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Aim To investigate the effect of Sijunzi Decoction on mRNA and protein expression related to growth and cell cycle in polyamine/HuR signaling pathway during small intestinal epithelial cell (IEC-6) proliferation, and to explore its mechanism on intestinal mucosal injury repair. Methods Sijunzi Decoction-containing serum (SJZD) was prepared from SD rats, the expression of HuR protein in cytoplasm and nucleus was analyzed by immunofluorescence and Western blot, the mRNA level of activating transcription factor-2 (A T F - 2), JunD and cyclin dependent kinase 4 (CDK4) were determined by real-time fluorescent quantitative PCR (RT-PCR), Western blot was used to detect protein level of HuR, ATF-2, JunD and CDK4, and flow cytometry was applied to analyse cell cycle distribution. Results Compared with the control group, the mRNA and protein expression of ATF-2 and JunD decreased, while the expression of Cdk4 mRNA and protein increased in SZJD group, and the proportion of G
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OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.
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Humans , Hepatic Stellate Cells/radiation effects , Interleukin-6 , NF-kappa B , NF-KappaB Inhibitor alpha , Phenotype , RNA , RNA, Circular/metabolism , Tumor Necrosis Factor-alpha , ELAV-Like Protein 1/metabolismABSTRACT
Mevalonate metabolism plays an important role in regulating tumor growth and progression; however, its role in immune evasion and immune checkpoint modulation remains unclear. Here, we found that non-small cell lung cancer (NSCLC) patients with higher plasma mevalonate response better to anti-PD-(L)1 therapy, as indicated by prolonged progression-free survival and overall survival. Plasma mevalonate levels were positively correlated with programmed death ligand-1 (PD-L1) expression in tumor tissues. In NSCLC cell lines and patient-derived cells, supplementation of mevalonate significantly up-regulated the expression of PD-L1, whereas deprivation of mevalonate reduced PD-L1 expression. Mevalonate increased CD274 mRNA level but did not affect CD274 transcription. Further, we confirmed that mevalonate improved CD274 mRNA stability. Mevalonate promoted the affinity of the AU-rich element-binding protein HuR to the 3'-UTR regions of CD274 mRNA and thereby stabilized CD274 mRNA. By in vivo study, we further confirmed that mevalonate addition enhanced the anti-tumor effect of anti-PD-L1, increased the infiltration of CD8+ T cells, and improved cytotoxic function of T cells. Collectively, our findings discovered plasma mevalonate levels positively correlated with the therapeutic efficacy of anti-PD-(L)1 antibody, and provided the evidence that mevalonate supplementation could be an immunosensitizer in NSCLC.
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Objective:To evaluate the effect of upregulation of HuR gene on the radiosensitivity of esophageal squamous cell carcinoma cell Kyse450. Methods:The HuR gene of Kyse450 cells was upregulated by lentivirus. At the same time, X-ray irradiation at a dose of 6 Gy was selected as the intervention condition. Western blot and qPCR were used to detect the expression levels of protein and RNA after Kyse450 transfection, respectively. CCK8 kit was employed to determine the cell proliferation rate. Clone formation assay was adopted to evaluate the ability of cell clone formation. Wound healing experiment and the Transwell test were performed to detect changes in cell migration. Results:CCK8 assay showed that the proliferation ability of cells was enhanced after upregulation of HuR gene, and this enhancement trend was more obvious after radiation. The plate cloning experiment showed that with the increase of radiation dose, the clone formation rates were decreased in both groups, but the clone formation rate in the overexpression group was higher than that in the control group. Wound healing experiment and Transwell test demonstrated that the wound healing rate and migration ability in the overexpression group were higher than those in the control group, and the difference was more significant after radiotherapy. Western blot showed that the levels of MMP9 and MMP2 at 24 h after radiotherapy in the overexpression group were higher than those in the control group. Conclusion:The upregulation of HuR can enhance the proliferation, cloning, migration capabilities and decrease the radiosensitivity of esophageal squamous cell carcinoma cells.
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Objective:To investigate the protective effect of glycyrrhizic acid (GA) from tumor necrosis factor-α+actinomycetes ketone （TNF-α+CHX） induced apoptosis in rat small intestine crypt epithelial cell line (IEC-6) via AU rich element mRNA binding protein HuR mediated posttranscription of p21 and the potential mechanism. Method:The cultured IEC-6 cells were observed. The experiment was divided into blank group, GA (60 μmol·L-1) group, TNF-α+CHX group and GA+TNF-α+CHX group. Cytoplasmic and nuclear HuR were measured by Western blot. The interaction of HuR and p21 mRNA was detected by biotin pull down and RNA IP. Luciferase activity was measured after transfection with construct with p21 3'-UTR cloned into downstream of luciferase reporter. Cell apoptosis was detected by real-time dynamic cell analyzer, p21 and cysteine proteinas-3 precursor protein（proCaspase-3） association was analysised by CO-IP. Result:After GA treatment for 48 h, cytoplasmic HuR protein expression increased（P<0.05）,the binding between HuR and p21 mRNA expression up regulated(P<0.05), luciferase activity increased(P<0.01), and p21 mRNA and protein expression also increased(P<0.05), while these results were abolished by HuR silencing with siRNA. GA enhanced p21 and procaspase3 interaction(P<0.05), and attenuated TNF-α+CHX induced apoptosis in IEC-6 cells. Conclusion:GA protected IEC-6 cells from TNF-α/CHX induced apoptosis via HuR mediated p21 posttranscription, which due to GA enhanced HuR binding to endogenous and recombinant p21 mRNA and increased p21 interaction with proCaspase3.
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Objective To compare the actions of Longbie Capsules and bone marrow mesenchymal stem cell (BMSC)transplantation in repairing the damaged cartilage of knee osteoarthritis(KOA)rats. Methods Thirty-six rats aged 4-6 weeks old were induced into KOA model(bilateral knees)by collagenase injection method. All of the modeled rats were randomly divided into model group(intragastric administration of normal saline), BMSC transplantation group(giving tail vein injection of 1 ×106 of BMSCs per time, 2 times every week), and Chinese medicine group (intragastric administration of Longbie Capsules of 7.5 g·kg-1·d-1),12 rats in each group. Six weeks later,the cartilage of rat bilateral knees was taken out. The pathological changes of cartilage were observed by hematoxylin-eosin(HE) staining method, and the protein and mRNA expression levels of Col2a1, X-linkedinhibitor of apoptosis protein (XIAP), HuR in rat knee cartilage were detected by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR), respectively. Results The HE staining results showed that the cartilage tissue surface was rough with more cracks, and the cartilage cells gathered with the cytoplasm collapsed and arranging disorderly in the model group. The number of chondrocytes was increased and cell surface was flat,and the cracks of the cartilage were decreased with the chondrocytes arranging uniformly in Chinese medicine group and BMSC transplantation group compared with the model group. The results of immunohistochemistry and qPCR detection showed that in Chinese medicine dosage group and BMSC transplantation group, the protein and mRNA expression levels of Col2a1,XIAP and HuR were significantly higher than those in the model group (P<0.05 or P<0.0001), but there was no significant difference between the two medication groups(P>0.05). Conclusion Longbie Capsules and BMSC transplantation can promote the secretion of Col2a1 in the cartilage tissue of KOA rats,improve cartilage, and their mechanism may be related with up-regulating apoptosis-related proteins HuR and XIAP.
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Objective To study the expression and roles of miR-31-5p in acute myeloid leukemia (AML).Methods miR-31-5p in AML patients were evaluated by real-time PCR;THP-1 cells were transfected with the miR-31-5p mimic and control, respectively.The effects of over-expression of miR-31-5p were examined by CCK-8 and FACS analysis;Dual-luciferase and Western blot were performed to detect target gene HuR expression.The effects of knock-down of HUR were also examined by CCK-8 and FACS analysis.Results miR-31-5p was down-regulated in AML patients compared to the normal control.Over-expression of miR-31-5p in THP-1 cells reduces cell proliferation and accelerates monocytic differentiation.miR-31-5p could target HuR, and knock-down of HuR inhibits cell proliferation and attenuates monocytic differentiation in THP-1 cells.Conclusions miR-31-5p may regulate AML cell proliferation and monocytic differentiation by targeting HuR.
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Objective:To investigate the molecular mechanism of PDGFC regulated by HuR in breast cancer cells.Methods:Through the software analysises,we first predicted the HuR-binding sites on the PDGFC 3'-UTR in breast cancer cells;An RNA-immunoprecipitation tested the interaction of HuR with the PDGFC mRN after adding PDGFC stimulation;Luciferase experiments tested the HuR-binding sites of PDGFC regulated by HuR through structuring five truncated of PDGFC 3'UTR.Results:We found five HuR-binding sites in the 3'UTR of PDGFC by software prediction;The RNA-immunoprecipitation showed the co-immunoprecipitation of HuR and PDGFC mRN after adding PDGFC stimulation confirming the direct association of HuR with the PDGFC;Luciferase experiments ofPDGFC mRNA 3'UTR showed that PDGFC regulated by the second and fourth HuR-binding sites.Conclusions:This study reveals the molecular mechanism of PDGFC regulated by HuR through binding to PDGFC mRNA 3'UTR in breast cancer cells,and provided rationale for the development of strategies in the clinical diagnosis and treatment for breast cancer.
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BACKGROUND: Nodular fasciitis is the most common reactive mesenchymal lesion to be misidentified as a type of sarcoma. HuR is an mRNA-binding protein that can stabilize cyclooxygenase-2 (COX-2) mRNA leading to COX-2 overexpression. The aim of this study is a comparison of the expressions of COX-2 and HuR and the relationships between their expressions and the clinicopathological parameters in nodular fasciitis and low-grade sarcoma. METHODS: We measured the expression of HuR and COX-2 in 21 cases of nodular fasciitis and 37 cases of low-grade sarcoma using immunohistochemistry. RESULTS: The frequency of cytoplasmic immunoreactivity for HuR was 5 of 21 cases of nodular fasciitis (23.8%) and 23 of 37 cases of low-grade sarcoma (62.1%) (p=.013). COX-2 expression was moderate or strong in nodular fasciitis (12/21, 57.1%) and in low-grade sarcoma (29/37, 78.4%) (p=.034). In addition, a significant difference existed between these two entities in terms of the relationship between moderate or strong COX-2 expression and HuR cytoplasmic immunoreactivity (p=.009). Moderate or strong COX-2 immunoreactivity correlated with nuclear (p=.016) or cytoplasmic HuR (p=.024) expression in low-grade sarcoma but not in nodular fasciitis. CONCLUSIONS: This study suggests that HuR and COX-2 expression may be useful to differentiate nodular fasciitis from low-grade sarcoma.
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Cyclooxygenase 2 , Cytoplasm , Fasciitis , Immunohistochemistry , RNA, Messenger , SarcomaABSTRACT
Objective To detect the expressions of HuR and COX-2 in epithelial ovarian carcinoma,and investigate the correlation of HuR and COX-2 expression. In addition, we attempt to seek the pathway to prevent the occurrence and development of epithelial ovarian cancer by combined analysis of various clinicopathologic characteristics. Methods The expressions of HuR and COX-2 in 68 epithelial ovarian carcinoma, 10 borderline ovarian tumors and 5 normal ovarian tissues were examined by S-P immunohistochemical method. The relationship of HuR and COX-2 expressions with clinicopathologic parameterwere evaluated by correlation analysis. Results(1) The expression of HuR in epithelial ovarian cancer tissue (76. 47% ,52/68) was significantly higher than that in borderline epithelial ovarian tumor tissues (30. 00% ,3/10) and normal ovarian tissues (0, x2 = 18. 873, Ps ＜ 0. 05), but there were no significant differences betweenthe expressions of HuR in borderline epithelial ovarian tumor and normal ovarian tissue(P ＞ 0. 05).(2) The positive expression rate of cytoplasmic HuR in epithelial ovarian carcinoma and borderline epithelial ovarian tumor were 45.60% (31/68) and 10. 00% (1/10) respectively,but normal ovarian tissues showed no staining of HuR. We found no significant differences between the expression of cytoplasmic HuR in epithelial ovarian carcinoma and borderline epithelial ovarian tumor or normal ovarian tissue(x2 = 7. 999 ,P =0. 018).(3) The positive expression rate of cytoplasmic HuR in epithelial ovarian carcinoma of FIGO stage Ⅲ - Ⅳ was significantly higher than that of stage Ⅰ - Ⅱ(56. 09% vs. 29. 63%, x2 = 4. 598, P = 0. 032). The positive expression rates of cytoplasmic HuR in epithelial ovarian carcinoma of histological grade 1,2,3 were 10. 00%,46. 67% ,57. 14% respectively, which showed significant difference in the comparison among the three groups (x2 =6. 627 ,P =0. 036). (4) The positive expression rates of COX-2 in epithelial ovarian cancer (67. 64%)and borderline epithelial ovarian tumor tissues (60. 00%) were significantly higher than that in normal ovarian tissues (0, Ps ＜ 0. 05), but we found no significant difference in the comparison between the expression of malignant and borderline ovarian tumors. Statistical analysis showed that the positive expression rate of COX-2 in epithelial ovarian carcinoma was correlated with FIGO stage and lymph node metastasis. (5)There was a significantly positive correlation between cytoplasmic HuR and COX-2 expressions in epithelial ovarian carcinoma. Conclusion The expressions of HuR and COX-2 increased in the epithelial ovarian carcinoma, and the cytoplasmic expression of HuR was significantly correlated with the expression of COX-2. These results suggested that increased cytoplasmic expression of HuR and COX-2 expression might play important roles in the initiation and development of epithelial ovarian carcinoma.
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Objective To study human lung adenocarcinoma cell line A-549 treated with antagonist and agonist of potassium channel how to affect metastasis of A-549 and its mechanism. Methods Invasion and migration capability of A-549 in vitro was evaluated by using transwell chamber model. Alteration of cytoskeleton was observed through immunofluorescence. Western blotting were used to detect the protein expression of Ezrin and HuR in A-549 cell lines while Glibenclamde and Pinacidil were applied to them. Results In the presence of the antagonist Glibenclamide, migration of A-549 was inhibited by (57.18±5.46)% and invasion was inhibited by (54.92±3.72)% in the transwell assay, meanwhile A-549 manifested disorder of microtubule and more orderly microfilament. And agonist of the potassium channel had an contrary effect on A-549. Ezrin and HuR protein were successfully down-regulated in A-549 treated with Glibenclamide and upregulated in A-549 treated with pinacidil. Conclusion Functional alterations of the potassium channel affects capability of migration and invision of A-549, which is associated with different expression of ezrin and HuR protein that modify cytoskeleton.
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PURPOSE: HuR, human family embryonic-lethal abnormal vision-like protein, can bind to mRNA and stabilizes the nucleic acid in the cytoplasm, resulting in more efficient translation. HuR is predominantly present in the nucleus and shuttles between the nucleus and cytoplasm. HuR stabilizes cyclooxygenase-2 (Cox-2) mRNA in several cancers, including breast, stomach, lung and brain cancer. MATERIALS AND METHODS: We investigated the expression and cellular location of HuR, as well as evaluated Cox-2 expression in 79 colorectal cancer patients with the use of immunohistochemical methods. The biological implications of HuR localization and Cox-2 expression in colorectal carcinoma were evaluated. RESULTS: Nuclear HuR expression was observed in 59 (74.7%) tumors and cytoplasmic HuR expression was seen in 25 (31.6%) tumors. Cox-2 immunoreactivity was noted in 42 (53%) tumors. The expression of cytoplasmic HuR was significantly associated with Cox-2 expression (p=0.004). Cytoplasmic expression of HuR showed a correlation with lymphatic invasion (p=0.025) and the presence of a lymph node metastasis (p=0.027). The presence of nuclear HuR showed no correlation with Cox-2 expression or any other of the clinicopathological parameters that were examined. CONCLUSION: These results suggest that cytoplasmic translocation of HuR is associated with Cox-2 expression for some colorectal carcinomas.
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Humans , Brain Neoplasms , Breast , Colon , Colonic Neoplasms , Colorectal Neoplasms , Cyclooxygenase 2 , Cytoplasm , Lung , Lymph Nodes , Neoplasm Metastasis , RNA, Messenger , StomachABSTRACT
BACKGROUND: Embryonic lethal abnormal vision (ELAV)-like protein HuR is known to stabilize mRNA through binding AU-rich elements in the 3'-untranslated region. Recent studies show that HuR expression is associated with the expression of several genes including cyclooxygenase-2 (COX-2). HuR exists predominantly in the nucleus, but cytoplasmic translocation of HuR is thought to be more important for its activity. COX-2 is a well-known enzyme that promotes tumor growth. METHODS: To evaluate the correlation of HuR and COX-2 expression, we analyzed expression of HuR and COX-2 in 91 cases of breast cancer using immunohistochemistry. RESULTS: Nuclear and cytoplasmic expression of HuR was seen in 76 (83.5%) and 19 (20.9%) of 91 cases respectively. COX-2 immunoreactivity was seen in 54 (59.4%) cases. Cytoplasmic HuR expression showed significant correlation with COX-2 expression (p=0.001). Nuclear HuR showed no correlation with COX-2 expression or other clinicopathological parameters. COX-2 expression is significantly associated with tumor grade (p=0.028). COX-2 (p=0.092) and cytoplasmic (p=0.569) and nuclear HuR (p=0.247) expression showed no correlation with survival. CONCLUSIONS: These results suggest that cytoplasmic HuR expression is associated with COX-2 expression in breast cancer and cytoplasmic location of HuR might contribute to the stabilization of COX-2 mRNA.
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Breast NeoplasmsABSTRACT
PURPOSE: The purpose of this study was to identify the effects of meridian massage on menopausal symptoms and Shin-Hur in middle-aged menopausal women. METHOD: The research design was a nonequivalent control group pre-post experimental design. The subjects of the study were middle-aged women who had had no menstruation in the last 12 months after the last menstrual bleeding. Cards of invitation on bulletin boards of several apartments were placed to recruit the subjects. The cards of invitation included: purpose of the study, eligibility criteria, method and period. Eighteen women in the experimental group and 16 women in the control group were conveniently assigned, respectively. The experimental group received 20 min meridian massage 3 times per week for 4 weeks. The menopausal symptoms and Shin-hur were measured and compared between the two groups before and after the intervention. Data were analyzed with the SPSS program by Fisher's exact test, Wilcoxon Sign Rank test, Mann Whitney U-test and Spearman's rank correlation. RESULT: The experimental group showed a significant decrease of menopausal symptoms (U=77.00, p=.020) and Shin-Hur (U=76.00, p=.017). There was a significantly positive correlation between menopausal symptoms and Shin-Hur (r=.497, p=.003). CONCLUSION: Meridian massage was effective in improving menopausal symptoms and Shin-Hur in middle-aged menopausal women. Thus it can be useful as a nursing intervention for menopausal women.
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Female , Humans , Middle Aged , Abdomen , Acupressure , Acupuncture Points , Massage/methods , Meridians , Postmenopause , Women's HealthABSTRACT
BACKGROUND: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. METHODS: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. RESULTS: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-alphatreatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNA- protein complexes was mapped by 2-dimensional gel electrophoresis. CONCLUSION: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-alphaup-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.