Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 1: The expression of ERα is negatively correlated with LY6K. (A and B) Expression of LY6K and ERα mRNA and protein was observed by qRT-PCR using specific primers and western blotting. h18s rRNA and β-actin were used for endogenous control. (C and D) Ectopic expression of LY6K in MCF7 and T47D downregulated ERα transcriptional activity and protein expression. (E and F) Ectopic expression of ERα in MCF7-ADR and MDA-MB-468 reduce LY6K mRNA and protein expression. Data are a mean ± S.D. (error bars) of three independent experiment in triplicate. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Activity Assay

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 2: The regulation of ERα and LY6K expression in a miRNA-dependent manner. (A and B) Gene expression level was determined on both mRNA and Protein levels after treatment with Ago2 siRNA combined with overexpression of LY6K for 48 hr. The expression of mRNA and protein was analyzed by qRT-PCR and western blotting. (C and D) LY6K expression in response to knock-down of AGO2 by treating with control and ERα for 48 hr. The expression of mRNA and protein was investigated by qRT-PCR and western blotting. Data are a mean ± S.D. (error bars) of three independent experiment in triplicate. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Over Expression, Quantitative RT-PCR, Western Blot, Knockdown, Control

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 3: Validation of primary and mature miRNAs induced by LY6K or ERα. (A) Validation of upregulated primary miRNA (pri-miRNA) expression in T47D/LY6K was compared with T47D/Mock (left) and MCF7-ADR/Mock compared with MCF7- ADR/ERα (right) by using qRT-PCR. Each primary miRNA produces mature miRNAs (Pri-miR-29a/b1: miR-29b, Pri-miR29b2/c: miR-29b and miR-29c, Pri-miR-194-2/192: miR-192-5p and miR-194, Pri-miR-194-1: miR-194, Pri-miR-34a: miR-34a, Pri-miR-500a: miR-500a-5p or -3p). (B) Verification of miRNA expression induced by LY6K in ERα-positive breast cancer cell lines, MCF7 (left) and T47D (right). (C) Verification of mature miRNA induced by ERα in ERα-negative breast cancer cell lines, MCF7-ADR (left) and MDA- MB-468 (right). Data are a mean ± S.D. (error bars) of three independent experiment in triplicate. ***p < 0.001; **p < 0.01; *p < 0.05.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 4: miR-192-5p directly binds ERα 3′UTR and regulates its expression. (A) Gene structure of ESR1 showing the predicted target site of miR-192-5p in its 3′-UTR. (B) Luciferase assay in MCF7 cells showed miR-192-5p dependent repression of wild type (WT) ERα 3′UTR (left), whereas mutation (MT) of the miR-192-5p predicted binding site did not affect repression (right). (C) Re-expression of miR-192-5p repressed the mRNA and protein expression of ERα in MCF7 and T47D. (D) mRNA and Protein expression of LY6K was determined in Human LY6K over-expressing stable cell line by qRT-PCR and western blot. (E) Inhibition of miR-192-5p expression increases ERα expression in T47D/LY6K stable cells. Mimic, miR-192-5p mimic treated cell; inhibitor, miR-192 inhibitor treated cell. Data present mean ± S.D. (error bars) of three independent experiments in triplicate. ***p < 0.001; **p < 0.01.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Expressing, Luciferase, Mutagenesis, Binding Assay, Stable Transfection, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 5: LY6K is a direct target of miR-500a-3p and down regulates its expression. (A) Gene structure of LY6K showing the two predicted target sites of miR-500a-3p in its 3′UTR. (B) Dual Luciferase assay in HEK293T cells showed ERα (left) and miR-500a- 3p (right) dependent inhibition of wild type (WT) LY6K 3′-UTR, whereas the mutant type (MT) seed sequence restored luciferase activity. (C) Ectopic expression of miR-500a-3p represses LY6K mRNA and protein expression in MCF7-ADR (left) and MDA-MB-468 (right). NC, miRNA negative control; mimic, miRNA mimic. Data represent mean ± S.D. (error bars) of three independent experiment in triplicate. ***p < 0.001.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Expressing, Luciferase, Inhibition, Mutagenesis, Sequencing, Activity Assay, Negative Control

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 6: miR-192-5p and miR-500a-3p mediates tamoxifen sensitivity in breast cancer. (A) Cell viability after transfection with miR-negative control inhibitor (NC) and miR-192-5p inhibitor in stably overexpressing LY6K (T47D/LY6K) cells was measured 3 hours after treatment with 4-OHT in a dose-dependent manner. (B) Caspase-3 activity was determined in T47D/LY6K transfected with miR-negative control inhibitor (NC) or miR-192-5p inhibitor after incubating with vehicle or 4-OHT (1, 5, 10 μM) for 3 hours. (C) T47D cells with miR-192-5p mimic transfected and then treated with 4-OHT (5 μM) for 24 hr. Apoptotic cells were assayed by flow cytometry sub-G1 analysis. (D) Cell viability with miR-negative control (NC) or miR-500a-3p was measured after 24 hr incubation with various doses of 4-OHT in MCF7-ADR. (E) Caspase-3 activation was increased by miR-500a-3p after 48 hr treatment with vehicle or 4-OHT. (F) MCF7-ADR cells with miR-500a-3p mimic transfected and then treated with 4-OHT (10 μM) for 24 hr. Apoptotic cells were determined by flow cytometry (sub-G1 analysis of PI staining). The graph shows the mean ± S.D. (error bars) of three independent experiments. ***p < 0.001; **p < 0.01; *p < 0.05; ns, non-specific.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Transfection, Negative Control, Stable Transfection, Activity Assay, Flow Cytometry, Incubation, Activation Assay, Staining

Journal: Oncotarget

Article Title: miRNAs involved in LY6K and estrogen receptor α contribute to tamoxifen-susceptibility in breast cancer.

doi: 10.18632/oncotarget.9950

Figure Lengend Snippet: Figure 7: miR-192-5p and miR-500a-3p involved in LY6K and ERα are related to tamoxifen resistance in breast cancer patients. (A) The relative LY6K expression depending on ER status in kao-breast cancer from oncomine™. (B) miR-192-5p expression in non-recurrence and recurrence primary breast tumors after tamoxifen treatment. The data were obtained from GSE 46823 (C) miR-500a-3p expression showed non-significant differences between non-recurrence and recurrence primary breast tumors after tamoxifen treatment. The data were obtained from GSE 46823. (D) Low expression of miR-500a-3p was correlated with poor survival outcomes in breast cancer patients received tamoxifen mono-therapy. (E) Hypothetical schematic pathway image. The mechanism for miR-192-5p and miR-500a-3p effects on tamoxifen susceptibility through the regulation of target genes in breast cancer. *P < 0.05; ns, non-significant.

Article Snippet: Co-transfection with plasmid and small interfering RNA MCF7 and T47D cells were transfected with Argonatue 2 siRNA (SMARTpooled, Dharmacon) and human LY6K clone purchased from Origene Technologies using Lipofectamin 2000 (Invitrogen, USA) for 48 hours following by the manufacturer’s instructions.

Techniques: Expressing

Journal: Risk Management and Healthcare Policy

Article Title: Impact of Diabetes Mellitus on the Immunity of Tuberculosis Patients: A Retrospective, Cross-Sectional Study

doi: 10.2147/rmhp.s354377

Figure Lengend Snippet: Figure 2 The comparison of lymphocyte subsets between TB-NDM and TB-DM patients. The absolute counts of total T lymphocytes (A), CD4+ T lymphocytes (B), CD8+

Article Snippet: IGRA Based on Enzyme-Linked Immunospot Assays (ELISPOT) First, lymphocytes in the blood samples were isolated using a Human Lymphocyte Isolation Kit (Solarbio, Beijing, China), following the manufacturer’s instructions.

Techniques: Comparison

Journal: Risk Management and Healthcare Policy

Article Title: Impact of Diabetes Mellitus on the Immunity of Tuberculosis Patients: A Retrospective, Cross-Sectional Study

doi: 10.2147/rmhp.s354377

Figure Lengend Snippet: Figure 3 The comparison of lymphocyte subsets among TB-NDM, TB-DM patients with euglycemia and TB-DM patients with hyperglycemia. The absolute counts of total T lymphocytes (A), CD4+ T lymphocytes (B), CD8+ T lymphocytes (C), NK cells (D), NKT cells (E), and B lymphocytes (F) among TB-NDM, TB-DM patients with euglycemia and TB-DM patients with hyperglycemia were compared with Ordinary one-way ANOVA test or Kruskal–Wallis nonparametric test according to the data normality and homogeneity of variances. The data were shown as mean ± SD, and P value < 0.05 was considered a significant difference.

Article Snippet: IGRA Based on Enzyme-Linked Immunospot Assays (ELISPOT) First, lymphocytes in the blood samples were isolated using a Human Lymphocyte Isolation Kit (Solarbio, Beijing, China), following the manufacturer’s instructions.

Techniques: Comparison

Journal: Risk Management and Healthcare Policy

Article Title: Impact of Diabetes Mellitus on the Immunity of Tuberculosis Patients: A Retrospective, Cross-Sectional Study

doi: 10.2147/rmhp.s354377

Figure Lengend Snippet: Figure 4 The comparison of the hospital LOS and absolute counts of lymphocyte subsets in TB-NDM patients and TB-DM patients. The absolute counts of total T lymphocytes (A), CD4+ T lymphocytes (B), CD8+ T lymphocytes (C), NK cells (D), NKT cells (E), and B lymphocytes (F) among TB-NDM with hospital LOS ≤2 weeks (TB-NDM-L≤2), TB-NDM with hospital LOS > 2 weeks (TB-NDM-L>2), TB-DM with euglycemia and hospital LOS ≤2 weeks (TB-DME-L≤2), TB-DM with euglycemia and hospital LOS > 2 weeks (TB-DME-L>2), TB-DM with hyperglycemia and hospital LOS ≤2 weeks (TB-DMH-L≤2), and TB-DM with hyperglycemia and hospital LOS > 2 weeks (TB-DMH-L>2) were compared with Ordinary one-way ANOVA test or Kruskal–Wallis nonparametric test according to the data normality and homogeneity of variances. The data were shown as mean ± SD, and P value < 0.05 was considered a significant difference.

Article Snippet: IGRA Based on Enzyme-Linked Immunospot Assays (ELISPOT) First, lymphocytes in the blood samples were isolated using a Human Lymphocyte Isolation Kit (Solarbio, Beijing, China), following the manufacturer’s instructions.

Techniques: Comparison

Journal: Risk Management and Healthcare Policy

Article Title: Impact of Diabetes Mellitus on the Immunity of Tuberculosis Patients: A Retrospective, Cross-Sectional Study

doi: 10.2147/rmhp.s354377

Figure Lengend Snippet: Figure 5 Linear regression analysis of the relationship between FBG level and absolute counts of lymphocyte subsets in TB-DM patients. The potential association between the FBG level and the absolute counts of total T lymphocytes (A), CD4+ T lymphocytes (B), CD8+ T lymphocytes (C), NK cells (D), NKT cells (E), or B lymphocytes (F) in TB-DM patients was analyzed with simple linear regression. P-value < 0.05 was considered as a significant linear relationship.

Article Snippet: IGRA Based on Enzyme-Linked Immunospot Assays (ELISPOT) First, lymphocytes in the blood samples were isolated using a Human Lymphocyte Isolation Kit (Solarbio, Beijing, China), following the manufacturer’s instructions.

Techniques: