miR-199a-5p Regulates Urothelial Permeability and May Play a Role in Bladder Pain Syndrome
2012; Elsevier BV; Volume: 182; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2012.10.020
ISSN1525-2191
AutoresKatia Monastyrskaya, Verónica Sánchez-Freire, Ali Hashemi Gheinani, David J. Klumpp, Eduard B. Babiychuk, Annette Draeger, Fiona C. Burkhard,
Tópico(s)Urinary Tract Infections Management
ResumoDefects in urothelial integrity resulting in leakage and activation of underlying sensory nerves are potential causative factors of bladder pain syndrome, a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. Herein, we identified the microRNA miR-199a-5p as an important regulator of intercellular junctions. On overexpression in urothelial cells, it impairs correct tight junction formation and leads to increased permeability. miR-199a-5p directly targets mRNAs encoding LIN7C, ARHGAP12, PALS1, RND1, and PVRL1 and attenuates their expression levels to a similar extent. Using laser microdissection, we showed that miR-199a-5p is predominantly expressed in bladder smooth muscle but that it is also detected in mature bladder urothelium and primary urothelial cultures. In the urothelium, its expression can be up-regulated after activation of cAMP signaling pathways. While validating miR-199a-5p targets, we delineated novel functions of LIN7C and ARHGAP12 in urothelial integrity and confirmed the essential role of PALS1 in establishing and maintaining urothelial polarity and junction assembly. The present results point to a possible link between miR-199a-5p expression and the control of urothelial permeability in bladder pain syndrome. Up-regulation of miR-199a-5p and concomitant down-regulation of its multiple targets might be detrimental to the establishment of a tight urothelial barrier, leading to chronic pain. Defects in urothelial integrity resulting in leakage and activation of underlying sensory nerves are potential causative factors of bladder pain syndrome, a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. Herein, we identified the microRNA miR-199a-5p as an important regulator of intercellular junctions. On overexpression in urothelial cells, it impairs correct tight junction formation and leads to increased permeability. miR-199a-5p directly targets mRNAs encoding LIN7C, ARHGAP12, PALS1, RND1, and PVRL1 and attenuates their expression levels to a similar extent. Using laser microdissection, we showed that miR-199a-5p is predominantly expressed in bladder smooth muscle but that it is also detected in mature bladder urothelium and primary urothelial cultures. In the urothelium, its expression can be up-regulated after activation of cAMP signaling pathways. While validating miR-199a-5p targets, we delineated novel functions of LIN7C and ARHGAP12 in urothelial integrity and confirmed the essential role of PALS1 in establishing and maintaining urothelial polarity and junction assembly. The present results point to a possible link between miR-199a-5p expression and the control of urothelial permeability in bladder pain syndrome. Up-regulation of miR-199a-5p and concomitant down-regulation of its multiple targets might be detrimental to the establishment of a tight urothelial barrier, leading to chronic pain. The bladder urothelium (UE) forms a lining of the urinary tract, separating the urinary reservoir from the underlying tissues and preventing the entry of noxious substances and pathogens. Apart from being just a passive barrier, it plays an important role in the sensory function of the bladder.1Birder L.A. More than just a barrier: urothelium as a drug target for urinary bladder pain.Am J Physiol Renal Physiol. 2005; 289: F489-F495Crossref PubMed Scopus (151) Google Scholar The structure of the UE is highly specialized, reflecting its ability to integrate mechanical and sensory input during bladder filling and to transmit this information to the connective tissue, nerves, and muscle layers. To adjust to the changing bladder volume during filling and micturition, the UE is stratified and ranges from four or five cells thick when the urinary bladder is empty to two or three cells thick when it is distended.2Apodaca G. The uroepithelium: not just a passive barrier.Traffic. 2004; 5: 117-128Crossref PubMed Scopus (251) Google Scholar The uppermost umbrella cell layer is formed by large polyhedral cells interconnected by tight and adherens junctions (TJs and AJs, respectively). TJ and AJ proteins of the umbrella cells regulate urothelial permeability, and their presence, amount, and distribution are the key factors determining the degree of tightness.3Acharya P. Beckel J. Ruiz W.G. Wang E. Rojas R. Birder L. Apodaca G. Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium.Am J Physiol Renal Physiol. 2004; 287: F305-F318Crossref PubMed Scopus (186) Google Scholar The umbrella cells forming the upper layer have transepithelial electrical resistance (TER) of >300,000 Ωcm2, making the UE one of the tightest epithelia of the body.4Lewis S.A. Everything you wanted to know about the bladder epithelium but were afraid to ask.Am J Physiol Renal Physiol. 2000; 278: F867-F874PubMed Google Scholar In addition, the bladder is coated with a thin mucinous layer composed of sulfonated glycosaminoglycans and glycoproteins.5Lilly J.D. Parsons C.L. Bladder surface glycosaminoglycans is a human epithelial permeability barrier.Surg Gynecol Obstet. 1990; 171: 493-496PubMed Google Scholar The integrity of the UE is indispensable for the healthy bladder.6Birder L.A. de Groat W.C. Mechanisms of disease: involvement of the urothelium in bladder dysfunction.Nat Clin Pract Urol. 2007; 4: 46-54Crossref PubMed Scopus (277) Google Scholar Mechanical or chemical damage, and bacterial infection, can lead to a compromised UE, allowing urine to penetrate into the interstitium.7Parsons C.L. The role of the urinary epithelium in the pathogenesis of interstitial cystitis/prostatitis/urethritis.Urology. 2007; 69: 9-16Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar Epithelial dysfunction resulting in a leaky UE has been suggested as a possible cause of bladder pain syndrome (BPS)/interstitial cystitis (IC), a clinical syndrome of pelvic pain, nocturia, and/or urinary urgency/frequency in the absence of a specific cause.8van de Merwe J.P. Nordling J. Bouchelouche P. Bouchelouche K. Cervigni M. Daha L.K. Elneil S. Fall M. Hohlbrugger G. Irwin P. Mortensen S. van Ophoven A. Osborne J.L. Peeker R. Richter B. Riedl C. Sairanen J. Tinzl M. Wyndaele J.J. Diagnostic criteria, classification, and nomenclature for painful bladder syndrome/interstitial cystitis: an ESSIC proposal.Eur Urol. 2008; 53: 60-67Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar The loss of epithelial integrity is a predominant histopathologic finding in biopsy samples from patients with BPS.9Tomaszewski J.E. Landis J.R. Russack V. Williams T.M. Wang L.P. Hardy C. Brensinger C. Matthews Y.L. Abele S.T. Kusek J.W. Nyberg L.M. Biopsy features are associated with primary symptoms in interstitial cystitis: results from the interstitial cystitis database study.Urology. 2001; 57: 67-81Abstract Full Text Full Text PDF PubMed Google Scholar In patients with BPS, the molecular markers for bladder permeability and proteoglycan core proteins have been shown to be down-regulated.10Slobodov G. Feloney M. Gran C. Kyker K.D. Hurst R.E. Culkin D.J. Abnormal expression of molecular markers for bladder impermeability and differentiation in the urothelium of patients with interstitial cystitis.J Urol. 2004; 171: 1554-1558Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Increased permeability and decreased TJ formation has been observed in bladder urothelial monolayers grown from biopsy samples from patients with IC compared with controls.11Zhang C.O. Wang J.Y. Koch K.R. Keay S. Regulation of tight junction proteins and bladder epithelial paracellular permeability by an antiproliferative factor from patients with interstitial cystitis.J Urol. 2005; 174: 2382-2387Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Therefore, it seems possible that urothelial damage is a preceding feature of this disorder and might be a causative factor in the pathogenesis of BPS. Recently, we showed that mRNA levels of the TJ proteins ZO-1, JAM-1, occludin, and tight claudins, normally present in water-impermeable epithelia and abundant in normal UE, were significantly down-regulated in bladder biopsy samples from patients with BPS, indicating a compromised TJ structure and possibly increased permeability of the UE.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar MicroRNAs (miRNAs) are quickly gaining recognition for their role in many biologic processes and disease states.13Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15350) Google Scholar, 14Farazi T.A. Spitzer J.I. Morozov P. Tuschl T. miRNAs in human cancer.J Pathol. 2011; 223: 102-115Crossref PubMed Scopus (806) Google Scholar miRNAs are endogenous noncoding single-stranded RNAs of approximately 22 nucleotides that regulate gene expression by posttranscriptional mechanisms following sequence-specific binding to the 3′ untranslated regions (3′UTRs) of their mRNA targets.13Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15350) Google Scholar, 15Lagos-Quintana M. Rauhut R. Lendeckel W. Tuschl T. Identification of novel genes coding for small expressed RNAs.Science. 2001; 294: 853-858Crossref PubMed Scopus (3853) Google Scholar miRNAs exhibit imperfect complementarity with mRNAs, allowing them to regulate multiple genes and, thus, complicating efforts to predict and functionally validate their targets.16Thomson D.W. Bracken C.P. Goodall G.J. Experimental strategies for microRNA target identification.Nucleic Acids Res. 2011; 39: 6845-6853Crossref PubMed Scopus (433) Google Scholar miRNAs play an important role in the regulation of cell adhesion and cell migration17Valastyan S. Weinberg R.A. Roles for microRNAs in the regulation of cell adhesion molecules.J Cell Sci. 2011; 124: 999-1006Crossref PubMed Scopus (83) Google Scholar, 18Huang S. He X. microRNAs: tiny RNA molecules, huge driving forces to move the cell.Protein Cell. 2010; 1: 916-926Crossref PubMed Scopus (24) Google Scholar: a subset of miRNAs, including miR-17, miR-29, miR-124, and miR-200, modulate cell adhesion pathways by controlling the expression levels of cytoskeletal regulatory proteins and cell-cell and cell-matrix adhesion molecules. The role of miRNAs in TJ formation is less well-established, although there is evidence that miR-212 influences intestinal permeability by interfering with ZO-1 protein in Caco-2 cells,19Tang Y. Banan A. Forsyth C.B. Fields J.Z. Lau C.K. Zhang L.J. Keshavarzian A. Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcoholic liver disease.Alcohol Clin Exp Res. 2008; 32: 355-364Crossref PubMed Scopus (232) Google Scholar and tumor necrosis factor α–induced miR-122a causes degradation of occludin in enterocytes and increases epithelial permeability.20Ye D. Guo S. Al-Sadi R. Ma T.Y. MicroRNA regulation of intestinal epithelial tight junction permeability.Gastroenterology. 2011; 141: 1323-1333Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar In patients with irritable bowel syndrome, miR-29a was increased, leading to a concomitant decrease of glutamine synthetase levels and directly influencing intestinal permeability through glutamine-dependent signaling pathways.21Zhou Q. Souba W.W. Croce C.M. Verne G.N. MicroRNA-29a regulates intestinal membrane permeability in patients with irritable bowel syndrome.Gut. 2010; 59: 775-784Crossref PubMed Scopus (205) Google Scholar To identify the miRNAs influencing urothelial permeability, we used an immortalized human ureteral cell line, TEU-2, expressing ZO-1, occludin, JAM-1, and claudins 1, 2, 4, and 8 on differentiation into a multilayered culture.22Rickard A. Dorokhov N. Ryerse J. Klumpp D.J. McHowat J. Characterization of tight junction proteins in cultured human urothelial cells.In Vitro Cell Dev Biol Anim. 2008; 44: 261-267Crossref PubMed Scopus (36) Google Scholar, 23Klumpp D.J. Weiser A.C. Sengupta S. Forrestal S.G. Batler R.A. Schaeffer A.J. Uropathogenic Escherichia coli potentiates type 1 pilus-induced apoptosis by suppressing NF-κB.Infect Immun. 2001; 69: 6689-6695Crossref PubMed Scopus (98) Google Scholar Taking 500 Ωcm2 as a reference,4Lewis S.A. Everything you wanted to know about the bladder epithelium but were afraid to ask.Am J Physiol Renal Physiol. 2000; 278: F867-F874PubMed Google Scholar we performed TER measurements in differentiating TEU-2 cells transfected with selected pre-miRNAs previously shown to be up-regulated in patients with BPS.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Herein, we show for the first time, to our knowledge, that miRNA expression influences urothelial permeability and might underlie the changes in the bladder UE observed in BPS. Polyclonal antibodies against claudin 1 (catalog No. 51-9000), occludin (catalog No. 71-1500), JAM-1 (catalog No. 36-1700), and Velis (MALS-3 or LIN7C) (catalog No. 51-5600) were from Invitrogen (Carlsbad, CA). Monoclonal anti-PALS1 (G-5) antibody (sc-365411) was from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488–labeled phalloidin was from Molecular Probes (Invitrogen). Restriction endonucleases, Taq polymerase, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). Pre-miR miRNA precursors and validated small-interfering RNAs (siRNAs) were from Ambion (Applied Biosystems, Foster City, CA). Chemicals were from Sigma-Aldrich (St. Louis, MO). Permission to conduct this study was obtained from the Ethics Committee of the Canton of Bern (KEK 146/05), and all the participants gave written informed consent. Patient selection and evaluation was described previously.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Patients were divided into two groups: the control group consisted of asymptomatic patients undergoing cystoscopy for other reasons (eg, stent placement for stone disease or microhematuria evaluation), and the BPS group consisted of patients with pain (for >3 months) considered to be located in the bladder and/or frequency, urgency, and nocturia. Cold cup biopsy samples from the bladder dome were collected from 8 controls and 28 patients with BPS in a previously published study.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar All the patients with BPS had bladder pain for >3 months, accompanied by frequency, urgency, or nocturia. No patient had an increased postvoid residual. Urodynamic studies showed a median cystometric bladder volume of 200 mL (range, 60 to 1000 mL). One patient had a high-capacity bladder with a volume >1000 mL, and two patients had a normal capacity; the remaining patients had low-capacity bladders. Histopathologic evaluation showed chronic inflammation (lymphoplasmocytic infiltration, interstitial edema, and/or hyperemia of the blood vessels with dilated lumina) in all but 4 patients, and 17 demonstrated increased mast cells in smooth muscle (SM) (≥20/mm2). The samples were processed and evaluated by quantitative real-time PCR (qPCR) as described previously.24Sanchez Freire V. Burkhard F.C. Schmitz A. Kessler T.M. Monastyrskaya K. Structural differences between the bladder dome and trigone revealed by mRNA expression analysis of cold-cut biopsies.BJU Int. 2011; 108: E126-E135Crossref PubMed Scopus (9) Google Scholar For morphologic studies, samples from the dome were obtained from four patients (three women and one man; mean age, 65 years; age range, 52–74 years) undergoing radical cystectomy for bladder cancer who had no lower urinary tract symptoms aside from hematuria. The samples localized well away from the tumor. The immortalized human urothelial cell line TEU-223Klumpp D.J. Weiser A.C. Sengupta S. Forrestal S.G. Batler R.A. Schaeffer A.J. Uropathogenic Escherichia coli potentiates type 1 pilus-induced apoptosis by suppressing NF-κB.Infect Immun. 2001; 69: 6689-6695Crossref PubMed Scopus (98) Google Scholar was maintained in serum-free EpiLife medium supplemented with human keratinocyte growth supplement and antibiotics (Cascade Biologics, Portland, OR). Differentiation of TEU-2 cells was achieved by the addition of serum and Ca2+ as previously described.22Rickard A. Dorokhov N. Ryerse J. Klumpp D.J. McHowat J. Characterization of tight junction proteins in cultured human urothelial cells.In Vitro Cell Dev Biol Anim. 2008; 44: 261-267Crossref PubMed Scopus (36) Google Scholar For TER measurements, the cells were cultured in 12-well plates with inserts (BD Falcon, Franklin Lakes, NJ). Pre-miR miRNA precursors for miR-199a-5p, miR-320a, and miR-328 and a validated Cy3-labeled negative control were from Ambion (Applied Biosystems). The reverse transfections were performed in 12-well plates with and without inserts using Lipofectamine 2000 reagent (Invitrogen) or siPORT NeoFX transfection agent (Applied Biosystems). The transfected cells were incubated at 37°C for 24, 48, or 72 hours before TER measurements and mRNA isolation. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium containing 2 mmol/L glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal calf serum. HEK293 cells were transiently transfected with reporter plasmids and pre-miR miRNA precursors using Lipofectamine 2000 reagent (Invitrogen) and were assayed for luciferase activity 24 hours after transfection. The immortalized human bronchial epithelial cell line 16HBE14o- was a gift from Prof. Dieter Gruenert (University of California at San Francisco, San Francisco, CA). The cells were cultured on CELL+-coated Petri dishes (Sarstedt Inc., Newton, NC) in minimal essential medium containing 2 mmol/L glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal calf serum and were transfected with pre-miR miRNA precursors using Lipofectamine 2000 reagent (Invitrogen). Primary cultures of human bladder UE were established and maintained in CnT-18 medium (CELLnTEC, Bern, Switzerland) as described previously.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar For differentiation, the UE cells were grown on 0.4-μm-pore inserts (BD Falcon) and were induced by incubating in CnT-21 medium supplemented with 2 mmol/L Ca2+ and 10% fetal calf serum. All the cell cultures were incubated at 37°C, 85% humidity, and 5% CO2. Cell proliferation and metabolic activity was assessed using alamarBlue reagent (Invitrogen) following the manufacturer’s instructions. Cells were cultured in 0.4-μm-pore inserts (BD Falcon). TER was measured using the Millicell-ERS device (Millipore, Billerica, MA). For each insert, TER was measured in four equidistant locations, and an average of the four measurements taken as the TER value at the selected time point. The resistance values are presented as unit area resistance (unit area resistance = TER × cm2 = Ωcm2). Cells grown on cell culture inserts with a 0.4-μm pore size (BD Falcon) were fixed with 4% paraformadehyde, permeabilized in 0.05% Triton X-100 in PBS, and incubated with the primary antibodies for 1 hour. Cells were washed and incubated with the Cy3- or Alexa Fluor 488–conjugated secondary antibodies and DAPI stain. Inserts were mounted in PBS-gelvatol and were examined using an Axiovert 200M microscope with laser scanning module LSM 510 META (Carl Zeiss MicroImaging GmbH, Jena, Germany). TEU-2 cells were cultured on 0.4-μm-pore inserts (BD Falcon) either as monolayers or in differentiation medium for 72 hours. The insert filters were cut out and fixed in 2.5% glutaraldehyde in Na+ cacodylate buffer and were embedded in Epon 812 resin for electron microscopy processing. Epon-embedded ultrathin sections were examined using a Zeiss 400 electron microscope (Carl Zeiss MicroImaging GmbH). Bladder UE and SM cells were selectively isolated using the PALM laser microdissection platform (Carl Zeiss MicroImaging GmbH). Optimal cutting temperature compound–embedded frozen bladder tissue was sectioned at a thickness of 10 μm and was mounted onto PALM membrane slides. The slides were stained using the Arcturus HistoGene LCM frozen section staining kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The PALM MicroBeam system was used to obtain pure populations of either urothelial cells (collected from the upper half of the normal bladder UE) or bladder SM cells (collected from the detrusor muscle bundles) until at least 10,000 cells were collected for each sample. The microdissected cells were transferred to lysis buffer (provided in the mirVana miRNA isolation kit; Ambion, Austin, TX) for miRNA extraction. Total RNA was isolated using the mirVana miRNA isolation kit (Ambion) as described previously.12Sanchez Freire V. Burkhard F.C. Kessler T.M. Kuhn A. Draeger A. Monastyrskaya K. MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.Am J Pathol. 2010; 176: 288-303Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Sanchez Freire V. Burkhard F.C. Schmitz A. Kessler T.M. Monastyrskaya K. Structural differences between the bladder dome and trigone revealed by mRNA expression analysis of cold-cut biopsies.BJU Int. 2011; 108: E126-E135Crossref PubMed Scopus (9) Google Scholar To separate the bladder UE from the underlying stroma, postoperative tissue was treated with 5 mg/mL of dispase II overnight at 4°C. The reverse transcription reactions were performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) with random hexamer primers. TaqMan primers (occludin Hs00170162_m1, claudin 1 Hs00221623_m1, JAM-1 Hs00170991_m1, 18S Hs99999901_s1, sirtuin 1 Hs01009006_m1, Rho GAP21 Hs00372683_m1, SRF Hs00182371_m1, E-cadherin Hs00170423_m1, N-cadherin Hs00169953_m1, fibronectin Hs01549976_m1, vimentin Hs0018584_m1, and Snail Hs00195591_m1) for real-time qPCR were from Applied Biosystems. Quantification of mature miR-199a-5p and endogenous control miRNA RNU48 was performed using TaqMan assays 000498 and 001006 with supplied assay-specific RT primers (Applied Biosystems). The exon junction–spanning primers for SYBR green qPCR were designed by using Primer-BLAST software (NCBI) and were synthesized by Microsynth AG (Balgach, Switzerland). The following primer pairs (forward and reverse) were used (all based on human sequences): 28S rRNA (endogenous control), 5′-TCTGACTTAGAGGCGTTCAGTCATAAT-3′ and 5′-GTTGTTGCCATGGTAATCCTGCTCAGT-3′; LIN7C, 5′-GAGCGAACGCTACTGCAAAGGCTA-3′ and 5′-ATCTCCACGTTTGAGGCCCCCA-3′; GEF12, 5′-ATCACCGACAGGTTTCCCCTCAA-3′ and 5′-TACGCAACGCTGAACAAGACCAT-3′; ARHGAP12, 5′-CCAAAGCACCAAGATACAGCCAGCA-3′ and 5′-GAACCCTGCAACACCGCCCAA-3′; ARHGAP29, 5′-CGAGATGGAGCGCCGTGGAT-TTC-3′ and 5′-GTTGACCTGATGCCCAAGCACG-3′; ROC-K1, 5′-GACTTTGGGCGTCCGAGCGG-3′ and 5′-CCAAA-GCATCCAATCCATCCAGCAA-3′; PALS1, 5′-CTGCAG-TTCCTCATACAACCCGGA-3′ and 5′-TCTGCCTCGAAT-GCTTGCCGC-3′; RND1, 5′-AGCGCACTCAAGAAGTGGAGGAC-3′ and 5′-GCCTGCTTCTGGTGGGACAGC-3′; PVRL1, 5′-ATCACAGAATTCCCCTACACCCCG-3′ and 5′-CCCCCAATGATGGCCGTGGG-3′; CLTC, 5′-GCAGCACTGGGACTTGAGCGA-3′ and 5′-TGCAGCTCTTAG-CACCTTGGGA-3′; and PXN, 5′-CAGGGCCTGGAGCAA-AGAGCG-3′ and 5′-TTCCCCTGGGCCATGAACCCTC-3′. qPCR was performed in triplicate using the 7900HT fast real-time PCR system (Applied Biosystems). The CT values obtained after real-time qPCR were normalized to the 18S rRNA when performing TaqMan qPCR and to the 28S rRNA when performing SYBR green qPCR. CT values of miR-199a-5p TaqMan assay were normalized to RNU48. The end products of all PCR reactions were analyzed on a 4% low melting point agarose gel to validate the fragment size. Luciferase reporter plasmids pSGG_3UTR containing 3′UTRs of occludin, claudin 1, and JAM-1 (F11R) as well as R01 negative control vector were purchased from SwitchGear Genomics Inc. (Menlo Park, CA). The constructs were cotransfected with Renilla luciferase expression vector pRenilla-TK-luc and pre-miR miRNA precursor molecules or negative control miRNA precursors (Ambion). Firefly and Renilla luciferase assays were performed 24 hours after transfection using a dual-luciferase reporter assay kit (Promega Corp., Madison, WI) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla expression for each sample. 3′UTR sequences of human LIN7C and ARHGAP12 mRNAs containing putative miR-199a-5p binding sites were PCR amplified from TEU-2 cDNA and cloned into pmirGLO vector (Promega Corp.). For generation of a reporter vector bearing a 2072-bp human LIN7C 3′UTR fragment with three putative miR-199a-5p binding sites, the following primers were used: forward 5′-AATAAGCTAGCTGGTGGTGGGAAGTAGCTGAAGCA-3′; reverse 5′-AATAACTCGAGGGCAAAGGGGGCAGAATGATGACA-3′ (Microsynth AG). For generation of a reporter vector containing a 1148-bp human ARHGAP12 3′UTR fragment with a putative miR-199a-5p binding site, the following primers were used: forward 5′-ATAAACTCGAGGCTAGCGGGAAGGGGAGAGTCGAGATGTGTG-3′; reverse 5′-ATAAACTCGAGTCTTCAACAGACCATGCTCCCTGTA-3′ (Microsynth AG). NheI and XhoI restriction sites are shown in italics. A pmirGLO vector containing the miR-199a-5p target sequence 5′-GAACAGGTAGTCTGAACACTGGG-3′ was constructed and used as a positive control. A vector containing a mismatched sequence (shown in italics), 5′-GAACAGGTAGTCTGAACGGTAT-G-3′, was used to verify the specificity of miR binding, and the empty pmirGLO vector was used as negative control. Following the similar cloning strategy, putative miR-199a-5p binding sites from the 3′UTRs of ARHGAP12: position 279, 5′-TTCTAACCAGTCATATACACTGGA-3′; ARHGAP12 mismatch (mutated nucleotides shown in bold), 5′-TTCTAACCAGTCATATCATTGACA-3′; LIN7C: 1182 site, 5′-AATATTTCATTTGTCCACTGGAT-3′; 1527 site, 5′-ATTAAGGTATTGAACACTGGA-3′; and 2542 site, 5′-TATAAAATGATCTGATTACACTGGA-3′; PALS1 (alias MPP5): position 1476, 5′-ACCCAGAGCTAAACACTGGA-3′; RND1: position 71, 5′-GATGAGACAATTTAGGACACT-GGA-3′; and PVRL1 (alias nectin 1): position 346, 5′-CTCCCCCGCCCCCCTACACTGGA-3′ were inserted as linkers between NheI and XhoI sites of pmirGLO vector. To detect luciferase activity, dual-luciferase reporter assay (Promega Inc.) was used, and the activity was normalized to Renilla luciferase expressed from the pmirGLO vector. In each assay, 2 × 106 TEU-2 cells were transfected with pre-miR miRNA precursor for miR-199a-5p and a validated Cy3-labeled negative control as described previously herein. The 5 × 104 transfected cells were plated separately on cell culture inserts with a 0.4-μm pore size (BD Falcon) to control differentiation by TER, and the rest were cultured on standard tissue culture dishes and induced to differentiate as described previously herein. Seventy-two hours after differentiation, cells were harvested and lysed in assay/sample buffer (Cell Biolabs Inc., San Diego, CA), and protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). The sample concentration was adjusted to 1 mg/mL to normalize the amount of starting material, and RhoA and Rac1 activity was determined using a small GTPase pull-down assay (RhoA/Rac1/cdc42 activation assay combination kit; Cell Biolabs Inc.), following the manufacturer’s instructions. Unless otherwise stated, all the procedures were performed at 4°C or on ice. The cell lysates were analyzed by SDS-PAGE, followed by Western blot analysis with specific antibodies. Image analysis to estimate the protein content of the individual bands after SDS-PAGE and Western blot analysis was performed using a PowerLook 1120 scanner and ImageQuant TL software version 2003 (Amersham Biosciences Europe GmbH; Freiburg, Germany). Total RNA was isolated from differentiated TEU-2 cells, and cDNA was produced by reverse transcription with random hexamer primers as described previously herein. The coding sequence of LIN7C was PCR amplified using forward primer 5′-ATAAAGCTAGCATGGCGGCGCTAGGGGAA-3′ and reverse primer 5′-ATAAAGGATCCGGTCTGTTCCTGCGTTTTG-3′. The coding sequence of PALS1 was PCR amplified using forward primer 5′-ATAAAGCTAGCATGACAACATCCCATATGAATG-3′ and reverse primer 5′-ATAAAGGATCCCCTCAGCCAAGTGGATGGTAC-3′. Cloning NheI and BamHI sites are shown in italics. PCR fragments were inserted into pCDH-EF1-T2A-copGFP vector (System Biosciences, Mountain View, CA) to produce pCDH-LIN7C and pCDH-PALS1 lentiviral vectors. The cloning vector without insert was used as a control (pCDH-GFP). HEK293FT cells (System Biosciences) were plated at 50% confluency on 10-cm dishes and were transfected with 12 μg of each of the pCDH-based lentiviral vectors [LIN7C, PALS1, and green fluorescent protein (GFP)], 8 μg of packaging pPAX2, and 4 μg of pMD2.G plasmids using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instruc
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