OBJECTIVETo study the clinicopathological significance of the expression of calreticulin (CRT) protein and mRNA in pancreatic ductal adenocarcinoma (PDAC).

METHODSThe expression of CRT protein in 33 paired paraffin embedded PDAC specimens and adjacent non-cancerous pancreatic tissues were detected by immunohistochemistry. Western blot and RT-PCR were used to examine the expression of CRT protein and mRNA in 12 paired fresh PDAC specimens and adjuvant non-cancerous pancreatic tissues. The relationship between the protein expression and clinicopathological features was analyzed.

RESULTSCRT expression was much higher in 33 PDAC tissues than that in paired adjacent non-cancerous pancreatic samples (t = 2.323, P = 0.027). CRT was over expressed in 16 PDAC tissues, but only in 8 adjuvant non-cancerous pancreatic tissues (48.5% vs. 24.2%). The expression of CRT protein had no correlation with tumor position (χ(2) = 1.588, P = 0.208), differentiation (χ(2) = 1.517, P = 0.218), TNM stage (χ(2) = 2.528, P = 0.112) and lymph node metastasis (χ(2) = 1.963, P = 0.161), but had statistic significancy with the prognosis of the patients (χ(2) = 4.080, P = 0.043). The median survival time in the patients with high expression of CRT protein was longer than that in the patients with low expression. The expression of CRT mRNA was higher in PDAC than that in non-cancerous tissues detected by RT-PCR (t = 2.539, P = 0.025), but no significant difference was found in protein level (t = 1.292, P = 0.223).

CONCLUSIONSCRT is up-regulated in PDAC and may be a prognosis factor for patients with PDAC.

Calreticulin ; metabolism ; Carcinoma, Pancreatic Ductal ; metabolism ; Humans ; Immunohistochemistry ; Pancreatic Neoplasms ; metabolism ; Prognosis

Animals ; Calreticulin ; metabolism ; Male ; Muscle, Skeletal ; metabolism ; Oxidative Stress ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley

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.
Humans ; MicroRNAs/genetics* ; Ki-67 Antigen/metabolism* ; Calreticulin/metabolism* ; Prognosis ; Lymphoma, Large B-Cell, Diffuse/genetics* ; Lactate Dehydrogenases/metabolism*

Calreticulin (CRT) is an essential Ca(2+)-binding chaperone existing in endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and is involved in intracellular Ca(2+) homeostasis and protein folding. Ischemic postconditioning (I-postC), a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning (H-postC). Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows (n=4): control, hypoxia/reoxygenation (H/R), H-postC, AS, AS + H/R, and AS + H-postC. Morphological studies, lactate dehydrogenase (LDH) activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca(2+) concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate (PNPP) was used as substrate to measure calcineurin (CaN) activity. The expression of CRT, CaN, nuclear factor kappa B (NFκB) and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein (CHOP) were detected by Western blot. The results were as follows. (1) H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively (P<0.05). (2) H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate and LDH leakage increased by 1.74% and 13.6% in AS + H-postC group, respectively (P<0.05), but intracellular Ca(2+) concentration, CaN activity, and expression of CaN and NFκB did not change significantly (P>0.05), suggesting that CRT participates in endogenous protection, not through Ca(2+)-CaN pathway. (3) H-postC inhibited the expression of pro-apoptosis proteins such as Bax and CHOP, but induced up-regulation of anti-apoptosis protein Bcl-2. Inhibition of CRT by AS-ODNs partly inhibited the changes in apoptosis-related proteins expression induced by H-postC, suggesting that CRT participates in the anti-apoptosis effect of H-postC through regulating expression of apoptosis-related proteins. These results indicate that CRT up-regulation induced by H-postC is involved in the cardioprotection through regulating expression of apoptosis-related proteins, not through Ca(2+)-CaN pathway in neonatal cardiomyocytes.
Animals ; Apoptosis ; Calcineurin ; metabolism ; Calreticulin ; metabolism ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Ischemic Postconditioning ; Myocytes, Cardiac ; metabolism ; Oxygen ; metabolism ; Rats ; Up-Regulation

OBJECTIVES: In this study, we explored how adiponectin mediated urotensin II (UII)‍-induced tumor necrosis factor-‍α (TNF-‍α) and α‍-smooth muscle actin (α‍-SMA) expression and ensuing intracellular signaling pathways in adventitial fibroblasts (AFs). METHODS: Growth-arrested AFs and rat tunica adventitia of vessels were incubated with UII and inhibitors of signal transduction pathways for 1‍‒‍24 h. The cells were then harvested for TNF-α receptor (TNF-‍α-R) messenger RNA (mRNA) and TNF-‍α protein expression determination by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Adiponectin and adiponectin receptor (adipoR) expression was measured by RT-PCR, quantitative real-time PCR (qPCR), immunohistochemical analysis, and cell counting kit-8 (CCK-8) cell proliferation experiments. We then quantified TNF-α and α-SMA mRNA and protein expression levels by qPCR and immunofluorescence (IF) staining. RNA interference (RNAi) was used to explore the function of the adipoR genes. To investigate the signaling pathway, we applied western blotting (WB) to examine phosphorylation of adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK). In vivo, an adiponectin (APN)‍-knockout (APN-KO) mouse model mimicking adventitial inflammation was generated to measure TNF-α and α‍-SMA expression by application of qPCR and IF, with the goal of gaining a comprehensive atlas of adiponectin in vascular remodeling. RESULTS: In both cells and tissues, UII promoted TNF-α protein and TNF-α-R secretion in a dose- and time-dependent manner via Rho/protein kinase C (PKC) pathway. We detected marked expression of adipoR1, T-cadherin, and calreticulin as well as a moderate presence of adipoR2 in AFs, while no adiponectin was observed. Globular adiponectin (gAd) fostered the growth of AFs, and acted in concert with UII to induce α-SMA and TNF-α through the adipoR1/T-cadherin/calreticulin/AMPK pathway. In AFs, gAd and UII synergistically induced AMPK phosphorylation. In the adventitial inflammation model, APN deficiency up-regulated the expression of α-SMA, UII receptor (UT), and UII while inhibiting TNF-‍α expression. CONCLUSIONS From the results of our study, we can speculate that UII induces TNF‍-‍α protein and TNF-‍α‍-R secretion in AFs and rat tunica adventitia of vessels via the Rho and PKC signal transduction pathways. Thus, it is plausible that adiponectin is a major player in adventitial progression and could serve as a novel therapeutic target for cardiovascular disease administration.
Mice ; Rats ; Animals ; Adventitia/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Calreticulin/metabolism* ; Vascular Remodeling ; AMP-Activated Protein Kinases/metabolism* ; Cells, Cultured ; RNA, Messenger/genetics* ; Inflammation

BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats

OBJECTIVETo observe the effect of angiotensin II (Ang II) on calreticulin (CRT) expression and its association with mitochondrial dysfunction in cardiomyocytes.

METHODSPrimary neonatal rat cardiomyocytes were randomly divided into CRT siRNA group, control siRNA group, control group, Ang II+ CRT siRNA group, Ang II+ control siRNA group and Ang II group. The cell surface area, protein synthesis rate, mitochondrial membrane potential level, enzyme activities, and CRT expression were observed.

RESULTSCompared with those in the control group, the cell surface area and protein synthesis rate were both increased and mitochondrial membrane potential level and enzyme activities decreased in Ang II groups. CRT expression was significantly down-regulated in Ang II+ CRT siRNA group with increased cell surface area, protein synthesis rate, mitochondrial membrane potential level and enzyme activities as compared with those in Ang II+ control siRNA group.

CONCLUSIONAng II up-regulates CRT expression to induce mitochondrial injury, which may be an important mechanism of myocardial hypertrophy.

Angiotensin II ; pharmacology ; Animals ; Calreticulin ; metabolism ; Cardiomegaly ; Cells, Cultured ; Membrane Potential, Mitochondrial ; Mitochondria ; pathology ; Myocytes, Cardiac ; pathology ; Protein Biosynthesis ; RNA, Small Interfering ; Rats

OBJECTIVETo study the protection of stress protein calreticulin (CRT) in rat skeletal muscle during the adaptation mechanism of calmodulin in the course of heat treatment.

METHODSIncreased heat treatment program would be applied, 40 SD rats were randomly divided into the quiet control group C (n = 8) and heat-treated group H (n = 32), then the heat treatment group would be divided into immediately group (H1), 24-hour post-heat treatment group (H2), 48 -hour post-heat treatment group (H3) and six days post-heat treatment group (H4) (n = 8).

RESULTSAfter heat treatment, the Ca(2+)-ATP activity in rat skeletal muscle sarcoplasmic reticulum in H2 group reached the highest value compared with that in the quiet control group C (P < 0.01), and the value in H1 group showed significant differences compared with control group C (P < 0.05); The Ca(2+)-ATP activity in mitochondrial had the highest value in H1 group, compared with the quiet control group C (P < 0.05), while the Ca2+ concentration in rat skeletal muscle sarcoplasmic reticulum had the highest in group H2, followed by H1 group, both showing significant difference compared with the quiet control group (P < 0.05); The Ca2+ concentration in mitochondrial was high in H1 and H2 group than that of the quiet control group C, and the value in H3 and H4 group was lower than that of the quiet control group C, which had no difference; After heat treatment, the expression of stress proteins of CRT from H1, H2 and H3 group in rat skeletal muscle increased significantly compared with quiet group C.

CONCLUSIONIn the process of increased heat treatment, calreticulin played the regulatory role on the imbalance of calcium homeostasis in skeletal muscle cells, and the adaptation protection from the thermal stimulation could have the very good effect on muscle.

Adaptation, Physiological ; Animals ; Calcium ; metabolism ; Calreticulin ; physiology ; Heat Stress Disorders ; metabolism ; physiopathology ; Male ; Mitochondria ; metabolism ; Muscle, Skeletal ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

Both in vivo and cultured cardiomyocyte experiments were performed to investigate the alteration of expression of calreticulin (CRT) during the delayed cardioprotection induced by hypoxic preconditioning (HPC) and the intracellular signal transduction mechanisms of the alteration. (1) Wistar rats were randomly divided into three groups: sham operation group (Sham), myocardial infarction (MI) group induced by left coronary artery ligation and HPC+MI group (4-hour HPC 24 h before MI). Twenty-four hours, 14 d and 28 d after left coronary artery ligation, myocardial function, infarction size and the area at risk were measured. Western blot was used to detect the expression of CRT, the activity of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK). (2) Cultured cardiomyocytes from neonatal Sprague-Dawley (SD) rat were divided into six groups: hypoxia/reoxygenation (H/R), HPC, HPC+H/R, p38 MAPK inhibitor SB203580+HPC+H/R (SB+HPC+H/R), SAPK inhibitor SP600125+HPC+H/R (SP+HPC+H/R) and control. Survival rate and apoptosis rate of cardiomyocytes 6 h after H/R and activities of lactate dehydrogenase (LDH) in culture medium in each group were measured. Western blot was used to detect the expression of CRT and activities of p38 MAPK and SAPK. The results are as follows: (1) During in vivo experiment, compared with MI group, HPC significantly improved +dp/dt(max) and -dp/dt(max), reduced infarction size and the area at risk. HPC dramatically changed the expression of CRT. CRT expression in HPC+MI group was 206% of that in MI group (P<0.05) 24 h after infarction, especially in the area at risk. However, 28 d after operation, the expression of CRT decreased by 57%. Correlation analysis indicated a positive correlation between CRT expression and myocardial function (r=0.9867, P<0.05), and negative correlation between CRT expression and infarction size (r=-0.9709, P<0.05). (2) In cultured cardiomyocytes, HPC attenuated cell injury induced by H/R. CRT expression increased moderately to 222% of control (P<0.05) during HPC, but increased dramatically to 503% of control (P<0.05) after H/R. HPC reduced H/R-induced CRT up-regulation to 56% of that in H/R group (P<0.05). Correlation analysis indicated that CRT expression induced by HPC had a positive correlation with p38 MAPK activity (r=0.9021, P<0.05), but a negative correlation with SAPK activity (r=-0.8211, P<0.05). Both in vivo and in vitro results indicate that HPC protects myocardium from ischemia or H/R injury. p38 MAPK is possibly involved in the up-regulation of CRT induced by HPC, while SAPK has a negative influence.
Animals ; Calreticulin ; metabolism ; Cells, Cultured ; Ischemic Preconditioning, Myocardial ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Myocardium ; pathology ; Myocytes, Cardiac ; metabolism ; Rats, Sprague-Dawley ; Rats, Wistar ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo generate a sensitive tool for noninvasive monitoring of a therapeutic gene vasostatin.

METHODSWe fused the bioluminescent reporter gene firefly luciferase to the therapeutic transgene vasostatin and ensured that these two proteins would not interrupt each other and kept their own natural character.

RESULTSWe therefore examined clones of PC3 cells stably expressing fusion gene and positive controlfluc with bioluminescence. In vivo imaging of PC3-Fluc subcutaneous tumors showed that the mean tumor bioluminescence increased in animals over several weeks.

CONCLUSIONNoninvasive monitoring facilitates the detection of gene expression in vivo and in vitro.

Animals ; Calreticulin ; genetics ; metabolism ; Cell Line, Tumor ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases, Firefly ; genetics ; metabolism ; Luminescent Measurements ; Neoplasm Transplantation ; Peptide Fragments ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism