LILRB4 on multiple myeloma cells promotes bone lesion by p-SHP2/NF-κB/RELT signal pathway.

BACKGROUND: Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion. METHODS: The conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma. RESULTS: We comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient's imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma. CONCLUSIONS: Our findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion.

Targeting the Immunoglobulin IGSF9 Enhances Antitumor T-cell Activity and Sensitivity to Anti-PD-1 Immunotherapy.

Immune checkpoints modulate the immune response and represent important immunotherapy targets for cancer treatment. However, as many tumors are resistant to current immune checkpoint inhibitors, the discovery of novel immune checkpoints could facilitate the development of additional immunotherapeutic strategies to improve patient responses. Here, we identified increased expression of the adhesion molecule immunoglobulin superfamily member 9 (IGSF9) in tumor cells and tumor-infiltrating immune cells across multiple cancer types. IGSF9 overexpression or knockout in tumor cells did not alter cell proliferation in vitro or tumor growth in immunocompromised mice. Alternatively, IGSF9 deficient tumor cells lost the ability to suppress T-cell proliferation and exhibited reduced growth in immunocompetent mice. Similarly, growth of tumor cells was reduced in IGSF9 knockout syngeneic and humanized mice, accompanied by increased tumor-infiltrating T cells. Mechanistically, the extracellular domain (ECD) of IGSF9 bound to T cells and inhibited their proliferation and activation, and the tumor-promoting effect of IGSF9 ECD was reversed by CD3+ T-cell depletion. Anti-IGSF9 antibody treatment inhibited tumor growth and enhanced the antitumor efficacy of anti-programmed cell death protein 1 immunotherapy. Single-cell RNA sequencing revealed tumor microenvironment remodeling from tumor promoting to tumor suppressive following anti-IGSF9 treatment. Together, these results indicate that IGSF9 promotes tumor immune evasion and is a candidate immune checkpoint target. SIGNIFICANCE: IGSF9 is an immune checkpoint regulator that suppresses T-cell activation in cancer and can be targeted to stimulate antitumor immunity and inhibit tumor growth.

Correction to: LILRB4 ITIMs mediate the T cell suppression and infiltration of acute myeloid leukemia cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LILRB4 ITIMs mediate the T cell suppression and infiltration of acute myeloid leukemia cells.

We recently demonstrated that leukocyte Ig-like receptor 4 (LILRB4) expressed by monocytic acute myeloid leukemia (AML) cells mediates T-cell inhibition and leukemia cell infiltration via its intracellular domain. The cytoplasmic domain of LILRB4 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs); the tyrosines at positions 360, 412, and 442 are phosphorylation sites. Here, we analyzed how the ITIMs of LILRB4 in AML cells mediate its function. Our in vitro and in vivo data show that Y412 and Y442, but not Y360, of LILRB4 are required for T-cell inhibition, and all three ITIMs are needed for leukemia cell infiltration. We constructed chimeric proteins containing the extracellular domain of LILRB4 and the intracellular domain of LILRB1 and vice versa. The intracellular domain of LILRB4, but not that of LILRB1, mediates T-cell suppression and AML cell migration. Our studies thus defined the unique signaling roles of LILRB4 ITIMs in AML cells.

Disrupting LILRB4/APOE Interaction by an Efficacious Humanized Antibody Reverses T-cell Suppression and Blocks AML Development.

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration.

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.

A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML.

To effectively improve treatment for acute myeloid leukemia (AML), new molecular targets and therapeutic approaches need to be identified. Chimeric antigen receptor (CAR)-modified T cells targeting tumor-associated antigens have shown promise in the treatment of some malignancies. However, CAR-T cell development for AML has been limited by lack of an antigen with high specificity for AML cells that is not present on normal hematopoietic stem cells, and thus will not result in myelotoxicity. Here we demonstrate that leukocyte immunoglobulin-like receptor-B4 (LILRB4) is a tumor-associated antigen highly expressed on monocytic AML cells. We generated a novel anti-LILRB4 CAR-T cell that displays high antigen affinity and specificity. These CAR-T cells display efficient effector function in vitro and in vivo against LILRB4+ AML cells. Furthermore, we demonstrate anti-LILRB4 CAR-T cells are not toxic to normal CD34+ umbilical cord blood cells in colony-forming unit assays, nor in a humanized hematopoietic-reconstituted mouse model. Our data demonstrate that anti-LILRB4 CAR-T cells specifically target monocytic AML cells with no toxicity to normal hematopoietic progenitors. This work thus offers a new treatment strategy to improve outcomes for monocytic AML, with the potential for elimination of leukemic disease while minimizing the risk for on-target off-tumor toxicity.

Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown. METHODS: Immunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2. RESULTS: In this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression. CONCLUSION: Our data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.

Target genes regulated by transcription factor E2F1 in small cell lung cancer.

Previously, we have reported that transcription factor E2F1 expression is up-regulated in approximately 95% of small cell lung cancer tissue samples and closely associated with invasion and metastasis, but few studies have investigated specific target genes regulated by E2F1 in this disease. The aim of this study was to clarify the target genes controlled by E2F1 in the small cell lung cancer cell line H1688. The results of chromatin immunoprecipitation sequencing (ChIP-seq) showed that total 5 326 potential target genes were identified, in which 4 700 were structural genes and 626 long non-coding RNAs (lncRNAs). Gene Ontology (GO) and enrichment map analysis results indicated that these target genes were associated with three main functions: (1) cell cycle regulation, (2) chromatin and histone modification, and (3) protein transport. MEME4.7.0 software was used to identify the E2F1 binding DNA motif, and six motifs were discovered for coding genes and lncRNAs. These results clarify the target genes of E2F1, and provide the experimental basis for further exploring the roles of E2F1 in tumorigenesis, development, invasion and metastasis, recurrence, and drug resistance in small cell lung cancer.

Expression of ADAM12 is regulated by E2F1 in small cell lung cancer.

Our previous study reported that ADAM12 was highly expressed in small cell lung cancer (SCLC) and could be an effective marker for diagnosis and prognosis. Yet, the reason for the high expression of ADAM12 in SCLC requires further elucidation. Transcription factor E2F1 has been receiving increasing attention due to the complexity and diversity of its function in cancer. In the present study, the expression of ADAM12 was significantly decreased following silencing of E2F1 expression by siRNA, thus indicating that E2F1 may regulate the expression of ADAM12 at the level of transcription. Chromatin immunoprecipitation-to-sequence analysis identified three binding sites for E2F1 in the locus for ADAM12. They were Chr10: 128010444-128011026, located in the intron of ADAM12, named seq0; Chr10: 128076927­128078127, located in the promoter of ADAM12, named seq1; and Chr10: 128086195­128086876, located in the upstream 20 kb from the transcription start site of ADAM12, named: seq2. Dual­luciferase reporter experiments revealed that seq1 not seq0 and seq2 was able to promote the expression of luciferase. Notably, co-transfection of E2F1 significantly increased the activity of seq1 not seq0 and seq2, but quantitative polymerase chain reaction results showed that seq0, seq1 and seq2 could recruit E2F1, indicating that the influence of E2F1 in regulating the expression of ADAM12 was complex. Sequence analysis clarified that seq1 was a part of the ADAM12 promoter, yet the functions of seq0 and seq2 were unknown. Fusion fragments containing seq0-seq1 or seq2-seq1 were analyzed in luciferase constructs. Compared with seq1 alone, the activities of these fusion fragments were non-significantly reduced. The activities of fusion fragments were significantly decreased following co-transfection with E2F1. Thus, the present findings support the conclusion that the E2F1 transcription factor regulates the expression of ADAM12 by binding differential cis-acting elements.

Differential regulation of MMPs by E2F1, Sp1 and NF-kappa B controls the small cell lung cancer invasive phenotype.

BACKGROUND: E2F1 transcription factor plays a vital role in the regulation of diverse cellular processes including cell proliferation, apoptosis, invasion and metastasis. E2F1 overexpression has been demonstrated in small cell lung cancer (SCLC), and extensive metastasis in early phase is the most important feature of SCLC. In this study, we investigated the involvement of E2F1 in the process of invasion and metastasis in SCLC by regulating the expression of matrix metalloproteinases (MMPs). METHODS: Immunohistochemistry was performed to evaluate the expression of E2F1 and MMPs in SCLC samples in a Chinese Han population. The impact of E2F1 on invasion and metastasis was observed by transwell and wound healing experiments with depletion of E2F1 by specific siRNA. The target genes regulated by E2F1 were identified by chromatin immunoprecipitation (ChIP)-to-sequence, and the expressions of target genes were detected by real time PCR and western blotting. The dual luciferase reporter system was performed to analyze the regulatory relationship between E2F1 and MMPs. RESULTS: E2F1 is an independent and adverse prognosis factor that is highly expressed in SCLC in a Chinese Han population. Knockdown of E2F1 by specific siRNA resulted in the downregulation of migration and invasion in SCLC. The expressions of MMP-9 and -16 in SCLC were higher than other MMPs, and their expressions were most significantly reduced after silencing E2F1. ChIP-to-sequence and promoter-based luciferase analysis demonstrated that E2F1 directly controlled MMP-16 expression via an E2F1 binding motif in the promoter. Although one E2F1 binding site was predicted in the MMP-9 promoter, luciferase analysis indicated that this binding site was not functionally required. Further study demonstrated that E2F1 transcriptionally controlled the expression of Sp1 and p65, which in turn enhanced the MMP-9 promoter activity in SCLC cells. The associations between E2F1, Sp1, p65, and MMP-9 were validated by immunohistochemistry staining in SCLC tumors. CONCLUSIONS: E2F1 acts as a transcriptional activator for MMPs and directly enhances MMP transcription by binding to E2F1 binding sequences in the promoter, or indirectly activates MMPs through enhanced Sp1 and NF-kappa B as a consequence of E2F1 activation in SCLC.

ADAM-12 as a diagnostic marker for the proliferation, migration and invasion in patients with small cell lung cancer.

Small cell lung cancer (SCLC) is highly aggressive and is characterized by malignant metastasis. Approximately 90% of patients die due to extensive metastasis. The extracellular matrix (ECM) is a natural barrier that can prevent cellular invasion and metastasis. Therefore, degradation of the ECM must take place in order for extensive metastasis to occur. A disintegrin and metalloprotease (ADAM) is a multi-domain protease that plays an important role in tumorigenesis, as well as tumor development, invasion and metastasis. However, there have been few reports on the expression and role of ADAMs in SCLC. In the current study, the expression and role of ADAMs in SCLC proliferation, invasion and metastasis was investigated. A total of 150 SCLC tissue samples were examined by immunohistochemistry for ADAMs expression. ADAM-12 was found to be abundantly expressed in 72.67% samples and other ADAMs were found to be expressed in 10% to 40% of samples. ADAM-12 levels in serum and urine, from 70 SCLC patients and 40 normal controls, were also measured using ELISA. ADAM-12 expression was significantly higher in SCLC patients than in healthy controls and in patients with extensive disease compared to those with more limited disease. Silencing the expression of ADAM-12 in H1688 cells through the use of specific siRNA significantly reduced cellular proliferation, invasion and metastasis. Supplementing the expression of ADAM-12-L or -S in H345 cells, significantly enhanced cellular proliferation, invasion and metastasis. Animal models with metastatic SCLC also exhibited increased expression of ADAM-12 along with enhanced invasion and metastasis. In brief, ADAM-12 is an independent prognostic factor and diagnostic marker, and is involved in the proliferation, invasion and metastasis of SCLC.

Effect of dynamic DNA methylation and histone acetylation on cPouV expression in differentiation of chick embryonic germ cells.

As a crucial pluripotency-related factor, the epigenetic regulation of Oct4 has been studied intensively in mammalians. However, its dynamic changes of DNA methylation and histone modification in avians remain poorly understood. In the present study, we first described the alterations of DNA methylation and histone acetylation in the promoter of chicken PouV (cPouV; the homologue of Oct4 in avian) during chick embryonic germ (EG) cell differentiation. The epigenetic modification analysis showed that DNA methylation in the cPouV promoter increased obviously, while histone acetylation decreased dramatically detected by chromatin immunoprecipitation assay in the process of differentiation. Gene expression analysis detection indicated that the levels of DNA methyltransferase 3a (Dnmt 3a), Dnmt 3b, and histone deacetylase 3 (HDAC 3) transcripts were significantly high, whereas the relative abundance of Dnmt 1, histone acetyltransferase (HAT), and cPouV mRNA was significantly decreased during the conversion of EG to embryoid body-like structures (EBs), which was correlated with the increased level of methylation and reduced level of H3 acetylation. Moreover, in vitro methylation assay indicated that the reporter gene was remarkably inhibited by the methylated promoter of cPouV. To further understand the effect of epigenetic modifiers on cPouV expression, we performed an analysis of EB cells treated with trichostatin A (TSA), Aza-2'-deoxycytidine (Aza), or TSA plus Aza (TSA/Aza). We observed that the effect of TSA/Aza is more sensitive to the reactivation of cPouV compared with TSA or Aza, indicating that these epigenetic inhibitors can function synergistically to facilitate the reprogramming process. The present study provided evidences that a critical role for cPouV activation/repression by DNA methylation and/or histone modifications is involved in the pluripotency maintenance and differentiation process of chick EG.

Knockdown of AGR2 induces cellular senescence in prostate cancer cells.

Anterior-gradient 2 (AGR2), overexpressed in many tumors including prostate cancer (PCa), is implicated in stimulation of cell proliferation, adhesion, anti-apoptosis and cell cycle regulation. Here, a potential role of AGR2 in cellular senescence was investigated. We first observed that AGR2 was overexpressed in Chinese Han PCa tissues and had a positive correlation with cyclin D1 and p-Rb but not with p16(INK4a). AGR2 expression profiles varied among cell lines, with PC3 cells being the highest level, LNCaP and DU145 relatively less. The expression of cyclin D1 showed similar pattern to the AGR2 in cell lines. Knockdown of AGR2 caused a decrease in cell viability in PC3 cells, whereas forced expression of AGR2 led to an increased cell proliferation of LNCaP and DU145 cells. Importantly, AGR2 depletion resulted in accumulation of cells at the G(0)/G(1) phase and induction of cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal) positive cells. Senescent response to AGR2 silencing was also evidenced by elevated γH2AX and fluorescent punctuate formation of tri-methyl-histone H3 in AGR2-depleted cells. Further studies indicated that LNCaP underwent a p21(CIP1)-dependent cellular senescence in response to AGR2 depletion that requires inactivation of ERK signaling, whereas PC-3 was also p21(CIP1) dependent but involved in suppression of PI3K/Akt. Unlike LNCaP and PC-3, senescent response of DU145 was found to be mainly p27(KIP1) dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling. Thus, these findings suggest a novel role of AGR2 in regulation of cellular senescence.

[Cyclin D1 regulates lung cancer invasion and metastasis].

Cyclin D1, as a regulatory factor in cell cycle, is highly expressed in many tumors, such as lung cancer, breast cancer and thyroid cancer. The aim of the present study was to study the role of Cyclin D1 in invasion and metastasis of lung cancer cells. Lung adenocarcinoma cell line A549 and squamous cell line SK-MES-1 were selected as the objects, because A549 expresses Cyclin D1 highly, and SK-MES-1 expresses lowly. Nude mice were injected with A549 or SK-MES-1 via tail vein, and were sacrificed after 4 weeks for cancer tissue isolation. The harvested cancer cells were reinjected into another nude mouse. After one more time of such seeding, highly metastatic lung cancer model was established. After A549 and SK-MES-1 were transfected with Cyclin D1 RNAi and expression vector respectively, transwell migration assay was used to analyze transferring capacity of lung cancer cells. Western blot was used to detect Cyclin D1 and WNT/TCF pathway proteins expressions in parental cell lines and cancer tissue from metastasis model animals. The results showed that, along with the increase of seeding times, lung cancer cells from model animals, no matter A549 or SK-MES-1, exhibited augmented metastasis activity and up-regulated Cyclin D1 expression. The transferring capacity was weakened significantly in A549 cells where the Cyclin D1 was interfered by RNAi, and it was enhanced significantly in SK-MES-1 cells which were transfected with the expression vector of Cyclin D1. The expressions of WNT/TCF pathway proteins, including ß-catenin, lymphoid enhancer-binding factor (LEF) and T cell factor (TCF), increased significantly in highly metastatic model animals. The parental cell lines showed lower expressions of WNT/TCF pathway proteins compared with cancer tissue from metastasis model animals. These results suggest that Cyclin D1 is closely related with the invasion and metastasis of lung cancer cells, and the WNT/TCF signal pathway may promote the expression of Cyclin D1.

[Anti-sense nucleic acid of CyclinD1 induces apoptosis of lung adenocarcinoma cancer cell A549].

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.

p53 status and its prognostic role in extrahepatic bile duct cancer: a meta-analysis of published studies.

The dysfunction of p53 is the most common genetic alteration in human cancer. A variety of studies have investigated the clinicopathologic correlation of p53 and its impact on patient survival in different types of cancer. For extrahepatic bile duct cancer (EBDC), however, the results were limited and conflicting. In this study, we performed an investigation to confirm whether there was a correlation between p53 status and some routine parameters. To further observe the impact of p53 on the survival of EBDC patients, a meta-analysis based on published studies was conducted. Candidate studies were searched from PubMed, EMBASE, and ISI Web of Science. Our results demonstrated that there were significant correlations between p53 expression and some clinicopathological parameters. Furthermore, the pooled results of the meta-analysis showed that the combined hazard ratio (HR) estimate for overall survival (OS) was 1.53 (95% CI, 1.10-2.14) and 1.23 (95% CI, 0.93-1.75) in univariate and multivariate analysis, respectively. In conclusion, the high level of p53 appears to be an effective prognostic factor to OS of EBDC patients. However, some limitations unavoidable in this meta-analysis and problems of previous p53 studies in EBDC mean that further studies are necessary before significant conclusions can be made.

Acute stress and chronic stress change brain-derived neurotrophic factor (BDNF) and tyrosine kinase-coupled receptor (TrkB) expression in both young and aged rat hippocampus.

PURPOSE: The purpose of this study is to explore the dynamic change of brainderived neurotrophic factor (BDNF) mRNA, protein, and tyrosine kinase-coupled receptor (TrkB) mRNA of the rat hippocampus under different stress conditions and to explore the influence of senescence on the productions expression. MATERIALS AND METHODS: By using forced-swimming in 4 degrees C cold ice water and 25 degrees C warm water, young and aged male rats were randomly divided into acute stress (AS) and chronic mild repeated stress (CMRS) subgroups, respectively. BDNF productions and TrkB mRNA in the hippocampus were detected by using Western-blotting and reverse transcription-polymerase chain reaction (RT-PCR), separately, at 15, 30, 60, 180, and 720 min after the last stress session. RESULTS: The short AS induced a significant increase in BDNF mRNA and protein in both age groups, but the changes in the young group were substantially greater than those of the aged group (p < 0.005). The CMRS resulted in a decrease in BDNF mRNA and protein, but a significant increase in TrkB mRNA in both young and age groups. The expression of BDNF mRNA and protein in the AS groups were higher than in the CMRS groups at 15, 30, and 60 min after stress. CONCLUSION: The results indicated that the up/down-regulation of BDNF and TrkB were affected by aging and the stimulus paradigm, which might reflect important mechanisms by which the hippocampus copes with stressful stimuli.

Regulating A549 cells growth by ASO inhibiting miRNA expression.

MicroRNAs (miRNAs) have a profound impact on cell processes, including proliferation, apoptosis, and stress responses. We aimed to explore the role of antisense oligonucleotide (ASO) to induce proliferation or apoptosis of A549 cancer cells by inhibiting the expression of miRNAs. After A549/HBE/293T cells were treated with ASO, cells proliferation/apoptosis, and their relevant oncogenes/tumor suppressor genes were detected by light and electron microscopy, real-time PCR, enzyme-linked immunosorbent assay, etc. The results showed that ASO could inhibit the expression of miRNAs effectively. miR-16, miR-17, miR-34a-c, and miR-125 served as tumor suppressor miRNAs, while miR-20, miR-106, and miR-150 acted as oncogenic miRNAs. Our results also indicated that miR-16/34a-c, miR-17-5p, miR-125, miR-106, and miR-150 were the upstream factors, which could regulate the expression of BCL-2, E2F1, E2F3, RB1, and P53, respectively. After A549 cells treated with ASO for 24 h and different concentrations of anti-cancer drug (cisplatin or demethylcantharidin) were added into culture medium, the results indicated the percentage of alive cells in group treated with both ASO-106 (or ASO-150) and anti-cancer drug was lower than that in group treated with ASO, or anti-cancer drug, or both ASO-16 (or ASO-34a) and anti-cancer drug. In conclusion, ASO (specific to oncogenic miRNAs) could induce A549 cells apoptosis by inhibiting oncogenic miRNAs, and could increase chemotherapy sensitivity of A549 cells to anti-cancer drug, which holds great promise to lung cancer therapy.

Familial congenital cyanosis caused by Hb-M(Yantai)(α-76 GAC → TAC, Asp → Tyr).

Methemoglobin (Hb-M) is a rare hemoglobinopathy in China. We hereby report on a family living in Yantai, East China, with congenital cyanosis due to Hb-M mutation. The proband, a 65-year-old female, presented 63% oxygen saturation. Both Hb-M concentration and arterial oxygen saturation remained unchanged, even following intravenous treatment with methylene blue. There was also no change in blood-color (chocolate-brown) after adding 0.1% KCN. A fast-moving band (Hb-X) in hemolysates was found by cellulose acetate electrophoresis, the Hb-X/Hb-A ratio exceeding 10%. GT transition at 131nt of exon 2, although present in one of the α(2) -globin alleles, was not found in α(1) -globin alleles as a whole. This mutation leads to the aspartic acid to tyrosine substitution (Asp76Tyr). In this family, the novel mutation in the α(2) -globin gene resulted in a rare form of congenital cyanosis due to Hb-M. This hemoglobin was named Hb-M (Yantai) .