Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Application of virus-induced gene silencing technology to investigate the phytochrome metabolism mechanism: a review / 生物工程学报

Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.

Silenciamiento génico en insectos plaga que afectan la industria agrícola usando ARN de interferencia / Gene silencing in pest insects that affect the agricultural industry using interference RNA

Resumen Los insectos plaga, son especies de organismos vivos que en forma constante se encuentran en poblaciones altas, ocasionando daños económicos en los cultivos. Generalmente, suele tratarse de especies puntuales, por lo general, sólo una o dos, que pueden causar gran afectación económica en el sector de la agricultura. En las últimas 3 décadas se ha venido desarrollando el concepto de un proceso biológico, detectado en eucariotas ampliamente, mediante el que se pueden silenciar genes, a partir de ARN de doble cadena (ARNdc). Esta maquinaria se ha investigado para conocer su funcionamiento y buscar potenciales aplicaciones que podrían tener en el campo de la biotecnología. En varios estudios se encontró que el silenciamiento de genes se debe a las interacciones enzimáticas intracelulares citoplasmáticas con moléculas de ARN pequeñas (ARNsi), que actúan sobre el ARN mensajero (ARNm) intracelular, impidiendo que este se traduzca a proteína. Mediante este mecanismo se busca silenciar genes específicos en insectos plaga, que sean esenciales para que el insecto pueda vivir y de esa manera evitar la proliferación de la plaga. Este artículo recopila los estudios realizados acerca del ARN de interferencia, referidos al mecanismo genético de los insectos, como alternativa para su control.

AbstractPest insects are species of living organisms that are constantly found in high populations,causing economic crops damage. Generally, it tends to be specific species, usually onlyone or two, which can cause great economic damage in the agricultural sector. In thelast 3 decades, the concept of a biological process has been developed, widely detected in eukaryotes, by which genes can be silenced, from double-stranded RNA (dsRNA). This machinery has been investigated to understand its operation and to look for potential applications that it could have in the field of biotechnology. In several studies it was found that gene silencing is due to cytoplasmic intracellular enzymatic interactions with small RNA molecules (siRNA), which act on intracellular messenger RNA (mRNA), preventing it from translating a protein. Through this mechanism, the aim is to silence specific genes in pest insects, which are essential for the insect to live and thus prevent the proliferation of the pest. This article compiles the studies carried out on RNA interference, referring to the genetic mechanism of insects, as an alternative for its control.

Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism / 中华肿瘤杂志

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

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.

Therapeutic effect of gene silencing peptidyl arginine deaminase 4 on pulmonary interstitial lesions induced by collagen-induced arthritis mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the therapeutic effect of gene silencing peptidyl arginine deaminase 4 (PAD4) on pulmonary interstitial lesions induced by collagen-induced arthritis (CIA) mice, and possible mechanisms.@*METHODS@#A CIA mouse model was established in DBA/1 mice, followed by a tail vein injection of the virus solution prepared by the PAD4-siRNA expression vector once a week for 8 times. The mice were sacrificed at the end of the experiment. The expression of PAD4 mRNA in lungs was detected by real-time quantitative PCR (qRT-PCR). The expression of PAD4 protein was detected by tissue immunohistochemistry. Cell culture was performed by spleen tissue. Flow cytometry changes in the ratio of Tfh cells to Tfr cells were examined; lung staining was performed in the lungs to observe changes in lung pathology.@*RESULTS@#(1) Compared with the blank group, the expression of PAD4 mRNA in the lung tissue of the model group increased, the difference was statistically significant (P < 0.05). PAD4 mRNA in the lung tissue of the CIA mice after PAD4-siRNA treatment. The expression level was significantly lower than that of the model group and the negative control group, and the difference was statistically significant (P < 0.05). (2) Red fluorescence was less in the lung tissue of the blank group, while more red fluorescence was observed in the inflammatory cell infiltration area and trachea around the lung tissue of the model group and the negative control group, and the red fluorescence of the three groups after PAD4-siRNA treatment was significantly reduced; (3) Compared with the blank group, the proportion of Tfh cells in the model group increased, the difference was statistically significant (P < 0.05), the proportion of Tfh cells in spleen cells of the CIA mice after PAD4-siRNA treatment was significantly lower than that of the model group and the negative control group, the difference was statistically significant (P < 0.05); compared with the blank group, in the mouse spleen cells in the model group the proportion of Tfr cells was slightly decreased, but the difference was not statistically signifi-cant. The proportion of Tfr cells in the spleen cells of the mice increased after PAD4-siRNA treatment, but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group (P < 0.05); (4) The proportion of Tfh/Tfr in the spleen cells of the model group was increased, compared with the blank group, the difference was statistically significant (P < 0.05); the ratio of Tfh/Tfr in the three groups after PAD4-siRNA treatment all decreased, the difference was statistically significant (P < 0.05); (5) Compared with the blank group, the alveolar wall of the lung tissue of the model group was thickened, the inflammatory cell infiltration was increased, and the lung tissue destruction and inflammatory infiltration of the CIA mice were decreased after PAD4-siRNA treatment. The degree of reduction was reduced.@*CONCLUSION@#Gene silencing of PAD4 can reduce the proportion of Tfh cells, increase the proportion of Tfr cells, reverse the proportion of Tfh/Tfr, and reduce the degree of interstitial lesions and inflammatory infiltration of lung tissue.

Síndrome X frágil: presentación clínica, patología y tratamiento / Fragile X syndrome: clinical presentation, pathology and treatment

Resumen El síndrome X frágil es la condición monogenética que produce más casos de autismo y de discapacidad intelectual. La repetición de tripletes CGG (> 200) y su metilación conllevan el silenciamiento del gen FMR1. La proteína FMRP (producto del gen FMR1) interacciona con los ribosomas, controlando la traducción de mensajeros específicos y su pérdida produce alteraciones de la conectividad sináptica. El tamizaje de síndrome X frágil se realiza por reacción en cadena de la polimerasa. La recomendación actual de la Academia Americana de Pediatría es realizar pruebas a quienes presenten discapacidad intelectual, retraso global del desarrollo o antecedentes familiares de afección por la mutación o premutación. Países hispanos como Colombia, Chile y España reportan altas prevalencias de síndrome X frágil y han creado asociaciones o corporaciones nacionales de X frágil que buscan acercar a los pacientes a redes disponibles de diagnóstico y tratamiento.

Abstract Fragile X syndrome is the monogenetic condition that produces more cases of autism and intellectual disability. The repetition of CGG triplets (> 200) and their methylation entail the silencing of the FMR1 gene. The FMRP protein (product of the FMR1 gene) interacts with ribosomes by controlling the translation of specific messengers, and its loss causes alterations in synaptic connectivity. Screening for fragile X syndrome is performed by polymerase chain reaction. Current recommendation of the American Academy of Pediatrics is to test individuals with intellectual disability, global developmental retardation or with a family history of presence of the mutation or premutation. Hispanic countries such as Colombia, Chile and Spain report high prevalence of fragile X syndrome and have created fragile X national associations or corporations that seek to bring patients closer to available diagnostic and treatment networks.

PPP3CA silence regulates MET process, cell apoptosis, proliferation and migration in metanephric mesenchyme cells / 生物工程学报

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Pioneering studies on monogenic central precocious puberty

ABSTRACT Pubertal timing in humans is determined by complex interactions including hormonal, metabolic, environmental, ethnic, and genetic factors. Central precocious puberty (CPP) is defined as the premature reactivation of the hypothalamic-pituitary-gonadal axis, starting before the ages of 8 and 9 years in girls and boys, respectively; familial CPP is defined by the occurrence of CPP in two or more family members. Pioneering studies have evidenced the participation of genetic factors in pubertal timing, mainly identifying genetic causes of CPP in sporadic and familial cases. In this context, rare activating mutations were identified in genes of the kisspeptin excitatory pathway (KISS1R and KISS1 mutations). More recently, loss-of-function mutations in two imprinted genes (MKRN3 and DLK1) have been identified as important causes of familial CPP, describing novel players in the modulation of the hypothalamic-pituitary-gonadal axis in physiological and pathological conditions. MKRN3 mutations are the most common cause of familial CPP, and patients with MKRN3 mutations present clinical features indistinguishable from idiopathic CPP. Meanwhile, adult patients with DLK1 mutations present high frequency of metabolic alterations (overweight/obesity, early onset type 2 diabetes and hyperlipidemia), indicating that DLK1 may be a novel link between reproduction and metabolism. Arch Endocrinol Metab. 2019;63(4):438-44

The pathways of cell cycle regulation in retinoblastoma / As vias de regulação do ciclo celular em retinoblastoma

Retinoblastoma is a childhood ocular tumor often caused by the biallelic inactivation of the RB1 gene affecting children up to 5 years of age. A retinoblastoma protein (pRB), encoded by the tumor suppressor gene RB1, is responsible for the regular progression of the G1 phase to the phase S of the cell cycle. This protein forms a complex with the transcriptional factor E2F causing the cell cycle to remain in the G0/G1 stage. With a phosphorylation of cyclin-dependent kinases (CDK), the phosphorylation of the RB protein is activated and the complex formed with E2F is disrupted, with the advancement of the cell cycle to an S phase and cell proliferation. All the control of cell proliferation is regulated not only by the complex formed by RB and E2F proteins, but also by other proteins that participate in and/or interfere in this cell division control mechanism, such as mdm2, mdm4 and p21 proteins.

O retinoblastoma é um tumor ocular infantil ocasionado, frequentemente, pela inativação bialélica do gene RB1 acometendo crianças até os 5 anos de idade. A proteína retinoblastoma (pRB), codificada pelo gene supressor tumoral RB1, é responsável por regular a progressão da fase G1 para a fase S do ciclo celular. Essa proteína forma um complexo com o fator transcricional E2F fazendo com que o ciclo celular permaneça no estágio G0/G1. Com a fosforilação de quinases dependentes de ciclinas, a fosforilação da proteína RB é ativada e o complexo formado com o E2F é desfeito, havendo o avanço do ciclo celular para a fase S e a proliferação celular. Todo esse controle da proliferação celular é regulado não só pelo complexo formado pela proteína RB e E2F, mas também por outras proteínas que participam e/ou interferem neste mecanismo de controle da divisão celular, como, por exemplo, as proteínas mdm2, mdm4, p21

Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells. METHODS AND RESULTS: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells. CONCLUSIONS: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

Effects of MD2 gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes.@*METHODS@#The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1β and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels.@*RESULTS@#The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P＜0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P＜0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1β , IL-6(P＜0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P＜0.01).@*CONCLUSION@#This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.

Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1 / 中国医学科学院学报

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(HO),calcium-sensitive receptor agonist gadolinium trichloride(GdCl),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of HO.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without HO.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with HO,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the HO group and HO+GdCl group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of HO concentration were similar.HO-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of HO.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the HO +siRNA treatment group and the HO treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.

Effect of Silencing and Overexpression of LNK Gene on STAT3 Expression in THP-1 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of LNK gene silencing and overexpression on the expression of STAT3 gene in human monocytic leukemia cells (THP-1).@*METHODS@#THP-1 cells were cultured, and the lentivirus was used as a vector to silence and overexpres the LNK gene stably. After transfection for 72 hours, the GFP expression levels were observed by inverted fluorescence microscopy. The lentiviral transfection efficiencies were detected by flow cytometry. The effects of LNK silencing and overexpression were confirmed, and the expression of STAT3 mRNA was detected by RT-PCR. The protein levels of LNK and STAT3 were detected by Western blotting.@*RESULTS@#The GFP expression level of THP-1 cells reached more than 85% after transfection with lentivirus for 72 hours, and the transfection efficiency of cells was above 99%. mRNA expressions levels of LNK and STAT3 in LNK silencing group were signifycantly lower than those in control group, while LNK and STAT3 mRNA levels in the LNK overexpression group was significantly higher than those in control group (P＜0.05). The protein expression levels of LNK and STAT3 in LNK silencing group were significantly lower than those in control group, while that in LNK overexpression group was significantly higher than that in control group (P＜0.05).@*CONCLUSION@#The THP-1 cell line with LNK gene silencing and overexpression has been successfully established. The LNK gene silencing resulted in decrease of STAT3 expression; LNK gene overexpression and leads to inereases of STAT3 expression indicating that LNK participates in the regulation of STAT3.

Silencing RRM1 gene reverses paclitaxel resistance in human breast cancer cell line MCF- 7/R by inducing cell apoptosis / 南方医科大学学报

OBJECTIVE@#To investigate the effects of ribonucleotide reductase catalytic subunit M1 (RRM1) gene silencing on drug resistance of human breast cancer cell line MCF-7/R.@*METHODS@#We established a paclitaxel-resistant breast cancer MCF-7 cell line (MCF-7/R) by exposing the cells to high-concentration paclitaxel in a short time. Small interfering RNAs (siRNAs) targeting RRM1 were designed to silence RRM1 expression in human breast cancer MCF-7/R cells. MTT assay was used to detect the IC values and the sensitivity to paclitaxel in the cells with or without siRNA transfection. The changes in the proliferative activity of MCF7 and MCF-7/R cells following RRM1 gene silencing were evaluated using EdU assay. Flow cytometry was used to analyze the cell apoptosis and cell cycle changes. We assessed the effect of RRM1 gene silencing and paclitaxel on the tumor growth in a nude mouse model bearing subcutaneous xenografts with or without siRNA transfection.@*RESULTS@#We detected significantly higher expressions of RRM1 at both the mRNA and protein levels in the drug-resistant MCF- 7/R cells than in the parental MCF-7 cells ( < 0.01). Transfection with the specific siRNAs significantly reduced the expression of RRM1 in MCF-7/R cells ( < 0.05), which showed a significantly lower IC value of paclitaxel than the cells transfected with the negative control siRNA ( < 0.05). RRM1 silencing significantly inhibited the proliferation ( < 0.01) and enhanced the apoptosis-inducing effect of paclitaxel in MCF-7/R cells ( < 0.001); RRM1 silencing also resulted in obviously reduced Akt phosphorylation, suppressed Bcl-2 expression and promoted the expression of p53 protein in MCF-7/R cells. In the tumor-bearing nude mice, the volume of subcutaneously transplanted tumors was significantly smaller in MCF-7/R/siRNA+ PTX group than in the other groups ( < 0.001).@*CONCLUSIONS@#RRM1 gene silencing can reverse paclitaxel resistance in human breast cancer cell line MCF-7/R by promoting cell apoptosis.

- gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism.@*METHODS@#CD4 T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA.@*RESULTS@#The CD4 T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals ( < 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein ( < 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment ( < 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups ( < 0.05).@*CONCLUSIONS@#Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells probably as the result of enhanced IL-2 secretion and T lymphocyte activation.

Mig- gene silencing inhibits vasculogenic mimicry formation and invasion of glioma U251 cells by suppressing MEK/ERK signaling / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effects of silencing migration-inducing gene-7 (Mig-7) on vasculogenic mimicry formation, migration and invasion of human glioma cells and whether MEK/ERK signaling pathway mediates these effects.@*METHODS@#Human glioma U251 cells were infected by lentiviral vectors carrying a small interfering RNA targeting Mig-7 gene (sh-Mig-) or a negative control shRNA (sh-NC), and real-time quantitative PCR was used to detect the expression level of Mig- mRNA in the cells. Three-dimensional culture and Transwell chamber invasion assay were used to observe the effect of Mig- gene silencing on vasculogenic mimicry formation and invasion ability of the U251 cells. Western blotting was performed to detect the changes in the protein expression levels of MEK/ERK in the infected cells.@*RESULTS@#We successfully obtained a U251 cell line with stable low expression of Mig- gene using RNA interference technique. Compared with the cells infected with sh-NC lentivirus and the non- infected cells, U251 cells infected with the lentiviral vector carrying sh-Mig- showed significantly decreased expression level of Mig- ( < 0.01) with obviously lowered vasculogenic mimicry formation and invasion abilities ( < 0.05). Mig- silencing also significantly lowered the expressions of MEK and ERK proteins in U251 cells ( < 0.05).@*CONCLUSIONS@#Silencing of Mig-7 gene inhibits vasculogenic mimicry formation and invasion of U251 cells possibly by suppressing MEK/ERK signaling, suggesting the important role of Mig-7 gene in vasculogenic mimicry formation and invasion of human glioma cells.

Effect of Silencing LNK Gene on the Expression of EPO and EPOR in THP-1 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of silencing LNK gene on the expression of EPO and EPOR in acute myeloid leukemia cells (THP-1).@*METHODS@#THP-1 cells were cultured. The lentivirus was used as a vector to silence the LNK gene stably. After 72 hours of infection, GFP expression level was detected by the fluorescent inverted microscopy. The lentiviral Infection efficiencies were monitored by flow cytometry. The LNK silencing effect was confirmed. The mRNA expressions of EPO and EPOR were detected by RT-PCR. The protein levels of LNK, EPO and EPOR were detected by Western blot.@*RESULTS@#At the time-point of 72 hours after lentivirus infection, the expression level of GFP was above 85% detected by fluorescent inverted microscopy. The infection efficiency was above 99% by flow cytometry. mRNA expressions of LNK, EPO and EPOR in LNK silencing group were signifycantly lower than those in control group (P＜0.05). The protein levels of LNK, EPO and EPOR in LNK silencing group were significantly lower than those in the control group (P＜0.05).@*CONCLUSION@#THP-1 cell line of LNK gene silencing has been successfully established,the LNK gene has been silenced, the expression of EPO and EPOR decrease, indicating that LNK may participate in the regulation of EPO and EPOR.