Phorbol Dibutyrate (PDB) Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) May Involve Down‐Regulation of MicroRNAs (miRs)

Abstract

In colonic epithelia, the concerted action of apical membrane Cl− channels and basolateral membrane Na+/K+‐ATPase, Na+‐K+‐2Cl− cotransporters and K+ channels, account for net Cl− secretion. CFTR, the major apical Cl− channel, is regulated by protein kinases (PK) A and C. We had previously showed in human colonic T84 cells that the cAMP activator, prostaglandin E1 (PGE1,10nM), stimulated Cl− secretion via CFTR, measured as short circuit current (ΔISc, μA/cm2) and this was attenuated by both acute and prolonged pretreatment with the PKC activator, phorbol dibutyrate (PDB, 100nM). Further, 2h of pretreatment with PDB significantly reduced CFTR mRNA. Since microRNAs (miRs) are known to modulate proteins by either degrading mRNA, or by inhibiting translation, we initially tested whether 6 specific CFTR related miRs: −145, −331, −384, −494, −1246 and 1290 were regulated by PDB. However, the expressions of these miRs were not significantly altered by PDB (FASEB 29:855.5,’15). In this study, we further explored the molecular basis of the attenuation in Cl− secretion. We first confirmed, by Western blotting, that CFTR protein expression is down regulated at 2h PDB treatment (n=2). In addition, the mRNA (60%, n=3) and protein expression for NKCC was also decreased. Since CFTR 3′ UTR contains almost 500 recognized miR target sites, we next undertook a detailed algorithm screening process involving TargetScan, miRDB, EMBL‐EBI and Diana to identify further possible CFTR miR regulators. We hypothesized that the miRs identified through algorithmic screening would be up‐regulated after PDB treatment, leading to a decrease in CFTR expression. We specifically screened miRs: −362, −433, −509, −600, −607, −619, −631, and −944, which were identified by at least 3 algorithms. After treating T84 cells with ±100nM PDB for 2h, 6h, and 24h, RNA was isolated using TriZol Reagent and cDNA was synthesized using an Invitrogen cDNA synthesis kit. We compared the expression levels of these miRs using an Invitrogen miRNA qRT‐PCR Kit with U6 snRNA as an internal control. Based on three separate experiments each (p<0.05), the results show that miR −607 (Control: 1.37±0.18, 2h: 0.51±0.08, 6h: 0.77±0.09, 24h: 0.55±0.18), miR‐619 (Control: 2.04±0.92, 2h: 0.58±0.05, 6h: 0.42±0.06, 24h: 0.44±0.04), and miR‐944 (Control: 1.50±0.30, 2h: 0.46±0.07, 6h: 0.36±0.08, 24h: 0.40±0.13) expressions were significantly down‐regulated at all measured time‐points. miR‐631 (Control: 1.24±0.18) expression was significantly down‐regulated at 2h (0.60±0.05) and 6h (0.58±0.04) but not at 24h (0.78±0.22). The results with miR‐607 are in contrast to studies in Caco2 cells, where an increase in miR‐607 leads to a decrease in CFTR expression (Biochem J. 438, p.25–32 2011). The other miRs tested did not show significant alteration of expression at any measured time‐point. Thus, PDB treatment reduces CFTR and NKCC expression and down regulates specific miRs. We speculate that perhaps there is an intermediary protein, the target of miRs: −607, −619, and −944, that alters CFTR/NKCC expression.Support or Funding InformationAmerican Physiological Society Undergraduate Research Excellence Fellowship (UGREF)