Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene

OBJECTIVE@#To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene.@*METHODS@#RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells.@*RESULTS@#The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01).@*CONCLUSIONS@#miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.

Effect and molecular mechanism of mir-146a on proliferation of lung cancer cells by targeting and regulating MIF gene

Objective To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene. Methods RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells. Results The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01). Conclusions miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.

Activated Notch1 reduces myocardial ischemia reperfusion injury in vitro during ischemic postconditioning by crosstalk with the RISK signaling pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ischemic postconditioning (IPost), able to significantly attenuate myocardial ischemia reperfusion injury, is dependent on RISK signaling. Studies have shown that Notch signaling repairs damaged myocardium, and this study aimed to investigate the effect of Notch signaling in myocardial IPost.</p><p><b>METHODS</b>We used H9c2 cells to establish the myocardial IPost and Hypoxia/Reoxygenation (H/R) model in vitro. which were randomly divided into control, H/R, IPost, Hepatocyte growth factor (HGF)+IPost and DAPT+IPost, N1ICD+IPost, miRNA+IPost, and Mock treatment groups. The myocardial cell viability was assessed by MTT, the cell apoptosis was detected using Annexin V/PI double staining and flow cytometry analyses. The expression of N1ICD, Hes1, PTEN Phospho-Akt/Akt, Phospho-GSK-3β/GSK-3β were detected by Western blotting. Finally, we assessed the changes in ψm using the potential-sensitive dye JC-1 and measured using flow cytometry analyses.</p><p><b>RESULTS</b>The Notch1 signaling is activated by HGF and ectopic expression of N1ICD during myocardial IPost, which increased myocardial cell viability, prevented cardiomyocyte apoptosis, and reduced loss of the mitochondrial membrane potential. However, myocardial ischemia reperfusion injury was increased in IPost when Notch1 signaling was inhibited using DAPT or with knockdown by Notch1-miRNA. Western blotting found that PTEN was down-regulated by Hes1 when Notch1 was activated, which consequently promoted Akt and GSK-3β phosphorylation.</p><p><b>CONCLUSIONS</b>Notch1 crosstalk with RISK signaling may be dependent on PTEN, which plays a cardioprotective role during IPost. This mechanism could provide a promising therapeutic target for the treatment of ischemic heart disease.</p>

Roles of targeting Ras/Raf/MEK/ERK signaling pathways in the treatment of esophageal carcinoma / 药学学报

Ras is best known for its ability to regulate cell growth, proliferation and differentiation. Mutations in Ras are associated with the abnormal cell proliferation which can result in incidence of all human cancers. Extracellular signal-regulated kinase (ERK) is a downstream effector of Ras and plays important roles in prognosis of tumors. Recently, evidence has gradually accumulated to demonstrate that there are other effectors between Ras and ERK, these proteins interact each other and constitute the thorough Ras/Raf/MEK/ERK signaling pathway. The pathway has profound effects on incidence of esophageal carcinoma and clinical applications of some chemotherapeutic drugs targeting the pathway. Further understanding of the relevant molecular mechanisms of Ras/Raf/MEK/ERK signaling pathway can be helpful for the development of efficient targeting therapeutic approaches which contribute to the treatment of esophageal cancer. In this article, roles of Ras/Raf/MEK/ERK signaling pathway in esophageal carcinoma as well as pharmacological targeting point in the pathway are reviewed.

Heat shock protein 70 gene transfection protects rat myocardium cell against anoxia-reoxygeneration injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A number of studies suggest that the expression of heat shock protein 70 (HSP(70)) induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP(70) expression in acute myocardial anoxia/reoxygeneration (A/R) injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP(70) into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison.</p><p><b>METHODS</b>The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n = 8): control group, A/R group, HS + A/R group and pCDNA HSP(70) + A/R group. A liposome-coated HSP(70) pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP(70) mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetrazolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incubation and the changes in cells ultrastructure were examined. NF-kappaB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry.</p><p><b>RESULTS</b>Compared with viability in the A/R group ((35.4 +/- 6.9)%) the cell viability in the HS + A/R group ((72.8 +/- 11.6)%) and the pCDNA HSP(70) + A/R group ((76.3 +/- 12.2)%) was improved significantly (P < 0.05). The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS + A/R group and pCDNA HSP(70) + A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS + A/R group and pCDNA HSP(70) + A/R group. HSP(70) mRNA and protein were slightly expressed in the myocardiocytes of the A/R group. However, obvious overexpression was observed in the HS + A/R group and in the pCDNA HSP(70) + A/R group (P < 0.01). And there was a significant difference between the HS + A/R group and the pCDNA HSP(70) + A/R group in the expression of HSP(70) mRNA and protein (P < 0.01). A high activity of NF-kappaB (5.76 +/- 0.64) was detected in the A/R group. But in the HS + A/R group there was a statistically significant decrease in the activity of NF-kappaB compared with the A/R group (3.11 +/- 0.52 vs 5.76 +/- 0.64, P < 0.01). The same statistically significant difference was also observed in the pCDNA HSP(70) + A/R group and A/R group (2.83 +/- 0.49 vs 5.76 +/- 0.64, P < 0.01).</p><p><b>CONCLUSIONS</b>Overexpression of HSP(70) alone by gene transfection leads to protection for cardiac myocyte against anoxia-reoxygeneration. These cardioprotective effects were related to the reduction in activation of NF-kappaB.</p>