Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Control, Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Primer used for RT-qPCR

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques:

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: DNMT3B upregulation is responsible for the hypermethylation of the MYH11 promoter. A , the expression of DNMT3A and DNMT3B in GC was queried in StarBase Pan-cancer platform; B , the enrichment ability of DNMT3A and DNMT3B on MYH11 promoter examined by ChIP-qPCR; C , DNMT3B mRNA expression in GC tumor tissues and their adjacent tissues by RT-qPCR; D , correlation of DNMT3B expression with MYH11 promoter methylation levels in tumor tissues analyzed by Pearson’s correlation analysis (r = 0.623, p < 0.001); E , correlation of DNMT3B expression with MYH11 expression in tumor tissues (r = − 0.609, p < 0.001); F , transfection efficiency of oe-DNMT3B in GC cells by RT-qPCR; G , the effects of DNMT3B overexpression on the methylation level of MYH11 promoter examined by qMSP; H , effects of DNMT3B overexpression on MYH11 expression by RT-qPCR. Each assay was performed at least three times. Statistical significance was analyzed by paired t test (panel C) or two-way ANOVA (panels B, F, G and H) and Tukey’s multiple range tests

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Expressing, Quantitative RT-PCR, Methylation, Transfection, Over Expression

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Mechanism diagram. Overexpression of DNMT3B in GC inhibited MYH11 expression by promoting methylation of the MYH11 promoter, thereby attenuating the repressive effect of MYH11 on TNFRSF14 transcriptional activity and promoting GC progression

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Over Expression, Expressing, Methylation, Activity Assay

Journal: Oncology Reports

Article Title: Circular RNA NOX4 promotes the development of colorectal cancer via the microRNA-485-5p/CKS1B axis

doi: 10.3892/or.2020.7758

Figure Lengend Snippet: CircNOX4 depletion restrains the tumor growth in a murine xenograft model in vivo . (A) BALB/c-nu female nude mice were arbitrarily divided into sh-NC and sh-circNOX4 groups (n=7 mice/group) and subcutaneously inoculated with SW480 cells stably transfected with sh-NC or sh-circNOX4. The size of CRC tumors was detected every week for five weeks. (B) Representative images and mean weights of tumors dissected from the mice in the sh-NC and sh-circNOX4 groups. The tumors were weighed 5 weeks post-inoculation. (C and D) The levels of (C) circNOX4 and (D) miR-485-5p in the tumor tissues were examined by reverse transcription-quantitative PCR. (E) Western blot assay was conducted to detect the protein expression levels of CKS1B in CRC tumor tissues. *P<0.05 vs. sh-NC. circNOX4, circular RNA NADPH oxidase 4; miR, microRNA; CRC, colorectal cancer; sh, short hairpin RNA; NC, negative control; CKS1B, CDC28 protein kinase regulatory subunit 1B.

Article Snippet: Small interfering (si)RNA targeting circNOX4 (si-circNOX4; sense, 5′-UAGCUUAUUGCAUAUGUAGAG-3′ and antisense, 5′-CUACAUAUGCAAUAAGCUAGG-3′), siRNA negative control (si-NC; sense, 5′-AACAGGCACACGUCCCAGCGU-3′ and antisense, 5′-ACGCUGGGACGUGUGCCUGUU-3′), pGIPZ-short hairpin (sh)RNA targeting circNOX4 (sh-circNOX4), pGIPZ-shRNA negative control (sh-NC), miR-485-5p mimic (miR-485-5p; 5′-AGAGGCUGGCCGUGAUGAAUUC-3′), miRNA mimic negative control (miR-NC; 5′-UUCUCCGAACGUGUCACGUUU-3′), miR-485-5p inhibitor (anti-miR-485-5p; 5′-CUCCGACCGGCACUACUUAAG-3′) and its negative control (anti-miR-NC; 5′-UUGUACUACACAAAAGUACUG-3′), CKS1B ectopic expression plasmid (CKS1B) and a pcDNA empty vector (vector) were acquired from Shanghai Genepharma Co., Ltd. Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 1 µg plasmid or 0.5 µM oligonucleotides into SW480 and SW620 CRC cells when cell confluency reached ~80%.

Techniques: In Vivo, Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, shRNA, Negative Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Knockdown of mitofilin inhibits autophagy and facilitates starvation-induced apoptosis in HeLa cells

doi: 10.22038/ijbms.2019.36173.8617

Figure Lengend Snippet: Knockdown of mitofilin inhibits basal autophagy activity. HeLa cells were transfected with mitofilin specific short hairpin RNA (shRNA) and selected with G418. Mitofilin expression level in the established stable cell line was detected at mRNA (A) and protein (B) expression levels. β-actin was used as internal control. (C) The LC3-II conversion was detected with western blotting. For the measurement of autophagy flux, cells were pretreated with 20 mM NH 4 Cl. (D) The LC3-II puncta distribution was assessed with immunofluorescence (400X), and (E) the LC3 puncta per cell were counted. Pretreatment of NH 4 Cl was used to evaluate the autophagy flux. Images are representative of three independent experiments. Data are expressed as mean±SD. * P <0.05

Article Snippet: Short hairpin RNA (shRNA) for mitofilin (5’-GCTAAGGTTGTATCTCAGTAT-3’) and its negative control were cloned into pGPU6/Neo (Genepharma, shanghai, China).

Techniques: Activity Assay, Transfection, shRNA, Expressing, Stable Transfection, Western Blot, Immunofluorescence