BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment fo...BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.展开更多
With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to ...With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.展开更多
Previous studies show that actin-binding Rho activating protein （Abra） is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central ne...Previous studies show that actin-binding Rho activating protein （Abra） is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.展开更多
BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric ...BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.展开更多
AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativer...AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.展开更多
Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive ...Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.展开更多
目的:探索肝癌缺失因子1(deleted in liver cancer 1,DLC-1)对耐恩杂鲁胺前列腺癌(prostate cancer,PCa)细胞增殖,侵袭和凋亡的影响以及作用机制。方法:体外以不同浓度的恩杂鲁胺药物培养人前列腺癌lncap及C4-2细胞株6个月以上,构建恩...目的:探索肝癌缺失因子1(deleted in liver cancer 1,DLC-1)对耐恩杂鲁胺前列腺癌(prostate cancer,PCa)细胞增殖,侵袭和凋亡的影响以及作用机制。方法:体外以不同浓度的恩杂鲁胺药物培养人前列腺癌lncap及C4-2细胞株6个月以上,构建恩杂鲁胺耐药细胞系lncap-R,C4-2-R。CCK-8法检测其IC50,验证恩杂鲁胺耐药株是否构建成功。利用慢病毒转染lncap-R,C4-2-R,以敲低和过表达DLC-1,采用克隆实验、Transwell实验和流式细胞术检测DLC-1对耐恩杂鲁胺的前列腺癌细胞的增殖、侵袭和凋亡的影响。结果:与对照组相比,过表达组DLC-1显著抑制了2个耐药株的增殖和侵袭进程。机制上,WB实验结果表明DLC-1表达与2个耐药株中Rho蛋白的表达呈负相关。Rho抑制剂rhosin逆转了因敲低DLC-1后lncap-R细胞凋亡减少的情况。结论:DLC-1可以下调耐药株中的Rho蛋白的表达,并抑制耐药株的增殖和侵袭。展开更多
基金Supported by National Natural Science Foundation of China,No.82260581Guangxi Zhuang Autonomous Region Health Committee Scientific Research Project,No.Z20201147+3 种基金Guangxi Medical University Education and Teaching Reform Project,No.2021XJGA02Undergraduate Teaching Reform Project of Guangxi Higher Education,No.2023JGB163Guangxi Medical University Teacher Teaching Ability Development Project,No.2202JFA20China Undergraduate Innovation and Entrepreneurship Training Program,No.S202310598170.
文摘BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.
基金Supported by National Natural Science Foundation of China(31472260)
文摘With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.
基金supported by the National Natural Science Foundation of China,No.30971532Ph.D.Programs Foundation of Ministry of Education of China,No.20090162110063+1 种基金the Natural Science Foundation of Hunan Province,No.09JJ5015the Scientific Research Program of Hunan Provincial Higher Education Institutes,No.110541
文摘Previous studies show that actin-binding Rho activating protein （Abra） is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.
基金supported by a grant from the National Natural Science Foundation of China (30972920)
文摘BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.
文摘AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.
基金supported by the National Natural Science Foundation of China,No.31170941the Fundamental Research Funds for the Central Universities,No.21612424the Science and Technology Planning Project of Guangdong Province,No.2010B031600102
文摘Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.
文摘目的:探索肝癌缺失因子1(deleted in liver cancer 1,DLC-1)对耐恩杂鲁胺前列腺癌(prostate cancer,PCa)细胞增殖,侵袭和凋亡的影响以及作用机制。方法:体外以不同浓度的恩杂鲁胺药物培养人前列腺癌lncap及C4-2细胞株6个月以上,构建恩杂鲁胺耐药细胞系lncap-R,C4-2-R。CCK-8法检测其IC50,验证恩杂鲁胺耐药株是否构建成功。利用慢病毒转染lncap-R,C4-2-R,以敲低和过表达DLC-1,采用克隆实验、Transwell实验和流式细胞术检测DLC-1对耐恩杂鲁胺的前列腺癌细胞的增殖、侵袭和凋亡的影响。结果:与对照组相比,过表达组DLC-1显著抑制了2个耐药株的增殖和侵袭进程。机制上,WB实验结果表明DLC-1表达与2个耐药株中Rho蛋白的表达呈负相关。Rho抑制剂rhosin逆转了因敲低DLC-1后lncap-R细胞凋亡减少的情况。结论:DLC-1可以下调耐药株中的Rho蛋白的表达,并抑制耐药株的增殖和侵袭。
