Journal: Aging (Albany NY)

Article Title: Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

doi: 10.18632/aging.202785

Figure Lengend Snippet: Upregulation of MIAT in hypoxic cardiomyocytes and myocardial tissues from the MI mice. ( A ) MIAT expression pattern in the cardiomyocytes determined by RT-qPCR normalized to GAPDH. ( B ) The cardiomyocyte apoptosis detected by flow cytometry. ( C ) RT-qPCR determination of MIAT expression pattern in the cardiomyocytes in mice 28 d after MI modeling (n = 10). ( D ) The apoptosis of cardiomyocytes in mice 28 d after MI modeling showed by TUNEL staining (scale bar = 50 μm). ( E ) The ATP content in hypoxic cardiomyocytes after alteration of MIAT. ( F ) The apoptosis of hypoxic cardiomyocytes after alteration of MIAT analyzed by flow cytometry. * p < 0.05. The above data were all measurement data, and expressed as mean ± standard deviation. The unpaired t test was used for comparison between two groups, and one-way ANOVA was applied for comparison between multiple groups followed by Tukey’s post hoc test. All the data was collected from 3 independent experiments respectively.

Article Snippet: The primary antibodies diluted by the addition of TBST containing 1% skim milk powder were added in a drop-wise manner and incubated with the membrane at 4° C overnight, including rabbit polyclonal antibody to caspase-3 (1 : 2000, ab13847, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to Bcl-2 associated X (Bax) (1 : 2000, ab32503, Abcam), rabbit monoclonal antibody to B cell lymphoma 2 (Bcl-2) (1 : 2000, ab32124, Abcam), rabbit polyclonal antibody to EGR2 (1 : 2000, NB110-59723, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal antibody to GAPDH (1 : 4000, 10494-1-AP, Proteintech, IL, USA).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, TUNEL Assay, Staining, Standard Deviation, Comparison

Journal: Aging (Albany NY)

Article Title: Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

doi: 10.18632/aging.202785

Figure Lengend Snippet: Overexpression of miR-10a-5p decreased cardiomyocyte apoptosis through inhibiting EGR2. ( A ) The target genes of miR-10a-5p predicted using Starbase, miRWalk, and RAID databases and the up-regulated genes in MI-related dataset GSE23294 and the potential binding sites of miR-10a-5p on EGR2. ( B ) The luciferase activity measured by dual-luciferase reporter gene assay. ( C ) Expression of EGR2 in myocardial tissues normalized to GAPDH determined by RT-qPCR (n = 10). ( D ) The expression pattern of EGR2 in hypoxic cardiomyocytes normalized to GAPDH determined by RT-qPCR. ( E ) The expression pattern of EGR2 in cardiomyocytes normalized to GAPDH determined by Western blot analysis. ( F ), Apoptosis of cardiomyocytes after EGR2 silencing determined by flow cytometry. ( G ) The expression patterns of EGR2, cleaved-caspase-3, Bax and Bcl-2 in cardiomyocytes after EGR2 silencing normalized to GAPDH determined by Western blot analysis. ( H ) The caspase-3 activity in hypoxic cardiomyocytes after EGR2 silencing. ( I ) The ATP content in hypoxic cardiomyocytes after EGR2 silencing. ( J ) The expression pattern of EGR2 in the hypoxic cardiomyocytes after alteration of EGR2 and/or miR-10a-5p normalized to GAPDH determined by RT-qPCR. ( K ) The caspase-3 activity in hypoxic cardiomyocytes after alteration of EGR2 and/or miR-10a-5p determined by RT-qPCR. ( L ) The ATP content in hypoxic cardiomyocytes after alteration of EGR2 and/or miR-10a-5p. * p < 0.05. The above data were all measurement data, and expressed as mean ± standard deviation. The unpaired t test was adopted for comparison between two groups. One-way ANOVA was adopted for comparison among multiple groups with Tukey’s post hoc test. All data was generated from 3 independent experiments respectively.

Article Snippet: The primary antibodies diluted by the addition of TBST containing 1% skim milk powder were added in a drop-wise manner and incubated with the membrane at 4° C overnight, including rabbit polyclonal antibody to caspase-3 (1 : 2000, ab13847, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to Bcl-2 associated X (Bax) (1 : 2000, ab32503, Abcam), rabbit monoclonal antibody to B cell lymphoma 2 (Bcl-2) (1 : 2000, ab32124, Abcam), rabbit polyclonal antibody to EGR2 (1 : 2000, NB110-59723, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal antibody to GAPDH (1 : 4000, 10494-1-AP, Proteintech, IL, USA).

Techniques: Over Expression, Binding Assay, Luciferase, Activity Assay, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Standard Deviation, Comparison, Generated

Journal: Aging (Albany NY)

Article Title: Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

doi: 10.18632/aging.202785

Figure Lengend Snippet: MIAT competitively binds to miR-10a-5p to upregulate EGR2 and further increase apoptosis of cardiomyocytes under exposure to hypoxia. ( A ) The expression of MIAT, miR-10a-5p and EGR2 in cardiomyocytes after inhibition of MIAT and/or miR-10a-5p determined by RT-qPCR. ( B ) Apoptosis of cardiomyocytes after inhibition of MIAT and/or miR-10a-5p detected by flow cytometry. ( C ) Western blot showing the expression patterns of cleaved-caspase-3 and caspase-3 in hypoxic cardiomyocytes after inhibition of MIAT and/or miR-10a-5p. The expression level is normalized to GAPDH. ( D ) The caspase-3 activity of cardiomyocytes under hypoxic conditions after inhibition of MIAT and/or miR-10a-5p. ( E ) The ATP content in cardiomyocytes after inhibition of MIAT and/or miR-10a-5p under hypoxic conditions. ( F ) The expression patterns of MIAT, miR-10a-5p and EGR2 in cardiomyocytes after MIAT and/or EGR2 overexpression determined by RT-qPCR normalized to GAPDH and U6. ( G ) The apoptosis of cardiomyocytes under hypoxic conditions after MIAT and/or EGR2 overexpression detected by flow cytometry. ( H ) The expression of cleaved-caspase-3 and pro-caspase-3 in the hypoxic cardiomyocytes after MIAT and/or EGR2 overexpression normalized to GAPDH determined by Western blot analysis. ( I ) The caspase-3 activity of cardiomyocytes under hypoxic conditions after MIAT and/or EGR2 overexpression. ( J ) The ATP content in cardiomyocytes under hypoxic conditions in response to MIAT and EGR2 overexpression relative to ATP content after EGR2 overexpression alone (fold). * p < 0.05, the above data were all measurement data, and expressed as mean ± standard deviation. The data comparison among multiple groups was performed using one-way ANOVA and Tukey’s post hoc test. The experiment was repeated 3 times independently.

Article Snippet: The primary antibodies diluted by the addition of TBST containing 1% skim milk powder were added in a drop-wise manner and incubated with the membrane at 4° C overnight, including rabbit polyclonal antibody to caspase-3 (1 : 2000, ab13847, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to Bcl-2 associated X (Bax) (1 : 2000, ab32503, Abcam), rabbit monoclonal antibody to B cell lymphoma 2 (Bcl-2) (1 : 2000, ab32124, Abcam), rabbit polyclonal antibody to EGR2 (1 : 2000, NB110-59723, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal antibody to GAPDH (1 : 4000, 10494-1-AP, Proteintech, IL, USA).

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Flow Cytometry, Western Blot, Activity Assay, Over Expression, Standard Deviation, Comparison

Journal: Aging (Albany NY)

Article Title: Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

doi: 10.18632/aging.202785

Figure Lengend Snippet: MIAT silencing improves cardiomyocyte function in MI mice by inhibiting EGR2 through miR-10a-5p upregulation. ( A ) EF, FS, LVEDD, and LVESD values in mice detected by small animal echocardiogram after MI modeling with alteration of MIAT, miR-10a-5p or EGR2. ( B ) Myocardial infarct size identified by HE staining after alteration of MIAT and miR-10a-5p (scale bar = 50 μm). ( C ) The expression patterns of MIAT, miR-10a-5p and EGR2 in the cardiac tissues of MI mice after alteration of MIAT and miR-10a-5p determined by RT-qPCR. ( D ) The expression patterns of cleaved caspase-3 and EGR2 normalized to GAPDH in the myocardial tissues of MI mice after alteration of MIAT and miR-10a-5p determined by Western blot analysis. ( E ) The apoptosis in myocardial tissues after alteration of MIAT and miR-10a-5p showed by TUNEL staining (scale bar = 50 μm). The red signal represented TUNEL-positive cells. ( F ) The collagen deposition in the myocardial tissues of MI mice after alteration of MIAT and miR-10a-5p showed by Masson staining (scale bar = 50 μm). The blue color represented collagen. ( G ) The myocardial infarct size in mice after alteration of MIAT and EGR2 showed by HE staining (scale bar = 50 μm). ( H ) The expression patterns of MIAT, miR-10a-5p and EGR2 in cardiac tissues of MI mice after alteration of MIAT and EGR2 determined by RT-qPCR. ( I ) Western blot showing the expression patterns of cleaved caspase-3, pro-caspase-3 and EGR2 in mouse myocardial tissues after alteration of MIAT and EGR2. The expression level is normalized to GAPDH. ( J ) The apoptosis in myocardial tissues of MI mice after alteration of MIAT and EGR2 determined by TUNEL staining (scale bar = 50 μm). The red signal represented TUNEL-positive cells. ( K ) The collagen deposition in the myocardial tissues of MI mice after alteration of MIAT and EGR2 showed by Masson staining (scale bar = 50 μm), and blue referred to collagen. The animals were selected blindly, and all values were measured on the same animal for 6 consecutive cardiac cycles. * p < 0.05. The above data were all measurement data, and expressed as mean ± standard deviation. The data of multiple groups were compared by one-way ANOVA and Tukey’s post hoc test. n = 10.

Article Snippet: The primary antibodies diluted by the addition of TBST containing 1% skim milk powder were added in a drop-wise manner and incubated with the membrane at 4° C overnight, including rabbit polyclonal antibody to caspase-3 (1 : 2000, ab13847, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to Bcl-2 associated X (Bax) (1 : 2000, ab32503, Abcam), rabbit monoclonal antibody to B cell lymphoma 2 (Bcl-2) (1 : 2000, ab32124, Abcam), rabbit polyclonal antibody to EGR2 (1 : 2000, NB110-59723, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal antibody to GAPDH (1 : 4000, 10494-1-AP, Proteintech, IL, USA).

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, TUNEL Assay, Standard Deviation

Journal: Aging (Albany NY)

Article Title: Silencing long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis

doi: 10.18632/aging.202785

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: The primary antibodies diluted by the addition of TBST containing 1% skim milk powder were added in a drop-wise manner and incubated with the membrane at 4° C overnight, including rabbit polyclonal antibody to caspase-3 (1 : 2000, ab13847, Abcam Inc., Cambridge, UK), rabbit monoclonal antibody to Bcl-2 associated X (Bax) (1 : 2000, ab32503, Abcam), rabbit monoclonal antibody to B cell lymphoma 2 (Bcl-2) (1 : 2000, ab32124, Abcam), rabbit polyclonal antibody to EGR2 (1 : 2000, NB110-59723, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal antibody to GAPDH (1 : 4000, 10494-1-AP, Proteintech, IL, USA).

Techniques: