Detection of Calreticulin as a Candidate Prognostic Biomarker in Invasive Breast Carcinoma from a Biological Scaffold-Based 3D Co-culture System

@#Introduction: Breast cancer is the most common cancer in women and the world’s second leading cause of death in women, after lung cancer. Calreticulin (CRT), an endoplasmic reticulum (ER) multipurpose protein, has been proposed as a potential biomarker for breast cancer. However, reports on the correlation between CRT expression and cell invasiveness in breast cancer micro-tissues are scarce. Thus, in the current study, we analyzed the potential correlation between CRT and invasiveness of breast cancer in a biological scaffold-based 3D co-culture system. Methods: MCF7, MDA-MB-231 and MCF-10A breast cell lines were co-cultured in a 3-dimensional (3D) system with MRC-5 lung fibroblast cell line in the cell density ratio of 3:1. Thereafter, calreticulin gene and protein expression levels were determined based on quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. Moreover, via RT-qPCR analysis, the gene expression levels of calreticulin-related candidate metastasis genes in breast cancer micro-tissues were carried out. Results: The results showed occasional foci of lumen-like morphology in the non-cancerous breast micro-tissues and the formation of solid clusters for breast cancer micro-tissues. Moreover, immunohistochemistry results revealed protein expression of calreticulin in non-cancerous and cancerous breast micro-tissues with cytoplasmic and nucleic acid localizations. It was found that PCMT1 and ER-α genes were significantly downregulated (p < 0.01) in invasive breast cancer micro-tissues. Conclusion: This study suggests that CRT and CRT-related candidate metastasis genes may potentially serve as prognostic biomarkers in invasive breast carcinoma.

The Expression and Correlation of miR-195, miR-125 and Calreticulin in Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To analyze the expression and correlation of microRNA-195 (miR-195), miR-125 and calreticulin in diffuse large B-cell lymphoma (DLBCL).@*METHODS@#From April 2020 to April 2021, 80 DLBCL patients with complete data archived by the Pathology Department of Handan First Hospital and The Second Hospital of Hebei Medical University were selected as the study group, and 70 patients with reactive lymph node hyperplasia were selected as the control group. The expressions of miR-195 and miR-125 were detected by real-time fluorescence quantitative PCR, and the expression of calreticulin was detected by Western blot. Pearson correlation was used to analyze the correlation between miR-195, miR-125, calreticulin and DLBCL, and ROC curve was used to analyze the predictive value of miR-195, miR-125 and calreticulin for DLBCL.@*RESULTS@#Compared with the control group, the expression of miR-195 decreased but miR-125 and calreticulin increased in the study group (P<0.001). The expression levels of miR-195, miR-125 and calreticulin were not related to sex, age, primary site and B symptoms of patients with DLBCL, but related to immunophenotype, Ann Arbor stage, lactate dehydrogenase, IPI score, nodule involvement and Ki-67 index. The expression of miR-195 decreased and the expression of miR-125 and calreticulin increased in DLBCL paitents with non-germinal center source, Ann Arbor stage III-IV, lactate dehydrogenase > 245 U/L, IPI score 3-5, nodule involvement≥2 and Ki-67 index≥75% (P<0.05). Pearson correlation analysis showed that miR-195 and miR-125 were negatively correlated (r=-0.536, P=0.001), miR-195 and calreticulin were negatively correlated (r=-0.545, P=0.001), while miR-125 and calreticulin were positively correlated (r=0.523, P=0.001). ROC curve showed that compared with the single diagnosis of miR-195, miR-125 and calreticulin, the combination of the three items had higher predictive value for DLBCL (P<0.001).@*CONCLUSION@#The expression of miR-195 decreases and the expression of miR-125 and calreticulin increase in patients with DLBCL. Along with the increase of disease stage and IPI score, the decrease of miR-195 and the increase of miR-125 and calreticulin aggravate gradually. The three items may participate in the occurrence and progress of DLBCL.

miR-148a-3p improves mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT expression / 西安交通大学学报(医学版)

【Objective】 To evaluate the effects of miR-148a-3p on calreticulin (CRT) expression and mitochondrial function in cardiomyocytes incubated with high glucose. 【Methods】 miR-148a-3p minic and inhibitor were used to intervene the H9c2 cardiomyocytes of rats. The expression of CRT protein was detected. Then the cells were divided into control group, high-glucose group (HG), HG +miR-148a-3p minic group, HG + miR-148a-3p minic + TG (CRT agonist) group, HG + miR-148a-3p inhibitor group, and HG + miR-148a-3p inhibitor + CRT- (CRT-siRNA) group. The content of adenosine triphosphate (ATP) and the level of reactive oxygen species (ROS), the activity of mitochondrial respiratory chain complex enzyme and apoptotic rate were detected. 【Results】 miR-148a-3p minic significantly inhibited the expression of CRT protein in cardiomyocytes, while miR-148a inhibitor increased the expression of CRT. miR-148a-3p minic inhibited the decrease of ATP production, the increase of ROS production and cell apoptosis, and the inactivity of mitochondrial respiratory chain complex enzyme in cardiomyocytes induced by high glucose, while TG weakened the above effects of miR-148a-3p minic. miR-148a inhibitor aggravated the mitochondrial injury and apoptosis of cardiomyocytes induced by high glucose, but the effects of miR-148a-3p inhibitor were partially blocked by CRT-siRNA. 【Conclusion】 miR-148a-3p negatively regulates the expression of CRT in cardiomyocytes and protects the mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT.

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

@#Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Methods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

Preliminary application experience of disk microfluidic chip for detecting CALR gene mutation in patients with cerebral infarction / 中华检验医学杂志

Objective:To establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

Calreticulin promotes thapsigargin induced epithelial mesenchymal transition in pancreatic cancer cells / 中华普通外科杂志

Objective:To investigate the effect of CRT on thapsigargin (TG)-induced epithelial mesenchymal transition (EMT) of pancreatic cancer (PC) cells.Methods:Immunohistochemistry was used to investigate the differential CRT expression in PC tissues. Western blot (WB) and transwell were used to detect the effect of CRT silencing on TG induced EMT phenotype. Fluro-4/AM and confocal microscopy were used to detect intracellular calcium level in PC cells.Results:CRT was overexpressed in PC tissues ( P<0.01). Overexpression of CRT was positively associated with lymph node metastasis ( P=0.017) and UICC stage ( P=0.021) of PC patients, and negatively associated with E-cadherin expression ( P=0.013). High CRT and low E-cad expression contributed to the poor prognosis of PC patients ( P=0.023). In PC cells, TG induced EMT phenotype was reversed by siRNA-mediated CRT silencing. TG induced EMT was significantly reversed by CRT silencing in vitro. Conclusions:CRT mediates TG induced intracytoplasmic Ca 2+, and ultimately promotes EMT of PC cells.

Clinical significance of expression of tumor immunogenic cell death related molecules / 国际肿瘤学杂志

Tumor immunogenic cell death is a type of regulatory cell death, which is driven by stress including chemotherapy drugs, radiotherapy, oncolytic virus, nano carrier drugs and photodynamic force. It can induce specific immune response to tumor death cell antigen. The further study can provide theoretical basis and new ideas for anti-tumor immunity and clinical immunotherapy of tumor.

A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer

The immune checkpoint blockade therapy has profoundly revolutionized the field of cancer immunotherapy. However, despite great promise for a variety of cancers, the efficacy of immune checkpoint inhibitors is still low in colorectal cancer (CRC). This is mainly due to the immunosuppressive feature of the tumor microenvironment (TME). Emerging evidence reveals that certain chemotherapeutic drugs induce immunogenic cell death (ICD), demonstrating great potential for remodeling the immunosuppressive TME. In this study, the potential of ginsenoside Rg3 (Rg3) as an ICD inducer against CRC cells was confirmed using in vitro and in vivo experimental approaches. The ICD efficacy of Rg3 could be significantly enhanced by quercetin (QTN) that elicited reactive oxygen species (ROS). To ameliorate in vivo delivery barriers associated with chemotherapeutic drugs, a folate (FA)-targeted polyethylene glycol (PEG)-modified amphiphilic cyclodextrin nanoparticle (NP) was developed for co-encapsulation of Rg3 and QTN. The resultant nanoformulation (CD-PEG-FA.Rg3.QTN) significantly prolonged blood circulation and enhanced tumor targeting in an orthotopic CRC mouse model, resulting in the conversion of immunosuppressive TME. Furthermore, the CD-PEG-FA.Rg3.QTN achieved significantly longer survival of animals in combination with Anti-PD-L1. The study provides a promising strategy for the treatment of CRC.

Análisis de expresión de calreticulina en vena safena parva / Calreticulin expression analysis in small saphenous vein

La proteína chaperona Calreticulina (CRT), ha sido identificada en retículo endoplásmico (RE) y últimamente en la matriz extracelular (MEC) de predentina y arterias, atribuyéndole diferentes funciones extracelulares entre las que destacan la adhesión celular, regulación de la MEC y prevención en la formación de trombos. El objetivo del estudio fue identificar la presencia de CRT en MEC de vena safena parva. Se extrajo una muestra de vena safena parva de un espécimen masculino y luego fue procesada por medios histológicos e inmunohistoquímicos para identificar su presencia. Mediante técnicas de inmunohistoquímica se pudo evidenciar la presencia de CRT en la MEC de la adventicia de vena safena parva. La presencia de CRT en MEC de safena parva orienta a que CRT tienen funciones de tipo extracelular en esta localización, pero es necesario realizar estudios más precisos para dilucidar sus principales funciones en la zona.

Calreticulin (CRT) protein, has been identified in the endoplasmic reticulum (ER) and lately in the extracellular matrix (ECM) of predentine and arteries. It is responsible for different extracellular functions, such as cell adhesion, ECM regulation, and the prevention of thrombosis. The aim was to identify the presence of CRT in ECM of small saphenous vein. A sample of small saphenous vein from a male specimen was extracted and then processed by histological and immunohistochemical assays to identify its presence. The presence of CRT in the ECM of the small saphenous vein was observed by immunohistochemical techniques. The presence of CRT in the small saphenous vein ECM, indicates that CRT have extracellular functions in this area, however, more precise studies are necessary to determine its main functions.

Expresión de calreticulina en placenta canina / Calreticulin expression in canine placenta

La placenta es un anexo embrionario de los mamíferos que tiene por función principal el intercambio de nutrientes y gases y proteger al concepto de un potencial daño inmune provocado por diferencias alogénicas en los Complejos Principales de Histocompatibilidad paternos. Se han descrito diversas proteínas asociadas a su función, siendo Calreticulina una de ellas. Si bien existen estudios de la presencia de Calreticulina en placenta humana, no existen reportes de esta proteína en la placenta canina. Se obtuvieron muestras de placenta canina de las que se extrajo el contenido proteico total y se determinó la presencia de Calreticulina por western blot e inmunohistoquímica. Los resultados mostraron presencia de Calreticulina en placenta canina con un peso molecular aparente de 60 kDa, concordante con lo descrito para la molécula por otros autores. El análisis inmunohistoquímico mostró que Calreticulina canina está presente principalmente en el trofoblasto de las vellosidades, no existiendo diferencias en cuanto a su localización al compararla con placenta humana, pese a sus diferencias morfológicas e histológicas. Esta información permitirá establecer un protocolo estandarizado de extracción de Calreticulina desde placenta, así como orientar acerca de los posibles roles de esta molécula en la placenta.

The placenta is an embryonic organ present in mammals, whose main functions are the exchange of nutrients and gases and to protect the fetus from potential immune damage mediated by paternal and maternal allogeneic differences in the Major Histocompatibility Complex. Several proteins associated with its function have been described, being Calreticulin one of them. Although there are studies on the presence of Calreticulin in human placenta, there are no reports of this protein in canine placenta. Samples from canine placenta were obtained, proteins extracted and Calreticulin was subsequently detected by western blot and immunohistochemistry. The results showed the presence of Calreticulin in canine placenta with an apparent molecular weight of 60 kDa, in agreement with the results from other authors. The immunohistochemical analysis showed that canine Calreticulin is present mainly in the trophoblast of the villi, and there is no difference in its localization when compared with a blood-filled placenta such as human one, despite its morphological and histological differences. We also propose a standardized protocol for the extraction of Calreticulin from placenta, given its abundant expression in this organ. Future studies are aimed at elucidating possible roles of this protein in placenta.

Calreticulina: aspectos moleculares y técnicas para su detección / Calreticulin: molecular aspects and techniques for its detection

Las neoplasias mieloproliferativas crónicas (NMPC) son enfermedades clonales caracterizadas por un aumento en el número de células maduras circulantes; estas incluyen: policitemia vera (PV), trombocitemia esencial (TE), mielofibrosis primaria (MFP), entre otras. Una de las principales características moleculares de estas tres entidades es la ausencia del gen de fusión BCR/ABL. La primera mutación relacionada directamente con estas neoplasias fue detectada en el gen JAK2; a partir de su descubrimiento, otras mutaciones en los genes del receptor de trombopoyetina (MPL) y calreticulina (CALR) han sido fuertemente relacionadas con la presentación de la enfermedad. La calreticulina es una proteína del retículo endoplásmico con diversas funciones a nivel celular como la homeostasis del calcio y la actividad de chaperona. Hasta la fecha se ha identificado un gran número de mutaciones en el gen CALR. La mayoría de ellas son inserciones y deleciones que generan cambios a nivel proteico con implicaciones importantes en el curso clínico y pronóstico de las neoplasias. Debido a su alta frecuencia y fuerte asociación con las NMPC, las mutaciones de CALR se incluyen como criterio mayor para el diagnóstico de estas entidades. Por este motivo, se han desarrollado varias técnicas encaminadas a la detección rápida, eficiente, sensible y especifica de esta mutación como: la secuenciación, el análisis de fragmentos y el análisis de fusión de alta resolución. El conocimiento e implementación de estas técnicas en los laboratorios clínicos constituye un avance importante para el diagnóstico y la evolución de los pacientes(AU)

Chronic myeloproliferative neoplasms (NMPC) are clonal diseases characterized by an increase in the number of mature circulating cells; these diseases include: polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (MFP) among others. One of the main molecular characteristics of these three entities is the absence of the BCR/ABL fusion gene. The first mutation related to this group of neoplasms was detected in the JAK2 gene; since its discovery, other mutations in thrombopoietin receptor (MPL) and calreticulin (CALR) genes have been strongly related with the presentation of the disease. Calreticulin is an endoplásmic reticulum protein with different functions in the cell such as calcium homeostasis and the chaperone activity. To date, a large number of mutations have been identified in CALR gene most of them are insertions and deletions that generate changes in the protein that generate important implications in the clinical course and prognosis of neoplasms. Due to its high frequency and strong association with NMPC, CALR mutations are included as a major criteria for the diagnosis of these entities. For this reason, several techniques have been developed aimed at the rapid, efficient, sensitive and specific detection of this mutation as: sequencing, fragment analysis and high resolution fusion analysis. The knowledge and implementation of these techniques in clinical laboratories is an important advance for the diagnosis and in the evolution of patients(AU)

Significance of calreticulin gene mutations classification in BCR-ABL1 negative myeloproliferative neoplasms / 白血病·淋巴瘤

Objective@#To investigate calreticulin (CALR) gene mutations classification in BCR-ABL1 negative myeloproliterative neoplasms (MPN), and its relationship with clinical manifestations.@*Methods@#Genomic DNA polymerase chain reaction (PCR) amplification product Sanger sequencing method was used to detect the mutation of exon 9 of CALR gene in 236 patients with BCR-ABL1 negative MPN (excluding polycythemia vera and negative CALR mutations) in Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from November 2015 to November 2018. The mutations were classified into 52 bp deletion (type 1) mutation, 5 bp insertion (type 2) mutation and other mutation types according to PCR sequencing analysis. The clinical characteristics of the carriers with two kinds of mutations in 198 patients with essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) were compared. For the types of mutations that could not be determined, they were classified according to the α-helix propensity score of the mutant protein peptide chain or the degree of retention of the negatively charged amino acid residues, and the differences between the two classification methods were also compared.@*Results@#Among 236 patients, the CALR gene type 1 or type 2 mutation was detected in 206 cases (87.3%), including 173 ET patients (99 cases of type 1 mutation and 74 cases of type 2 mutation) and 33 PMF patients (28 cases of type 1 mutation and 5 cases of type 2 mutation). The CALR non-type 1 or non-type 2 mutation was detected in 30 cases, including 25 ET patients and 5 PMF patients. Among 173 ET patients with CALR gene mutation, the white blood cell count (WBC) of patients with type 1 mutation was higher than that of patients with type 2 mutation [(8.6±2.7)×109/L vs. (7.6±2.4)×109/L, t = 2.45, P = 0.015]. Among 33 PMF patients with CALR gene mutation, the age of patients with type 1 mutation was older than that of patients with type 2 mutation [(58±13) years old vs. (41±16) years old, t = 2.51, P = 0.018]. According to the α-helix propensity score of mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues, 27 kinds of non-type 1 or non-type 2 mutations were classified by using sequencing method, and there were differences between the two methods. According to the α-helix propensity score of the mutant protein peptide chain, the proportion of type 1/type 1-like mutation in PMF patients was higher than that in ET patients [78.9% (30/38) vs. 56.6% (112/198), P < 0.01]. According to the degree of retention of negatively charged amino acid residues in the mutant protein peptide chain, the isoelectric point (pI) value of the mutant protein peptide chain was higher than that of the wild type sequence. The pI value of the type 1-like mutant protein peptide chain was higher than that of the type 2-like mutation (11.79±0.15 vs. 10.02±0.42, t = 11.51, P < 0.01).@*Conclusions@#Type 1 mutated ET patients may be closely related to the high risk of myelofibrosis transformation. The results of the classification of CALR mutations are different according to the α-helix propensity score of the mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues. Further study is necessary to identify the pathogenesis of MPN caused by CALR mutation, and to determine the relationship between mutation type and prognosis of disease.

Molecular genetics of BCR-ABL1 negative myeloproliferative neoplasms in India

Introduction: Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. Materials and Methods: One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. Results: Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. Discussion and Conclusion: We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance.

Progress of CALR gene mutation in myeloproliferative neoplasm / 白血病·淋巴瘤

Myeloproliferative neoplasm (MPN) is a kind of clone hematopoietic stem cell disease characterized by one or more myeloid cell lines hyperproliferation, including bcr-abl gene positive chronic myeloid leukemia (CML) and bcr-abl negative MPN, the later representing polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A big stride has been made since the discovery of JAK2 and MPL gene mutations. However, the exact genetic basis of JAK2/MPL mutation double negative in MPN patients is still unclear. It has been reported recently that a new CALR mutation is discovered in the JAK2/MPL unmutated MPN patients who show unique clinical presentations, which provides a new diagnostic and prognosis-accessing criteria. The paper reviews CALR mutation and genetic mechanism mediating MPN.

The expression of calreticulin on gallbladder carcinoma tissues and its effect on the biological behavior in gallbladder cancer cells / 中华普通外科杂志

Objective To explore the expression of calreticulin (CRT) in gallbladder cancer tissue and its effect on the biological behavior in gallbladder cancer GBC-SD cells.Methods Immunohistochemistry and RT-qPCR were applied to detect the expression of CRT.Small interfering RNA was transfected into gallbladder cancer GBC-SD cells and Western blotting were used to detect the expression of CRT.The proliferation was determined by using cell counting kit-8 (CCK-8) and clone assays.Flow cytometry were applied to detect the apoptosis and cell cycle.Migration was detected by wound healing and transwell assays,respectively.The expression of p-Akt and MMP-9 were detected by using Western blotting.Results Expression of CRT in gallbladder cancer tissues is higher than adjacent cancer tissues and chronic cholecystitis tissues(t =5.571,P ＜ 0.05).The relative growth rate in the siCRT-1,siCRT-2 experimental group for 24 hours,48 hourrs were 71.5％ ±6.3％,79.5％ ±2.7％;62.6％ ± 8.8％,55.6％ ±2.6％,respectively.The apoptosis rate in the blank group,the negative control group,siCRT-1 and siCRT-2 group were 3.0％ ± 1.8％,4.7％ ± 1.3％,13.6％ ± 1.0％,20.0％ ± 4.0％,respectively.Wound healing assays showed that the wound closure ratio in the blank group,negative control group,siCRT-1 and siCRT-2 group were(0.67 ±0.02),(0.58 ±0.02),(0.22 ±0.01),(0.37 ±0.04),respectively.Transwell experiments showed that the numbers of migration of GBC-SD cells in the blank group,negative control group,siCRT-1 and siCRT-2 group were (302 ± 11),(297 ± 15),(178 ± 10),(165 ± 12),respectively,compared with the blank group and the negative control group,the relative growth rate for 24 hours and 48 hours was significantly lower,the apoptosis rate was higher,the numbers of migration was lower (F =29.310,118.618,69.651,144.515,190.145,P ＜ 0.05).Compared with the blank group and the negative control group,the expression of p-Akt and MMP-9 decreased after down-regulating the expression of CRT.Conclusions The expression of CRT in gallbladder cancer tissue was higher.CRT downregulation mediated changes of biological behaviors in gallbladder cancer may be associated with p-Akt/MMP-9 signal pathway.

CALR mutation in patients with Ph negative chronic myeloproliferative neoplasms and its clinical significance / 中国综合临床

Objective To observe the CALR mutation in patients with Ph negative chronic myeloproliferative neoplasms(MPNs) and its clinical significance. Methods From January 2012 to January 2015,the clinical data of ninety-seven patients with chronic myeloproliferative neoplasms was retrospectively analyzed and followed up to analyze different types of MPNs, including the clinical characteristics and gene mutation of polycythemia vera(PV),essential thrombocythemia(ET)and primary myelofibrosis (PMF).The hematological parameters and prognosis of patients with different mutation types were compared ( Cox regression model). Results Among the patients,the incidence of JAK2 mutation was the highest,64. 95% (63/97), followed by CALR mutation ( 19. 59% ( 19/97 ) ) and triple negative ( 10. 31% ( 10/97 ) ) . The incidence of MPL mutation was 5. 15% (5/97),which was the lowest and CALR mutations in ET and PMF were 28. 57%(10/35) and 28. 13% (9/32),respectively. The difference was not statistically significant (χ2 =1. 616,P>0. 05);the CALR gene mutation was not detected in PV patients. Compared with the JAK2 mutation, the hemoglobin,leukocyte and neutrophils in the patients with CALR mutation were lower (P<0. 05),PLT levels were lower in CALR-mutant ET patients ( P<0. 017) ,whereas platelet levels in CALR-mutant PMF patients were higher (P<0. 017). The incidence of disease progression in JAK2 and CALR mutation was 47. 62% (30/63)and 31. 58% (6/19) (χ2=1. 525,P>0. 05). The risk of disease progression in patients with CALR mutation was significantly lower than that of JAK2 mutation ( HR=0. 46,95%CI 0. 26-0. 98,P<0. 05) . Conclusion The clinical characteristics of MPNs patients with different gene mutations are different. The prognosis of MPNs patients with CALR mutation is better than that of JAK2 mutation.

CRT promoted c-FLIP expression via NF-κB pathway in rheumatoid arthritis fibroblast-like synoviocytes / 天津医药

Objective To investigate the molecular mechanisms of upregulated expression of cellular Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein(c-FLIP)by calreticulin(CRT)in patients with rheumatoid arthritis (RA). Methods The semi-quantitative analysis and localization of c-FLIP in RA and osteoarthritis (OA)synovium were detected by immunohistochemistry.The fibroblast-like synoviocytes(FLS)were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and OA patients,and cultured as an in vitro experiment model.The expressions of c-FLIP in RA and OA synovial fibroblasts were detected by immunofluorescence and Western blot assay. Whether CRT influenced c-FLIP expression and its molecular mechanism were explored by Western blot assay. Results The high expression of c-FLIP was found in RA synovium, mainly in the lining and sublining areas of FLS and vascular endothelial cells detected by immunohistochemistry.Meanwhile,weak staining of c-FLIP was observed in OA synovium.The expression of c-FLIP was significantly higher in RA synovium than that of OA synovium(t=11.717,P<0.001).Results of immunofluorescence and Western blot assay showed that c-FLIP was mainly located in cytoplasm, and which was higher expressed in FLS of RA than that of OA. The increased c-FLIP expression and phosphorylation of NF-κB were detected after being co-incubated with exogenous CRT (0, 10, 50, 100 μg/L), in dose-dependent manner. The effect of CRT upregulating c-FLIP expression was blocked by NF-κB inhibitor BAY 11-7082.Conclusion CRT can increase c-FLIP expression at least partly through NF-κB pathway in RA,which may provide therapeutic target for the treatment of RA.

Expression of calreticulin and its relationship with autoantibodies and inflammatory cytokines in early rheumatoid arthritis / 天津医药

Objective To investigate the expression of calreticulin and its correlation with autoantibodies and inflammatory cytokines in patients with early rheumatoid arthritis (RA). Methods Serum samples were obtained from 106 patients with early active RA, 95 patients with stable RA, 85 osteoarthritis (OA) and 80 healthy controls (HC). Serum levels of calreticulin, anti- cyclic peptide antibody (CCP), interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were measured by enzyme-linked immnuosorbent assay (ELISA). The serum level of rheumatoid factor (RF) was detected by immunoturbidimetry. The correlations between serum calreticulin and inflammatory cytokines were evaluated using Spearman's rank correlation test. Results Serum levels of calreticulin were significantly higher in patients with early active RA [(5.84±2.62)μg/L] than those in patients with stable RA [(4.26±1.42)μg/L], patients with OA [(3.92±1.10)μg/L] and HC [(3.86 ± 0.91)μg/L] (P<0.001). There were no statistical differences in serum levels of calreticulin between stable RA, OA and HC groups (P>0.05). Serum levels of calreticulin were significantly higher in RF-positive RA patients than those of RF-negative RA patients [(6.12±2.87)μg/L vs. (4.92±1.22)μg/L, P=0.045]. Serum calreticulin levels were also significantly higher in anti-CCP-positive RA patients than those of anti-CCP-negative RA patients [(6.39±2.93)μg/L vs. (4.69±1.17)μg/L, P=0.002]. The serum level of calreticulin was positively correlated with IL-1β (rs=0.386, P=0.009), IL-6 (rs=0.405, P=0.006) and TNF-α(rs=0.428, P=0.003) in early active RA patients. Conclusion The elevated serum level of calreticulin is related to autoantibodies and inflammatory cytokines in early RA patients, suggesting that calreticulin can be used as a potential biomarker for early diagnosis and prognosis of RA.

Calreticulin promotes migration and invasion of nasopharyngeal carcino-ma cells by inducing cell EMT / 中国病理生理杂志

AIM:To observe the expression of calreticulin(CRT)in nasopharyngeal carcinoma tissues,ana-lyze the significance of clinical pathology and the influence on epithelial -mesencymal transition(EMT)of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues,and the significance of clinical pathology was evaluated.The calreticulin gene-specific small interfering RNA was constructed,and then was transfected into the NPC cell line CNE 2 using the cationic liposome meth-od.The effect of CRT on the morphological changes of the CNE 2 cells was observed under light microscope.The effect of CRT on the cell migration and invasion abilities of the CNE 2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin,vimentin,transforming growth factor(TGF)-βand matrix metallopro-teinase(MMP)-9 in the CNE2 cells was determined by Western blot.RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29%(11/57),which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69%(43/52).The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis(P<0.05).Knockdown of CRT expression made the CNE 2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change,arranged more compact,and the migration and invasion abilities were significantly decreased(P<0.05).Knockdown of CRT expression resulted in significant increase in the protein expression of E -cadhe-rin,and the decreases in the protein expression of vimentin, TGF-βand MMP-9 in the CNE2 cells(P<0.05).CON-CLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with naso -pharyngeal carcinoma stage and lymph node metastasis.Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.

Chemotherapy and tumor immunogenic cell death / 国际肿瘤学杂志

Chemotherapy resist is the problem for clinic,and some researches find that chemotherapy with anthracycline and oxaliplatin not only induces the tumor cell apoptosis,but also the celt immunogenic cell death (ICD) by inducing the tumor cell apoptosis and releasing three kinds of signals:exposure of calreticulin on the cell surface to stimulate the dendritic cell (DC) to engulf,and the secretion of adenosine triphosphate to recruit DC to enter into tumor bed,and the release of the high mobility group B1 to promote DC to steadily bind with dying tumor cell to induce specific T cell antitumor immune response.It is with great meaning to promote the chemotherapy protocol by studying the ICD induced by chemotherapy.