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1.
Braz. j. med. biol. res ; 54(4): e10117, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153531

ABSTRACT

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.


Subject(s)
Humans , Animals , Female , Rabbits , Endometriosis/genetics , RNA, Long Noncoding/genetics , RNA, Small Interfering/genetics , Cell Proliferation/genetics , Endometrium , Mice, Nude
2.
Article in Chinese | WPRIM | ID: wpr-815607

ABSTRACT

@#Objective: To investigate the effect of Notch4 regulatingATF2 (activating transcription factor 2) on the invasion and migration of pancreatic cancer (PC) cells and its possible mechanism. Methods:Atotal of 60 pairs of PC tissues and corresponding para-cancerous tissues that surgically resected at Taizhou University Hospital during February 2015 and July 2019 were collected for this study. The expression of Notch4 was detected by immunohistochemistry. siRNA technology was used to knock down Notch4 gene expression in PC cell lines (MiaPaCa-2 and PANC-1). Transwell assay was used to analyze the effect of Notch4 knockdown on cell invasion and migration. qPCR and Western blotting (WB) were used to detect the effects of Notch4 knockdown on mRNA and protein expressions of Notch4 and ATF2. Results: Compared with para-cancerous tissues, the expression of Notch4 in PC tissues significantly higher (P<0.01). After Notch4 siRNA transfection, the mRNA and protein expressions of Notch4 and ATF2 in MiaPaCa-2 and PANC-1 cells significantly decreased (all P<0.01). Compared with Control siRNA group, the migration and invasion ability of PC cells in Notch4 siRNA groupsignificantlyreduced(allP<0.01).Conclusion:Notch4ishighlyexpressedinPCtissues.Knockdown of Notch4 can down-regulate the expression ofATF2 at the transcriptional level, thereby inhibiting the invasion and migration ability of PC cells.

3.
Chinese Journal of Biotechnology ; (12): 1414-1421, 2020.
Article in Chinese | WPRIM | ID: wpr-826835

ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.


Subject(s)
CRISPR-Cas Systems , DNA Ligase ATP , Genetics , Gene Expression Regulation , Genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Ku Autoantigen , Genetics
4.
Article in Chinese | WPRIM | ID: wpr-863746

ABSTRACT

Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism.Methods PMVECs were cultured in vitro.When the cells were in the logarithmic growth phase,the cell were ransfected with lentivirus,and a stable low-expression ERRα cell line was constructed.The cells were divided into four groups:Ctr group (normal control group),Ctr+LPS group (normal celI+LPS treatment group),shERRα1 (shERRα1 gene knockdown group),and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group).After 20 μg/mL LPS stimulated cells in the control group and shERRal group for 6,12 and 24 h,cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group,and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid.After 12 h LPS stimulation,the expression levels of ERRα and NF-κB related proteins (p-p65,p65,P-IKBα,IKBα) were measured by Western blot.Pairwise comparisons were performed with SNK-q test (two-tailed),and multiple-group comparisons were performed with one-way ANOVA.The non-parametric test of rank transformation was used when homogeneity of variance were not met.P value<0.05 was considered significantly different.Results Compared with the control group,ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01).At 6,12 and 24 h after LPS stimulation,compared with the control group,the cell proliferation ability (%) of the shERRαl+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supematant were significantly increased.The change was most obvious after 12 h stimulation.Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly,while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRαl+LPS group (all P<0.05).Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation.

5.
Article in Chinese | WPRIM | ID: wpr-843437

ABSTRACT

Objective • To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods • A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nuclease-deactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results • Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion • This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.

6.
Article in Chinese | WPRIM | ID: wpr-746048

ABSTRACT

Objective To investigate the regulation of Treg/Th17 axis by mannan-binding lectin (MBL) in mice with Candida albicans ( C. albicans ) infection. -ethods MBL gene-knockdown (MBL-/-) C57BL/6 mice and wild-type (WT) C57BL/6 mice were divided into four groups. C. albicans strains (2×107 CFU) were injected intraperitoneally into the mice of infection groups. Paraffin sections of mouse liver and kidney tissues were prepared on 3 d. Histopathological changes were observed with hematox-ylin and eosin ( HE) and Periodic acid-Schiff ( PAS) staining. Flow cytometry was performed to analyze Th17 and Treg cells in mice on 7 d. The levels of IL-10 and IL-17A were measured by ELISA. CD4+T cells were obtained from spleen cells by magnetic sorting. Expression of Foxp3 and RORγt at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Results The mouse model of C. albicans infection was established successfully. After infection, the MBL-/- mice had higher percentages of Th17 cells, but lower percentages of Treg cells than the WT mice. ELISA results showed that compared with the WT mice with C. albicans infection, the MBL-/- mice had significantly increased IL-17A and decreased IL-10 after infection. Moreover, the expression of RORγt at both mRNA and protein levels was up-regula-ted, while that of Foxp3 was down-regulated in the MBL-/- mice than in the WT mice following infection. Conclusions MBL regulates the immune balance of Treg/Th17 cells in mice infected with C. albicans through promoting the differentiation of CD4+ T cells into Treg cell subsets and inhibiting the differentiation into Th17 cell subsets.

7.
Chinese Journal of Immunology ; (12): 398-402, 2018.
Article in Chinese | WPRIM | ID: wpr-702741

ABSTRACT

Objective:To establish a mouse macrophage line lacking NLRP3.Methods:A GFP and neomycin dual selection marker vector which contains an efficient shRNA-coding insert for mouse NLRP3,was constructed and transfected into macrophages (RAW264.7) to select the stable clone cells in G418-contained medium.Then,the expanded clone cells that retain strong GFP expression were further sorted using the popular flow cytometry.The obtained cell mix (herein termed RAWNKD) were passaged and maintained for further identification,including observation of GFP marker,especially quantitative PCR (RT-qPCR) to confirm knockdown of NLRP3 in the generated RAWNKD cells which were challenged with LPS and ATP or not.Results:Over 80% of RAWNKD cells expressed GFP,and little NLRP3 mRNA was detected in RAWNKD cells,notably no obvious increase in NLRP3 mRNA was observed when the RAWNKD cells were challenged by LPS and ATP.Conclusion:The macrophage line lacking NLRP3 was successfully established,and such macrophage deficient in NLRP3 inflammasome is a valuable cell model for investigating the activation of NLRP3 inflammasome,especially signaling in inflammation mediated by NLRP3 inflammasome.

8.
Article in Chinese | WPRIM | ID: wpr-496208

ABSTRACT

Objective:To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3)expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets.Methods:The research includes two groups:sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line;shN is the control group in which CMTM3 is negatively knocked down.The expression of CMTM3 was detected by Western blot.The mi-gration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay.The invasion ability of PC3 was detected by Matrigel assay.Results were obtained from at least three indivi-dual experiments.Results:The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0 ±0.000 4 vs.0.490 0 ±0.055 7,P <0.001)detected by Western blot.It also had statistical significance in Matrigel assays (248.6 ±4.5 vs.113.0 ±3.3),Transwell (203.6 ±1.9 vs.103.0 ± 1.2)and Wound healing assays (95.0 ±2.9 vs.33.0 ±1.5)that knockdown of CMTM3 promoted mi-gration,and invasion of PC3 cells in vitro (P <0.001).Conclusion:Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells,and knocking down CMTM3 affects migration,and invasion ability in PC3 cells.

9.
Article in Chinese | WPRIM | ID: wpr-504814

ABSTRACT

Objective Lamins are the major components of nuclear lamina underneath the inner nuclear membrane (INM).Lamins express in most cells and are involved in the whole process of growth, also play a major role in cell stability and embryonic development.Mutant in human LMNA gene may lead to a series of disorders, which are similar to progeria or other aging-associate syndrome.In this study, we report a new lmna knockdown animal model generated in our laboratory in order to provide a useful tool for studying laminopathies.Methods Two plasmids tagged to zebrafish lmna gene were designed based on morpholino oligonucleotides technology.Co-microinjected the plasmids into zebrafish embryos to knockdown lmna gene.Imagining and western blot detection were used to identify the mutants.Results Two different proteins, Lamin A/C, were expressed in the zebrafish embryos.Two plasmids lmna-MO and lmna-EGFP-pCS 2 + were generated and co-microinjected into embryos.The results of imagining and western blot showed that the expression of lmna gene was downregulated in the zebrafish embryos.Conclusions Lamin A/C are expressed in zebrafish.lmna gene can be knocked down by the injection of lmna-MO and lmna-EGFP-pCS 2 +.This new animal model may be a powerful tool for study on laminopathies.

10.
Korean Journal of Anatomy ; : 285-292, 2009.
Article in English | WPRIM | ID: wpr-653605

ABSTRACT

Intranasal administration provides a method of bypassing the blood brain barrier, which separates the systemic circulating system and central interstitial fluid, and directly delivering drugs to the central nervous system. This method also circumvents first-pass elimination by the liver and gastrointestinal tract. In the present study, the authors investigated intranasal siRNA delivery efficiency by using FITC-labeled transfection control siRNA and a genespecific siRNA. The localization of fluorescence-tagged siRNA revealed that siRNA was delivered to cells in the olfactory bulb and that the level of the siRNA target gene (alpha B-crystallin) was significantly reduced in the same area. siRNA was delivered to processes as well as nuclei and cytoplasm. At 12 hrs after intranasal delivery, siRNA-mediated target gene reduction was observed in other more distally located brain regions, for example, in the amygdala, entorhinal cortex, and hypothalamus. Target gene knockdown was demonstrated by double immunohistochemistry, which demonstrated alpha B crystallin expression depletion in more than 70% of cells at 12 hrs after the intranasal delivery. siRNA-mediated target gene suppression was detected not only in neurons but in glia, for example, astrocytes. These results indicate that intranasal siRNA delivery offers an efficient means of reducing specific target genes in certain regions of the brain and of performing gene knockdown-mediated therapy.


Subject(s)
Administration, Intranasal , alpha-Crystallin B Chain , Amygdala , Astrocytes , Blood-Brain Barrier , Brain , Central Nervous System , Cytoplasm , Entorhinal Cortex , Extracellular Fluid , Gastrointestinal Tract , Gene Knockdown Techniques , Hypothalamus , Immunohistochemistry , Liver , Neuroglia , Neurons , Olfactory Bulb , RNA, Small Interfering , Transfection
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