Association between interpregnancy interval and gestational diabetes mellitus: A cohort study of the National Vital Statistics System 2020.

OBJECTIVE: To assess the association between interpregnancy interval (IPI) and gestational diabetes mellitus (GDM). METHODS: Data of this retrospective cohort study were obtained from the National Vital Statistics System (NVSS) 2020. The participants were divided into different groups according to different IPI (<6, 6-11, 12-17, 18-23, 24-59 (reference), 60-119, ≥120 months). Multivariate logistic models were constructed to evaluate the association between IPI and GDM. Subgroup analysis was further performed. RESULTS: A total of 1 515 263 women were included, with 123 951 (8.18%) having GDM. Compared with the 24-59 months group, the <6 months (odds ratio [OR] 0.64, 95% confidence interval [CI] 0.46-0.90, P = 0.009), 12-17 months (OR 0.96, 95% CI 0.94-0.98, P < 0.001), and 18-23 months (OR 0.94, 95% CI 0.93-0.96, P < 0.001) groups had a significantly lower risk of GDM, while the 60-119 months (OR 1.13, 95% CI 1.11-1.15, P < 0.001) and ≥120 months (OR 1.18, 95% CI 1.15-1.21, P < 0.001) groups had a significantly higher risk of GDM. No significant difference was observed in the risk of GDM between the 6-11 and 24-59 months groups (P = 0.542). The PI-GDM association varied across different groups of age, pre-pregnancy body mass index, pre-pregnancy smoking status, history of cesarean section, history of preterm birth, prior terminations, and parity. CONCLUSION: An IPI of 18-23 months may be a better interval than 24-59 months in managing the risk of GDM.

Transcriptomic and antiviral analyses of PoIFN-Delta5 against porcine enteric viruses in porcine intestinal epithelial cells.

The interferon-delta family was first reported in domestic pigs and belongs to the type I interferon (IFN-I) family. The enteric viruses could cause diarrhea in newborn piglets with high morbidity and mortality. We researched the function of the porcine IFN-delta (PoIFN-δ) family in the porcine intestinal epithelial cells (IPEC-J2) cells infected with porcine epidemic diarrhea virus (PEDV). Our study found that all PoIFN-δs shared a typical IFN-I signature and could be divided into five branches in the phylogenic tree. Different strains of PEDV could induce typical IFN transitorily, and the virulent strain AH2012/12 had the strongest induction of porcine IFN-δ and IFN-alpha (PoIFN-α) in the early stage of infection. In addition, it was found that PoIFN-δ5/6/9/11 and PoIFN-δ1/2 were highly expressed in the intestine. PoIFN-δ5 had a better antiviral effect on PEDV compared to PoIFN-δ1 due to its higher induction of ISGs. PoIFN-δ1 and PoIFN-δ5 also activated JAK-STAT and IRS signaling. For other enteric viruses, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV), PoIFN-δ1 and PoIFN-δ5 both showed an excellent antiviral effect. Transcriptome analyses uncovered the differences in host responses to PoIFN-α and PoIFN-δ5 and revealed thousands of differentially expressed genes were mainly enriched in the inflammatory response, antigen processing and presentation, and other immune-related pathways. PoIFN-δ5 would be a potential antiviral drug, especially against porcine enteric viruses. These studies were the first to report the antiviral function against porcine enteric viruses and broaden the new acquaintances of this type of interferon though not novelly discovered.

CircCCNB1 Knockdown Blocks the Progression of Cervical Cancer by Acting as Competing Endogenous RNA in the miR-370-3p/SOX4 Pathway.

OBJECTIVE: Cervical cancer is one of the leading causes of cancer-related death in women, which has been shown to be associated with the deregulation of circular RNAs (circRNAs). The aim of this study was to determine the role of circRNA cyclin B1 (circCCNB1) in cervical cancer. METHODS: The expression of circCCNB1, microRNA-370-3p (miR-370-3p), and SRY-box transcription factor 4 (SOX4) mRNA was detected by quantitative real-time PCR (qPCR). Functional experiments, including colony formation assay, EdU assay, transwell assay and flow cytometry assay, were performed. Lactate production and glucose uptake were examined to assess glycolysis metabolism. The protein levels of glycolysis-related markers and SOX4 were detected by western blot. The interaction between miR-370-3p and circCCNB1 or SOX4 was verified by dual-luciferase reporter, RIP, and pull-down assay. Xenograft assay was performed to monitor the role of circCCNB1 in animal models. RESULTS: CircCCNB1 was highly expressed in cervical cancer tissues and cells (squamous cell carcinoma and adenocarcinoma cells). The knockdown of circCCNB1 inhibited cell proliferation, migration, invasion and glycolysis metabolism, and induced cell apoptosis. CircCCNB1 functioned as miR-370-3p sponge to suppress miR-370-3p expression and function. Moreover, circCCNB1 inhibited the expression of miR-370-3p to increase the expression of SOX4. MiR-370-3p inhibition reversed the effects of circCCNB1 knockdown and thus promoted cell proliferation, migration, invasion and glycolysis. SOX4 overexpression reversed the effects of miR-370-3p restoration and thus promoted cell proliferation, migration, invasion and glycolysis. CONCLUSION: CircCCNB1 knockdown blocks cervical cancer development by targeting the miR-370-3p/SOX4 pathway.

Trends in surgical approaches and adnexal surgeries during hysterectomies for benign diseases between 2015-2021.

Background: Hysterectomy is a widely used surgical approach for benign gynecological conditions, although recently there have been differences in the surgical route selected in different regions. Aims: To estimate recent temporal trends, this study collected data on surgical approaches and adnexal surgeries during hysterectomies for benign diseases at a single institute from 2015 to 2021. Materials and methods: We retrospectively analyzed data from Xiangyang No.1 People's Hospital, Hubei University of Medicine in Xiangyang, China, and identified 1828 women who underwent hysterectomies for benign gynecologic conditions performed with or without bilateral salpingectomy (BS) or bilateral salpingo-oophorectomy (BSO) between January 2015 and December 2021. Results: There was an upward trend in the performance of hysterectomy and hysterectomy with BS, and there was a difference in the trends of concomitant adnexal surgery between AH, TLH, and VH, especially in TLH with BS. Patient characteristics data demonstrated that the most frequent indication for hysterectomy was leiomyoma, especially in women aged 45 to 65. Compared to AH, TLH, and VH, the operative bleeding, duration of surgery, and length of hospital stays of patients undergoing TLH with BS and BSO was the lowest. The surgical approach to benign diseases has changed dramatically due to a growing proportion of patients choosing minimally invasive procedures. The laparoscopic approach is becoming popular due to its capacity to decrease intraoperative blood loss and reduce hospitalization. Conclusions: We should put more emphasis on surgical training for the TLH approach and help gynecologic surgeons provide the proposed added benefit of BS to their patients.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Effects of Malania oleifera Chun Oil on the Improvement of Learning and Memory Function in Mice.

BACKGROUND: The fruits of Malania oleifera Chun & S. K. Lee have been highly sought after medically because its seeds have high oil content (>60%), especially the highest known proportion of nervonic acid (>55%). Objective of the Study. The objective was to explore the effects of different doses of Malania oleifera Chun oil (MOC oil) on the learning and memory of mice and to evaluate whether additional DHA algae oil and vitamin E could help MOC oil improve learning and memory and its possible mechanisms. METHODS: After 30 days of oral administration of the relevant agents to mice, behavioral tests were conducted as well as detection of oxidative stress parameters (superoxide dismutase, malondialdehyde, and glutathione peroxidase) and biochemical indicators (acetylcholine, acetyl cholinesterase, and choline acetyltransferase) in the hippocampus. RESULTS: Experimental results demonstrated that MOC oil treatment could markedly improve learning and memory of mouse models in behavioral experiments and increase the activity of GSH-PX in hippocampus and reduce the content of MDA, especially the dose of 46.27 mg/kg. The addition of DHA and VE could better assist MOC oil to improve the learning and memory, and its mechanism may be related to the inhibition of oxidative stress and restrain the activity of AChE and also increase the content of ACh. CONCLUSION: Our results demonstrated that MOC oil treatment could improve learning and memory impairments. Therefore, we suggest that MOC oil is a potentially important resource for the development of nervonic acid products.

Alpha-linolenic acid protects against lipopolysaccharide-induced acute lung injury through anti-inflammatory and anti-oxidative pathways.

Alpha-linolenic acid (ALA), an important component of polyunsaturated fatty acids (PUFAs), possesses potent anti-inflammatory properties. To date, the effects of ALA on acute lung injury (ALI) remains unknown. This study was designed to investigate the potential protective effects of ALA on LPS-induced ALI and the underpinning mechanisms. An animal model of ALI was established via intratracheally injection of lipopolysaccharide (LPS, 1 mg/kg). We found that lung wet/dry weight ratio and protein concentration in Bronchoalveolar lavage fluid (BALF) were dramatically decreased by ALA pretreatment. Treatment with ALA significantly alleviated the infiltration of total cells and neutrophils, while increased the number of the macrophages. ALA significantly inhibited the secretion of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) and increased anti-inflammatory cytokine. Moreover, we found that the levels of myeloperoxidase (MPO) and malondialdehyde (MDA) were highly increased in LPS-induced ALI, while the activities of glutathione (GSH) and superoxide dismutase (SOD) were decreased, which were reversed by ALA. ALA attenuated LPS-induced histopathological changes and apoptosis. Furthermore, ALA significantly inhibited the phosphorylation of IκBα and NF-κB (p65) activation in ALI. ALA showed anti-inflammatory effects in mice with LPS-induced ALI. NF-κB pathway may be involved in ALA mediated protective effects.

[The Establishment of a Three-color Flow Cytometry Approach for the Scoring of Micronucleated Reticulocytes in Rat Bone Marrow].

OBJECTIVE: To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow. METHODS: In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time. RESULTS: A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7). CONCLUSION: With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway.

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.

The large-scale process of microbial carbonate precipitation for nickel remediation from an industrial soil.

Microbial carbonate precipitation is known as an efficient process for the remediation of heavy metals from contaminated soils. In the present study, a urease positive bacterial isolate, identified as Bacillus cereus NS4 through 16S rDNA sequencing, was utilized on a large scale to remove nickel from industrial soil contaminated by the battery industry. The soil was highly contaminated with an initial total nickel concentration of approximately 900 mg kg-1. The soluble-exchangeable fraction was reduced to 38 mg kg-1 after treatment. The primary objective of metal stabilization was achieved by reducing the bioavailability through immobilizing the nickel in the urease-driven carbonate precipitation. The nickel removal in the soils contributed to the transformation of nickel from mobile species into stable biominerals identified as calcite, vaterite, aragonite and nickelous carbonate when analyzed under XRD. It was proven that during precipitation of calcite, Ni2+ with an ion radius close to Ca2+ was incorporated into the CaCO3 crystal. The biominerals were also characterized by using SEM-EDS to observe the crystal shape and Raman-FTIR spectroscopy to predict responsible bonding during bioremediation with respect to Ni immobilization. The electronic structure and chemical-state information of the detected elements during MICP bioremediation process was studied by XPS. This is the first study in which microbial carbonate precipitation was used for the large-scale remediation of metal-contaminated industrial soil.

Suppressor of cytokine signaling 3 plays an important role in porcine circovirus type 2 subclinical infection by downregulating proinflammatory responses.

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated diseases and usually evokes a subclinical infection, without any obvious symptoms, in pigs. It remains unclear how PCV2 leads to a subclinical infection. In this study, we found that peripheral blood mononuclear cells (PBMCs) from PCV2-challenged piglets with no significant clinical symptoms exhibited increased expression of suppressor of cytokine signaling (SOCS) 3, but no significant changes in the expression of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α; this differed from piglets that displayed significant clinical symptoms. IL-6- and TNF-α-mediated signalings were inhibited in PBMCs from subclinical piglets. Elevated SOCS3 levels inhibited IL-6- and TNF-α-mediated NF-kappa-B inhibitor alpha degradation in PBMCs and PK-15 cells. SOCS3 production was also increased in PCV2-infected PK-15 porcine kidney cells, and IL-6 and TNF-α production that was induced by PCV2 in PK-15 cells was significantly increased when SOCS3 was silenced by a small interfering RNA. SOCS3 interacted with signal transducer and activator of transcription 3 and TNF-associated receptor-associated factor 2, suggesting mechanisms by which SOCS3 inhibits IL-6 and TNF-α signaling. We conclude that SOCS3 plays an important role in PCV2 subclinical infection by suppressing inflammatory responses in primary immune cells.

Blockade of CXC chemokine receptor 3 on endothelial cells protects against sepsis-induced acute lung injury.

BACKGROUND: CXCR3, a G-protein coupled chemokine receptor, has been shown to play a critical role in recruiting inflammatory cells into lungs in several studies. However, its roles in polymicrobial septic acute lung injury (ALI) is yet unknown. Therefore, the purpose of this study was to elucidate the protective effects of CXCR3 blockade on pulmonary microvascular endothelial cells (PMVECs) in septic ALI and explore potential mechanisms. MATERIALS AND METHODS: ALI was induced by polymicrobial sepsis through cecal ligation and puncture surgery. The expression of CXCR3 on pulmonary microvascular endothelial cells was measured 24 h after cecal ligation and puncture surgery. In addition, the protective effects of neutralizing antibody were detected, including protein concentration, inflammation cell counts, lung ​wet-to-dry ratio, and lung damages. In human umbilical vein endothelial cells (HUVECs) culture condition, CXCR3 expression was measured after exposure to tumor necrosis factor-α. The permeability and apoptosis ratio were detected through CXCR3 gene silencing on HUVECs. The p38 mitogen-activated protein kinase (MAPK) was analyzed with Western blot. RESULTS: CXCR3 expression was upregulated both in vivo and in vitro. After CXCR3 neutralizing antibody administrated intraperitoneally, the protein concentration, inflammatory cell counts in BALF and lung ​wet-to-dry ratio were decreased significantly, as well as the lung tissue damages. In vitro, CXCR3 gene silencing inhibited tumor necrosis factor-α and CXCL10-induced hyperpermeability and apoptosis in HUVECs. In addition, p38 mitogen-activated protein kinase activation was essential for CXCR3-mediated apoptosis. CONCLUSIONS: CXCR3 blockade exerts protective effects on ALI at least partly by inhibiting endothelial cells apoptosis and decreasing the leakage of protein-rich fluid and inflammatory cells. Blockade of CXCR3 may be a promising therapeutic strategy for severe sepsis-induced ALI.

Systematic analysis of berberine-induced signaling pathway between miRNA clusters and mRNAs and identification of mir-99a ∼ 125b cluster function by seed-targeting inhibitors in multiple myeloma cells.

BACKGROUND: Berberine (BBR) is a natural alkaloid derived from a traditional Chinese herbal medicine. However, the exact mechanisms underlying the different effects of berberine on MM cells have not been fully elucidated. METHODS: A systematic analysis assay integrated common signaling pathways modulated by the 3 miRNA clusters and mRNAs in MM cells after BBR treatment. The role of the mir-99a ∼ 125b cluster, an important oncomir in MM, was identified by comparing the effects of t-anti-mirs with complete complementary antisense locked nucleic acids (LNAs) against mature mir-125b (anti-mir-125b). RESULTS: Three miRNAs clusters (miR-99a ∼ 125b, miR-17 ∼ 92 and miR-106 ∼ 25) were significantly down-regulated in BBR-treated MM cells and are involved in multiple cancer-related signaling pathways. Furthermore, the top 5 differentially regulated genes, RAC1, NFκB1, MYC, JUN and CCND1 might play key roles in the progression of MM. Systematic integration revealed that 3 common signaling pathways (TP53, Erb and MAPK) link the 3 miRNA clusters and the 5 key mRNAs. Meanwhile, both BBR and seed-targeting t-anti-mir-99a ∼ 125b cluster LNAs significantly induced apoptosis, G2-phase cell cycle arrest and colony inhibition. CONCLUSIONS: our results suggest that BBR suppresses multiple myeloma cells, partly by down-regulating the 3 miRNA clusters and many mRNAs, possibly through TP53, Erb and MAPK signaling pathways. The mir-99a ∼ 125b cluster might be a novel target for MM treatment. These findings provide new mechanistic insight into the anticancer effects of certain traditional Chinese herbal medicine compounds.

Integrative analysis of differential miRNA and functional study of miR-21 by seed-targeting inhibition in multiple myeloma cells in response to berberine.

BACKGROUND: Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells. RESULTS: We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells. CONCLUSIONS: Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.

VEGF depletion enhances bcr-abl-specific sensitivity of arsenic trioxide in chronic myelogenous leukemia.

The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment.

Targeted inhibition of VEGF-modulated survival and arsenic sensitivity in acute myeloid leukemia (AML).

Vascular endothelial growth factor (VEGF) is a specific growth factor for tumor-associated angiogenesis, and is also involved in leukemogenesis; however, its exact role in leukemia development remains elusive. In this study we used antisense oligonucleotide (AS) to manipulate VEGF function in acute myeloid leukemia (AML). HL60 and primary AML cells were transfected with VEGF AS (0.3 µmol/l). Cell proliferation and survival were assessed using the trypan blue exclusion assay. The viability of cells was determined using MTT. The IC50 values of arsenic trioxide (ATO) in HL60 cell were calculated by ICp software. The results showed that VEGF AS effectively inhibited AML cell proliferation and survival 72 hours post-transfection and exhibited time dependence. The IC50 value of ATO was significantly down-regulated by VEGF AS in HL60. Meanwhile, VEGF AS alone induced cell apoptosis, and promoted ATO-induced apoptosis. There is synergistic inhibitory effects between AS and ATO. VEGF AS down-regulated VEGF protein level in the supernatants and cellular VEGF mRNA level by western blot and real-time PCR. Therefore, targeted inhibition of VEGF suppressed survival and increased arsenic sensitivity in AML.

miR-181a post-transcriptionally downregulates oncogenic RalA and contributes to growth inhibition and apoptosis in chronic myelogenous leukemia (CML).

MicroRNAs (miRNAs) are a class of short RNAs that regulate gene expression through either translational repression or mRNA cleavage. miRNA-181a (miR-181a), one of the many miRNAs conserved among vertebrates, is differentially expressed in a variety of leukemia. However, its function in leukemia, particularly chronic myelogenous leukemia (CML), is poorly understood. Here we have reported the identification of miR-181a targets by combining TargetScan software prediction and expression profiling through overexpression of miR-181a mimic in leukemic K562 cells. Four overlapping genes were found to be the likely targets of miR-181a. Among the four genes, RalA is a downstream molecule of bcr-abl fusion protein in ras signaling pathway. However, its role in CML remains elusive. Luciferase reporter and Western blot assays confirmed that RalA is a direct target of miR-181a. overexpression of miR-181a effectively suppresses cell growth and induces G2-phase arrest and apoptosis partially by targeting RalA in leukemic K562 cells. Using the KEGG database combined with recent publications, downstream signaling pathway of RalA was graphed by cytoscape software. Therefore, our study is the first to report that RalA is directly regulated by miR-181a and plays an important role in CML. The approach of computational prediction combined with expression profiling might be valuable for the identification of miRNA targets in animal.

Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells.

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the bcr-abl gene. RalA, one of the Ras superfamily of small GTPases, is a downstream molecule of bcr-abl fusion protein in ras signaling pathway, but its role in CML is poorly understood. Here, we first detected RalA level in CML cells, which is highly expressed and distributed mainly in the cytoplasm and/or partially in endomembrane. Next, siRNA was used to deplete RalA expression for elucidating its function. The results showed that siRNA RalA effectively inhibited cell viability, induced apoptosis and enhanced sensitivity of arsenic trioxide (ATO), and there are some synergistic effects of anti-CML between RalA siRNA and ATO. Finally, we found that ATO also could downregulate protein level of bcr-abl in K562 and KCL-22. Our research provides evidence that RalA might also serve as linchpin modulators in leukemia, and combinatorial therapies of dual inhibition of bcr-abl and ras signaling pathways have a great potential in treatment of CML.

miRNA-21 regulates arsenic-induced anti-leukemia activity in myelogenous cell lines.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cellular sensitivity to anti-cancer drugs. miRNA-21 (miR-21), one of the most prominent miRNAs in the genesis and progression of many human cancers, has been rarely characterized in myelogenous leukemia. Arsenic trioxide (ATO) was successfully used in the treatment of acute promyelocytic leukemia (APL) etc. However, cytotoxicity or insensitivity is a major concern in the successful treatment of leukemia. Here, we used a specific precursor miRNA-21 (pre-miR-21) or anti-miRNA-21 oligonucleotide (AMO-miR-21) to study sensitivity of HL60 and K562 cells to ATO. Cell viability and cell cycle were evaluated by MTT assay and PI assay using flow cytometry, respectively. Levels of miR-21 and its target PDCD4 were quantified by real-time PCR and/or western blot. AMO-miR-21 or ATO alone led to growth inhibition, apoptosis and G1 phase arrest of cell cycle. Apoptotic cells were confirmed morphologically with Hoechst staining. Moreover, there was somewhat synergistic effect of AMO-miR-21 and ATO in growth inhibition and apoptosis promotion. Meanwhile, enforced pre-miR-21 expression increased resistance to ATO, nevertheless not affecting cell growth alone. Dual-luciferase reporter vector containing two tandem PDCD4 3' UTR validated that PDCD4 was directly up-regulated by miR-21. Therefore, miRNA-21 by targeting PDCD4 may play a functional role in modulating ATO-induced cell death, and strategy using AMO-miR-21 and its combination with ATO may be useful as a myelogenous leukemia therapy.

Anti-miR-21 oligonucleotide enhances chemosensitivity of leukemic HL60 cells to arabinosylcytosine by inducing apoptosis.

Drug insensitivity or resistance is a major obstacle for successful treatment of acute myeloid leukemia. MicroRNAs (miRNAs) are small non-coding RNA molecules. Increasing evidence suggests that miRNAs modulate cellular sensitivity to anticancer drugs. We used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize leukemic HL60 cells to arabinosylcytosine (Ara-C) by down-regulating miR-21. AMO-miR-21 alone effectively inhibited HL60 cell viability as measured by MTT assays and induced apoptosis as evaluated by flow cytometry, whereas AMO-miR-21 in combination with Ara-C enhanced HL60 cells to Ara-C-sensitivity and promoted Ara-C-induced apoptosis. Levels of miR-21 and its target PDCD4, quantified by real-time PCR, showed that expression of miR-21 was significantly decreased after AMO-miR-21 treatment. PDCD4 as a direct target of miR-21 in leukemic HL60 cells was confirmed by the dual-luciferase reporter assay. Our study suggests that AMO-miR-21 significantly sensitizes HL60 cells to Ara-C by inducing apoptosis and these effects of AMO-miR-21 may be partially due to its up-regulation of PDCD4. Therefore, exploiting synergistic effects between AMO-miR-21 and Ara-C might be an effective clinical strategy for leukemia chemotherapy.