Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Study flow diagram. UVM/UM, uveal melanoma; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment; LASSO, least absolute shrinkage and selection operator; PRRX1, paired related homoeobox 1; ROC, receiver operating characteristic; WB, Western blot; siRNA, small interfering RNA; GSEA, Gene Set Enrichment Analysis; GEO, Gene Expression Omnibus; TMB, tumor mutation burden.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Selection, Western Blot, Small Interfering RNA, Gene Expression, Mutagenesis

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: The overall prognostic performance of PRRX1 expression in two UM cohorts. Kaplan–Meier analyses of (A) OS in TCGA cohort and (B) MFS in the GEO cohort, the numbers shown below the survival curves are the number of patients at risk at the specified year. Distribution of PRRX1 expression levels in the (C) TCGA and (D) GEO cohorts, the dotted line represented the median PRRX1 expression and divided the patients into low-expression (green dots) and high-expression groups (red dots). Percentages of (E) deceased patients (red dots) in the TCGA cohort and (F) patients with metastasis (red dots) in the GEO cohort as a function of PRRX1 expression level.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Prognostic predictive ability of PRRX1 expression. (A) Univariate and multivariate Cox analyses of PRRX1 expression and seven other clinicopathological parameters in the TCGA cohort. (B) Time-dependent ROC analysis for prediction of OS in the TCGA cohort. (C) ROC analyses of PRRX1 expression and other seven clinicopathological parameters for predicting OS in the TCGA cohort. (D) Univariate and multivariate Cox analyses of PRRX1 expression and six other clinicopathological parameters in the GEO cohort. (E) Time-dependent ROC analysis for prediction of MFS in the GEO cohort. (F) ROC analyses of PRRX1 expression and other six clinicopathological parameters for predicting MFS in the GEO cohort. Corresponding AUC of each curve is displayed in the lower right corner of each figure, the higher the AUC value, the better the predictive power of the parameter.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: (A) Association between PRRX1 expression level and OS in patients with 33 types of cancer from the TCGA database, red dots represent the possible risk factors while blue represents potential protective factors, abbreviations for all cancer types are in Table S4 . (B) Correlations between PRRX1 expression and chromosome 3/8q copy number, the density curve on the x and y-axes represents the distribution trend of themselves. The blue line indicates the best fitted linear models and the shaded area indicates the 95% confidence interval. (C) GSEA plot of the top 20 ranked Hallmark sets of genes associated with high PRRX1 expression, as determined by comparing high and low PRRX1 expression groups (FDR < 0.25, p < 0.05).

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Analysis of immunologic characteristics associated with PRRX1 expression levels. (A) Comparisons of the levels of expression of immune checkpoint genes in the high and low PRRX1 groups (** p < 0.01; *** p < 0.001). (B) Correlation between PRRX1 expression level and TMB. (C) Correlation between PRRX1 expression and tumor immune/stromal cell populations in the UM microenvironment: T cells, CD8 T cells, cytotoxic lymphocytes, B lineage cells, NK cells, cells of the monocytic lineage, myeloid dendritic cells, neutrophils, endothelial cells, and fibroblasts, the x-axis represents the expression levels of PRRX1 , and the y axis is the abundance of TME cells. The density curve on the x and y-axes represents the distribution trend of themselves. The blue line indicates the best fitted linear models and the shaded area indicates the 95% confidence interval.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Knockdown of PRRX1 in UM cell suppresses EMT, migration and invasion in vitro . (A) GSEA results revealed that PRRX1 expression significantly positively correlated with the EMT signatures (NES = 1.6, p < 0.001, FDR = 0.002). (B) WB showed PRRX1 protein level with three different PRRX1 –siRNA treatment, relative expression levels of proteins were normalized based on the β-actin levels. (C) The influence of PRRX1 knockdown on the expression level of EMT-related factors (Snail, N-cadherin and E-cadherin) were analyzed using WB, relative expression levels of proteins were normalized based on the β-actin levels (left: representative images of three independent experiments; right: quantitative analyses, n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001, paired Student’s t-test). (D) Wound-healing assay was used to detect cell migration ability in si-NC and si- PRRX1 UM cells (left: representative images of three independent experiments; right: quantitative analyses, n = 3, * p < 0.05, paired Student’s t-test). (E) Transwell assays showed the effect of PRRX1 knockdown on UM cell invasion (left: representative images of three independent experiments; right: quantitative analyses, n = 3, *** p < 0.001, unpaired Student’s t-test).

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Knockdown, Migration, In Vitro, Expressing, Wound Healing Assay

Journal: The Journal of International Medical Research

Article Title: Downregulation of PTEN mediates bleomycin-induced premature senescence in lung cancer cells by suppressing autophagy

doi: 10.1177/0300060520923522

Figure Lengend Snippet: PTEN played a key role in mediating bleomycin-induced premature senescence in lung cancer cells. Following transfection with PTEN siRNA or upregulation of PTEN, A549 cells were treated with bleomycin (1 µg/mL) for 5 days. (a) siRNA-mediated PTEN knockdown was confirmed by western blot analysis. (b) p16 and p21 protein expression levels were detected by western blot analysis, and (c) mRNA expression levels were detected by polymerase chain reaction (PCR). (d) siPTEN accelerated the inhibition of A549 cell proliferation by bleomycin. (e) Cells treated with bleomycin for 5 days were examined by SA-β-gal activity assay and SA-β-gal staining (original magnification ×200). (f) PTEN protein overexpression was confirmed by western blot analysis. (g, h) p16 and p21 protein and mRNA levels were measured by western blotting and reverse transcription-PCR, respectively. Values given as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. (n = 3). siPTEN, silence of PTEN; upPTEN, upregulation of PTEN; BLM, bleomycin.

Article Snippet: The lentiviral vectors were constructed by GENCHEM, Inc. (CA, USA) and loaded with the targeting gene and non-targeting control sequences for silencing PTEN , including PTEN small interfering (si) RNA (siPTEN).

Techniques: Transfection, Knockdown, Western Blot, Expressing, Polymerase Chain Reaction, Inhibition, Activity Assay, Staining, Over Expression, Reverse Transcription

Journal: The Journal of International Medical Research

Article Title: Downregulation of PTEN mediates bleomycin-induced premature senescence in lung cancer cells by suppressing autophagy

doi: 10.1177/0300060520923522

Figure Lengend Snippet: PTEN accelerated premature senescence by inhibiting autophagy in bleomycin-treated lung cancer cells. (a) Cells were treated with bleomycin, proteins were extracted, and LC3 and p62 expression were detected by western blotting. (b) Protein levels of LC3 and p62 were measured by western blotting following silencing of PTEN. (c) A549 cells were treated with 100 nmol/L rapamycin for 24 hours, and the medium was then changed to fresh DMEM with or without bleomycin (1 µg/mL) for 5 days. (d) p16 and p21 protein levels were measured by western blotting. (e) A549 cells were subjected to SA-β-gal activity assay and SA-β-gal staining (original magnification ×200). Values given as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. (n = 3). RAPA, rapamycin.

Article Snippet: The lentiviral vectors were constructed by GENCHEM, Inc. (CA, USA) and loaded with the targeting gene and non-targeting control sequences for silencing PTEN , including PTEN small interfering (si) RNA (siPTEN).

Techniques: Expressing, Western Blot, Activity Assay, Staining

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: The Relationship Between HEIH Expression and Clinicopathologic Features of Retinoblastoma Patients

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: HEIH up-regulation was associated with TNM stage, optic nerve invasion and choroidal invasion in retinoblastoma. ( A ) Relative expression level of HEIH in retinoblastoma cell lines (Y79 and SO-Rb50) and normal retinal epithelial cell line ARPE-19. ( B ) Relative expression level of HEIH in 35 retinoblastoma tissues and 7 normal retinal tissues. ( C ) Relative expression level of HEIH in retinoblastoma patients with different tumor ICRB stages. ( D ) Relative expression level of HEIH in retinoblastoma patients with or without optic nerve invasion. ( E ) Relative expression level of HEIH in retinoblastoma patients with or without choroidal invasion. ( F ) The 35 retinoblastoma patients were divided into two groups (high HEIH and low HEIH) according to the median value of HEIH expression. Then we analyzed the correlation of up-regulation of HEIH with overall survival of the patients used Kaplan-Meier method and Log rank test. *P<0.05; **P<0.01.

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: HEIH knockdown inhibited retinoblastoma cell proliferation, migration and invasion. ( A ) Relative expression level of HEIH in Y79 and SO-Rb50 cells transfected with si-HEIH or siRNA control. ( B ) Cell viability was detected using the trypan blue exclusion method in Y79 and SO-Rb50 cells transfected with si-HEIH or siRNA control. ( C and D ) Cell growth capacity was evaluated using the colony formation assay. ( E and F ) Effects of HEIH knockdown on cell migration were determined using wound-healing assays. ( G and H ) Effects of HEIH knockdown on cell invasion were determined using transwell invasion assays. **P<0.01.

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Knockdown, Migration, Expressing, Transfection, Control, Colony Assay

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: HEIH negatively regulated miR-194-5p expression in retinoblastoma. ( A ) Bioinformatics analysis revealed that HEIH contained putative binding site for miR-194-5p using starBase v3.0. ( B and C ) Relative luciferase activity in Y79 ( B ) and SO-Rb50 ( C ) cells co-transfected with wt-HEIH or mut-HEIH with miR-194-5p mimics or miRNA negative control. ( D ) Relative expression level of miR-194-5p in Y79 and SO-Rb50 cells transfected with si-HEIH or siRNA control. ( E ) Relative expression level of HEIH in Y79 and SO-Rb50 cells transfected with miR-194-5p mimics or miRNA negative control. ( F ) Relative expression level of miR-194-5p in 35 retinoblastoma tissues and 7 normal retinal tissues. ( G ) Relative expression level of miR-194-5p in retinoblastoma cell lines (Y79 and SO-Rb50) and normal retinal epithelial cell line ARPE-19. ( H ) Pearson correlation analysis between HEIH and miR-194-5p level in retinoblastoma tissues. **P<0.01.

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Negative Control, Control

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: HEIH positively regulated WEE1 expression via miR-194-5p. ( A ) Predicted binding site of miR-194-5p in WEE1 3ʹUTR using TargetScan v7.2. ( B and C ) Relative luciferase activity in Y79 ( B ) and SO-Rb50 ( C ) cells co-transfected with wt- WEE1 or mut- WEE1 with miR-194-5p mimics or miRNA negative control. ( D and E ) WEE1 protein levels in Y79 and SO-Rb50 cells transfected with si-HEIH and miR-194-5p inhibitor or miRNA negative control. ( F ) Relative expression level of WEE1 in 35 retinoblastoma tissues and 7 normal retinal tissues. ( G ) Pearson correlation analysis between HEIH and WEE1 mRNA level in retinoblastoma tissues. **P<0.01.

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Negative Control

Journal: OncoTargets and therapy

Article Title: Long Noncoding RNA HEIH Promotes Proliferation, Migration and Invasion of Retinoblastoma Cells Through miR-194-5p/WEE1 Axis

doi: 10.2147/OTT.S268942

Figure Lengend Snippet: Overexpression of WEE1 reversed malignant phenotypes inhibition of retinoblastoma cells induced by HEIH knockdown. ( A ) and ( B ) Western blot was performed to validate WEE1 overexpression. ( C ) Cell viability was detected using the trypan blue exclusion method in Y79 and SO-Rb50 cells co-transfected si-HEIH or siRNA control with pcDNA 3.1- WEE1 or pcDNA 3.1-NC. ( D ) Cell growth capacity was evaluated using the colony formation assay. ( E ) Effects of WEE1 overexpression on cell migration inhibition-induced by HEIH knockdown were determined using wound-healing assays. ( F ) Effects of WEE1 overexpression on cell invasion inhibition-induced by HEIH knockdown were determined using transwell invasion assays. **P<0.01.

Article Snippet: Human normal retinal epithelial cell line ARPE-19 and retinoblastoma cell lines (Y79 and SO-Rb50) were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in 5% CO2. si-RNA targeting HEIH, and corresponding negative control si-NC, miR-194-5p mimics, inhibitor and negative control, WEE1 overexpressing vector pcDNA3.1- WEE1 and negative control pcDNA3.1 were supplied by RiboBio (Guangzhou, China).

Techniques: Over Expression, Inhibition, Knockdown, Western Blot, Transfection, Control, Colony Assay, Migration

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Primer sequences of each gene.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Sequencing

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: The miR-30b-5p profile was heightened in the joint tissues of OA patients and correlated with pro-inflammatory responses. The joint tissues of 15 non-OA patients and 40 OA patients were collected. ( A – G ) The levels of miR-30b-5p, IL-1β, IL-6, TNF-α, IL-18, SIRT1 and FoxO3a in joint tissues were compared by RT-qPCR. ( H – M ) Pearson analysis determined the correlation between miR-30b-5p, inflammatory cytokines and SIRT1/FoxO3a in OA patients’ joint tissues. *** P <0.001 (vs.Normal group).

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: The influence of inhibiting miR-30b-5p on OA rats. miR-30b-5p inhibitors were added to the knee joint cavity of OA rats. ( A ) The miR-30b-5p profile was measured by RT-qPCR. ( B ) HE and Safranin O staining were used to observe the morphological differences in cartilage tissue, and the number of injured chondrocytes was counted. ( C ) The levels of IL-1β and TNF-α were compared by RT-qPCR. ( D , E ) WB was implemented to test the profiles of Bax, Cleaved-Caspase3, MMP3 and MMP13. ( F ) The level of NF-κB in the cartilage tissue was gauged by IHC. ( G ) Expression of NF-κB, SIRT1/FoxO3a in the cartilage tissue was compared by WB. ** P <0.01, *** P <0.001. N=5.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Quantitative RT-PCR, Staining, Expressing

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: SIRT1 targeted miR-30b-5p. ( A, B ) The target association between miR-30b-5p and SIRT1 was predicted in the ENCORI database. ( C , D ) The targeted affinity between miR-30b-5p and SIRT1 was validated by the dual-luciferase reporter assay and RIP experiment, respectively. ( E , F ) The profiles of SIRT1 and FoxO3a were further evaluated by RT-qPCR and WB. ns P >0.05, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Luciferase, Reporter Assay, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Overexpressing SIRT1 weakened the miR-30b-5p-mediated effect. ( A , B ) SIRT1 mimics were transfected into IL-1β-treated HC-A cells, and the transfection validity was verified by RT-qPCR and WB. ( C , D ) The expression of SIRT1 /FoxO3a in IL-1β-treated HC-A cells was assessed by RT-qPCR and WB. ( E , F ) CCK8 and flow cytometry monitored cell viability and apoptosis, respectively. ( G ) Profiles of Bax and Cleaved-Caspase3 were measured by WB. ( H ) RT-qPCR monitored the expression of IL-1β and TNF-α. ( I , J ) The profiles of MMP3, MMP13 and NLRP3-ASC- cleaved Caspase1 were verified by WB. NS P >0.05, * P <0.05, ** P <0.01, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Inhibiting SIRT1/FoxO3a curbed the protection of miR-30b-5p knockdown on IL-1β-mediated HC-A cells. ( A – D ) si-SIRT1 and si-FoxO3a were transfected into IL-1β-treated HC-A cells and their transfection effects were verified by RT-qPCR and WB. ( E , F ) The SIRT1/FoxO3a expression in IL-1β-treated HC-A cells was checked by RT-qPCR and WB, respectively. ( G , H ) The CCK8 method and flow cytometry were utilized to gauge cell viability and apoptosis. ( I ) The levels of IL-1β and TNFα were monitored by RT-qPCR. ( J – L ) The expression of Bax, cleaved Caspase3, MMP3, MMP13 and NLRP3-ASC-cleaved Caspase1 was examined by WB. nsP>0.05, **P<0.01, ***P<0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Inhibition of NF-κB reduced miR-30b-5p levels and IL-1β-mediated HC-A cell injury. BAY 11-7082 was added to IL-1β-mediated HC-A cells and miR-30b-5p mimics were given. ( A , B ) NF-κB and miR-30b-5p expression was monitored by WB and RT-qPCR, respectively. ( C , D ) Cell proliferation and apoptosis were determined by CCK8 and flow cytometry, respectively. ( E ) Expression of Bax and Cleaved-Caspase3 was determined by WB. ( F , G ) RT-qPCR and WB were conducted to compare the levels of IL-1β, TNF-α, MMP3 and MMP13. ( H , I ) The expression of SIRT1 and FoxO3a was assessed by RT-qPCR. ( J ) WB detected the expression of NLRP3-ASC-cleaved Caspase1 inflammasomes. ** P <0.01, *** P <0.001. N=3.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: NF-κB inducible miR-30b-5p aggravates joint pain and loss of articular cartilage via targeting SIRT1-FoxO3a-mediated NLRP3 inflammasome

doi: 10.18632/aging.203466

Figure Lengend Snippet: Graphical abstract. In OA chondrocytes, NF-κB-mediated miR-30b-5p activates NLRP3 inflammasomes by targeting and abating the SIRT1/FoxO3a expression, thereby aggravating joint pain and articular cartilage damage in OA patients.

Article Snippet: SIRT1 overexpression plasmid (SIRT1) and its corresponding negative control (vector), small inference RNA (si-RNA) SIRT1 (si-SIRT1), si-FoxO3a, miR-30b-5p mimics, miR-30b-5p inhibitors (miR-30b-5p-in), and the negative controls (si-NC and miR-NC) were constructed and synthesized by Guangzhou FulenGen Co., Ltd., China The NF-κB inhibitor BAY 11-7082 (Article No. HY-13453) was bought from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing