Circulating microRNA-196a as a candidate diagnostic biomarker for chronic hepatitis C.

Previous studies have demonstrated the inhibitory effect of microRNA (miR)-196a on hepatitis C virus (HCV) expression in human hepatocytes. However, the clinical implications of aberrant miR-196a expression and the application of circulating miR-196a in the diagnosis and management of chronic hepatitis C (CHC) require further investigation. The present study aimed to examine the possibility of using serum miR-196a as a biomarker for CHC. The Affymetrix miRNA array platform was used for miRNA expression profiling in adenovirus (Ad)-HCV core-infected (HepG2-HCV) and Ad-enhanced green fluorescence protein (EGFP)-infected HepG2 cells (HepG2-control). miR-196a downregulation and levels were analyzed using stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the sera of 43 patients with CHC and 22 healthy controls. A total of six miRNAs were identified as significantly different (≥ 1.5 fold; P ≤ 0.05) between the two groups. Of note, significant miR-196a downregulation was observed in HepG2-HCV as compared with HepG2­EGFP. Furthermore, as compared with that of the healthy control group, serum miR-196a was demonstrated to be significantly lower in patients with CHC. In addition, analysis of the receiver operating characteristic (ROC) curve for serum miR-196a revealed an area under the ROC curve of 0.849 (95% confidence interval, 0.756-0.941; P<0.001) with 81.8% sensitivity and 76.7% specificity in discriminating chronic HCV infection from healthy controls at a cut-off value of 6.115 x 10(-5), demonstrating significant diagnostic value for CHC. However, no correlation was identified between serum miR-196a and alanine aminotransferase, aspartate aminotransferase or HCV-RNA. In conclusion, the present study identified circulating miR-196a as a specific and noninvasive candidate biomarker for the diagnosis of CHC.

[Effect of the methylation enzyme inhibitors of 5-aza-2-deoxycytidine on the TGF-beta/smad signal transduction pathway in human keloid fibroblasts].

OBJECTIVE: To investigate the effect of 5-aza-2-deoxycytidine on the TGF-beta/smad signal transduction pathway in human keloid fibroblasts (KFSs). METHODS: Firstly, immunohistochemical method was used to detect the positive expression rate of phospho-smad2 and phospho-smad3 in the specimens of 15 cases of keloid and 15 cases of normal skin. The keloid fibroblasts were cultured in vitro with 5-aza-2-deoxycytidine(experimental group) or with DMEM (control group). The effect of 5-aza-2-deoxycytidine on the cell cycle and apoptosis of fibroblasts was analysed with flow cytometry ( FCM). Transforming growth factor (TGF)-beta1, Smad7, phospho-smad2 and phospho-smad3 were analyzed by Western Blot, and Immunofluorescence. RESULTS: It was found that the positive expression of phospho-smad2 and phospho-smad3 in keloid were higher than those in normal skin. The FCM showed that the proportion of cells in G0/G1 stage was increased, and so does the proportion of apoptosis cells in keloid fibroblasts intervened by 5-aza-2-deoxycytidine. The expression of TGF-beta1, phospho-smad2 and phospho-smad3 protein were significantly suppressed while the expression of smad7 protein increased in keloid fibroblasts with 5-aza-2-deoxycytidine. In addition, 5-aza-2-deoxycytidine reversed phosphorylation and nuclear translocation of smad2 and smad3. CONCLUSIONS: 5-aza-2-deoxycytidine, methylase inhibitors, inhibits cell proliferation and promotes apoptosis of KFSs, which may be associated with the suppression of TGF-beta/smad signal pathway.

[Expression and significance of DNMT1 in human keloid fibroblast].

OBJECTIVE: To investigate the expression and significance of DNA methyltransferase 1 (DNMT1) in different parts and formation time of keloid fibroblasts. METHODS: Specimen were collected from fifty-three patients with keloid. The keloids were grouped according to formation time. Immunohistochemistry was used to assay the expression of DNMT1 in normal skin, invasive part, proliferative part and aged part. RT-PCR was adopted to assay the expression of DNMT1 mRNA in each group. RESULTS: DNMT1 is mainly distributed in the nucleus of fibroblasts. The expression of DNMT1 protein and DNMTl mRNA in keloid (72%) is higher than that in normal skin fibroblasts (8%) (P = 0.001). The expression rate of DNMT1 protein and DNMT1 mRNA in shorter formation time group (100%, Score:10.47 +/- 1.85) is higher than a longer formation time group (45%,Score: 7.71 +/- 1.80) (P = 0.007). The expression rate of DNMT1 protein and DNMT1 mRNA in the invasive part of keloid (100%, Score: 10.47 +/- 1.85) is higher than the proliferative part (78%, Score: 6.63 +/- 1.75) and aged part (38%, Score: 5.53 +/- 1.55) (P = 0.001). CONCLUSIONS: DNMT1 plays an important role in keloid. The expression of DNMT1 tends to decrease along with the increase of the formation time in keloid. It is also related to the part of keloid. DNMT1 plays an important role in the process of keloid growth and is closely related to infiltrative growth in keloid.

[The relationship between scarless wound healing and the expression of CD68 and CD3 in the immunocytes in fetal skin].

OBJECTIVE: To investigate the difference in the distribution, amount and morphology of immunocytes in fetal skin, normal adult skin and hypertrophic scar, and to probe into their roles in fetal scarless wound healing from the dermatological and immunological point of view. METHODS: Skin specimens obtained from 10 fetuses of induced labor (16 to 33 weeks gestation) due to incipient abortion, 7 adults, and 18 hypertrophic scars in different stages were collected for the detection of the expression and distribution of CD68 (the surface marker of macrophages) and CD3 (the surface marker of T-lymphocytes) with immunohistochemical assay. RESULTS: The cells with positive expression of CD68 (CD68+ macrophages) in fetal skin [(5 +/- 6)/per 400 x visual field] were significantly lower than those in normal adult skin [(23 +/- 4) per/400 x visual field, P <0. 01], and they were obviously lower in normal skin than those in hypertrophic scar [(38 +/- 16)/per 400 x visual field, P < 0.01]. Along with their increase in gestational age, the CD68+ macrophages increased gradually. The cells increased in amount sharply during 24 - 28 gestational weeks, and then the increase slowed down after the 28th gestational week. The lymphocytes with CD3+ expression were not found in all the fetal stages, but were found in small amounts in adult skin [(24 +/- 8)/per 400 x visual field] which were mainly located in the epithelial basal lamina. But there were much more CD3+ lymphocytes [(69 +/- 25)/per 400 x visual field] in the HS, assembling usually in sheet form, and were chiefly distributed in dermal papillary layer around the small vessels in the shape of oversleeve. The cells were much more than those in normal adult skin (P <0.01) in terms of number and pigmentation intensity. CONCLUSION: The low content of CD68+ macrophages in fetal skin might be related to certain extent to the scarless skin wound healing. At the same time, the scarless skin wound healing in fetus could be related to the lack of CD3+ lymphocytes in fetal skin.