Gnificance of difference in mean values was assessed by analysis of variance or Student’s t test. Only p values ,0.05 were considered statistically significant.Lens Protein Enzymatic Digestion for Advanced Glycation Endproduct AnalysisFor AGE analysis by LC/MS, 1 mg of lens protein extract was enzymatically digested in Chelex treated phosphate buffer with sequential additions of peptidase (Sigma P7500), protease K, pronase and aminopeptidase M (Roche, IN) as 115103-85-0 biological activity described earlier [12]. Corresponding enzyme blanks were incubated without added protein as a background control. Protein concentration was determined using the ninhydrin assay, as described earlier [12].AcknowledgmentsWe thank Christopher Strauch for LC/MC analyses of advanced 18334597 glycation and oxidation products.Author ContributionsConceived and designed the experiments: XF VMM. Performed the experiments: XF XL SH BW. Analyzed the data: XF VMM. Contributed reagents/materials/analysis tools: MLR. Wrote the paper: XF VMM.
CFTR is a chloride channel that is primarily expressed at the apical surface of airway epithelial cells and is involved in the control of airway surface fluid homeostasis [1]. Absence of functional CFTR is known to cause Cystic Fibrosis with lungrelated problems being the leading cause of mortality [2]. CFTR expression can be regulated at the transcriptional and posttranscriptional levels. CFTR interacts with many proteins that can affect its stability, degradation, and/or processing [3]. On the other hand, few copies of CFTR mRNA have been found in airway epithelial cells [4] suggesting that translational repression and/or mRNA degradation would strongly impact the amount of CFTR protein. MicroRNAs (miRNAs) are short non-coding RNAs of about 22 nucleotides [5], which mainly function by translational repression and/or mRNA degradation by binding to the 39 Untranslated Region (UTR). Therefore, down-regulation of miRNAs will result in increased protein expression of the targeted gene(s) whereas upregulation of miRNAs will lead to suppression of the targeted protein(s). Deregulation of miRNAs has been found in many diseases including lung KDM5A-IN-1 web cancer and chronic obstructive pulmonary disease (COPD) [6,7]. Up to 30 of human protein coding genesmay be regulated by miRNAs [8]. Some pathological conditions lead to the loss of certain miRNAs such as Let-7 members in cancer. A single miRNA can target several mRNAs and multiple miRNAs can target the same gene. It was only recently that CFTR was found to be regulated by miRNAs [9,10]. In this study, we investigated the effect of airway pollutants (cigarette smoke and cadmium) on miRNAs predicted to target CFTR in vitro in human airway epithelial cells as well as in vivo in the lung of smoke exposed mice and COPD patients. We also determined the role of miR-101 and miR-144 in regulating CFTR expression.Materials and Methods Ethics StatementThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Laboratory Animal Care and Use Committee (ILACUC) of the Ohio State University (protocol number: 2007A0168-R1).MiR-101 and -144 Regulate CFTR ExpressionTissue Culture and ReagentsThe human bronchial epithelial cell line 16HBE14o- (HBE), a gift from Dr. Gruenert [11], was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine, 10 FBS and penicillin (100 U/ml) and strep.Gnificance of difference in mean values was assessed by analysis of variance or Student’s t test. Only p values ,0.05 were considered statistically significant.Lens Protein Enzymatic Digestion for Advanced Glycation Endproduct AnalysisFor AGE analysis by LC/MS, 1 mg of lens protein extract was enzymatically digested in Chelex treated phosphate buffer with sequential additions of peptidase (Sigma P7500), protease K, pronase and aminopeptidase M (Roche, IN) as described earlier [12]. Corresponding enzyme blanks were incubated without added protein as a background control. Protein concentration was determined using the ninhydrin assay, as described earlier [12].AcknowledgmentsWe thank Christopher Strauch for LC/MC analyses of advanced 18334597 glycation and oxidation products.Author ContributionsConceived and designed the experiments: XF VMM. Performed the experiments: XF XL SH BW. Analyzed the data: XF VMM. Contributed reagents/materials/analysis tools: MLR. Wrote the paper: XF VMM.
CFTR is a chloride channel that is primarily expressed at the apical surface of airway epithelial cells and is involved in the control of airway surface fluid homeostasis [1]. Absence of functional CFTR is known to cause Cystic Fibrosis with lungrelated problems being the leading cause of mortality [2]. CFTR expression can be regulated at the transcriptional and posttranscriptional levels. CFTR interacts with many proteins that can affect its stability, degradation, and/or processing [3]. On the other hand, few copies of CFTR mRNA have been found in airway epithelial cells [4] suggesting that translational repression and/or mRNA degradation would strongly impact the amount of CFTR protein. MicroRNAs (miRNAs) are short non-coding RNAs of about 22 nucleotides [5], which mainly function by translational repression and/or mRNA degradation by binding to the 39 Untranslated Region (UTR). Therefore, down-regulation of miRNAs will result in increased protein expression of the targeted gene(s) whereas upregulation of miRNAs will lead to suppression of the targeted protein(s). Deregulation of miRNAs has been found in many diseases including lung cancer and chronic obstructive pulmonary disease (COPD) [6,7]. Up to 30 of human protein coding genesmay be regulated by miRNAs [8]. Some pathological conditions lead to the loss of certain miRNAs such as Let-7 members in cancer. A single miRNA can target several mRNAs and multiple miRNAs can target the same gene. It was only recently that CFTR was found to be regulated by miRNAs [9,10]. In this study, we investigated the effect of airway pollutants (cigarette smoke and cadmium) on miRNAs predicted to target CFTR in vitro in human airway epithelial cells as well as in vivo in the lung of smoke exposed mice and COPD patients. We also determined the role of miR-101 and miR-144 in regulating CFTR expression.Materials and Methods Ethics StatementThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Laboratory Animal Care and Use Committee (ILACUC) of the Ohio State University (protocol number: 2007A0168-R1).MiR-101 and -144 Regulate CFTR ExpressionTissue Culture and ReagentsThe human bronchial epithelial cell line 16HBE14o- (HBE), a gift from Dr. Gruenert [11], was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine, 10 FBS and penicillin (100 U/ml) and strep.
