[Relationship between the Expression of miRNA181a-5p and the Imbalance of Treg/Th17 in Primary Immune Thrombocytopenia].

OBJECTIVE: To investigate the role of relationship between the expression of miRNA181a-5p and imbalance of Treg/Th17 in the pathogenesis of primary immune thrombocytopenia(ITP), which contributes to clarify the mechanism of T cell immune imbalance in ITP patients. METHODS: Peripheral blood was collected from 37 ITP patients, concluding 21 untreated patients and 16 effectively treated patients, and 19 healthy controls; Peripheral blood mononuclear cells (PBMC) were isolated and the expression of miRNA181a-5p and Notch1 was analyzed by RT-PCR. The proportion of Th17 subsets and Treg cells in the peripheral circulation was detected by flow cytometer (FCM). Clinical data of ITP group was collected, including age, platelet count and disease course. RESULTS: The expression of miR-181a-5p was significantly decreased in ITP group than that of healthy control group (P＜0.01). After effective treatment, the expression of miR-181a-5p was significantly higher than that of ITP group (P＜0.05), but still significantly lower than that of healthy control group (P＜0.01); The expression of Notch1 was significantly increased in ITP group and effectively treated group than that of healthy control group (P＜0.01). There was no significant difference in proportion of Treg cells in ITP group, effectively treated group and healthy control group (P＞0.05). The proportion of Th17 subsets in ITP group was significantly increased than that of healthy control group (P＜0.05), while the ratio of Treg/Th17 was significantly decreased (P＜0.05). There was a positive correlation between the expression of miR-181a-5p and ratio of Treg/Th17 in ITP group (r=0.555). CONCLUSION: The expression of miR-181a-5p is significantly decreased in ITP patients, which is closely related to the imbalance of Treg/Th17 cells. After effective treatment, the expression of miR-181a-5p can be significantly corrected, but still failed to reach the level of healthy people. While the expression of Notch1 is significantly increased in ITP patients, and could not reach the level of healthy people after effective treatment.

CD103-CD23+ classical hair cell leukemia: A case report and review of the literature.

INTRODUCTION: This case report is presented to improve our understanding of the atypical immunophenotype of hairy cell leukemia. PATIENT CONCERNS: A 58-year-old woman presented to our department with fatigue for >10 days. DIAGNOSIS: The patient was diagnosed with an increased proportion of abnormal lymphocytes in peripheral blood and bone marrow smear, positive for CD11c, CD19, CD20, CD22, CD25, CD123, CD200, and Kappa, partial expression of CD23, but no expression of CD103, positive for BRAF V600E mutation. INTERVENTIONS AND OUTCOMES: Cladribine combined with rituximab achieved complete remission of minor residual disease negativity. CONCLUSION: Hairy cell leukemia is rare, and the diagnosis and differential diagnosis should be made by combining the patient's medical history, clinical manifestations, immunophenotype, gene detection, and other means. Purine nucleoside analogs are the first-line treatments.

Successful treatment using immunotherapy in combination with chemotherapy for metastatic squamous cell carcinoma of unknown primary origin with bulky abdominal mass: A case report.

RATIONALE: Cancer of unknown primary (CUP) means that the primary focus cannot be found after preliminary clinical evaluation. It accounts for 2.3% to 5% of newly diagnosed cancer cases. Due to the lack of standard treatment, CUP is usually associated with poor prognosis and is the third to fourth most common cause of cancer-related deaths. PATIENT CONCERNS: We report the case of a 42-year-old female patient who was admitted to the hospital for intermittent right abdominal pain and abdominal distension. Abdominal computed tomography (CT) showed a large abdominal mass of unknown origin, which was difficult to resect due to its close relationship with surrounding tissues. Twenty days later, the patient had enlarged left supraclavicular lymph nodes, and percutaneous biopsy revealed squamous cell carcinoma. In addition, next-generation sequencing (NGS) of tissue and blood samples showed immune-related mutations and PD-L1 expression. DIAGNOSES: The patient was diagnosed with metastatic squamous cell carcinoma of unknown primary origin, with a bulky abdominal mass. INTERVENTIONS: The patient was treated with carboplatin, albumin-binding paclitaxel, and immune checkpoint inhibitor (carilizumab). After 6 cycles, the patient was switched to maintenance treatment with carilizumab. OUTCOMES: The general condition of the patient improved, and the lesion was significantly reduced. The treatment efficacy was assessed as partial remission according to Response Evaluation Criteria in Solid Tumors. The patient benefited from immunotherapy combined with chemotherapy. LESSONS: There is no recommended standard treatment for most CUPs, which leads to their poor prognoses. By performing NGS for patients and targeting immune-related positive predictors, immunotherapy combined with chemotherapy may prolong the overall survival of patients. This case report suggests that immunotherapy combined with chemotherapy is feasible and effective in patients with CUP.

The expression, modulation and use of cancer-testis antigens as potential biomarkers for cancer immunotherapy.

Cancer-testis antigens (CTA) are tumor antigens, present in the germ cells of testes, ovaries and trophoblasts, which undergo deregulated expression in the tumor and malignant cells. CTA genes are either X-linked or autosomal, favourably expressed in spermatogonia and spermatocytes, respectively. CTAs trigger unprompted humoral immunity and immune responses in malignancies, altering tumor cell physiology and neoplastic behaviors. CTAs demonstrate varied expression profile, with increased abundance in malignant melanoma and prostate, lung, breast and epithelial cell cancers, and a relatively reduced prevalence in intestinal cancer, renal cell adenocarcinoma and malignancies of immune cells. A combination of epigenetic and non-epigenetic agents regulates CTA mRNA expression, with the key participation of CpG islands and CpG-rich promoters, histone methyltransferases, cytokines, tyrosine kinases and transcriptional activators and repressors. CTA triggers gametogenesis, in association with mutated tumorigenic genes and tumor repressors. The CTAs function as potential biomarkers, particularly for prostate, cervical, breast, colorectal, gastric, urinary bladder, liver and lung carcinomas, characterized by alternate splicing and phenotypic heterogeneity in the cells. Additionally, CTAs are prospective targets for vaccine therapy, with the MAGE-A3 and NYESO-1 undergoing clinical trials for tumor regression in malignant melanoma. They have been deemed important for adaptive immunotherapy, marked by limited expression in normal somatic tissues and recurrent up-regulation in epithelial carcinoma. Overall, the current review delineates an up-dated understanding of the intricate processes of CTA expression and regulation in cancer. It further portrays the role of CTAs as biomarkers and probable candidates for tumor immunotherapy, with a future prospect in cancer treatment.

Let-7c regulated epithelial-mesenchymal transition leads to osimertinib resistance in NSCLC cells with EGFR T790M mutations.

Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) have shown promise against non-small cell lung cancers (NSCLCs) in clinics but the utility is often short-lived because of T790M mutations in EGFR that help evade TKIs' action. Osimertinib is the third and latest generation TKI that targets EGFRs with T790M mutations. However, there are already reports on acquired resistance against Osimertinib. Recent work has revealed the role that miRNAs, particularly tumor suppressor let-7c, play in the invasiveness and acquired resistance of NSCLCs, but the mechanistic details, particularly in Osimertinib resistance, remain elusive. Using two cells lines, H1975 (endogenous T790M mutation) and HCC827-T790M (with acquired T790M mutation), we found that let-7c is a regulator of EMT, as well as it affects CSC phenotype. In both the cell lines, transfection with pre-let-7c led to reversal of EMT as studied through EMT markers e-cadherin and ZEB1. This resulted in reduced proliferation and invasion. Conversely, reduced expression of let-7c through anti-let-7c transfections significantly increased proliferation and invasion of lung cancer cells. Expression of let-7c was functionally relevant as EMT correlated with resistance to Osimertinib. High let-7c expression reversed EMT and made cells sensitive to Osimertinib, and vice versa. WNT1 and TCF-4 were found to be two targets of let-7c which were epigenetic suppressed by let-7c through increased methylation. In vivo, pre-let-7c inhibited while anti-let-7c potentiated tumor growth and WNT1 and TCF-4 were downregulated in xenografts with pre-let-7c. Silencing of both WNT1 and TCF-4 resulted in potentiation of Osimertinib action. Our results suggest an important role of let-7c in regulating EMT and the resulting Osimertinib resistance in T790M NSCLCs. More clinical studies need to be performed to fully understand the translational relevance of this novel mechanism.

Dieckol inhibits non-small-cell lung cancer cell proliferation and migration by regulating the PI3K/AKT signaling pathway.

Non-small-cell lung cancer (NSCLC) is one of the most prevalent type of lung cancers with an increased mortality rate in both developed and developing countries worldwide. Dieckol is one such polyphenolic drug extracted from brown algae which has proven antioxidant and anti-inflammatory properties. In the present study, we evaluated the anticancer property of dieckol against NSCLC cell line A549. The LC50 value of dieckol was found to be 25 µg/mL by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the antiapoptotic property of dieckol was analyzed by dual staining technique with acridine orange/propidium iodide (AO/PI) stains. It was further confirmed with flow cytometry analysis with Annexin FITC and JC-1 staining and the anti-invasive property was assessed by Transwell assay. The molecular mechanism of dieckol anticancer activity was confirmed by estimating the levels of caspases and by estimating the signaling proteins of Pi3K/AKT/mTOR signaling pathway using the immunoblotting technique. Our data suggest that dieckol is potent anticancer agent, it effectively inhibits the invasive and migratory property A549 cells and it also induces apoptosis via inhibiting Pi3K/AKT/mTOR signaling, activating the tumor suppressor protein E-cadherin signifying that dieckol is potent natural anticancer drug to treat NSCLC.

[Analysis of miRNA differential expression in peripheral blood cells of primary immune thrombocytopenia patients].

This study was purposed to clarify the difference of microRNA (miRNA) expression in the peripheral blood cells of patients with primary immune thrombocytopenia (ITP) and normal controls. Exqion miRCURY(TM) microarray was used to investigate differentially expressed miRNA of peripheral blood cells obtained from affected ITP patients and the healthy controls. Cluster analysis was used to identify miRNA expression profile between the ITP patients and the healthy controls. Real-time PCR was used for validation. The results showed that a total of 159 miRNA were found to be differentially expressed in ITP patients compared to the controls, with 79 up-regulated and 80 down-regulated. Based on these differentially expressed miRNA, a tree with clear distinction between the controls and ITP patients was generated by cluster analysis. Real-time PCR confirmed microarray analysis results. It is concluded that differentially expressed miRNA were found in the peripheral blood cells from ITP patients, which may be potential novel biomarkers for ITP as well as help to elucidate pathogenic mechanisms of ITP.

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3 (glycoprotein IIb/IIIa). PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family. OBJECTIVES: To determine the molecular basis of GT and characterize the mutation by in vitro expression studies. RESULTS: Analysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of beta3, exposed on her platelet surface, but a complete absence of alphaIIbbeta3. Sequence analysis revealed a novel C470A transversion in exon 4 of the alphaIIb gene predicting a Pro126His alteration in the blade 2 of the alphaIIb beta propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alphaIIb and wild-type beta3 failed to express alphaIIbbeta3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature alphaIIb. Immunoprecipitation with antibody against beta3 demonstrated pro-alphaIIb in the cells expressing the mutant alphaIIbbeta3, indicating pro-alphaIIbbeta3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-alphaIIbbeta3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker. CONCLUSIONS: A novel Pro126His mutation in alphaIIb compromised transport of the pro-alphaIIbbeta3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired alphaIIbbeta3 transport is responsible for the thrombasthenia in this patient.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alphaII b P126H mutation: an in vitro experiment].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha IIb P126H mutation. METHODS: Eukaryotic vector of alpha IIb P126H was constructed by PCR site-directed mutagenesis and then co-transfected with eukaryotic vector PCDM8 II a expressing the subunit beta3 into human renal epithelial cells of the line 293& and Chinese hamster ovarian cancer cells of the line CHO after sequencing. The membrane expression of alpha IIb P126H mutant was analyzed by flow cytometry and the whole expression was confirmed by Western blotting. The alpha IIb P126H mutant subcellular localization was determined by laser confocal scanning microscopy. RESULTS: The 293T cells cotransfected with cDNAs of mutated alpha IIb and wild-type beta3 failed to express alpha II bbeta3 on the cell surface as shown by FACS. Western blotting of the cell lysate showed no detectable mature alpha IIb. Immunofluorescence studies demonstrated proa II bbeta3 complex colocalized with an endoplasmic reticulum (ER) marker, but showed minimal colocalization with an Golgi marker. CONCLUSION: The P126H mutation in alpha IIb prevents transport of the pro-alpha II bbeta3 complex from ER to the Golgi body, thus hindering its maturation and surface expression. The impaired alpha II bp33 transport is responsible for the thrombasthenia.

[Analysis of clinical features and genotype in three Chinese pedigrees with Glanzmann thrombasthenia].

OBJECTIVE: To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees. METHODS: Platelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls. RESULTS: Three probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3. CONCLUSIONS: Compound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation. METHODS: All exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy. RESULTS: The alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi. CONCLUSIONS: The L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.

[Antithrombotic mechanisms of holothurian glycosaminoglycan extracted from sea cucumber].

OBJECTIVE: To investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber. METHODS: Human endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity. RESULTS: GAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG. CONCLUSIONS: GAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.