Effect of Dimethyl Fumarate (DMF) on T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the effect and possible mechanism of dimethyl fumarate (DMF) on T-cell acute lymphoblastic leukemia (T-ALL), and provide experimental and theoretical basis for the clinical treatment of T-ALL.@*METHODS@#Jurkat cells were treated with different concentrations of DMF for 24 hours, and then the proportion and absolute count of Ki67-positive Jurkat cells were analyzed by flow cytometry. Meanwhile, the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and E3 ubiquitin ligase HACE1 in Jurkat cells treated with DMF for 24 hours were evaluated by Western blot. Nrf2 proteins were co-immunoprecipitated in Jurkat cells, and then HACE1 protein was assessed by Western blot. Plasmids of Flag-Nrf2 and different gradients of Flag-HACE1 were transfected into HEK293T cells, and the levels of Flag-Nrf2 were detected by Western blot after 48 hours.@*RESULTS@#DMF could significantly inhibit the proportion and absolute count of Ki67-positive Jurkat cells, and DMF inhibited the proliferation of Jurkat cells in a dose-dependent manner (r=0.9595, r=0.9054). DMF could significantly up-regulate the protein levels of Nrf2 and E3 ubiquitin ligase HACE1 in Jurkat cells (P<0.01, P<0.01). HACE1 physically interacted with Nrf2 in Jurkat cells. Overexpression of Flag-HACE1 significantly increased the protein level of Flag-Nrf2 in a dose-dependent manner (r=0.9771).@*CONCLUSION@#DMF inhibits the proliferation of T-cell acute lymphoblastic leukemia cell. The mechanism may be that, DMF significantly up-regulates the protein levels of Nrf2 and E3 ubiquitin ligase HACE1, and HACE1 interacts with Nrf2 and positively regulates Nrf2 protein level.

Ginsenoside Rg_1 protects PC12 cells against Aβ-induced injury through promotion of mitophagy by PINK1/parkin activation / 中国中药杂志

Amyloid β-protein(Aβ) deposition in the brain is directly responsible for neuronal mitochondrial damage of Alzheimer's disease(AD) patients. Mitophagy, which removes damaged mitochondria, is a vital mode of neuron protection. Ginsenoside Rg_1(Rg_1), with neuroprotective effect, has displayed promising potential for AD treatment. However, the mechanism underlying the neuroprotective effect of Rg_1 has not been fully elucidated. The present study investigated the effects of ginsenoside Rg_(1 )on the autophagy of PC12 cells injured by Aβ_(25-35) to gain insight into the neuroprotective mechanism of Rg_1. The autophagy inducer rapamycin and the autophagy inhi-bitor chloroquine were used to verify the correlation between the neuroprotective effect of Rg_1 and autophagy. The results showed that Rg_1 enhanced the viability and increased the mitochondrial membrane potential of Aβ-injured PC12 cells, while these changes were blocked by chloroquine. Furthermore, Rg_(1 )treatment increased the LC3Ⅱ/Ⅰ protein ratio, promoted the depletion of p62 protein, up-regulated the protein levels of PINK1 and parkin, and reduced the amount of autophagy adaptor OPTN, which indicated the enhancement of autophagy. After the silencing of PINK1, a key regulatory site of mitophagy, Rg_1 could not increase the expression of PINK1 and parkin or the amount of NDP52, whereas it can still increase the LC3Ⅱ/Ⅰ protein ratio and promote the depletion of OPTN protein which indicated the enhancement of autophagy. Collectively, the results of this study imply that Rg_1 can promote autophagy of PC12 cells injured by Aβ, and may reduce Aβ-induced mitochondrial damage by promoting PINK1-dependent mitophagy, which may be one of the key mechanisms of its neuroprotective effect.

Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase / 中国医学科学院学报

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.

Ubiquitin-conjugating enzyme UBE2Q2 participates in HUWE1-mediated protection on renal tubulointerstitial fibrosis / 生理学报

The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.

Speckle type POZ adaptor protein (SPOP) and its role in cancer / La proteína adaptadora Speckle-type POZ (SPOP) y su papel en cáncer

Abstract Proteasomal degradation is an essential regulatory mechanism for cellular homeostasis maintenance. The speckle-type POZ adaptor protein (SPOP) is part of the ubiquitin ligase E3 cullin-3 RING-box1 complex, responsible for the ubiquitination and proteasomal degradation of biomolecules involved in cell cycle control, proliferation, response to DNA damage, epigenetic control, and hormone signaling, among others. Changes in SPOP have been associated with the development of different types of cancer, since it can act as a tumor suppressor mainly in prostate, breast, colorectal, lung cancer and liver cancer, due to point mutations and/or reduced expression, or as an oncogene in kidney cancer by protein overexpression. In endometrial cancer it has a dual role, since it can act as a tumor suppressor or as an oncogene. SPOP is a potential prognostic biomarker and a promising therapeutic target.

Resumen La degradación proteosómica es un mecanismo de regulación esencial para el mantenimiento de la homeostasis celular. La proteína adaptadora Speckle-type POZ (SPOP) hace parte del complejo ubiquitin ligasa E3 cullin-3 RING-box1, encargado de la ubiquitinación y degradación proteosomal de biomoléculas involucradas en el control del ciclo celular, proliferación, respuesta al daño de ADN, control epigenético, señalización hormonal, entre otros. Las alteraciones en SPOP han sido asociadas al desarrollo de diferentes tipos de cáncer, ya que puede actuar como supresor tumoral principalmente en cáncer de próstata, mama, colorrectal y pulmón, debido a mutaciones puntuales y/o expresión reducida o como oncogén en cáncer riñón por sobreexpresión de la proteína. En cáncer endometrial tiene un rol dual, ya que puede actuar como supresor tumoral o como oncogén. SPOP es considerado como un potencial biomarcador pronóstico y un objetivo terapéutico prometedor.

A novel lncRNA-mRNA-miRNA signature predicts recurrence and disease-free survival in cervical cancer

Cervical cancer (CC) patients have a poor prognosis due to the high recurrence rate. However, there are still no effective molecular signatures to predict the recurrence and survival rates for CC patients. Here, we aimed to identify a novel signature based on three types of RNAs [messenger RNA (mRNAs), microRNA (miRNAs), and long non-coding RNAs (lncRNAs)]. A total of 763 differentially expressed mRNAs (DEMs), 46 lncRNAs (DELs), and 22 miRNAs (DEMis) were identified between recurrent and non-recurrent CC patients using the datasets collected from the Gene Expression Omnibus (GSE44001; training) and The Cancer Genome Atlas (RNA- and miRNA-sequencing; testing) databases. A competing endogenous RNA network was constructed based on 23 DELs, 15 DEMis, and 426 DEMs, in which 15 DELs, 13 DEMis, and 390 DEMs were significantly associated with disease-free survival (DFS). A prognostic signature, containing two DELs (CD27-AS1, LINC00683), three DEMis (hsa-miR-146b, hsa-miR-1238, hsa-miR-4648), and seven DEMs (ARMC7, ATRX, FBLN5, GHR, MYLIP, OXCT1, RAB39A), was developed after LASSO analysis. The built risk score could effectively separate the recurrence rate and DFS of patients in the high- and low-risk groups. The accuracy of this risk score model for DFS prediction was better than that of the FIGO (International Federation of Gynecology and Obstetrics) staging (the area under receiver operating characteristic curve: training, 0.954 vs 0.501; testing, 0.882 vs 0.656; and C-index: training, 0.855 vs 0.539; testing, 0.711 vs 0.508). In conclusion, the high predictive accuracy of our signature for DFS indicated its potential clinical application value for CC patients.

DDB1- and CUL4-associated factor 8 plays a critical role in spermatogenesis / 医学前沿

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.

Degradation of SARS-CoV-2 receptor ACE2 by the E3 ubiquitin ligase Skp2 in lung epithelial cells / 医学前沿

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4%-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.

Effect of TRIM31 Gene Silencing on the Proliferation and Apoptosis of U266 Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism.@*METHODS@#The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively.@*RESULTS@#Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells.@*CONCLUSION@#TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.

Screening of interacting proteins of idiopathic gonadotropin-releasing hormone deficiency pathogenic gene RNF216 / 中华医学遗传学杂志

OBJECTIVE@#To screen proteins interacting with ring finger protein 216(RNF216) through yeast two hybrid experiment, and further clarify the role of RNF216 in the pathogenesis of gonadotropin-releasing hormone deficiency.@*METHODS@#A recombinant expression vector pGBKT7-RNF216 was constructed and transformed into yeast Y2HGold, which was hybridized with a human cDNA library in order to screen proteins interacting with RNF216. The interaction was verified in yeast Y2HGold.@*RESULTS@#A recombinant expression vector pGBKT7-RNF216 was successfully constructed and expressed in yeast Y2HGold. Filamin B (FLNB) was identified by yeast two hybrid experiment, and their interaction was verified in yeast Y2HGold.@*CONCLUSION@#An interaction between FLNB and RNF216 was identified through yeast two hybrid experiment. RNF216 may affect the proliferation and migration of GnRH neurons by regulating FLNB or FLNB/FLNA heterodimers.

Intellectual disability due to heterozygous c.40C>T variant of TRIP12 gene in a patient / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a patient with intellectual disability.@*METHODS@#Whole exome sequencing and Sanger sequencing were carried out for the patient. The result was verified in her family.@*RESULTS@#DNA sequencing revealed that the patient has carried a heterozygous nonsense c.40C>T (p.Arg14X) variant of the TRIP12 gene, which was de novo in origin. The variant was unrecorded in the Human Gene Mutation Database. Based on the American College of Medical Genetics and Genomics standards and guidelines, the variant was predicted to be pathogenic (PVS1+ PS2+ PP3).@*CONCLUSION@#The patient was diagnosed with autosomal dominant intellectual disability due to heterozygous c.40C>T variant of the TRIP12 gene.

Rosmarinic acid inhibits high glucose-induced cardiomyocyte hypertrophy by activating Parkin-mediated mitophagy / 南方医科大学学报

OBJECTIVE@#To evaluate the effect of rosmarinic acid (RA) on mitophagy and hypertrophy of cardiomyocytes exposed to high glucose (HG).@*METHODS@#Rat cardiomyocytes (H9c2) exposed to HG (25 mmol/L) were treated with 50 μmol/L RA or with both RA treatment and Parkin siRNA transfection, with the cells cultured in normal glucose (5.5 mmol/L) and HG as the controls. The expressions of PINK1, Parkin and LC3II/LC3I in the cells were detected by Western blotting. The formation of mitochondrial autophagosomes was observed by transmission electron microscope. Flow cytometry was employed to detect the level of reactive oxygen species (ROS) and apoptotic rate of the cells. The activities of respiratory chain complex enzymes were measured by spectrophotometry. Fluorescence enzyme labeling and @*RESULTS@#RA treatment significantly increased the expression levels of PINK1, Parkin and LC3-II/I (@*CONCLUSIONS@#RA can protect rat cardiomyocytes against oxidative stress injury and cardiomyocyte hypertrophy induced by HG by activating Parkin-mediated mitophagy.

Targeting Cullin-RING E3 ligases for anti-cancer therapy: efforts on drug discovery / 浙江大学学报·医学版

Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.

LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis

BACKGROUND: Osteoarthritis (OA) is one of the most common rheumatic diseases of which clinical symptoms includes swelling, synovitis and inflammatory pain, affect patients' daily life. It was reported that non-coding RNAs play vital roles in OA. However, the regulation mechanism of ncRNA in OA pathogenesis has not been fully elucidated. METHODS: The expression of SNHG7, miR-34a-5p and SYVN1 was detected using qRT-PCR in tissues, serum and cells. The protein expression of SYVN1, PCNA, cleavage-caspase 3, beclinl and LC3 were measured using western blot. The RNA immunoprecipitation (RIP), RNA pulldown, and luciferase reporter assays were used to verify the relationship between SNHG7, miR-34a-5p and SYVN1. The MTT and flow cytometry assay was performed to detected cell proliferation and cell apoptosis respectively. RESULTS: In this study, SNHG7 and SYVN1 expression were down-regulated, but miR-34a-5p was up-regulated in OA tissues and IL-1P treated cells compared with normal tissues and chondrocyte. Functional investigation revealed that up-regulated SNHG7 or down-regulated miR-34a-5p could promote cell proliferation and inhibit cell apoptosis and autophagy in OA cells. More than that, RIP, pulldown and luciferase reporter assay was applied to determine that miR-34a-5p was a target miRNA of SNHG7 and SYVN1 was a target mRNA of miR-34-5p. Rescue experiments showed that overexpression of miR-34a reversed high expression of SNHG7-mediated suppression of apoptosis and autophagy as well as promotion of proliferation, while its knockdown inhibited cell apoptosis and autophagy and promoted cell proliferation which could be impaired by silencing SYVN1. In addition, SNHG7 regulated SYVN1 through sponging miR-34a-5p. CONCLUSION: SNHG7 sponged miR-34a-5p to affect cell proliferation, apoptosis and autophagy through targeting SYVN1 which provides a novel sight into the pathogenesis of OA.

PELI3 mediates pro-tumor actions of down-regulated miR-365a-5p in non-small cell lung cancer

BACKGROUND: To analyze the relative expression of PELI3 and its mechanistic involvement in the non-small cell lung cancer (NSCLC). Methods: PELI3 expression in NSCLC tissue samples was determined by the immunohistochemistry. The transcripts abundance of PELI3 was measured with real-time PCR. The protein intensity was analyzed by western blot. The overall survival in respect to PELI3 or miR-365a-5p expression was plotted by the Kaplan-Meier's analysis. Cell growth was determined by colony formation assay. Cell viability was measured by MTT assay. The migration and invasion were evaluated by wound healing and transwell assay respectively. The regulatory effect of miR-365a-5p on PELI3 was interrogated with luciferase reporter assay. The direct binding between miR-365a-5p and PELI3 was analyzed by pulldown assay. RESULTS: PELI3 was aberrantly up-regulated in NSCLC both in vivo and in vitro. High level of PELI3 associated with poor prognosis. PELI3-deficiency significantly inhibited cell viability, colony formation, migration and invasion. We further identified that miR-365a-5p negatively regulated PELI3 in this disease. Ectopic expression of miR-365a-5p in both A549 and H1299 phenocopied PELI3-deficiency. Meanwhile, PELI3-silencing significantly abolished the pro-tumoral effect elicited by miR-365a-5p inhibition. CONCLUSIONS: Our results highlighted the importance of dysregulated miR-365a-5p-PELI3 signaling axis in NSCLC.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Identification of two novel Parkin gene mutations in a patient affected with Juvenile Parkinson's syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP).@*METHODS@#Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated.@*RESULTS@#The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1G>C, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic.@*CONCLUSION@#The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

Recurrent Angelman syndrome caused by a rare partial deletion of UBE3A gene / 中华医学遗传学杂志

OBJECTIVE@#To provide genetic testing for two brothers with mental retardation and epilepsy.@*METHODS@#Array comparative genomic hybridization (aCGH) was used to detect copy number variations in the two patients, their parents and maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was utilized to delineate the deleted region in the pedigree.@*RESULTS@#A 138 kb deletion in 15q11.2 region was detected by aCGH in both patients, which encompassed part of the UBE3A gene. MS-MLPA has narrowed down the region to exons 8 to 14 of the UBE3A gene. The same deletion was also found in their mother and grandfather.@*CONCLUSION@#The pathogenesis of this rare form of recurrent Angelman syndrome may be attributed to the partial deletion of maternal UBE3A gene.

Effect of Denticleless E3 Ubiquitin Protein Ligase on Proliferation and Colone Formation of Multiple Myeloma Cells / 中国医学科学院学报

Objective To determine the effect of denticleless E3 ubiquitin protein ligase(DTL)on the proliferation and clone formation of multiple myeloma(MM)cells and investigate the related mechanism. Methods Mononuclear cells were extracted from 34 MM patients.Mononuclear cells harvested from 14 healthy volunteers were used as controls.Quantitative polymerase chain reaction was used to detect the change of DTL at mRNA level.Furthermore,12 MM patients and 2 controls were selected,in whom the change of DTL at protein level was detected by Western blot.Human MM cell line RPMI8226 was divided into control(CON)group and DTL-short hairpin RNA(DTL-shRNA)group,which was infected with the CON and DTL-shRNA virus,respectively,for 48 hours.The infection efficiency was detected by using flow cytometry,the knock-down efficiencies at mRNA and protein levels were detected by quantitative polymerase chain reaction and Western blot,the change of cell counts in the next 0,24,48,72,96 hours were measured with CCK8 assay.The CON and DTL-shRNA cells were cultured in semisolid medium.Ten days later,inverted phase microscopy was used to measure the number of colones that contain more than 50 cells,annexin V/propidium iodide double staining to detect apoptosis,and propidium iodide staning to detect cell cycle.Finally,Western blot was empoyed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)in nuclear factor-κB(NF-κB)pathway and electrophoretic mobility shift assay(EMSA)to detect the NF-κB transcriptional ability. Results The DTL expression was(1.00±0.12)and(9.36±3.71),respectively in the bone marrow mononuclear cells of healthy volunteers and in the CD138+cells of MM patients(t=3.65,P=0.0024).DTL was also highly expressed in MM CD138+positive cells at protein level.After RPMI8226 was infected by CON and DTL-shRNA virus for 48 hours,green fluorescent protein-positive cells accounted for more than 90%.The relative expression of DTL was(1.00±0.01)and(0.21±0.04)(t=33.19,P<0.0001)at mRNA level and(0.52±0.13)and(0.11±0.02)at protein level(t=5.399,P=0.0057).CCK8 revealed that CON and DTL-shRNA cells proliferated by(1.00±0.03)vs.(1.00±0.02),(2.19±0.28)vs.(1.47±0.13),(3.50±0.14)vs.(2.24±0.19),(5.43±0.41)vs.(3.08±0.14),(7.42±0.17)vs.(4.29±013)after 0,24,48,72,and 96 hours(F=24.58,P=0.001).The number of colone containing more than 50 cells was in 76±4 in CON group and 0 in DTL-shRNA group(P<0.01).The proportion of G1 stage cells was(28.61±8.64)% in CON group and(57.25±10.37)% in DTL-shRNA group(t=3.675,P=0.0213).The proportion of annexin V+in CON and DTL-shRNA groups was(3.21±0.89)% vs.(34.71±18.68)%(t=2.895,P=0.0443).After RPMI8226 was infected with CON or DTL-shRNA virus for 48 hours,the relative expression of phosphorylation P65 was(1.52±0.14)vs.(0.82±0.11)(t=6.81,P=0.0024),the P65 relative expression was(0.25±0.04)vs.(0.24±0.08)(t=0.19,P=0.85),the CON and DTL-shRNA phosphorylation-IκBα relative expression was(0.19±0.03)vs.(0.13±0.02)(t=2.882,P=0.0449),and the IκBα was(0.22±0.05)vs.(1.01±0.06)(t=17.52,P<0.0001).Detection of the transcriptional ability of DTL-shRNA NF-κB by EMSA further confirmed the down-regulation of DTL suppressed the NF-κB transcriptional ability. Conclusions DTL is highly expressed in MM cells,and down-regulation of DTL suppresses the cell proliferation,inhibit the colony formation,and induce cell apoptosis and cell cycle arrest.The effect of DTL on the biological functions of MM cells is related to the change of NF-κB pathway.

Progress in genetic research on familiar aneurysms / 中南大学学报(医学版)

The subarachnoid hemorrhage (SAH) caused by ruptured intracranial aneurysms (IAs) is always a lethality. Increasing evidence suggests a familiar aggregation of IA occurrence, which may relate to genetics and there might be an increasing number of IAs in IA families when mutation of disease genes is aggregating. With the progress in the study of familiar intracranial aneurysms (FIAs), a large number of chromosome fragments are found to be related with IAs, such as 1p36, 5q31, 7q11, 14q22, 17cen, 19q13, Xp22. Further studies indicated that mutation of several genes could be the cause of FIAs, including TNFRSF13B, ANRIL, SOX17, ADAMTS15, RNF213 and LOXL2. The independent genetic epidemiologic study on aneurysm families can be used to discover the related genes more effectively, and to explore the mechanism of occurrence of IAs. It's also the precondition for the prevention of disease.