A growing number of studies have suggested microRNAs (miRNAs) are involved in the modulation of myocardial ischemia-reperfusion (MI/R) injury; however, the role of endogenous miRNAs targeting endothelial cells (ECs) and its interaction with ICAM-1 in the setting of MI/R remain poorly understood. Our microarray results showed that miR-146a, miR-146b-5p, miR-155*, miR-155, miR-497, and miR-451 were significantly upregulated, whereas, miR-141 and miR-564 were significantly downregulated in the ECs challenged with TNF-α for 6 h. Real-time PCR analyses additionally validated that the expression levels of miR-146a, miR-155*, and miR-141 were consistent with the microarray results. Then, ICAM-1 was identified as a novel target of miR-141 by Target Scan software and the reporter gene system. Further functional experiments showed that elevated levels of miR-141 inhibited ICAM-1 expression and diminished leukocytes adhesion to ECs in vitro. In an in vivo murine model of MI/R injury, pretreatment with miR-141 mimics through the tail vein downregulated the expression level of ICAM-1 in heart and attenuated MI/R injury as evidenced by decreased infarct size and decline of serum cardial troponin I (cTnI) and lactate dehydrogenase (LDH) concentration. The cardioprotective effects of miR-141 mimics may be attributed to the decreased infiltration of CD11b(+) cells and F4/80(+) macrophages into ischemic myocardium tissue. In conclusion, our results demonstrate that miR-141, as a novel repressor of ICAM-1, is involved in the attenuation of MI/R injury via antithetical regulation of ICAM-1 and inflammatory cells infiltration. Thus miR-141 may constitute a new therapeutic target in the setting of ischemic heart disease.
BackgroundMICA/B are major ligands for NK cell activating receptor NKG2D and previous studies showed that the serum level of soluble MICA (sMICA) is an independent prognostic factor for advanced human hepatocellular carcinoma. However, the correlation between cellular MICA/B expression pattern and human hepatocellular carcinoma progression has not been well explored. The unfolded protein response is one of the main causes of resistance to chemotherapy and radiotherapy in tumor cells. However, whether the UPR in HCC could regulate the expression levels of MICA/B and affect the sensitivity of HCC cells to NK cell cytolysis has not been established yet.MethodsMICA/B expression pattern was evaluated by immunohistochemistry and Kaplan-Meier survival analysis was done to explore the relationship between MICA/B expression level and patient survival. The protein and mRNA expression levels of MICA/B in SMMC7721 and HepG2 cells treated by tunicamycin were evaluated by flow cytometry, Western Blot and RT-PCR. The cytotoxicity analysis was performed with the CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay.ResultsMICA/B was highly expressed in human hepatocellular carcinoma and the expression level was significantly and negatively associated with tumor-node metastasis (TNM) stages. Patients with low level of MICA/B expression showed a trend of shorter survival time. The unfolded protein response (UPR) downregulated the expression of MICA/B. This decreased protein expression occurred via post-transcriptional regulation and was associated with proteasomal degradation. Moreover, decreased expression level of MICA/B led to the attenuated sensitivity of human HCC to NK cell cytotoxicity.ConclusionThese new findings of the connection of MICA/B, UPR and NK cells may represent a new concrete theory of NK cell regulation in HCC, and suggest that targeting this novel NK cell-associated immune evasion pathway may be meaningful in treating patients with HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0076-7) contains supplementary material, which is available to authorized users.
Calreticulin (CRT) is an endoplasmic reticulum luminal Ca(2+)-binding chaperone protein. By immunizing mice with recombinant fragment (rCRT/39-272), six clones of monoclonal antibodies (mAbs) were generated and characterized. Based on these mAbs, a microplate chemiluminescent enzyme immunoassay (CLEIA) system with a measured limit of detection of 0.09 ng/ml was developed. Using this CLEIA system, it was found that soluble CRT (sCRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals (only 9 were detectable, P < 0.0001). Among them, serum sCRT in the small cell lung cancer was lower than that in adenocarcinoma (P = 0.0085), while both were lower than that in the squamous cell carcinoma (P = 0.013, P = 0.0012, respectively). Moreover, it was found that sCRT in sera from the patients after chemotherapy was higher than that from the patients without chemotherapy (P = 0.042). Further study by immunohistochemistry showed that CRT was also highly expressed in the cytoplasm and on the membrane of the lung cancer cells, while there was a trace amount of CRT expression in normal lung cells. Correspondingly, the expression level of CRT on lung cancer cell membrane was associated with the tumor pathological grade. This study demonstrates that sCRT concentration in sera of lung cancer patients is higher than that in sera of healthy individuals, and CRT expression level on lung cancer cell membrane is associated with tumor pathological classification and grade. These findings suggest that CRT may be used as a biomarker in lung cancer prediction and diagnosis.
NK cells play important roles in anti-tumor immunity. CD226 is a major NK-cell activating receptor, which transduces activating signals after binding ligands CD155 andCD112. Here, we demonstrated that activated unfolded protein response (UPR) attenuated the sensitivity of human hepatocellular carcinoma cell (HCC) to NK-cell cytotoxicity by decreasing the expression level of CD226 ligand CD155 in HCC. The decreased expression level of CD155 was due to the involvement of the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1α (IRE1α) pathways. In addition, the IRE1α pathway contributed to the increased expression level of the ER-associated degradation (ERAD)-related molecule HRD1 and facilitated the degradation of CD155. Moreover, we found that low levels of CD155 expression were significantly associated with poor prognosis in patients with HCC. Thus, our results provide molecular, cellular, and clinical evidence demonstrating a novel NK cell-associated immune evasion mechanism, and indicate that targeting this immune evasion pathway may be meaningful in treating patients with HCC. Keywords: Cancer r Immune evasion r NK cellsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionOne of the main obstacles of cancer immunotherapy is tumor immune evasion. How to overcome the immune escape and resistance of tumors to immunologic surveillance and lead the body immune response effective on tumors is one of the most Correspondence: Prof. Lihua Chen e-mail: chenlh@fmmu.edu.cn important issues [1]. NK cells mediate direct cytotoxic activity against tumor cells and provide the first line of defense for the elimination of neoplastic cells. Previous studies revealed that abundant NK cells exist in the liver, and these NK cells are essential for anti-hepatoma activity [2]. However, although there are accumulated NK cells in hepatoma tissues and these accumulated * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 3758-3767 Clinical immunology 3759 Figure 1. TM induced UPR activation in hepatoma cells. (A) Whole-cell lysates from SMMC7721 and HepG2 cells were treated with TM (3 μmol/mL) for the indicated periods, and were then subjected to Western blot analysis for GRP78 expression. Equal loading was monitored by assessing GAPDH levels. (B) The RT-PCR products of XBP-1 mRNA from cells treated with TM were digested with Apa-LI for 90 min followed by electrophoresis. The longer fragment derived from the active form of XBP1 (sXBP-1) mRNA and the two shorter bands derived from the inactive form (uXBP-1) mRNA are indicated. Data are shown as means + SD from three samples/replicates and are representative of three independent experiments. **p < 0.01, unpaired two-tailed Student's t-test.NK cells showed higher cytotoxic activity than those in peripheral blood in vitro, the hepatoma cells still can escape NK cellmediated killing and survive. We conjec...
Natural killer (NK) cells play a critical role in antitumor immunity, and the activation of NK cells is regulated by a series of NK cell receptors. Here, we show that crosslinking CD226, an important NK cell receptor, with the anti-CD226 mAb LeoA1 on NKL cells, regulated the expression of several microRNA and transmembrane tumor necrosis factor-a. Among them, miR-30c-1* was noticed because overexpression of miR-30c-1* triggered upregulation of transmembrane tumor necrosis factor-a expression and enhanced NK cell cytotoxicity against hepatoma cell lines SMMC-7721 and HepG2. Furthermore, we proved that the inhibitory transcription factor HMBOX1, which depressed the activation of NK cells, was the direct target gene of miR-30c-1*. In conclusion, our results revealed a novel regulatory mechanism: miR-30c-1* promoted NK cell cytotoxicity against hepatoma cells by targeting HMBOX1. (Cancer Sci 2012; 103: 645-652)
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