Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Erratum: [Corrigendum] Cross regulation of signaling pathways in gastrointestinal stromal tumor.

[This corrects the article DOI: 10.3892/ol.2018.9494.].

A novel pathogenic mutation of MeCP2 impairs chromatin association independent of protein levels.

Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment.

The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.

Application and curative effect of laparoscopic purse-string sutures in the treatment of adult acute complicated appendicitis.

OBJECTIVE: To investigate the effect of laparoscopic purse-string sutures in adult complicated appendicitis treatment. METHODS: The data of 568 adult cases of complicated appendicitis treated by laparoscopic appendectomy at the Hefei Second People's Hospital, Anhui Province, China, from September 2018 to September 2021 were analysed retrospectively. The patients were divided into two groups: 295 cases in the laparoscopic purse-string suture treatment group (observation group) and 273 cases in the simple Hem-o-lok® clamp treatment group (control group). The baseline data collected included age, gender, preoperative body temperature, leukocyte count and percentage of neutrophils and the surgery time. The postoperative data collected included antibiotic treatment duration, drainage tube placement time and the incidence of complications. RESULTS: There were no significant differences in the baseline data of the two groups, including age, gender, preoperative body temperature, leukocyte count and neutrophil percentage (all P > 0.05). Compared with the control group, the postoperative hospital length of stay, duration of antibiotic treatment, the recovery time of peripheral white blood cell and neutrophil counts and the incidence of postoperative complications in the observation group were significantly decreased (P < 0.05). CONCLUSION: Purse-string sutures can effectively reduce the incidence of postoperative complications after a laparoscopic appendectomy for adult acute complicated appendicitis. There was faster postoperative recovery when patients' appendiceal stumps were treated with laparoscopic purse-string sutures.

Regulation of host factor γ-H2AX level and location by enterovirus A71 for viral replication.

Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent in vitro and in vivo data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.

The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C.

Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid­dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate­binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human NPC1 mutant induced pluripotent stem cell­derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.

Efficacy and safety of mesenchymal stem cell in Chinese patients with chronic renal failure: A pilot study in Shandong province, China.

This study designed to evaluate efficacy and safety profile of Mesenchymal stem cells (MSCs) versus Acetyl cysteine (NACys) in the Chinese patients with Chronic renal failure (CRF). The CRF patients having eGFR less than 60ml per minute per 1.73m2 randomly assigned to MSCs (N=100) or NACys (N=100) (1:1) for 8 weeks. MSCs administered as intravenous infusion of marrow-derived autologous MSCs (1 × 106 to 2 × 106/kg) reperfusion, whereas, another group received NACys 600mg orally twice a day for 8 weeks. The efficacy variables include: creatinine; cystatin C; TGF-ß levels; oxidants/reactive oxygen species production induced by TGF-ß; collagen levels (type 1 and 4); urinary albumin/creatinine ratio and Glomerular area. Safety was also assesed. Both the treatments significantly decreased creatinine, cystatin C and reactive oxygen species from baseline, however, reduction in creatinine, cystatin C, and reactive oxygen species level from baseline was significantly higher in patient treated with MSCs (N=100) as compared to NACys (N=100). Moreover, improvement in renal and systemic functional parameters from baseline was significantly higher in patient treated with MSCs as compared to NACys. Overall, MSCs offer significantly greater improvement in renal function as compared to NACys in Chinese CRF patients.

Circ_002059 suppresses cell proliferation and migration of gastric cancer via miR-182/MTSS1 axis.

Circular RNAs (circRNAs) play either oncogenic or tumor suppressive roles in gastric cancer (GC). A previous study demonstrated that circ_002059, a typical circRNA, was downregulated in GC tissues. However, the role and mechanism of circ_002059 in GC development are still unknown. In this study, the levels of circ_002059, miR-182, and metastasis suppressor-1 (MTSS1) were examined by real-time quantitative polymerase chain reaction and western blot analysis. Cell proliferation and migration were evaluated by MTT assay and Transwell migration assay, respectively. The interactions between miR-182 and circ_002059 or MTSS1 were analyzed by dual-luciferase reporter assay. A GC xenograft model was established to validate the role of circ_002059 in GC progression in vivo. Overexpression of circ_002059 significantly inhibited, whereas knockdown of circ_002059 notably facilitated, cell proliferation and migration in GC cells. MTSS1 was found to be a direct target of miR-182 and circ_002059 upregulated MTSS1 expression by competitively sponging miR-182. Transfection with miR-182 mimic and MTSS1 silencing abated the inhibitory effect of circ_002059 on GC progression. Circ_002059 inhibited GC cell xenograft tumor growth by regulating miR-182 and MTSS1 expression. Collectively, Circ_002059 inhibited GC cell proliferation and migration in vitro and xenograft tumor growth in mice, by regulating the miR-182/MTSS1 axis.

Downregulating circRNA_0044516 Inhibits Cell Proliferation in Gastric Cancer Through miR-149/Wnt1/ß-catenin Pathway.

BACKGROUND: Circular RNAs (circRNAs) play important roles in the progression of gastric cancer (GC). The Wnt1/ß-catenin pathway can promote the proliferation of GC cells. This study aimed to explore whether circRNA_0044516 can regulate the proliferation of GC cells by modulating the Wnt1/ß-catenin pathway. METHODS: The expression of circRNA_0044516, miR-149, Wnt1, and ß-catenin in GC tissues or cells was detected by qRT-PCR and western blot. Cell viability and apoptosis were measured by CCK-8 and flow cytometry assays, respectively. The interaction between circRNA_0044516 and miR-149 was determined by luciferase reporter and RNA pull-down assays. RESULTS: Upregulated circRNA_0044516 was found in GC tissues and cell lines. Downregulating circRNA_0044516 inhibited the viability and promoted apoptosis of GC cells. CircRNA_0044516 targeted miR-149, and its downregulation elevated miR-149 level in GC cells. Mechanistically, silencing circRNA_0044516 reduced the protein level of Wnt1 and ß-catenin through miR-149, and finally suppressed viability and contributed to apoptosis of GC cells. Moreover, circRNA_0044516 knockdown inhibited the tumor growth of HGC-27 cells in nude mice. CONCLUSIONS: Our results indicated an important role of circRNA_0044516 in GC and elucidated that downregulation of circRNA_0044516 inhibits the proliferation of GC cells through miR-149/Wnt1/ß-catenin.

Loss of MeCP2 Function Across Several Neuronal Populations Impairs Breathing Response to Acute Hypoxia.

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by loss of function of the transcriptional regulator Methyl-CpG-Binding Protein 2 (MeCP2). In addition to the characteristic loss of hand function and spoken language after the first year of life, people with RTT also have a variety of physiological and autonomic abnormalities including disrupted breathing rhythms characterized by bouts of hyperventilation and an increased frequency of apnea. These breathing abnormalities, that likely involve alterations in both the circuitry underlying respiratory pace making and those underlying breathing response to environmental stimuli, may underlie the sudden unexpected death seen in a significant fraction of people with RTT. In fact, mice lacking MeCP2 function exhibit abnormal breathing rate response to acute hypoxia and maintain a persistently elevated breathing rate rather than showing typical hypoxic ventilatory decline that can be observed among their wild-type littermates. Using genetic and pharmacological tools to better understand the course of this abnormal hypoxic breathing rate response and the neurons driving it, we learned that the abnormal hypoxic breathing response is acquired as the animals mature, and that MeCP2 function is required within excitatory, inhibitory, and modulatory populations for a normal hypoxic breathing rate response. Furthermore, mice lacking MeCP2 exhibit decreased hypoxia-induced neuronal activity within the nucleus tractus solitarius of the dorsal medulla. Overall, these data provide insight into the neurons driving the circuit dysfunction that leads to breathing abnormalities upon loss of MeCP2. The discovery that combined dysfunction across multiple neuronal populations contributes to breathing dysfunction may provide insight into sudden unexpected death in RTT.

Caspase-3 inhibitor inhibits enterovirus D68 production.

Enterovirus D68 (EVD68) is an emerging pathogen that recently caused a large worldwide outbreak of severe respiratory disease in children. However, the relationship between EVD68 and host cells remains unclear. Caspases are involved in cell death, immune response, and even viral production. We found that caspase-3 was activated during EVD68 replication to induce apoptosis. Caspase-3 inhibitor (Z-DEVD-FMK) inhibited viral production, protected host cells from the cytopathic effects of EVD68 infection, and prevented EVD68 from regulating the host cell cycle at G0/G1. Meanwhile, caspase-3 activator (PAC-1) increased EVD68 production. EVD68 infection therefore activates caspase-3 for virus production. This knowledge provides a potential direction for the prevention and treatment of disease related to EVD68.

CircRNA-PTN Sponges miR-326 to Promote Proliferation in Hepatocellular Carcinoma.

BACKGROUND: Mounting evidences reveal that circular RNAs (circRNAs) are critical to regulate biological behavior and process of tumor. Our objective is to explore the role of circRNA-PTN (circPTN) and explain the exact mechanism in hepatocellular carcinoma (HCC). METHODS: Real-time polymerase chain reaction assay was used to detect the level of circPTN and miR-326. The proliferation of cell was measured by CCK-8 assay and EdU assay. Western blot assay was performed to assess ErbB/PI3K expression. Luciferase and RNA pull-down assays were carried out to confirm the interaction between circPTN and miR-326. RESULTS: Our results indicated that circPTN was upregulated in human hepatocellular carcinoma tumor tissues and cell lines, compared with paratumor tissues and immortalized normal liver cell line. circPTN could significantly promote HCC tumor growth according to gain-and loss-of-function assays. Additionally, we determined that circPTN acted as a sponge through interacting with miR-326. Overexpression of miR-326 could rescue the cell proliferation inhibition and ErbB/PI3K downregulation in HCC cells by circPTN. Besides, the effects of miR-326 on HCC were missing when circPTN binding sites were mutated. CONCLUSION: Our study indicates that circPTN acts as an oncogenic factor via sponging miR-326 in HCC.

A CpG Methylation Classifier to Predict Relapse in Adults with T-Cell Lymphoblastic Lymphoma.

PURPOSE: Adults with T-cell lymphoblastic lymphoma (T-LBL) generally benefit from treatment with acute lymphoblastic leukemia (ALL)-like regimens, but approximately 40% will relapse after such treatment. We evaluated the value of CpG methylation in predicting relapse for adults with T-LBL treated with ALL-like regimens. EXPERIMENTAL DESIGN: A total of 549 adults with T-LBL from 27 medical centers were included in the analysis. Using the Illumina Methylation 850K Beadchip, 44 relapse-related CpGs were identified from 49 T-LBL samples by two algorithms: least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE). We built a four-CpG classifier using LASSO Cox regression based on association between the methylation level of CpGs and relapse-free survival in the training cohort (n = 160). The four-CpG classifier was validated in the internal testing cohort (n = 68) and independent validation cohort (n = 321). RESULTS: The four-CpG-based classifier discriminated patients with T-LBL at high risk of relapse in the training cohort from those at low risk (P < 0.001). This classifier also showed good predictive value in the internal testing cohort (P < 0.001) and the independent validation cohort (P < 0.001). A nomogram incorporating five independent prognostic factors including the CpG-based classifier, lactate dehydrogenase levels, Eastern Cooperative Oncology Group performance status, central nervous system involvement, and NOTCH1/FBXW7 status showed a significantly higher predictive accuracy than each single variable. Stratification into different subgroups by the nomogram helped identify the subset of patients who most benefited from more intensive chemotherapy and/or sequential hematopoietic stem cell transplantation. CONCLUSIONS: Our four-CpG-based classifier could predict disease relapse in patients with T-LBL, and could be used to guide treatment decision.

Impact of Nutritional Indices on the Survival Outcomes of Patients with Colorectal Cancer.

BACKGROUND: The effect of preoperative nutritional status on the survival of patients with colorectal cancer is still unknown. The purpose of our study was to examine the impact of the prognostic nutritional index (PNI), prealbumin (PAB) and the albumin to globulin ratio (AGR) on survival outcomes in patients with colon and rectal cancer. METHODS: Between January 2012 and December 2013, 361 patients with colorectal cancer who underwent curative surgery in the survey and various clinical and haematological parameters were recorded. The optimal cut-off values of the PNI, PAB and AGR were determined by MedCalc software, and Cox regression analysis was performed to investigate the effect of the PNI, PAB and AGR on the overall survival (OS) of patients with colon and rectal cancer. RESULTS: In patients with colon and rectal cancer, a high PNI, PAB, and AGR correlate with higher survival times. Receiver operating characteristic (ROC) curve analysis showed that at most time points, the PNI has a higher area under the curve (AUC) in predicting colon and rectal cancer OS. Multivariate Cox regression analysis showed that of the PNI, PAB and AGR, only the PNI was an independent risk factor for OS in patients with colon and rectal cancer. Patients with a high PNI were predicted to have higher OS (hazard ratio [HR]: 0.479; 95% confidence interval [CI]: 0.233-0.985; P = 0.045) in colon cancer and higher OS (HR: 0.225; 95% CI: 0.111-0.454; P < 0.001) in rectal cancer compared with patients with a low PNI. CONCLUSION: Preoperative PNI, PAB and AGR may be predictors of OS in patients with colon and rectal cancer after radical surgery, especially the PNI, which has a good ability to predict OS in both tumours.

Coxsackievirus A6 Induces Necroptosis for Viral Production.

Hand, foot, and mouth disease (HFMD) is a febrile exanthematous disease with typical or atypical symptoms. Typical HFMD is usually caused by enterovirus 71 (EV71) or coxsackievirus A16, while atypical HFMD is usually caused by coxsackievirus A6 (CA6). In recent years, worldwide outbreaks of CA6-associated HFMD have dramatically increased, although the pathogenic mechanism of CA6 is still unclear. EV71 has been established to induce caspase-dependent apoptosis, but in this study, we demonstrate that CA6 infection promotes a distinct pathway of cell death that involves loss of cell membrane integrity. Necrostatin-1, an inhibitor of necroptosis, blocks the cell death induced by CA6 infection, but Z-DEVD-FMK, an inhibitor of caspase-3, has no effect on CA6-induced cell death. Furthermore, CA6 infection up-regulates the expression of the necroptosis signaling molecule RIPK3. Importantly, necrostatin-1 inhibits CA6 viral production, as assessed by its ability to inhibit levels of VP1 protein and genomic RNA and infectious particles. CA6-induced necroptosis is not dependent on the generation of reactive oxygen species; however, viral 3D protein can directly bind RIPK3, which is suggestive of a direct mechanism of necroptosis induction. Therefore, these results indicate that CA6 induces a mechanism of RIPK3-dependent necroptosis for viral production that is distinct from the mechanism of apoptosis induced by typical HFMD viruses.

A gene-expression-based signature predicts survival in adults with T-cell lymphoblastic lymphoma: a multicenter study.

We aimed to establish a discriminative gene-expression-based classifier to predict survival outcomes of T-cell lymphoblastic lymphoma (T-LBL) patients. After exploring global gene-expression profiles of progressive (n = 22) vs. progression-free (n = 28) T-LBL patients, 43 differentially expressed mRNAs were identified. Then an eleven-gene-based classifier was established using LASSO Cox regression based on NanoString quantification. In the training cohort (n = 169), high-risk patients stratified using the classifier had significantly lower progression-free survival (PFS: hazards ratio 4.123, 95% CI 2.565-6.628; p < 0.001), disease-free survival (DFS: HR 3.148, 95% CI 1.857-5.339; p < 0.001), and overall survival (OS: HR 3.790, 95% CI 2.237-6.423; p < 0.001) compared with low-risk patients. The prognostic accuracy of the classifier was validated in the internal testing (n = 84) and independent validation cohorts (n = 360). A prognostic nomogram consisting of five independent variables including the classifier, lactate dehydrogenase levels, ECOG-PS, central nervous system involvement, and NOTCH1/FBXW7 status showed significantly greater prognostic accuracy than each single variable alone. The addition of a five-miRNA-based signature further enhanced the accuracy of this nomogram. Furthermore, patients with a nomogram score ≥154.2 significantly benefited from the BFM protocol. In conclusion, our nomogram comprising the 11-gene-based classifier may make contributions to individual prognosis prediction and treatment decision-making.

Bursectomy Versus Nonbursectomy for Gastric Adenocarcinoma: A Single-Center, Propensity-Score Matched Cohort Study in China.

Background: The therapeutic value of bursectomy remains controversial for patients with gastric cancer. Therefore, the purpose of our study was to explore the safety and survival benefits of bursectomy. Materials and Methods: A total of 943 patients with gastric adenocarcinoma were included in our study, and all patients were operated on by high-quality gastrointestinal surgeons. The factors associated with overall survival (OS) were determined using the Kaplan-Meier method. In addition, patients in the bursectomy group and nonbursectomy group were matched with 1:1 propensity score matching for sex, age, tumor location, type of operation, tumor size, degree of differentiation, and pathological stage to reduce the possibility of choice bias. Results: Among the 943 eligible patients, 188 (19.9%) underwent bursectomy and 755 (80.1%) did not. In all patients, the number of retrieved lymph nodes (P = .0596), blood loss volume (P = .0896), operation time (P = .0747), number of postoperative complications (P = .626), and OS in the bursectomy group were similar to those in the nonbursectomy group. After a stratified analysis of TNM grade and T stage, it was found that bursectomy could lead to survival benefits for patients with stage T4 disease (P = .0398). Conclusions: Bursectomy does not increase the amount of blood loss, operation time, or incidence of postoperative complications. This procedure is an extended and safe surgical method for gastric adenocarcinoma. Bursectomy does not improve the survival of all patients, but for patients with stage T4 disease, bursectomy can provide survival benefits.

LncRNA CASC9 Suppressed the Apoptosis of Gastric Cancer Cells through Regulating BMI1.

Long noncoding RNAs (lncRNAs) play important roles in regulating the apoptosis of gastric cancer (GC) cells. This study aims to investigate the underlying mechanism of lncRNA CASC9 in regulating the apoptosis of GC cells. The expressions of lncRNA and protein in GC tissues and cell lines were detected by qRT-PCR and western blot. GC cell apoptosis was detected by flow cytometry analysis. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to verify the interaction between CASC9 and BMI1. LncRNA CASC9 was upregulated in GC tissue and GC cells, and high CASC9 expression was positively correlated with TNM stage and lymph node metastasis. Silencing CASC9 promoted the apoptosis of GC cells. LncRNA CASC9 could interact with BMI1 and positively regulate BMI1 expression. Silencing CASC9 promoted the ubiquitination of BMI1. In addition, lncRNA CASC9 regulated the apoptosis of GC cells through BMI1. Furthermore, interfering CASC9 inhibited the tumor growth of GC. LncRNA CASC9 could interact with BMI1 to regulate the degradation of BMI1, thus to affect the apoptosis of GC cells and suppressed tumor growth.

The combined effect of oleonuezhenide and wedelolactone on proliferation and osteoblastogenesis of bone marrow mesenchymal stem cells.

BACKGROUND: Regulation of the survival and differentiation of bone marrow mesenchymal stem cells is an essential consideration in the development of targeted drugs for treatment of osteoporosis. PURPOSE: The present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide, two compounds from Chinese formula Er-Zhi-Wan, on osteoblastogenesis and the underlying molecular mechanisms. METHODS: MTT assay was taken to evaluate cell proliferation. The alkaline phosphatase (ALP) activity assay was used to determine the activity of ALP. Alizarin red S (ARS) staining was taken to indicate the intensity of the calcium deposits. Quantitative real-time PCR and Western blot were performed to the levels of Runx2, Osteocalcin, and Osterix expression in mouse bone marrow mesenchymal stem cells (BMSCs). Ovariectomized mouse model and bone histomorphometric analysis were also used to research the effects of wedelolactone and oleonuezhenide on bone loss caused by ovariectomy. RESULTS: Wedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization. Osteoblastogenesis-related marker genes including osteocalcin, Runx2, and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide. At the molecular level, oleonuezhenide did not affect GSK-3ß phosphorylation induced by wedelolactone, but elevated casein kinase 2-alpha (CK2α) expression, resulting in ß-catenin and Runx2 nuclear translocation. In addition, 30 µM wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 µM oleonuezhenide. These cells were protected by oleonuezhenide and maintained osteoblastic activity. Oleonuezhenide increased Wnt5a and CK2α expression. Wedelolactone-reduced extracellular signal-regulated kinase (ERK) phosphorylation was reversed by oleonuezhenide. In ovariectomized mice, administration of wedelolactone and oleonuezhenide prevented ovariectomy-induced bone loss by enhancing osteoblastic activity. CONCLUSION: These results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis. These two compounds could be developed as a combined therapeutic agent for osteoporosis.