Journal: Cell Journal (Yakhteh)

Article Title: Endoplasmic Reticulum Stress Induces miR-706, A Pro-Cell Death microRNA, in A Protein Kinase RNA-Like ER Kinase (PERK) and Activating Transcription Factor 4 (ATF4) Dependent Manner

doi: 10.22074/cellj.2020.6873

Figure Lengend Snippet: miR-706 tiggers ER stress dependent cell death trough CAAP1. A. Target scan prediction showing predicted binding of miR-706 and CAAP1 3ˊUTR. B. NIH3T3 cells were transfected with scrambled microRNA (control; ctrl) or mimic miR-706 (mimic). Total RNA was purified and CAAP1 expression was determined using qRT-PCR. C. NIH3T3 cells were transduced with antisense miR-706 (anti-706) and then incubated with Thapsigargin for 24 hours. Total RNA was purified and CAAP1 expression was determined using qRT-PCR. D. NIH3T3 cells were transduced with scrambled microRNA (ctrl), antisense miR-706 (anti-706) or antisense miR-706 and shCAAP1. The cells were then incubated with Thapsigargin for 24 hours. Cell death was assesed by Annexin V staining. E. Quantification of panel C. *; P<0.05, **; P<0.01 compared to control, and qRT-PCR; Quantitative reverse transcription polymerase chain reaction.

Article Snippet: The antibodies are: CAAP1 (NBP1-94020, Novus Biologicals, USA) and GAPDH (14C10, Cell Signaling Technology, USA).

Techniques: Binding Assay, Transfection, Control, Purification, Expressing, Quantitative RT-PCR, Transduction, Incubation, Staining, Reverse Transcription, Polymerase Chain Reaction

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: CAAP1 inhibits apoptosis and promotes autophagy in gastric cancer cells. Transfect CAAP1 expression plasmid or empty vector (control group) into MGC8-3 cells. (A-B): caspase3 and cleaved-caspase3 protein expressions were detected by western blot, and cleaved-caspase3 protein was statistically analyzed ( n = 3), * P < 0.05; (C-D): Cell dysfunction was detected by flow cytometry Death, and statistical analysis of cell apoptosis rate ( n = 3), * P < 0.05. Transfect si-CAAP1 or si-CAAP1 (control group) into AGS cells. (E-F): caspase3 and cleaved-caspase3 protein expressions were detected by western blot, and cleaved-caspase3 protein was statistically analyzed ( n = 3), * P < 0.05; (G-H): Cell dysfunction was detected by flow cytometry Death, and statistical analysis of cell apoptosis rate ( n = 3), * P < 0.05. Transfect CAAP1 or empty vector (control group) into MGC80-3 cells. (I-J): LC3B protein expression was detected by western blot, and the ratio of LC3BⅡ/LC3BⅠ was statistically analyzed ( n = 3), * P < 0.05; (K): Cell autophagy was detected using an autophagosome detection kit. Transfect si-CAAP1 or si-CAAP1 control (control group) into AGS cells. (L-M): LC3B protein expression was detected by western blot, LC3BⅠ and LC3BⅡ proteins were quantified, and the ratio of LC3BⅡ/LC3BⅠ was statistically analyzed ( n = 3), * P < 0.05; N: cells were detected using an autophagosome detection kit.

Article Snippet: Negative control mimic (NC mimic), miR-5100 mimic (mimic), Negative control inhibitor (NC inhibitor), miR-5100 inhibitor (inhibitor) Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1), siRNA-CAAP1 (si-CAAP1) were purchased from RIBOBIO biological.

Techniques: Expressing, Plasmid Preparation, Western Blot, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: miR-5100 inhibits CAAP1 protein expression by targeting CAAP1 3′UTR. A: Analysis of the TargetScanHuman ( http://www.targetscan.org/vert_72/ ) website shows that miR-5100 can target the 3′UTR of CAAP1. B: miR-5100 targets the base sequence and mutant sequence of CAAP1 3′UTR. C: miR-5100 minic or miR-5100 minic NC (control group) and WT-pmir-CAAP1 or MUT-pmir-CAAP1 were co-transfected into MGC80-3 cells. The luciferase reporter assay was used to detect whether miR-5100 targeted to bind to CAAP1 3′UTR ( n = 3); * P < 0.05, ** P < 0.01. D: miR-5100 minic or miR-5100 minic NC (control group) was transfected into AGSA cells, and CAAP1 protein expression was detected by Western blot ( n = 3), * P < 0.05. E: miR-5100 inhibitor or miR-5100 inhibitor NC (control group) was transfected into MGC80-3 cells, and CAAP1 protein expression was detected by western blot ( n = 3), * P < 0.05. F: MUT format of WT and miR-5100 sequences are shown. G: miR-5100 was pulled down by a CAAP1 probe or a random probe. MiR-5100 levels were analyzed by Northern blot. I indicates input (10% of sample is loaded); and P, precipitation (100% of sample is loaded). H: Cardiomyocytes were transfected with biotinylated WT-miR-5100 (Bio-mir-5100-WT) or a biotinylated mutant miR-5100 (Bio-mir-5100-MUT). Biotinylated microRNA that is not complementary to CAAP1 was used as a negative control (Bio-NC). CAAP1 expression level was analyzed by real-time RT-PCR, * P < 0.05.

Article Snippet: Negative control mimic (NC mimic), miR-5100 mimic (mimic), Negative control inhibitor (NC inhibitor), miR-5100 inhibitor (inhibitor) Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1), siRNA-CAAP1 (si-CAAP1) were purchased from RIBOBIO biological.

Techniques: Expressing, Sequencing, Mutagenesis, Transfection, Luciferase, Reporter Assay, Western Blot, Northern Blot, Negative Control, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: MKL1 targets the promoter of CAAP1 to promote the expression of CAAP1 protein. A: The base sequence of the CArG box of the wild type (WT-pGL3-CAAP1) and mutant (MUT-pGL3-CAAP1) on the CAAP1 promoter. B: MKL1 or empty vector (control group) was co-transfected with WT-pGL3-CAAP1 or MUT-pGL3-CAAP1 into MGC80-3 cells. The luciferase reporter gene test is used to detect whether MKL1 can target the CAAP1 promoter, * P < 0.05, ** P < 0.01. Transfect MKL1 or empty vector (control group) into AGS cells, (C-E): CAAP1 mRNA expression level was detected by RT-QPCR ( n = 3) ** P < 0.01, and CAAP1 protein expression level was detected by western blot, CAAP1 protein was quantitatively and statistically analyzed ( n = 3), ** P < 0.01. F: Co-transfect MKL1 or empty vector (control group) with WT-pGL3-miR-5100 or MUT-pGL3-miR-5100 into AGS cells and target CAAP1 according to the method described in “Experiment” The ChIP analysis of the primers by PCR showed the amount of DNA in each sample (2% of the input) in the second lane. Immunoprecipitation without primary antibody (No Ab) was used as a mock control, and IgG was used as a negative control.

Article Snippet: Negative control mimic (NC mimic), miR-5100 mimic (mimic), Negative control inhibitor (NC inhibitor), miR-5100 inhibitor (inhibitor) Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1), siRNA-CAAP1 (si-CAAP1) were purchased from RIBOBIO biological.

Techniques: Expressing, Sequencing, Mutagenesis, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells

doi: 10.1016/j.neo.2020.03.001

Figure Lengend Snippet: miR-5100 inhibits tumorigenesis in nude mice. A: Nude mice injected with two different MGC80-3 cell lines or gavage with paclitaxel after 28 days. The images shown represent results for 3 mice. B: Image shows tumor tissue isolated from nude mice with three mice in each group. C: Tumor growth curves of nude mice injected with two different MGC80-3 cell lines or administered paclitaxel after 28 days, and the tumor volume was estimated using calipers; * P < 0.05. D: The image shows the statistics of the weight of tumor tissue isolated from nude mice; the weight of the tumor tissue is measured with a balance; * P < 0.05. E: Representative H&E-stained sections of the tumor tissues isolated from mice. F: Immunohistochemical analysis of tumor tissue isolated from nude mice, the image shows the results of in-situ analysis of CAAP1 protein. G: Tumor tissues of miR-5100-plko.1 and plko.1 (control group) isolated from nude mice were analyzed for protein expression levels of CAAP1, caspase3, and LC3B by western blot. H: Tumor tissues of miR-5100-plko.1+Taxol group and plko.1+Taxol group (control group) isolated from nude mice were analyzed for protein expression levels of CAAP1, caspase3, and LC3B by western blot.

Article Snippet: Negative control mimic (NC mimic), miR-5100 mimic (mimic), Negative control inhibitor (NC inhibitor), miR-5100 inhibitor (inhibitor) Negative control siRNA (si-NC), siRNA-MKL1 (si-MKL1), siRNA-CAAP1 (si-CAAP1) were purchased from RIBOBIO biological.

Techniques: Injection, Isolation, Staining, Immunohistochemical staining, In Situ, Expressing, Western Blot