Journal: CytoJournal

Article Title: Exploring miR-3148’s impact on Krüppel-like factor 6-driven mitophagy and apoptosis in myocardial ischemic injury

doi: 10.25259/Cytojournal_209_2024

Figure Lengend Snippet: KLF6 is the target of miR-3148 and is down-regulated in AMI mice and OGD cardiomyocytes. (a) Hsa-miR-3148 targets were predicted in the miRDB, mirDIP and TargetScan databases. (b) The illustration of predicted 3'-UTR binding sites for KLF6 of miR-3148 in www.targetscan.org . (c) Luminescence levels were measured in cells transfected with luciferase reporter plasmids harboring either the WT (KLF6 WT) or MUT (KLF6 MUT) miR-3148 binding sites within the 3’-UTR of KLF6, comparing both the Control group and the group treated with miR-3148 mimic. (d and e) The expression levels of KLF6 were evaluated using WB analysis in myocardial tissues of mice. (f) The expression level of miR-3148 was measured by qRT-PCR after transfecting AC16 with miR-3148 mimic or miR-3148 inhibitor. (g and h) Up-regulation of miR-3148 inhibited KLF6 expression, (i and j) while its down-regulation promoted KLF6 expression. All analyses, n = 3; ✶ : P < 0.05. KLF6: Krüppel-like factor 6, AMI: Acute myocardial infarction, OGD: Oxygen and glucose deprivation, miRDB: MicroRNA target prediction database, mirDIP: MicroRNA data integration portal, 3’-UTR: 3’-untranslated region, WT: Wild type, MUT: Mutant, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, WB: Western blot.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following antibodies: Glyceraldehyde 3-phosphate dehydrogenase (1:1000, cat. no. ab8245; Abcam, USA), β-actin monoclonal antibody (β-actin; 1:20000, 66009-1-Ig, Proteintech, Wuhan, China), KLF6 (1:500, 14716-1-AP, Proteintech, Wuhan, China), PINK1 (1:1000, 23274-1-AP, Proteintech, Wuhan, China), PARK2/Parkin (1:2000, 14060-1-AP, Proteintech, Wuhan, China), microtubule-associated proteins 1A/1B light chain 3B (LC3)B (1:3000, ab192890, Abcam, USA), anti-sequestosome 1 (p62, 1:1000, K002179P, Solarbio, Beijing, China), Beclin1 (1:1000, K101553P, Solarbio, Beijing, China), cysteine-aspartic acid protease (Caspase) 3 (1:1000, K003262P, Solarbio, Beijing, China), Caspase 9 (1:1000, K109226P, Solarbio, Beijing, China), and Cyt C (1:1000, K107696P, Solarbio, Beijing, China).

Techniques: Binding Assay, Transfection, Luciferase, Control, Expressing, Quantitative RT-PCR, Mutagenesis, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: CytoJournal

Article Title: Exploring miR-3148’s impact on Krüppel-like factor 6-driven mitophagy and apoptosis in myocardial ischemic injury

doi: 10.25259/Cytojournal_209_2024

Figure Lengend Snippet: Suppression of KLF6 promoted the apoptosis of AC16 cells under OGD conditions and reduced mitophagy. (a) The expression level of KLF6 was measured by qRT-PCR after transfecting AC16 cells with si-KLF6. (b) CCK8 detection of cells activity. (c and d) The measurement of apoptosis in AC16 cells was conducted using Flow Cytometry. (e and f) After treatment with si-KLF6, the levels of apoptosis-related proteins, including Caspase 9, Caspase 3, and Cyt C, (g and h) and the levels of mitophagy-related proteins of PINK1, Parkin, Beclin1, p62, LC3 I and LC3 II, were evaluated through WB analysis. (i) The mRNA expression levels of PINK1 and Parkin were detected by qRT-PCR. All analyses, n = 3; ✶ : P < 0.05. KLF6: Krüppel-like factor 6, Caspase: Cysteine-aspartic acid protease, Cyt C: Cytochrome C, Parkin: Parkin RBR E3 ubiquitin protein ligase, CCK-8: Cell counting kit 8, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, OGD: Oxygen and glucose deprivation, si-KLF6: Small interfering RNA targeting KLF6, PINK1: PTEN-induced kinase 1, LC3: Microtubule-associated proteins 1A/1B light chain 3B, WB: Western blot, p62: Sequestosome 1, mRNA: Messenger RNA.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following antibodies: Glyceraldehyde 3-phosphate dehydrogenase (1:1000, cat. no. ab8245; Abcam, USA), β-actin monoclonal antibody (β-actin; 1:20000, 66009-1-Ig, Proteintech, Wuhan, China), KLF6 (1:500, 14716-1-AP, Proteintech, Wuhan, China), PINK1 (1:1000, 23274-1-AP, Proteintech, Wuhan, China), PARK2/Parkin (1:2000, 14060-1-AP, Proteintech, Wuhan, China), microtubule-associated proteins 1A/1B light chain 3B (LC3)B (1:3000, ab192890, Abcam, USA), anti-sequestosome 1 (p62, 1:1000, K002179P, Solarbio, Beijing, China), Beclin1 (1:1000, K101553P, Solarbio, Beijing, China), cysteine-aspartic acid protease (Caspase) 3 (1:1000, K003262P, Solarbio, Beijing, China), Caspase 9 (1:1000, K109226P, Solarbio, Beijing, China), and Cyt C (1:1000, K107696P, Solarbio, Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Ubiquitin Proteomics, CCK-8 Assay, Cell Counting, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA, Western Blot

Journal: CytoJournal

Article Title: Exploring miR-3148’s impact on Krüppel-like factor 6-driven mitophagy and apoptosis in myocardial ischemic injury

doi: 10.25259/Cytojournal_209_2024

Figure Lengend Snippet: miR-3148 regulated mitophagy and apoptosis though KLF6. (a) CCK-8 detection of cells activity. (b and c) The measurement of apoptosis in AC16 cells was conducted using flow cytometry. (d and e) The levels of apoptosis-related proteins of Caspase 9, Caspase 3, and Cyt C were evaluated through WB analysis. (f) The mRNA expression levels of PINK1 and Parkin were detected by qRT-PCR. All analyses, n = 3; ✶ : P < 0.05. (g and h) The levels of mitophagy-related proteins of PINK1, Parkin, Beclin1, p62, LC3 I and LC3 II, were evaluated through WB analysis. Cyt C: Cytochrome C, Parkin: Parkin RBR E3 ubiquitin protein ligase, CCK-8: Cell counting kit 8, LC3: Microtubule-associated proteins 1A/1B light chain 3B, WB: Western blot, Caspase: Cysteine-aspartic acid protease, PINK1: PTEN-induced kinase 1, KLF6: Krüppel-like factor 6, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, p62: Sequestosome 1, mRNA: Messenger RNA.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following antibodies: Glyceraldehyde 3-phosphate dehydrogenase (1:1000, cat. no. ab8245; Abcam, USA), β-actin monoclonal antibody (β-actin; 1:20000, 66009-1-Ig, Proteintech, Wuhan, China), KLF6 (1:500, 14716-1-AP, Proteintech, Wuhan, China), PINK1 (1:1000, 23274-1-AP, Proteintech, Wuhan, China), PARK2/Parkin (1:2000, 14060-1-AP, Proteintech, Wuhan, China), microtubule-associated proteins 1A/1B light chain 3B (LC3)B (1:3000, ab192890, Abcam, USA), anti-sequestosome 1 (p62, 1:1000, K002179P, Solarbio, Beijing, China), Beclin1 (1:1000, K101553P, Solarbio, Beijing, China), cysteine-aspartic acid protease (Caspase) 3 (1:1000, K003262P, Solarbio, Beijing, China), Caspase 9 (1:1000, K109226P, Solarbio, Beijing, China), and Cyt C (1:1000, K107696P, Solarbio, Beijing, China).

Techniques: CCK-8 Assay, Activity Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Ubiquitin Proteomics, Cell Counting, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: CytoJournal

Article Title: Exploring miR-3148’s impact on Krüppel-like factor 6-driven mitophagy and apoptosis in myocardial ischemic injury

doi: 10.25259/Cytojournal_209_2024

Figure Lengend Snippet: The mechanism of miR-3148 attenuated PINK1/Parkin-mediated mitophagy and aggravated apoptosis in myocardial ischemia injury. In cases of myocardial ischemia injury, there is an up-regulation of miR-3148 which exacerbates apoptosis and hinders mitophagy. MiR-3148 plays a crucial role in PINK1/Parkin-mediated mitophagy by suppressing the mitochondria-related gene KLF6 . Moreover, the decrease in KLF6 expression may impede the fusion between autophagosomes and lysosomes by reducing PINK1 accumulation on the outer membrane of mitochondria, resulting in reduced recruitment of Parkin. Consequently, this condition leads to an accumulation of p62, receptor proteins located on the outer membrane of mitochondria, causing a decline in Parkin ubiquitin products and impaired binding with LC3 for autophagy maturation. Therefore, inhibition of KLF6-mediated mitophagy contributes to the buildup of damaged mitochondria and promotes cellular apoptosis. Parkin: Parkin RBR E3 ubiquitin protein ligase, KLF6: Krüppel-like factor 6, PINK1: PTEN-induced kinase 1, p62: Sequestosome 1, LC3: Microtubule-associated proteins 1A/1B light chain 3B.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following antibodies: Glyceraldehyde 3-phosphate dehydrogenase (1:1000, cat. no. ab8245; Abcam, USA), β-actin monoclonal antibody (β-actin; 1:20000, 66009-1-Ig, Proteintech, Wuhan, China), KLF6 (1:500, 14716-1-AP, Proteintech, Wuhan, China), PINK1 (1:1000, 23274-1-AP, Proteintech, Wuhan, China), PARK2/Parkin (1:2000, 14060-1-AP, Proteintech, Wuhan, China), microtubule-associated proteins 1A/1B light chain 3B (LC3)B (1:3000, ab192890, Abcam, USA), anti-sequestosome 1 (p62, 1:1000, K002179P, Solarbio, Beijing, China), Beclin1 (1:1000, K101553P, Solarbio, Beijing, China), cysteine-aspartic acid protease (Caspase) 3 (1:1000, K003262P, Solarbio, Beijing, China), Caspase 9 (1:1000, K109226P, Solarbio, Beijing, China), and Cyt C (1:1000, K107696P, Solarbio, Beijing, China).

Techniques: Expressing, Membrane, Ubiquitin Proteomics, Binding Assay, Inhibition

Journal: Cardiovascular research

Article Title: Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

doi: 10.1093/cvr/cvv155

Figure Lengend Snippet: Figure 1 Klf6 haploinsufficiency resulted in decreased cardiac fibrosis and preserved cardiac function. (A and B) Klf6+/2 mice showed markedly de- creased interstitial cardiac fibrosis compared with wild-type mice after continuous Ang II stimulation for 14 days, and heart samples were stained with Masson’s trichrome. Representative figures are shown. Scale bar: 2.0 mm (upper panels), 500 mm (lower panels). (C–E) Klf6+/2 mouse hearts showed preserved left ventricular function and size, whereas wild-type mouse hearts showed decreased function and dilated left ventricular size by continuous Ang II infusion. (F) Whole heart mRNA was subjected to quantitative RT-PCR. Klf6+/2 mouse hearts showed decreased expression levels of Tgfb1, Ctgf, and Col1a. (G) Representative figures of wild-type and Klf6+/2 mouse hearts subjected to pressure-overload (TAC) and stained with Masson’s trichrome. Scale bar: 2.0 mm. (H and I) There was no significant difference between wild-type and Klf6+/2 mouse hearts in cardiac fibrosis and cardiac hypertrophy by TAC. HW, heart weight; BW, body weight. Data are presented as mean +SD. n ¼ 3–6 per group. *P , 0.05, **P , 0.01.

Article Snippet: For immunohistochemistry, after deparaffinization and blocking, mouse heart sections were incubated with KLF6 antibody (Santa Cruz Biotechnology or Biolegend).

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: Cardiovascular research

Article Title: Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

doi: 10.1093/cvr/cvv155

Figure Lengend Snippet: Figure 2 KLF6 expression increased in cardiomyocytes, but not in cardiac fibroblasts, after Ang II stimulation. (A and B) Immunohistochemistry showed KLF6 up-regulation (brown stained) in cardiomyocytes of wild-type, but not in Klf6+/2 mice, after Ang II stimulation. Scale bar: 200 mm. Note that KLF6 was up-regulated in stimulated myocardium, specifically in the nuclei of cardiomyocytes. (C and D) Primary cultured rat neonatal car- diomyocytes and cardiac fibroblasts were stimulated by Ang II and subjected to immunofluorescent staining. After stimulation, KLF6 (green) was select- ively expressed in cardiomyocytes. Green fluorescence, KLF6; red fluorescence, Troponin T. Scale bar: 50 mm. Data are presented as mean + SD. n ¼ 3–4 per group. **P , 0.01.

Article Snippet: For immunohistochemistry, after deparaffinization and blocking, mouse heart sections were incubated with KLF6 antibody (Santa Cruz Biotechnology or Biolegend).

Techniques: Expressing, Immunohistochemistry, Staining, Cell Culture

Journal: Cardiovascular research

Article Title: Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

doi: 10.1093/cvr/cvv155

Figure Lengend Snippet: Figure 3 Expression of KLF6 in cardiomyocytes is essential for cardiac fibrosis. (A–D) Conditional deletion of Klf6 in the cardiomyocyte (Klf6flox/2; aMHC-Cre) and cardiac fibroblast (Klf6flox/flox;Postn-Cre). Both mice were subjected to Ang II stimulation for 14 days, and cardiac fibrosis was analysed. Klf6 deletion in cardiomyocytes showed significantly less fibrosis than that in the cardiac fibroblast-specific Klf6-deleted mice in response to Ang II stimu- lation. Cardiac fibrosis area was calculated by fibrotic area/heart area, and then compared with control mice. Scale bar: 1.0 mm. (E and F) Klf6 protein and mRNA expression levels were significantly decreased in cardiomyocyte-specific Klf6-deleted mice heart. (F) Whole heart mRNA was subjected to quan- titative PCR. Cardiomyocyte-specific Klf6-deleted mouse hearts also showed decreased expression levels of Tgfb1, Ctgf, and Col1a. (G and H) Klf6+/2

Article Snippet: For immunohistochemistry, after deparaffinization and blocking, mouse heart sections were incubated with KLF6 antibody (Santa Cruz Biotechnology or Biolegend).

Techniques: Expressing, Control

Journal: Cardiovascular research

Article Title: Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes.

doi: 10.1093/cvr/cvv155

Figure Lengend Snippet: Figure 4 KLF6 suppressed TSP4 expression levels in response to Ang II stimulation. (A and B) Tsp4 mRNA expression levels after Ang II stimulation by qRT-PCR. Whole heart samples were normalized by Gapdh and then by basal expression levels of wild-type mice. Klf6+/2 and cardiomyocyte-specific Klf6-deleted mice heart showed remarkably increased TSP4 expression levels after Ang II stimulation. (C) TSP4 protein expression levels were assayed from wild-type or Klf6+/2 mouse whole heart samples after Ang II stimulation. Right bar graph shows quantification of TSP4 protein levels normalized by GAPDH. n ¼ 3. (D) TSP4 protein expression levels were assayed from Klf6flox/2 or Klf6flox/2;aMHC-Cre mouse whole heart samples after Ang II stimu- lation. Right bar graph shows quantification of TSP4 protein levels normalized by GAPDH. n ¼ 3. (E) Primary cultured neonatal rat cardiomyocytes were infected with adenoviral KLF6 and backbone adenovirus at 100 m.o.i. for 12 h. Cells were then stimulated with Ang II (10 mM) for 12 h. Cells and medium were subjected to western blot. GAPDH was used as protein-loading control for cell lysate. Right bar graphs indicate densitometric quantification of TSP4 levels (normalized by GAPDH in the cell lysate). Assays were done in triplicate. (F) KLF6 was directly recruited to the Tsp4 promoter region under Ang II stimulation. Chromatin immunoprecipitation was done with KLF6 antibody, and refined DNA samples were evaluated by PCR using primer pairs for the Tsp4 promoter region. Input was 5% of the chromatin sample. (G) The KLF6 expression vector (pCAG-KLF6) and the Tsp4 promoter reporter constructs were co-transfected and subjected to evaluation of luciferase reporter activity. Luciferase activity was normalized by protein concentration, and relative activities were calculated. Assays were done in triplicate. Data are presented as mean+ SD. *P , 0.05, **P , 0.01.

Article Snippet: For immunohistochemistry, after deparaffinization and blocking, mouse heart sections were incubated with KLF6 antibody (Santa Cruz Biotechnology or Biolegend).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Infection, Western Blot, Control, Chromatin Immunoprecipitation, Plasmid Preparation, Construct, Transfection, Luciferase, Activity Assay, Protein Concentration

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

doi: 10.1172/JCI63455

Figure Lengend Snippet: (A) A 3′ UTR reporter assay was used to verify the targets. Luciferase reporter activity of 8 3′ UTR constructs in HEK293T cells overexpressing miR-122 (293T-122) or mutant MIR122 (293T-122M). Aldoa and B2m are the positive and the negative controls, respectively. (B) Diagram depicting the seed region of MIR122, the mutated seed region of MIR122 (MIR122M), and the two binding site mutations (mu1 and mu2) within the 3′ UTR of Klf6. (C) Luciferase reporter activity of the Klf6–3′ UTR construct in 293T-GFP, 293T-122, or 293T-122M. (D) Luciferase reporter activity of the Klf6–3′ UTR constructs containing WT, mu1, or mu2 in 293T-GFP or 293T-122. The data are representative of 3 experiments. §P < 0.001. The hydrodynamic injection of shKlf6 reduced the expression of KLF6 (E and F) and reduced HSC cell activation and collagen deposition (F) in Mir122a–/– livers. Values in E represent the levels of KLF6 protein expression of mice with different treatments relative to the level in KO-shLacZ livers. Scale bars: 50 μm and 20 μm (insets). shLacZ is a control for RNA interference. (G) Quantitation of Sirius red staining. Ten different microscopic fields for each sample were evaluated with the MetaMorph software (Molecular Devices). (H) The serum level of TGF-β1 was reduced. n = 3 mice per group. †P < 0.01, §P < 0.001 for KO-shLacZ versus WT-shLacZ mice; ‡P < 0.01 for KO-shKlf6 versus KO-shLacZ mice. (I) Enlarged images of the insets of F. Scale bars: 10 μm. Expression of KLF6 is found in both the hepatocytes and HSCs (with ellipsoidal nucleus). Blue arrows, KLF6hi HSCs; yellow arrowhead, KLF6lo HSC.

Article Snippet: The immunohistochemical analysis used antibodies against F4/80, CD31, collagen I (Abcam), PCNA, vimentin, MTTP, E-cadherin (Cell Signaling Technology), desmin (Millipore), and KLF6 (Abgent).

Techniques: Reporter Assay, Luciferase, Activity Assay, Construct, Mutagenesis, Binding Assay, Injection, Expressing, Activation Assay, Quantitation Assay, Staining, Software