Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells.@*METHODS@#Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated.@*RESULTS@#Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing.@*CONCLUSION@#RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

Neutrophil extracellular traps activates focal adhesion kinase by upregulating MMP9 expression to promote proliferation and migration of mouse colorectal cancer cells / 细胞与分子免疫学杂志

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by TranswellTM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.

Knock-down of long intergenic noncoding RNA cyclooxygenase 2 (lincRNA-COX2) inhibits apoptosis and polarization into M1 in Listeria monocytogenes-infected macrophages / 细胞与分子免疫学杂志

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.

Circular RNA ame_circ_000115 regulates expression of genes in larval gusts of Apis mellifera ligustica stressed by Ascosphaera apis / 生物工程学报

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.

Role of biomimetic nanomaterials made from glioma cell- derived extracellular vesicles in targeted delivery of STAT3-siRNA / 中南大学学报(医学版)

OBJECTIVES@#Glioma is the most common primary intracranial tumor and there is still no ideal treatment at present. Gene therapy, as one of the new methods for treating glioma, has attracted attention in recent years. But its application in treating glioma is very limited due to lack of effective delivery vectors. This study aims to investigate the feasibility of biomimetic nanomaterials made from glioma cells-derived extracellular vesicles (EV) for targeted delivery of signal transducers and activators of transcription 3 (STAT3)-small interfering RNA (siRNA) in treating glioma.@*METHODS@#First, U251 glioma cells-derived extracellular vessel (EVU251) was extracted by ultra-centrifugal method. Nanoparticle tracking analysis was used to characterize the particle size distribution, the transmission electron microscope was used to analyze the morphology, and Western blotting was used to verify the expression of srface characteristic protein. The homing ability was verified by cell uptake assay after labeling EVU251 with membrane dye kit PKH67; the EVU251 contents were removed by a low permeability method and then EVMU251 was prepared through a microporous membrane. Finally, the biomimetic nanomaterials EVMU251@STAT3-siRNA were prepared by loading STAT3-SiRNA with electro-dyeing method. The real-time quantitative PCR was used to quantify the successful encapsulation of siRNA, and the encapsulation and drug loading rate was calculated; then Cy5-labeled siRNA was used to evaluate the ability of biomimetic nanomaterials (EVMU251@CY5-siRNA) to target U251 cells. Lysosomal escape ability of the biomimetic nanomaterial was evaluated by lysosomal dye lyso-tracker green. At last, the ability of EVMU251@STAT3-siRNA to knock down STAT3 gene and selective killing of U251 cells was detected by cell experiments in vitro.@*RESULTS@#The size of EVU251 ranged from 50 nm to 200 nm with a natural disc shape. The expression of extracellular vesicle marker proteins could be detected on the membrane of EVU251. The cell uptake assay demonstrated that it had homing ability to target U251 cells. After EVU251 was prepared as EVMU251@STAT3-siRNA, the particle size was (177.9±5.0) nm, the siRNA loading rate was (33.5±2.2)% and the drug loading rate was (3.24±0.21)%. The biomimetic nanomaterial EVMU251@STAT3-siRNA still had the ability to target U251 cells and successfully deliver siRNA to the cytoplasm without lysosomal degradation. The EVMU251@STAT3-siRNA can effectively knock down the expression of STAT3 gene and produce selective killing ability in U251 cells.@*CONCLUSIONS@#The biomimetic nanomaterials EVMU251@STAT3-siRNA made from glioma U251 cells-derived extracellular vesicles can knock down STAT3 gene of U251 cells and produce selective killing effect, which can provide a new idea for the treatment of glioma.

Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells / 中华肝脏病杂志

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

Inhibitory Effect of Interference with PD-L1 Expression on B-cell lymphoma in Mice / 中国实验血液学杂志

OBJECTIVE@#To investigate the potential inhibitory effect of interference with PD-L1 on B cell lymphoma in mice.@*METHODS@#Three shRNA vectors for mouse CD274 (PD-L1) were constructed and transiently transfected into 293T cells. RT-qPCR was used to validate the interference efficiency of CD274. The shRNA vector that interfere efficiently with CD274 expression was packaged by using lentivirus packaging system to generate shRNA lentivirus, and then transfected into A20 lymphoma cell line. The methyl thiazol terazolium (MTT) assay was used to detect proliferation after 48 h culture of CD274-sh A20 cells. Meanwhile, BALB/c mice were hypodermically infected with CD274-sh A20 cells. Infected mice were observed daily and assessed to visualize tumor by in vivo fluorescence imaging.@*RESULTS@#The proliferation rate of CD274-sh A20 cells in vitro was significantly lower than that of A20 cells (P<0.05). The tumor size detected by in vivo fluorescence imaging showed a significant reduce in tumor bearing mice with CD274-sh compared with other tumor bearing mice. And the weight and size of tumor in CD274-sh group were also significantly reduced compared with other group (P<0.05). Moreover, the survival time of tumor bearing mice in CD274-sh group was longer than that of the PD-L1 high expression group.@*CONCLUSION@#PD-L1 plays an important role in the incidence and the progression of lymphoma, and the shRNA-based PD-L1 knockdown can inhibit cell proliferation of A20 cells and partly suppress tumor growth.

Vascular endothelial growth factor induces inflammatory injury of pancreatic tissue by activating autophagy in hyperlipidemic acute pancreatitis rats / 生理学报

This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.

Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle / 中华医学杂志(英文版)

BACKGROUND@#Systemic lupus erythematosus (SLE) is a complex autoimmune disease, and the mechanism of SLE is yet to be fully elucidated. The aim of this study was to explore the role of two-pore segment channel 2 (TPCN2) in SLE pathogenesis.@*METHODS@#Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of TPCN2 in SLE. We performed a loss-of-function assay by lentiviral construct in Jurkat and THP-1 cell. Knockdown of TPCN2 were confirmed at the RNA level by qRT-PCR and protein level by Western blotting. Cell Count Kit-8 and flow cytometry were used to analyze the cell proliferation, apoptosis, and cell cycle of TPCN2-deficient cells. In addition, gene expression profile of TPCN2-deficient cells was analyzed by RNA sequencing (RNA-seq).@*RESULTS@#TPCN2 knockdown with short hairpin RNA (shRNA)-mediated lentiviruses inhibited cell proliferation, and induced apoptosis and cell-cycle arrest of G2/M phase in both Jurkat and THP-1 cells. We analyzed the transcriptome of knockdown-TPCN2-Jurkat cells, and screened the differential genes, which were enriched for the G2/M checkpoint, complement, and interleukin-6-Janus kinase-signal transducer and activator of transcription pathways, as well as changes in levels of forkhead box O, phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin, and T cell receptor pathways; moreover, TPCN2 significantly influenced cellular processes and biological regulation.@*CONCLUSION@#TPCN2 might be a potential protective factor against SLE.

Knockdown of lncRNA H19 suppresses endometriosis in vivo

The long noncoding RNA (lncRNA) H19 is involved in the pathogenesis of endometriosis by modulating the proliferation and invasion of ectopic endometrial cells in vitro, but related in vivo studies are rare. This study aimed to investigate the role of lncRNA H19 in a nude mouse model of endometriosis. Ectopic endometrial stromal cells (ecESCs) were isolated from ectopic endometrium of patients with endometriosis and infected with lentiviruses expressing short hairpin RNA (shRNA) negative control (LV-NC-shRNA) or lncRNA-H19 shRNA (LV-H19-shRNA). The ecESCs infected with LV-NC-shRNA and LV-H19-shRNA were subcutaneously implanted into forty 6- to 8-week-old female nude mice. The size and weight of the endometriotic implants were measured at 1, 2, 3, and 4 weeks after implantation and compared, and lncRNA H19 levels in endometriotic implants were evaluated using real-time polymerase chain reaction (RT-PCR). All nude mice survived the experimental period, and no significant differences in body weight were observed between the experimental group and the control group. All nude mice developed histologically confirmed subcutaneous endometriotic lesions with glandular structures and stroma after 1 week of implantation. The subcutaneous lesions in the LV-NC-shRNA group after 1, 2, 3, and 4 weeks of implantation were larger than those in the LV-H19-shRNA group, and lncRNA H19 levels in subcutaneous lesions in the LV-NC-shRNA group were significantly higher than those in the LV-H19-shRNA group. Knockdown of lncRNA H19 suppresses endometriosis in vivo. Further study is required to explore the underlying mechanism in the future.

Repair of osteoarthritis in animal model with exosomes derived from BMSCs transfected by the siRNA -Piezo1 through CT navigation / 中国骨伤

OBJECTIVE@#To investigate the effect of the exosomes from bone marrow mesenchymal stem cells (BMSCs) transfected with silence plasmid of Piezol small interference RNA (siRNA)on osteoarthritis (OA) animal model.@*METHODS@#Twenty male SD rats with specific pathogen free (SPF) were selected, ranging in age from 5.46 to 6.96 months, with a mean of (6.21± 0.75) months;and ranging in weight from 385.76 g to 428.66 g, with a mean of (407.21±21.45) g. BMSCs were extracted. The siRNA silencing plasmid of piezo1 was constructed by siRNA technology. After lentivirus was transfected into BMSCs, the exosomes were extracted. At the cellular level, BMSCs were divided into blank plasmid group and siRNA silencing plasmid group according to whether siRNA-Piezo1 was transfected or not. The osteogenic induction ability of siRNA-Piezo1 on BMSCs was detected by RT-PCR and Western blot. At the animal model level, the OA model was established by surgical resection of cruciate ligament of knee joint.According to different treatment schemes, SD rats were divided into 4 groups:blank control group, model group, BMSCs group and exosome group. SD rats in the blank control group were not treated. In the model group, the cruciate ligaments of rats were excised and OA animal model was established. In BMSCs group, BMSCs were injected into knee joint under CT guidance after OA model establishment, and the cell volume was 5×10@*RESULTS@#The lentivirus transfection efficiency was(92.11±4.22)%. RT-PCR showed that the relative expression of Piezo1 mRNA in blank plasmid group was 1.07±0.06, which was significantly different from that of 0.31±0.01 in siRNA silencing plasmid group (@*CONCLUSION@#Piezo1 siRNA silencing vector can promote the differentiation of BMSCs into chondrocytes and effectively inhibit the progression of OA, so as to delay the disease of OA.

Lentivirus mediated siRNA hsa-circ-0000885 transfection of BMSCs and osteoclast co-culture system on cell differentiation, proliferation and apoptosis / 中国骨伤

OBJECTIVE@#To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP).@*METHODS@#From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining.@*RESULTS@#The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (@*CONCLUSION@#The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.

Pathways and mechanisms of RNA interference mediated by viral siRNA / 生物工程学报

RNA interference (RNAi) is one of the important mechanisms to regulate gene expression in eukaryotes. One of the original functions of RNAi is to facilitate the antiviral strategy of host. Early studies reveal that invertebrates can use RNAi to resist viruses. However, if this mechanism exists in mammals is still controversial. The latest studies confirm that mammals do have the RNAi-based immunity, and researchers believe that RNAi-based antiviral immunity is a brand-new immunological mechanism that was neglected in the past. It is worthy to note that virus can also use RNAi to enhance its infectivity and immune escape in host cells. This review introduces the research history of RNAi-based antiviral immunity in animals and summarizes the main findings in this field. Last but not least, we indicate a series of unresolved questions about RNAi-based antiviral immunity, and explore the relationship between RNAi-based antiviral immunity and other innate immunological pathways. The virus-mediated RNAi pathway in animal is not only an interesting basic biology question, but also has important guiding roles in the development of antiviral drugs.

Effects of Arf6 downregulation on biological characteristics of human prostate cancer cells

ABSTRACT Objective To evaluate the effects of Arf6 downregulation on human prostate cancer cells. Materials and Methods The effects of Arf6 downregulation on cell proliferation, migration, invasion and apoptosis were assessed by MTT, BrdU, scratch, Transwell assays and flow cytometry respectively. AKT, p-AKT, ERK1/2, p-ERK1/2 and Rac1 protein expressions were detected by Western blot. Results Downregulating Arf6 by siRNA interference suppressed the mRNA and protein expressions of Arf6. The proliferation capacities of siRNA group at 48h, 72h, and 96h were significantly lower than those of control group (P <0.05). The migration distance of siRNA group at 18h was significantly shorter than that of control group (P <0.01). The number of cells penetrating Transwell chamber membrane significantly decreased in siRNA group compared with that of control group (P <0.01). After 24h, negative control and normal control groups had similar apoptotic rates (P >0.05) which were both significantly lower than that of siRNA group (P <0.01). After Arf6 expression was downregulated, p-ERK1/2 and Rac1 protein expressions were significantly lower than those of control group (P <0.05). Conclusion Downregulating Arf6 expression can inhibit the proliferation, migration and invasion of prostate cancer cells in vitro, which may be related to ERK1/2 phosphorylation and Rac1 downregulation.

Small interfering RNA targeting of keratin 17 reduces inflammation in imiquimod-induced psoriasis-like dermatitis / 中华医学杂志(英文版)

BACKGROUND@#Psoriasis is a common chronic inflammatory skin disease with 2% to 3% prevalence worldwide and a heavy social-psychological burden for patients and their families. As the exact pathogenesis of psoriasis is still unknown, the current treatment is far from satisfactory. Thus, there is an urgent need to find a more effective therapy for this disease. Keratin 17 (K17), a type I intermediate filament, is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis. Therefore, we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis. This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA (siRNA) on mice with imiquimod (IMQ)-induced psoriasis-like dermatitis.@*METHODS@#Eight-week-old female BALB/c mice were administered a 5% IMQ cream on both ears to produce psoriatic dermatitis. On day 3, K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days. The right ears of the mice were treated in parallel with negative control (NC) siRNA. Inflammation was evaluated by gross ear thickness, histopathology, the infiltration of inflammatory cells (CD3+ T cells and neutrophils) using immunofluorescence, and the expression of cytokine production using real-time quantitative polymerase chain reaction. The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance.@*RESULTS@#The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears, as evidenced by the alleviated ear inflammation phenotype, including decreased ear thickness, infiltration of inflammatory cells (CD3+ T cells and neutrophils), and inflammatory cytokine/chemokine expression levels (interleukin 17 [IL-17], IL-22, IL-23, C-X-C motif chemokine ligand 1, and C-C motif chemokine ligand 20) (P < 0.05 vs. the Blank or NC siRNA groups). Compared to the NC siRNA treatment, the K17 siRNA treatment resulted in increased K1 and K10 expression, which are characteristic of keratinocyte differentiation (vs. NC siRNA, K17 siRNA1 group: K1, t = 4.782, P = 0.0050; K10, t = 3.365, P = 0.0120; K17 siRNA2 group: K1, t = 4.104, P = 0.0093; K10, t = 4.168, P = 0.0042; siRNA Mix group: K1, t = 3.065, P = 0.0221; K10, t = 10.83, P < 0.0001), and decreased K16 expression, which is characteristic of keratinocyte proliferation (vs. NC siRNA, K17 siRNA1 group: t = 4.156, P = 0.0043; K17 siRNA2 group: t = 2.834, P = 0.0253; siRNA Mix group: t = 2.734, P = 0.0250).@*CONCLUSIONS@#Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis. Thus, gene therapy targeting K17 may be a potential treatment approach for psoriasis.

Silencing long non-coding RNA HIF1A-AS2 inhibits proliferation, invasion and migration of cervical cancer cells / 南方医科大学学报

OBJECTIVE@#To explore the inhibitory effects of silencing long non-coding RNA (LncRNA) HIF1A-AS2 on epithelialmesenchymal transition (EMT) and tumor stem cell-like phenotype in cervical cancer cells.@*METHODS@#We designed 3 shRNA constructs for silencing HIF1A-AS2 in CaSki cells, and the shRNA with the strongest interference effect was selected for subsequent experiment. CaSki cells were transfected with shRNA-NC or Sh-HIF1A-AS2, and the changes in cell viability, invasion ability, EMT, expressions of EMT-related proteins, formation of cell spheres and expressions of stem cell markers were detected.@*RESULTS@#Transfection with shRNA-NC and Sh-HIF1A-AS2 did not significantly affected the viability of CaSki cells (@*CONCLUSIONS@#Silencing HIF1A-AS2 can inhibit proliferation, invasion and migration of cervical cancer cells

Effect of small interfering RNA-mediated BIRC6 silencing on apoptosis and autophagy of renal cancer 786-O cells / 南方医科大学学报

OBJECTIVE@#To study the expression of BIRC6 in renal cancer tissues and investigate the effect of BIRC6 silencing on apoptosis and autophagy of 786-O cells.@*METHODS@#Twenty surgical specimens of renal cancer tissues and adjacent renal tissues were collected from Meizhou People's Hospital between February, 2016 and December, 2018 for detection of BIRC6 protein expression using immunohistochemistry. Renal cancer 786-O cells were transfected with a control small interfering RNA (siRNA) or BIRC6 siRNA @*RESULTS@#The expression of BIRC6 protein was significantly higher in renal cancer tissues than in the adjacent renal tissues. Western blotting showed that siRNA-mediated silencing of BIRC6 significantly lowered the expression of BIRC6 in 786-O cells. In the cells with BIRC6 silencing, treatment with 12.5, 25, 50, 100 and 200 μg/mL 5-FU resulted in significantly higher proliferation inhibition rates than in the cells transfected with the control siRNA (@*CONCLUSIONS@#Interference of BIRC6 mediated by siRNA can inhibit autophagy and promote 5-FU-induced apoptosis to enhance the sensitivity of 786-O cells to 5-FU.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA

Abstract The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

Ectasia ductal mamaria en niños, a propósito de un caso clínico: A case report / Mammary duct ectasia in children

Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.

Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.

Síndrome de Wiskott-Aldrich: Caso clínico / Wiskott-Aldrich syndrome: Case report

El síndrome de Wiskott-Aldrich es una inmunodeficiencia primaria; con una incidencia de 3,5 a 5,2 por cada millón de recién nacidos masculinos. Se caracteriza por tener un patrón de herencia recesiva ligada al cromosoma X. En estos pacientes; se ha descrito la tríada clásica de inmunodeficiencia; microtrombocitopenia y eczema. Presentamos un paciente de 5 años de edad; hispánico; con antecedentes de numerosas infecciones desde el primer año de vida. Actualmente; presenta desnutrición crónica; talla baja secundaria y retraso en el desarrollo del lenguaje. Se diagnosticó una mutación poco frecuente del gen asociado al síndrome de Wiskott-Aldrich.

The Wiskott-Aldrich syndrome is a rare X-linked recessive immunodeficiency, with an estimated incidence of 3.5 to 5.2 cases per million males. It is characterizedby immunodeficiency, microthrombocytopenia and eczema. We present a 5-year-old Hispanic male, with a medical history of numerous infectious diseases, compromised health, chronic malnutrition, language delay and failure to thrive. An infrequent mutation in the Wiskott-Aldrich syndrome gene was found.