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Background As the main cellular constituent of corneal epithelium,corneal epithelial cells play critical roles in regulating and controlling the migration, proliferation and differentiation of cells during the repair of damage.MicroRNA (miRNA) is endogenously expressed small non-coding RNAs, which participates in a variety of biological processes.Previous studies demonstrated that miR-204 is highly expressed in normal corneal epithelium,but its function is still unclear.Objective This study was to investigate the function and mechanism of miR-204 in corneal epithelial cell proliferation.Methods Corneal epithelial tissue was collected during the corneal refractive surgery from the patients with refractive error under the informed consent, and human corneal epithelial cells(HCECs) were cultured and passaged.The relative expressing levels of miR-204 mRNA in the normal corneal epithelium and HCECs were detected by real time quantitative PCR.Cultured HCECs were evenly divided into three groups.The liposome with miR-204 mimic was transfected into the cells of the miR-204 mimic group, and blank liposome was transfected in the cells of the positive control group,and regularly cultured cells served as the normal control group.Cell proliferation capability was evaluated by colony-forming assay, and the percentage of the cells in different cell cycles was analyzed by flow cytometry.Western blot assay was employed to detect the expression levels of p-RB, E2F1 ,p27,CyclinA, CDC2, p-CDC2 and CDK2 proteins in the cells.Results The relative expression levels of miR-204 mRNA were 1.077 ±0.268 in the normal corneal epithelium and 0.041 ±0.018 in the HCECs, showing a significant difference between them (t =7.700,P＜0.001).The cloning cell number was evidently decreased in the miR-204 mimic group in comparison with the positive control group and normal control group.The percentage of cells in the G1 phase was 47.75％ in the miR-204 mimic group, which was significantly higher than 37.23％ in the positive control group and 40.72％ in the normal control group.The expression levels of E2F1 and p27 proteins in the cells were elevated (t=14.87,25.11;both at P＜0.01) and those of CDK2 and p-CDC2 proteins were decreased (t=5.39,10.65;both at P＜0.01) in the miR-204 mimic group in comparison with the positive control group.Conclusions The overexpression of miR-204 in the normal corneas probably is associated with non-proliferation status of corneal epithelial cells.Transfection of miR-204 into corneal epithelial cells can inhibit the proliferation of corneal epithelial cells probably by up-regulating the expression of E2F1 and p27 and suppressing the expression of CDK2/CyclinA and p-CDC2/CyclinA,which lead to cell arrest in G1 phase.
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Objective To investigate the relationship between the sigmoid colon propagating contractions and the gut hormones cholecystokinin(CCK)and motilin(MTL)in the patients with irritable bowel syndrome(IBS),and to reveal the gut hormones' modulation to the sigmoid colon motility.Methods Twenty diarrea-predominent IBS patients(D-IBS),and sex/age matched 20 constipation-predominent IBS patients(C-IBS)and 15 healthy volunteers were enrolled in the study.From June 2005 to Mar.2006,the high amplitude propagating waves and loger time of the sigmoid colon was measured by means of a liquid-perfused capillary tube connected to a computer.Meanwhile,the concentrations of CCK,MTL of blood plasma and sigmoid colon mucosa were tested by means of immunoassay technology.Results The D-IBS group had higher amplitude of the sigmoid colon propagating waves and their time percentage than that of the C-IBS group and the control group(P
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Objective To reveal the changes of pre-and post-prandial sigmoid colon motility in irritable bowel syndrome (IBS) patients and healthy comtrols.Methods Twenty consitipation-predominant and sex/age mathched 20 diarrhea-predominant IBS patients and 15 healthy volunteers were enrolled in the experiment.Migrating long spike bursts,and nonmigrating long spike bursts were recorded by a water perfusing manometry apparatus connected with a computer.Results In fasting status,diarrhea-predominant IBS patients had higher motor index mainly due to the elevated wave amplitude and the percentage of the recording time of the migrating long spike bursts.The test meal significantly increased the amplitude and the number of migrating and nonmigrating long spike bursts in all subjects,especially in the volunteers in the first half hour.The gastrosigmoidocolic reflex of the diarrhea-predominant IBS patients lasted longer than that of the consitipation-predominant IBS patients and healthy controls,mainly due to the increased amplitude of migrating long spike bursts and the number of nonmigrating long spike bursts.Conclusion Diarrhea-predominant IBS patients have stronger sigmoid colon motility in fasting state.The gastrosigmoidocolic reflex appears earlier in healthy subjects than in IBS patients and it lasts longer in diarrhea-predominant IBS patients than in consitipation-predominant IBS patients and healthy controls.