Effect of miRNA-193b-5p-mediated decreased expression of transcriptional regulator CITED2 on melanogenesis / 中华皮肤科杂志

Objective:To investigate the effect of miRNA (miR) -193b-5p on melanogenesis and its possible mechanisms.Methods:Human primary melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision, and cultured in vitro. miR-NC mimics (miR-NC mimic group) and miR-193b-5p mimics (miR-193b-5p mimic group) were transfected into human primary melanocytes and human MNT1 melanoma cells, separately. After transfection, real-time quantitative PCR (RT-qPCR) was performed to determine the overexpression efficiency of miR-193b-5p at 48 hours, Western blot analysis to determine the expression of melanogenesis-related proteins tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) in human primary melanocytes and human MNT1 melanoma cells at 72 hours, and the melanin content in the above cells was determined by a sodium hydroxide solubilization method at 1 week. The target gene of miR-193b-5p was predicted by using Targetscan algorithms and verified by dual-luciferase reporter assay, and RT-qPCR and Western blot analysis were performed to analyze changes in mRNA and protein expression of the target gene respectively after the overexpression of miR-193b-5p. Two-independent-samples t test was used for comparisons between two groups. Results:In human primary melanocytes and human MNT1 melanoma cells, the miR-193b-5p expression levels were significantly higher in the miR-193b-5p mimic groups than in the miR-NC mimic groups ( t = 65.57, 22.49, respectively, both P < 0.001) , and the melanin content was significantly lower in the miR-193b-5p mimic groups (0.091 ± 0.007, 0.130 ± 0.004, respectively) than in the miR-NC mimic groups (0.117 ± 0.002, 0.188 ± 0.032, t = 5.98, 3.24, P < 0.01, < 0.05, respectively) . Western blot analysis showed that the expression of melanogenesis-related proteins TYR and MITF in both human primary melanocytes and human MNT1 melanoma cells was significantly lower in the miR-193b-5p mimic groups than in the miR-NC mimic groups (all P < 0.01) . TargetScan analysis and dual-luciferase reporter assay revealed a binding site for miR-193b-5p in the 3′ untranslated region of the transcriptional regulator CITED2. After up-regulation of miR-193b-5p expression in human primary melanocytes and human MNT1 melanoma cells, the CITED2 mRNA and protein expression levels significantly decreased compared with the miR-NC mimic groups (all P < 0.05) . Conclusion:miR-193b-5p overexpression can down-regulate the expression of melanogenesis-related proteins TYR and MITF, and then inhibit melanogenesis, which may be related to the targeted inhibition of CITED2 expression.

Mutation analysis of CITED2 gene in patients with situs inversus / 上海交通大学学报（医学版）

Objective • To analyze the correlation between CITED2 gene mutation and situs inversus. Methods • A total of 24 patients with situs inversus and 100 healthy controls were collected. The genomic DNA was isolated from their peripheral blood. PCR and Sanger sequecing were employed to analyze the exons of CITED2. The potential effect of the mutation was characterized by the software Sift, PolyPhen-2, PROVEAN and Mutation Taster. The Y. Zhang laboratory server was used to predict the three-dimensional structure of the protein, and the SWISS-PdbViewer was imported to see the impacts of the mutation on the protein structure. Results • A novel heterozygous CITED2 mutation c.418C>T (p.P140S) was identified in 1 patient with situs inversus, which was absent in all controls. The novel heterozygous p.P140S mutation was predicted to be pathogenic by SIFT and Mutation Taster. The SWISS-PdbViewer showed that the mutation p.P140S caused all three hydrogen bonds on the aspartic acid at position 137 to be disconnected, and an abnormal weak hydrogen bond was re-established between the serine at position 140 and the alanine at position 142. Conclusion • The novel heterozygous mutation c.418C>T (p.P140S) may affect the biological activity of CITED2 and may be related to situs inversus.

The role and underlying mechanism of CITED2-mediated insulin signaling pathway in the pathogenesis of diabetic vascular diseases / 中华内分泌代谢杂志

It is well established that cardiovascular diseases are the leading causes of diabetes-related death. Endothelial dysfunction is widely accepted as the initial and critical factor contributing to diabetic vascular diseases. Insulin resistance may result in vascular endothelial dysfunction, which in turn aggravates diabetic vascular diseases. Via PI3K/Akt signaling pathway, insulin inhibits the function of FoxOs, which, endothelial FoxO1 especially, exerts an important role in atherosclerosis and angiogenesis. In this regard, Wang et al. characterized 10 FoxO1 target genes regulated by insulin in endothelial cells, among which, CITED2, a transcriptional coregulator, was selected to extensively investigate its role and the underlying mechanism of insulin-regulated angiogenesis. CITED2 was significantly increased in vascular endothelial cells in diet-induced mice, ob/ob mice, as well as obese type 2 diabetic patients, all of those models or subjects are accompanied by insulin resistance. In endothelial cells, insulin significantly down-regulated CITED2 expression through insulin receptor-PI3K-Akt-FoxO1 pathway. Inhibition of CITED2 resulted in significant increases in proliferation and tube formation of endothelial cells. Overexpression of CITED2, however, repressed the transactivation of HIF. The study on the mouse model with hind limb ischemia showed that endothelial CITED2 deficiency gave rise to significant increases of expression of endothelin-1, a well-known HIF target gene, induced by ischemia or hypoxia, suggesting that CITED2 inhibited endothelial angiogenesis via suppressing HIF transactivation. In summary, insulin resistance accompanying obesity and type 2 diabetes leads to enhanced CITED2 expression, consequently impairing HIF signaling and proangiogenic capacity in endothelial cells. Inhibition of CITED2 will be a promising novel way to deal with ischemic cardiovascular diseases in diabetic patients.

Molecular mechanism and regulation in the formation of dextrocardia / 国际儿科学杂志

Dextrocardia is formed by the disruption of the normal left-right (L-R) asymmetry of the thoracoabdominal organs.Asymmetric expression of Nodal,Pitx2,Cited2 have affected L-R asymmetry of vertebrate significantly,and generated dextrocardia,mesocardia,ectopia cordis,heterotaxy and so on.Dextrocardia is often accompanied by congenital cardiac malformation.Therefore,it is so difficult to be treated that it has poor prognosis.This paper reveiws the molecular mechanism and it's regulation of dextrocardia for proceeding the genetic diagnosis and early intervention of dextrocardia sequentially in order to improve the survival rate and life quality of patients.