Journal: Cancer Science

Article Title: Programmed death ligand 1 regulates epithelial–mesenchymal transition and cancer stem cell phenotypes in hepatocellular carcinoma through the serum and glucocorticoid kinase 2/β‐catenin signaling pathway

doi: 10.1111/cas.15753

Figure Lengend Snippet: Programmed death ligand 1 (PD‐L1) regulates epithelial–mesenchymal transition ( EMT) and cancer stem cells ( CSCs ) through serum and glucocorticoid kinase 2 ( SGK2) /β‐catenin. (A) Expression of SGK2, PD‐L1, and Akt (phosphorylated at Ser473) in MHCC97H cells treated with 5 μM perifosine for 48 h or 120 h were detected by western blot analysis. (B) Using an anti‐PD‐L1 Ab, the PD‐L1 immunoprecipitates (IP) were purified and separated using SDS‐PAGE. Western blotting was utilized to determine whether SGK2 was present. Anti‐SGK2 Ab was used to purify the SGK2 immunoprecipitates, which were then separated using SDS‐PAGE. Western blotting was used to determine whether PD‐L1 was present. The negative control (NC) was normal IgG. (C) mRNA expression levels of PD‐L1 and SGK2 were detected in 20 hepatocellular carcinoma (HCC) specimens using RT‐PCR. Pearson's correlation method was used to evaluate the correlation of PD‐L1 and SGK2 in HCC specimens. (D) mRNA expression levels of SGK2 and β‐catenin in PD‐L1‐overexpressing or NC MHCC97H cells and PD‐L1‐knockdown or NC MHCC97H cells. (E) Luciferases activity was designed to detect β‐catenin‐mediated TCF/LEF transcription activity by TOPFlash/FOPFlash system assay in MHCC97H cells after transfection with shPD‐L1‐expressing vector or PD‐L1‐expressing vector. The luciferase activity with shPD‐L1 was divided by that with control sh‐NC; the luciferase activity with PD‐L1‐overexpressing Lentiviral vectors (LV‐PD‐L1) was divided by that with control vector ( n = 3). (F) Protein levels of β‐catenin, EMT markers E‐cadherin and N‐cadherin, and liver CSC marker CD133 in MHCC97H cells cotransfected with LV‐PD‐L1 or NC and CTNNB1 siRNA (siCTNNB1) or NC. (G) Protein levels of SGK2, PD‐L1, and β‐catenin signaling pathway proteins, EMT markers, and liver CSC markers in MHCC97H cells cotransfected with LV‐PD‐L1 or NC and siSGK2 or NC. (H) Protein levels of SGK2, PD‐L1, β‐catenin signaling pathway proteins, EMT markers, and liver CSC markers in MHCC97H cells cotransfected with shPD‐L1 or NC and pcDNA3.1‐SGK2 or NC. (I) Migration and invasion assays of MHCC97H cells cotransfected with LV‐PD‐L1 or NC and siSGK2 or NC. (J) Migration and invasion assays of MHCC97H cells cotransfected with shPD‐L1 or NC and pcDNA3.1‐SGK2 or NC. (K) Spheroid formation assay of MHCC97H cells cotransfected with LV‐PD‐L1 or NC and siSGK2 or NC. Scale bar, 100 μm. (L) Spheroid formation assay of MHCC97H cells cotransfected with shPD‐L1 or NC and pcDNA3.1‐SGK2 or NC. Scale bar, 100 μm. (M) Schematic diagram showing the effect of PD‐L1 on EMT and CSC phenotypes in HCC through the SGK2/β‐catenin signaling pathway. *p < 0.05. LV‐con, Control Lentiviral vectors; ns, not significant

Article Snippet: The cells were transfected using a Ribo FECTTM CP Transfection Kit (RiboBio), following the manufacturer's instructions.

Techniques: Expressing, Western Blot, Purification, SDS Page, Negative Control, Reverse Transcription Polymerase Chain Reaction, Knockdown, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Control, Marker, Migration, Tube Formation Assay

Journal: Journal of Bone Oncology

Article Title: MiR-193a regulates chemoresistance of human osteosarcoma cells via repression of IRS2

doi: 10.1016/j.jbo.2019.100241

Figure Lengend Snippet: IRS2 is a direct target of miR-193a-3p in OS cells. (A) Scheme of the luciferase reporter plasmid and the sequences of the WT and Mut 3′-UTR region of the IRS2 gene targeted by miR-193a-3p. The relative luciferase activity (fold-change) of the reporter with WT or Mut IRS2-UTR or with no UTR (Vec) was determined in the miR-193a-3p mimic (in SJSA-1)-, or antagomir (in G292)- or mock-transfected cells (B, C and D). Renilla luciferase activity of a cotransfected control plasmid was used as a transfection efficiency control. Representative results from three independent experiments are shown. Error bars represent the s.e.m. * p < 0.05 by Student's t -test.

Article Snippet: The riboFECT CP transfection kit, mimics, antagomiR, siRNA, and negative control (NC) RNA were supplied by ShangHai GenePharma, China.

Techniques: Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: American Journal of Cancer Research

Article Title: MicroRNA 421 induces the formation of high-invasive cell subsets of ovarian cancer from low-invasive cell subsets mediated by exosomes by activating the PI3K/AKT pathway

doi: 10.62347/UHEY7375

Figure Lengend Snippet: Diagnostic performance and the effect of miR-421 from exosomes on ALC. (A) The expression level of CA125 was significantly upregulated in serum exosomes of HOSGC patients. (B) The expression level of CA125 is not significantly related to clinical staging. (C) ROC curve analysis of CA125, miR-1910-3p, miR-378b, and miR-421 in the serum exosomes of HGSOC patients and benign patients. (D) The sensitivities and specificities of serum exosomal CA125, miR-1910-3p, miR-378b and miR-421. (E) ROC curve analysis of miR-1910-3p, miR-378b, and miR-421 and a combination of the CA125 in the serum exosomes of HGSOC patients and benign patients. (F) The sensitivities and specificities of miR-1910-3p, miR-378b, and miR-421 and a combination of the CA125 in the serum exosomes. (G) qRT-PCR results showed a significant upregulation of miR-421 expression in AHE after miR-421-mimic transfection. (H) Colony formation assay (274.80 ± 47.20 vs. 512.00 ± 28.15, P < 0.005), (I) CCK-8 assay, (J) EDU assay (63.57 ± 3.95 vs. 35.21 ± 5.45, P < 0.0001), (K) Wound healing assay (63.63 ± 5.15% vs. 27.52 ± 4.71%, P < 0.0001) and (L) Transwell Matrigel invasion/migration assay were showed that overexpression of miR-421 in AHE significantly promoted the proliferation, invasion, and migration ability of ALC compared to the control group. (M) WB experiment showed that overexpression of miR-421 in AHE significantly activated the PI3K/AKT signaling pathway of ALC compared to the control group. The data were presented as the mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. The scale bar equals 100 μm as indicated in the graph.

Article Snippet: The candidate DEM mimic/inhibitor (100 uM) and negative controls (100 uM) were transfected into AHC cells using the RiboFECT CP Transfection Kit (Ruibo Biology Co., Ltd.).

Techniques: Diagnostic Assay, Expressing, Quantitative RT-PCR, Transfection, Colony Assay, CCK-8 Assay, EdU Assay, Wound Healing Assay, Migration, Over Expression, Control