ELK1-Induced upregulation of long non-coding TNK2-AS1 promotes the progression of acute myeloid leukemia by EZH2-mediated epigenetic silencing of CELF2.

Acute myeloid leukemia (AML) is the second most common hematological malignancy after lymphoma in the world. Long non-coding RNAs (LncRNAs) have been suggested as key regulators of cancer development and progression in AML. As a member of lncRNA family, the biological role and mechanisms of tyrosine kinase non receptor 2 antisense RNA 1 (TNK2-AS1) in AML is still unclear. The expression of TNK2-AS1 was measured with RT-qPCR in AML cell lines. The changes of the proliferation, apoptosis, and differentiation in TNK2-AS1 shRNA-transfected HL-60 and THP-1 cells were detected with CCK-8, EdU, flow cytometry, Western blot, and NBT assays. Molecular control of TNK2-AS1 on CUGBP Elav-like family member 2 (CELF2) and ETS domain-containing protein-1 (ELK1) on TNK2-AS1 was assessed by chromatin immunoprecipitation (ChIP), RT-qPCR, Western blot, and RNA immunoprecipitation (RIP) assays. TNK2-AS1 expression was upregulated in AML cell lines and negatively correlated with survival patients. Knockdown of TNK2-AS1 markedly reduced AML cell proliferation and promoted apoptosis and differentiation. Likewise, TNK2-AS1 knockdown significantly suppressed tumor growth in vivo. Mechanistically, the upregulation of TNK2-AS1 was activated by transcription factor ELK1. We also uncovered that TNK2-AS1 exerted tumor-promoting effect through silencing CELF2 via binding with EZH2, thus activating PI3K/Akt pathway in AML cells. Elevated expression of TNK2-AS1 was induced by ELK1 and facilitated AML progression by suppressing CELF2 expression via EZH2-mediated epigenetic silencing, suggesting TNK2-AS1 may be a promising therapeutic target and prognostic marker for AML patients.

Hsa_circ_0056686, derived from cancer-associated fibroblasts, promotes cell proliferation and suppresses apoptosis in uterine leiomyoma through inhibiting endoplasmic reticulum stress.

Abnormal expression of circular RNAs (circRNAs) in cancer-associated fibroblasts (CAFs) is involved in the tumor-promoting ability of CAFs. Hsa_ circ_ 0056686 has been reported to affect leiomyoma size. The purpose of this study is to investigate the regulatory role of hsa_circ_0056686 in CAFs on uterine leiomyoma (ULM). The primary CAFs and corresponding normal fibroblasts (NFs) were isolated from the tumor zones of ULM tissues and adjacent, respectively. Hsa_circ_0056686 level was higher in CAFs than NFs, and also higher in ULM tissues than in adjacent tissues. CAFs-CM significantly increased the proliferation and migration and inhibited apoptosis of ULM cells, as confirmed by CCK-8, transwell, and flow cytometry assays. Moreover, conditioned medium (CM) from CAFs transfected with hsa_circ_0056686 shRNA (CAFssh-circ_0056686-CM) abolished CAFs-mediated proliferation, migration and apoptosis of ULM cells. CAFs-CM suppressed the expression of endoplasmic reticulum stress (ERS) marker proteins and induced the expression of extracellular matrix (ECM) marker proteins, thus suppressing ERS and increasing ECM accumulation, which could be declined by CAFssh-circ_0056686-CM. Meanwhile, knockdown of hsa_circ_0056686 reversed the inhibitory effects of CAFs-CM on brefeldin A-induced cell apoptosis. Luciferase gene reporter and RNA pull-down assays indicated that miR-515-5p directly bound with hsa_circ_0056686. MiR-515-5p overexpression restored the hsa_circ_0056686-shRNA-mediated malignant biological behaviors of ULM cells. Hsa_circ_0056686 contributed to tumor-promoting effects of CAFs in ULM, manifested by promoting ULM cell proliferation and migration and reducing ERS-induced apoptosis through sponging miR-515-5p.

Brain and testicular metabonomics revealed the protective effects of Guilingji on senile sexual dysfunction rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ), which has been used to treat male diseases in China for centuries, contains 28 Chinese herbs and was previously established as an effective treatment for male sexual dysfunction. However, its mechanism of action remains unclear. AIM OF THE STUDY: To explore the efficacy and mechanism of action of GLJ in improving senile sexual dysfunction (SSD) in aging rats. MATERIALS AND METHODS: An aging rat model of SSD was induced by the subcutaneous injection of d-galactose (300 mg⋅kg-1) and used to analyse the effects of GLJ (different concentrations of 37.5, 75, and 150 mg⋅kg-1) on the mating of aging rats. At the end of the 8th week, histopathological analysis of testicular tissues, assessment of the hypothalamic-pituitary-gonadal (HPG) axis hormone levels in serum or brain, and metabonomics analysis of the brain and testicular tissue with liquid chromatography-mass spectrometry was performed to explore the mechanism of action of GLJ. RESULT: After treatment with GLJ, the mount and ejaculation latency levels were increased in the treatment group than those in model group (P < 0.05), moreover, the testicular morphology was improved. Gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) levels in rats were also improved significant (P < 0.05) compared with those in the model group. Furthermore, the metabonomics results in the testicular and brain tissue showed that GLJ improved SSD by adjusting amino acid and lipid metabolism. CONCLUSION: This study integrated the complementary metabolic profiles of the target tissues. GLJ might affect SSD rats by regulating amino acid and lipid metabolism and may modulate sensitivity to the signaling pathway in the HPG axis. This study provides an essential basis for the broad clinical application of GLJ.

MiR-30c-5p Directly Targets MAPK1 to Regulate the Proliferation, Migration and Invasion of Adenomyotic Epithelial Cells in Adenomyosis.

The purpose of our study was to elucidate the functions of miR-30c-5p on adenomyosis for exploring novel treatment strategies. We first detected the expression of miR-30c-5p in clinical adenomyotic tissues and isolated endometrial cells from adenomyotic tissues. Next, gain and loss-of-function assays were performed to detect the effect of miR-30c-5p on adenomyotic endometrial cells. Further, luciferase assay and real-time polymerase chain reaction as well as western blot were conducted to investigate the potential target of miR-30c-5p; and transwell assay, wound-healing assay and CCK-8 assay were used to evaluate the effects of miR-30c-5p and its target on regulating biological functions of adenomyotic endometrial cells. Our results found that miR-30c-5p was down-regulated in both adenomyosis tissues and adenomyotic epithelial cells, which correlated with dysmenorrhea, longer duration of symptoms and more menstrual bleeding. Moreover, the overexpression of miR-30c-5p inhibited the proliferation, migration and invasion of adenomyotic epithelial cells, where miR-30c-5p knockdown had an opposite effect. Furthermore, we confirmed mitogen-activated protein kinase 1 (MAPK1) was one of the direct targets of miR-30c-5p, indicating its important role in miR-30c-5p-mediated suppression of proliferation, invasion and migration in adenomyotic epithelial cells. This study showed that the interaction of miR-30c-5p with MAPK1 can regulate the proliferation, invasion and migration in adenomyotic epithelial cells.

Attenuation of lipid accumulation in Bel-7402 cells through ADPN/AMPKα signaling stimulated by Fructus rosae laxae extract.

In this work, a comparison study was conducted on the contents of total flavonoids and hyperoside in different polarity extracts of Fructus rosae laxae (FRL). The lipid-lowering effect and mechanism of FRL ethyl acetate extract (FRLE) on the lipid accumulation model of Bel-7402 cells in vitro were studied. The results showed that the contents of total flavonoids and hyperoside in FRLE were significantly higher than those in the other polarity extracts. Compared with those in the model group, the levels of triglyceride and total cholesterol decreased, the activities of superoxide dismutase and lactate dehydrogenase increased, and the levels of inflammatory factors interleukin-6 and tumor necrosis factor-α decreased significantly in the cells intervened with FRLE. Moreover, FRLE can regulate lipid metabolism by activating the AMP-activated protein kinase α phosphorylation pathway and increasing the expression of adiponectin. PRACTICAL APPLICATIONS: Fructus rosae laxae (FRL) is an edible medicinal fruit with multiple biological activities, such as antioxidation, anti-inflammatory, and hepatoprotective properties. However, the lipid-lowering activity of FRL and its mechanism of action have not yet been investigated. Our data indicate that the FRL extract, which contains high levels of antioxidant and anti-inflammatory components, plays a beneficial role in regulating lipid metabolism disorders, mainly by regulating the expression of proteins involved in the ADPN/AMPK signaling pathway, and reduces the release of inflammatory factors. Thus, the FRL extract effectively reduces the accumulation of free fatty acids (FFA) in vitro and exhibits considerable potential for the prevention and treatment lipid metabolism disorders.

Chemical profliling of Dingkun Dan by ultra High performance liquid chromatography Q exactive orbitrap high resolution mass spectrometry.

Dingkun Dan (DKD) has been widely used for a variety of gynecological disease. However, the systematic analysis of the chemical constituents of DKD has not been well established because of the complexity of the formula and confidentiality. In this paper, liquid chromatography Q Exactive high resolution accurate mass spectrometry (UHPLC-QE-HRMS) with automated MetaboLynx analysis was established to characterize the chemical constituents of DKD. The analysis was performed on a Water Acquity UPLC® HSS T3 using a gradient elution system. Full scan ranged 100-1500 m/z in positive and negative ion mode combined with MS/MS fragmentation for top 5 ions was proposed for aiding the structural identification of the components. All of the peaks were tentatively characterized by not only comparing the retention time and MS data with those from reported literature and database, but also summarizing the fragmentation pathways and promoting to other ingredients identification. Additionally, the network pharmacology study had been used to analysis the identified ingredients and DKD's clinical diseases. In this work, a total of 121 components and isomers were characterized, including amino acids, phenolic acids, lactones, terpenoids, alkaloids, saponins, flavonoids, and other compounds. Network pharmacology analysis showed that identified compounds, such as ginsenosides and notoginsenosides, crocin I, echinacoside, rutin and verbascoside, could be responsible for the pharmacological activity of DKD by regulating the hormone with related metabolism pathways, estrogen signaling pathways and serotonergic synapse pathways. It could indicate that UHPLC-MS showed obvious superiority used to find the potential bioactive compounds of complicated TCM formula without the process of extraction and isolation.

Suppression of HSP70 inhibits the development of acute lymphoblastic leukemia via TAK1/Egr-1.

Acute lymphoblastic leukemia (ALL), usually treated with chemotherapy, has limited therapeutic effects and high toxicity. Upregulation of HSP70 induces tumor development, however, the molecular mechanism of HSP70 in ALL remains unclear. In our research, we aimed to investigate the role of HSP70 in ALL, specifically the molecular mechanisms underlying cell apoptosis and proliferation. We found that HSP70 expression in leukomonocytes from ALL patients was increased compared with the control group. HSP70 expression in NALM-6 and BE-13 was also up-regulated contrast with AHH-1. Inhibition of HSP70 significantly promoted cell apoptosis and suppressed cell proliferation in ALL cell lines. Suppression of HSP70 decreased TAK1 and increased Egr-1 protein expression. Further experiments indicated that overexpression of TAK1 ameliorated the effect of HSP70 inhibition on Egr-1 protein expression, cell apoptosis and proliferation. In order to determine whether the effect of HSP70 inhibition on apoptosis and proliferation of ALL cell lines could be achieved via regulation of Egr-1, we performed a loss-of-function experiment for Egr-1. Egr-1 suppression was found to reverse the effect of HSP70 inhibition on cell apoptosis and proliferation in ALL. Taken together, our results suggest that HSP70 inhibition upregulates Egr-1 via TAK1, inducing apoptosis and restricting proliferation in ALL cells.

Proteomics of acute heart failure in a rat post-myocardial infarction model.

The aim of the present study was to identify the mechanisms underlying the development of post-myocardial infarction (post-MI) heart failure. The left anterior descending coronary artery of rats was occluded to mimic human ischemic heart disease. Linear Trap Quadropole OrbiTrap mass spectrometry was used to profile the expressions of energy metabolism­associated and calcium­binding proteins in the post­MI and control groups. Using the online Protein Analysis Through Evolutionary Relationships classification system, 78 differentially expressed proteins were identified, including 50 downregulated proteins and 28 upregulated proteins in post­MI group when compared with the control group. The differentially expressed proteins were closely associated with energy metabolism, contractile function, calcium handling, pathological hypertrophy and cardiac remodeling. These results were further validated using western blotting. At different postoperative time points (1st and 14th day following surgery) during the progression of advanced heart failure post­MI, dynamic alterations in differential protein expression were identified. The expression of the vitamin D protein was significantly upregulated on the 1st day post­MI however, was then downregulated with progression of the disease on the 14th day post­MI. These results identified various target proteins associated with the disease, which may be used as diagnostic markers.

TLR-4/miRNA-32-5p/FSTL1 signaling regulates mycobacterial survival and inflammatory responses in Mycobacterium tuberculosis-infected macrophages.

Macrophages play a pivotal role in host immune response against mycobacterial infection, which is tightly modulated by multiple factors, including microRNAs. The purpose of the present study was to investigate the biological function and potential mechanism of miR-32-5p in human macrophages during Mycobacterium tuberculosis (M.tb) infection. The results demonstrated that miR-32-5p was robustly enhanced in THP-1 and U937 cells in response to M.tb infection. TLR-4 signaling was required for upregulation of miR-32-5p induced by M.tb infection. Additionally, the introduction of miR-32-5p strongly increased the survival rate of intracellular mycobacteria, whereas inhibition of miR-32-5p suppressed intracellular growth of mycobacteria during M.tb challenged. Furthermore, forced expression of miR-32-5p dramatically attenuated the accumulation of inflammatory cytokines IL-1ß, IL-6 and TNF-α induced by M.tb infection. Conversely, downregulated expression of miR-32-5p led to enhancement in these inflammatory cytokines. More importantly, our study explored that Follistatin-like protein 1 (FSTL1) was a direct and functional target of miR-32-5p. qRT-PCR and western blot analysis further validated that miR-32-5p negatively regulated the expression of FSTL1. Mechanistically, re-expression of FSTL1 attenuated the ability of miR-32-5p to promote mycobacterial survival. Meanwhile, miR-32-5p-mediated inhibition of the inflammatory cytokine production were completely reversed by overexpression of FSTL1. Collectively, our findings demonstrated a novel role of TLR-4/miRNA-32-5p/FSTL1 in the modulation of host defense against mycobacterial infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.

[Enhancing effect of compound Kusheng injection in combination with chemotherapy for patients with advanced non-small cell lung cancer].

OBJECTIVE: To explore the enhancing effect of compound Kusheg injection in chemotherapy for patients with stage III and IV non-small cell lung cancer (NSCLC). METHODS: A total of 286 patients with advanced NSCLC were enrolled in this study. The patients were treated with either compound Kusheng injection in combination with NP (NVB + CBP) chemotherapy (vinorelbine and carboplatin, n = 144), or with NP (NVB + CBP) chemotherapy alone (n = 142). The chemotherapy was performed for 4 cycles of 3 weeks, and the therapeutic efficacy was evaluated every 2 weeks. The following indicators were observed: levels of Hb, WBC, PLT and T cell subpopulations in blood, serum IgG level, short-term efficacy, adverse effects and quality of life. RESULTS: The gastrointestinal reactions and the myelosuppression in the combination chemotherapy group were alleviated as compared with the chemotherapy alone group, showing a significant difference (P < 0.05). CD(8)(+) cells were markedly declined in the combination chemotherapy group, and the CD(4)(+)/CD(8)(+) ratio showed an elevation trend in the chemotherapy alone group. The KPS scores and serum IgM and IgG levels were higher in the combination chemotherapy group than those in the chemotherapy alone group (P < 0.01 and P < 0.05). The serum lgA levels were not significantly different in the two groups. CONCLUSION: The compound Kusheng injection plus NP chemotherapy regimen shows better therapeutic effect, reduces adverse effects of chemotherapy and improves the quality of life in patients with stage III and IV NSCLC.