK1 had no direct effect on the kinase activity of PFKFB3, we aimed to determine whether there was direct binding between them at the protein level. If not, does the transcription factor c-MYC act as the bridge between these proteins? The protein interaction between ROCK1 and PFKFB3 was predicted by the protein interaction database STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results indicated that there was no direct binding between the ROCK1 and PFKFB3 proteins. However, direct binding interactions might occur between the ROCK1 and c-MYC proteins and between the c-MYC and PFKFB3 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). A co-IP assay was applied to verify these predictions in AsPC-1 cells. No direct binding between the ROCK1 and PFKFB3 proteins was observed, but a direct and strong binding interaction between the ROCK1 and c-MYC proteins was found. In addition, the results of the IF assay showed that c-MYC colocalized with ROCK1 in the cytoplasm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eROCK1 promotes glycolytic activity in pancreatic cancer cells through the c-MYC/PFKFB3 signaling axis\u003c/h2\u003e \u003cp\u003eIt has been reported that phosphorylation of c-MYC (Ser 62) inhibits its degradation by the ubiquitin‒proteasome pathway, thereby increasing the stability of the c-MYC protein. Thus, it was speculated that ROCK1 can enhance the stability of c-MYC, thus promoting the transcription of downstream target genes. AsPC-1 cells were transfected with the ROCK1 silencing plasmid, cultured for 48 h, and then treated with 10 \u0026micro;M MG-132 (a proteasome inhibitor) for 4 h. MG132 treatment significantly increased the accumulation of c-MYC protein compared with that in the untreated siNC group, indicating that c-MYC was indeed degraded through the proteasome pathway. However, when ROCK1 was silenced, MG132 treatment still reduced the accumulation of c-MYC protein compared with that in the untreated siNC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, CHX (10 mg/mL, a protein synthesis inhibitor) was used to treat AsPC-1 cells with ROCK1 silencing, and samples were collected for Western blot analysis after 2 h, 4 h and 8 h of treatment. The experimental results suggested that the c-MYC protein began to degrade significantly at the 4th hour in the control group but was obviously degraded at the 2nd hour after ROCK1 silencing, further demonstrating ROCK1\u0026rsquo;s stabilizing and protective effect on the c-MYC protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The above results suggest that ROCK1 binds to the c-MYC protein in the cytoplasm to promote the phosphorylation of c-MYC (Ser 62), thereby enhancing its stability and inhibiting its degradation by the proteasome pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relationship between c-MYC and the downstream enzyme PFKFB3 was further explored. GEPIA database analysis showed a significant positive correlation between the expression of these two genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Because c-MYC can promote the transcription of its downstream genes, the possible binding site of c-MYC to the promoter region of the PFKFB3-encoding gene was further predicted by the JASPAR and NCBI databases (Relative score\u0026thinsp;=\u0026thinsp;0.85568): GGGCATGTGCTC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This relationship was verified by a luciferase reporter assay. After transfection of corresponding plasmids into HEK293T cells, the luciferase activity of the group transfected with the pGL3-basic-PFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-c-MYC plasmids was significantly increased (1.5-fold) compared with that in the control group transfected with the pGL3-basic-PFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-NC plasmids. In cells transfected with pGL3-basic-mutPFKFB3 pro (-2000 to -1800) and pcDNA3.1(+)-c-MYC, the luciferase activity was decreased significantly compared with that in the experimental group. These results indicated that c-MYC bound to the promoter of the PFKFB3-encoding gene and promoted the transcription of this gene. Further validation was carried out in two PC cell lines, AsPC-1 and PANC-1. With an increase in the quantity of pcDNA3.1(+)-c-MYC plasmid transfected, there was a dose-dependent increase in luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results indicated that c-MYC can promote the transcription of the PFKFB3-encoding gene in a dose-dependent manner, thus promoting the protein expression of PFKFB3. To further explain the relationship among ROCK1, c-MYC and PFKFB3, the c-MYC inhibitor 10058-F4 was applied to treat AsPC-1 and PANC-1 cells after silencing ROCK1 or while treating them with the ROCK1 inhibitor GSK429286A. Treatment with 10058-F4 alone did not seem to effectively inhibit the protein expression of PFKFB3. However, when ROCK silencing or ROCK1 inhibitor treatment was combined with 10058-F4 treatment, the inhibitory effect on PFKFB3 was excellent, much better than the single effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-I). Collectively, these results suggest that ROCK1 binds to the c-MYC protein in the cytoplasm and promotes the phosphorylation of c-MYC (Ser 62), thereby enhancing the stability of the c-MYC protein and inhibiting its degradation. C-MYC could bind to the promoter of the PFKFB3-encoding gene to promote its mRNA transcription and subsequent protein expression, thus promoting tumor glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAsPC-1 tumor growth is effectively inhibited and GEM sensitivity is increased after ROCK1-silencing in nude mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether ROCK1 also has carcinogenic activity in vivo, the silencing efficiency of shRNA was first verified by Western blotting. The protein expression of ROCK1 was significantly inhibited after transduction with the shROCK1 lentiviral plasmid, indicating successful transduction of the shROCK 1 silencing plasmid. We then investigated whether ROCK1 deficiency enhanced the sensitivity of pancreatic cancer cells to GEM. AsPC-1 cells were transduced with shROCK1 and shNC lentiviral plasmids, plated and cultured with a concentration gradient of GEM (0, 1, 2.5, 5, 7.5 \u0026micro;M) for 48 h. Cytotoxicity was then detected by a MTT assay. Silencing ROCK1 significantly increased the sensitivity of AsPC-1 cells to GEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Then, the xenograft tumors in nude mice were analyzed. AsPC-1 cells transduced with shNC and shROCK1 were inoculated into nude mice (approximately 5 weeks old) under the left axilla, and the mice were weighed every four days. On day 44, the mean tumor volume was 181.2 mm in the shROCK1 group compared to 281.9 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e in the shNC group. Thus, silencing ROCK1 significantly suppressed the increase in the tumor volume in nude mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with that in the shNC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). These data suggest that silencing ROCK1 can effectively inhibit tumor growth in tumor-bearing mice. In vitro experiments have shown that the protein levels of ROCK1, p-c-MYC (Ser 62), and PFKFB3 are positively correlated; thus, we aimed to further validate the above results through in vivo experiments. Immunohistochemical analysis of different tumor tissues showed that the p-c-MYC (Ser 62) and PFKFB3 protein levels were also relatively low in tissue samples with low ROCK1 expression. Conversely, tumor tissues with high ROCK1 expression also exhibited increased protein levels of p-c-MYC (Ser 62) and PFKFB3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), consistent with the results of the in vitro experiments. Then, GEM sensitivity was analyzed in vivo. According to the above subcutaneous xenograft method, beginning when subcutaneous tumors began to form in nude mice on the 20th day, GEM (25 mg/kg, 50 mg/kg) was injected intraperitoneally every Monday and Thursday. The nude mice were sacrificed after 44 days, and the xenograft tumors were removed. The tumor volume in the shROCK1 group was significantly lower than that in the shNC group, indicating that the sensitivity of xenograft tumors to GEM (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was significantly increased after silencing of ROCK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent studies have shown that ROCK dysregulation is involved in the metastasis and progression of various malignant tumors. ROCK1 shows high kinase activity and protein expression levels during breast cancer metastasis and advanced breast cancer development, and a similar phenomenon has been observed in colorectal cancer \u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the relationship between ROCK1 expression and PC activity is unclear. In this study, we show that ROCK1 enhances the stability of c-MYC by promoting its phosphorylation (at Ser 62) and inhibiting its degradation by the ubiquitin‒proteasome pathway, thus promoting the transcription of the downstream key glycolytic enzyme PFKFB3 at the mRNA level and subsequently increasing its protein expression, thereby increasing glycolytic activity in PC cells. These results define a theoretical basis for finding important PC treatment strategies.\u003c/p\u003e \u003cp\u003eOur study revealed that ROCK1 was highly expressed in PC and associated with poor prognosis in patients. The GEPIA database and immunohistochemical analyses showed that the expression of ROCK1 was abnormally high in the tissues of PC patients. High expression of ROCK1 was found to be significantly negatively correlated with the prognosis of patients, indicating that ROCK1 is a key oncogenic factor in the development of PC. The qRT‒PCR results further confirmed that ROCK1 mRNA showed significantly high expression in AsPC-1 and PANC-1 cells. The other four PC cell lines showed low expression of ROCK1, indicating that ROCK1 expression differed among cell lines, an effect that may be related to cell genotype. The genes that regulate this phenomenon may need to be further verified by gene mutation or deletion assays. The immunofluorescence results showed that ROCK1 was distributed mainly in the cytoplasm, which is the main site of glycolysis.\u003c/p\u003e \u003cp\u003eTo verify the biological function of ROCK1 in the growth of PC cells, we evaluated the effects of ROCK1 expression changes on cell proliferation, migration and apoptosis. ROCK1 silencing significantly inhibited the growth of the two tested PC cell lines and effectively suppressed colony formation. After silencing ROCK1, the colony formation of AsPC-1 and PANC-1 cells was significantly inhibited, with inhibition rates of 51.3% and 15.8%, respectively. The flow cytometry results showed that ROCK1 silencing promoted the apoptosis of AsPC-1 and PANC-1 cells. The Transwell assay indicated that silencing ROCK1 significantly inhibited the migration of PC cells, with inhibition rates of 61.6% and 37.9% in AsPC-1 and PANC-1 cells, respectively. These results suggest that ROCK1 plays a role as an oncogenic factor in the development and progression of PC.\u003c/p\u003e \u003cp\u003eFurthermore, the mechanism by which ROCK1 promotes PC cell activity was explored. Since cancer cells utilize glycolysis as the main energy source for their rapid proliferation even under oxygen-rich conditions, the cytoplasm is the main site of glycolysis \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and it was speculated that ROCK1 may play a role as an oncogenic factor by affecting glycolysis in PC cells. The glucose uptake and lactic acid release assays showed that ROCK1 silencing significantly inhibited glucose uptake and lactic acid release in the two tested PC cell lines, indicating that ROCK1 can promote glycolytic activity in PC cells. The mRNA expression of glycolysis-related genes after silencing ROCK1 was further evaluated, and PFKFB3 and c-MYC mRNA expression was obviously downregulated in both tested PC cell lines. Studies have shown that cancer cells adapt to stressful conditions and continue to proliferate rapidly, in part because the actions of PFKFB3 are diverse and reversible \u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Moreover, the c-MYC protein has been reported to be involved in the regulation of tumor cell growth, cell cycle progression, metabolism, and angiogenesis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Western blot analysis showed that both silencing of ROCK1 and treatment with the ROCK1 inhibitor GSK429286A significantly reduced the protein levels of p-c-MYC (Ser 62) and PFKFB3, while ROCK1 did not directly affect the kinase activity of PFKFB3. The co-IP results showed that ROCK1 directly binds to c-MYC but not to PFKFB3. The immunofluorescence experiments also intuitively proved that ROCK1 and c-MYC were obviously colocalized in the cytoplasm. Subsequently, treatment of AsPC-1 cells with MG132 and CHX showed that ROCK1 enhanced the stability of c-MYC by promoting its phosphorylation (at Ser 62) and inhibiting its degradation. In addition, the luciferase reporter assay showed that c-MYC can promote the transcription of the PFKFB3-encoding gene in a dose-dependent manner. These results indicate that the ROCK1 protein promotes the glycolytic activity in PC cells through the c-MYC/PFKFB3 signaling axis, thereby promoting PC tumor growth.\u003c/p\u003e \u003cp\u003eFinally, our findings further verified the role and mechanism of ROCK1 in promoting the growth of PC in vivo. By establishing a nude mouse xenograft model of AsPC-1 cells, we found that silencing ROCK1 significantly reduced tumor volume and weight in nude mice. Immunohistochemical analysis of tumor tissues showed that the p-c-MYC (Ser 62) and PFKFB3 protein levels were also relatively high in tumor tissues with high ROCK1 expression and were also low in tumor tissues with low ROCK1 expression. The above results indicated that ROCK1 promoted the growth of PC in vivo, and the positive correlations of ROCK1 expression with p-c-MYC (Ser 62) and PFKFB3 expression were consistent with the results in vitro. However, the relationships between the protein levels of ROCK1, p-c-MYC (Ser 62) and PFKFB3 were verified only by immunohistochemistry at the animal level, and the changes in glycolysis in animals were not described in detail, although this will be the focus of subsequent research. Moreover, the mechanism by which ROCK1 affects enzymatic activity in the ubiquitin‒proteasome degradation pathway to stabilize c-MYC needs to be further studied.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings demonstrate that ROCK1 acts on PC cells through the c-MYC/PFKFB3 signaling axis to enhance glycolytic activity in PC cells, thereby enhancing the growth activity of PC. Moreover, knockdown of ROCK1 can also increase the sensitivity of PC cells to GEM. This paper indicates that ROCK1 is an effective therapeutic target for PC and that its positive regulation of glycolysis may provide an effective clinical therapeutic strategy for PC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the National Key Research and Development Program of China (2018YFA0902000) and the \u0026quot;Double First-Class\u0026quot; University project (CPU2022QZ09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuyang Pang: conceptualization, methodology and writing - original draft; Yuting Shen, Yanan Wang data curation, writing - reviewing and editing; Lingman Ma and Yiran Zhou supervision and funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed according to the protocols approved by the Ethics Committee of China Pharmaceutical University (approval no. SYXK 2021‑0011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eT K, LD W, T I, K T.-Pancreatic cancer. Lancet 2016;388:73\u0026ndash;85\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res 2014;74:2913\u0026ndash;2921\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eF S, F P.-Current role of endoscopic ultrasound in the diagnosis and management of. 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Semin Cancer Biol 2006;16:288\u0026ndash;302\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang C, Zhang S, Zhang Z, He J, Xu Y, Liu S. ROCK has a crucial role in regulating prostate tumor growth through interaction with c-Myc. Oncogene 2014;33:5582\u0026ndash;5591\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJozefczuk J, Adjaye J. Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol 2011;500:99\u0026ndash;109\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChelakkot C, Chelakkot VS, Shin Y, Song K. Modulating Glycolysis to Improve Cancer Therapy. Int J Mol Sci 2023;24\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJones BC, Pohlmann PR, Clarke R, Sengupta S. Treatment against glucose-dependent cancers through metabolic PFKFB3 targeting of glycolytic flux. Cancer Metastasis Rev 2022;41:447\u0026ndash;458\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBerquand A, Devy J. Multimodal Approach for Cancer Cell Investigation. Methods Mol Biol 2021;2350:289\u0026ndash;297\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXu D, Zhou Y, Xie X, et al. Inhibitory effects of canagliflozin on pancreatic cancer are mediated via the downregulation of glucose transporter\u0026ndash;1 and lactate dehydrogenase A. Int J Oncol 2020;57:1223\u0026ndash;1233\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBrzuzan P, Mazur-Marzec H, Florczyk M, et al. Luciferase reporter assay for small-molecule inhibitors of MIR92b-3p function: Screening cyanopeptolins produced by Nostoc from the Baltic Sea. Toxicol In Vitro 2020;68:104951\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXie X, Zhou Y, Wang X, et al. Enhanced antitumor activity of gemcitabine by polysaccharide-induced NK cell activation and immune cytotoxicity reduction in vitro/vivo. 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Clin Breast Cancer 2022;22:e604-e614\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLei L, Hong LL, Ling ZN, et al. A Potential Oncogenic Role for PFKFB3 Overexpression in Gastric Cancer Progression. Clin Transl Gastroenterol 2021;12:e00377\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiao DJ, Dickson RB. c-Myc in breast cancer. Endocr Relat Cancer 2000;7:143\u0026ndash;164\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDhanasekaran R, Deutzmann A, Mahauad-Fernandez WD, Hansen AS, Gouw AM, Felsher DW. The MYC oncogene-the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol 2022;19:23\u0026ndash;36\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ROCK1, c-MYC, PFKFB3, Glycolysis, Pancreatic cancer","lastPublishedDoi":"10.21203/rs.3.rs-3836816/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3836816/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. The coimmunoprecipitation assay confirmed the direct binding between ROCK1 and c-MYC, and the luciferase reporter assay clarified the binding between c-MYC and the promoter of the PFKFB3-encoding gene. These results were verified in animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased Gemcitabine (GEM) sensitivity in vivo and in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eROCK1 promotes glycolytic activity in PC cells and PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.\u003c/p\u003e","manuscriptTitle":"ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 17:05:25","doi":"10.21203/rs.3.rs-3836816/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e493d110-0f93-4312-8009-5278d52fc252","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-26T12:11:21+00:00","versionOfRecord":{"articleIdentity":"rs-3836816","link":"https://doi.org/10.1016/j.bbagen.2024.130669","journal":{"identity":"biochimica-et-biophysica-acta-bba-general-subjects","isVorOnly":true,"title":"Biochimica et Biophysica Acta (BBA) - General Subjects"},"publishedOn":"2024-07-01 12:11:21","publishedOnDateReadable":"July 1st, 2024"},"versionCreatedAt":"2024-01-08 17:05:25","video":"","vorDoi":"10.1016/j.bbagen.2024.130669","vorDoiUrl":"https://doi.org/10.1016/j.bbagen.2024.130669","workflowStages":[]},"version":"v1","identity":"rs-3836816","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3836816","identity":"rs-3836816","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}