Potential biomarkers: The hypomethylation of cg18949415 and cg22193385 sites in colon adenocarcinoma.

Overall cancer hypomethylation had been identified in the past, but it is not clear exactly which hypomethylation site is the more important for the occurrence of cancer. To identify key hypomethylation sites, we studied the effect of hypomethylation in twelve regions on gene expression in colon adenocarcinoma (COAD). The key DNA methylation sites of cg18949415, cg22193385 and important genes of C6orf223, KRT7 were found by constructing a prognostic model, survival analysis and random combination prediction a series of in-depth systematic calculations and analyses, and the results were validated by GEO database, immune microenvironment, drug and functional enrichment analysis. Based on the expression values of C6orf223, KRT7 genes and the DNA methylation values of cg18949415, cg22193385 sites, the least diversity increment algorithm were used to predict COAD and normal sample. The 100 % reliability and 97.12 % correctness of predicting tumor samples were obtained in jackknife test. Moreover, we found that C6orf223 gene, cg18949415 site play a more important role than KRT7 gene, cg22193385 site in COAD. In addition, we investigate the impact of key methylation sites on three-dimensional chromatin structure. Our results will be help for experimental studies and may be an epigenetic biomarker for COAD.

[MicroRNA-144 over-expression induced myocytes apoptosis].

OBJECTIVE: To investigate the effects of microRNA-144 (miR-144) expression on H9C2 (2-1) myocytes. METHODS: MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with Lipofectamine(TM) 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. RESULTS: Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84 ± 838.52) compared with negative (2.06 ± 0.73) and blank (1.00 ± 0.00) control group (all P < 0.01). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group, while no significant difference was found between the latter 2 groups. CONCLUSION: MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.

Chromatin accessibility landscape and regulatory network of high-altitude hypoxia adaptation.

High-altitude adaptation of Tibetans represents a remarkable case of natural selection during recent human evolution. Previous genome-wide scans found many non-coding variants under selection, suggesting a pressing need to understand the functional role of non-coding regulatory elements (REs). Here, we generate time courses of paired ATAC-seq and RNA-seq data on cultured HUVECs under hypoxic and normoxic conditions. We further develop a variant interpretation methodology (vPECA) to identify active selected REs (ASREs) and associated regulatory network. We discover three causal SNPs of EPAS1, the key adaptive gene for Tibetans. These SNPs decrease the accessibility of ASREs with weakened binding strength of relevant TFs, and cooperatively down-regulate EPAS1 expression. We further construct the downstream network of EPAS1, elucidating its roles in hypoxic response and angiogenesis. Collectively, we provide a systematic approach to interpret phenotype-associated noncoding variants in proper cell types and relevant dynamic conditions, to model their impact on gene regulation.

Effects of six compounds with different chemical structures on melanogenesis.

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D3, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 µmol·L-1in vivo and 100 µmol·L-1in vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.

[Expression of retinoid acid receptors in human scleral fibroblasts and regulation of growth of fibroblasts by retinoic acid].

OBJECTIVE: The aim of this study was to identify the presence of retinoid acid receptors in cultured human scleral fibroblasts and the effect of retinoic acid (RA) on the regulation of cell growth. METHODS: Human scleral fibroblasts from three donors were isolated and cultured in Dulbecco Modified Ealge's Medium. Cells from the third or fifth generation were used in the present study. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect the expression of retinoid acid receptor subtypes in the fibroblasts. RA was added to the culture medium at concentrations of 0.01, 0.10, 1.00, 10.00 and 100.00 nmol/L, separately. Cells were counted and statistically analyzed six days after being treated with RA. Each strain of the cells was examined three times. RESULTS: Cultured fibroblasts mainly showed a bipolar or spindle-shaped morphology with an average time of 2 to 3 days for each division. RT-PCR showed mRNA expression of six subtypes of RA receptors (RAR alpha, RAR beta, RAR gamma, RXR alpha, RXR beta, and RXR gamma). The proliferation of scleral fibroblasts was inhibited by 2.2%, 17.6%, 37.6%, 43.8% and 63.2% following the treatment with 0.01, 0.10, 1.00, 10.00 and 100.00 nmol/L of RA, respectively. Cell proliferation in RA-treated samples reduced significantly as compared to the control samples (P < 0.05) when the RA concentration exceeded 0.10 nmol/L. CONCLUSIONS: Six retinoid acid receptor subtypes are present in cultured human scleral fibroblasts and the growth of scleral fibroblasts was inhibited significantly by retinoic acid. These findings indicate that the sclera is a potential site of action for the retinoid acid during progression of myopia.

[Expression of dopamine receptor D2 and adenosine receptor A2A in human retinal pigment epithelium].

OBJECTIVE: The aim of this study was to identify the presence of dopamine receptor D2 and adenosine receptor A2A in human retinal pigment epithelium (RPE) in order to determine whether the RPE is a potential site of action for these two receptors. METHODS: RPE Cells were isolated and cultured. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect the expression of D2 and A2A receptors in the RPE. RESULTS: RT-PCR analysis showed that mRNA of D2 and A2A receptors were expressed in the RPE. D2 and A2A receptors were distributed in the RPE by immunocytochemistry. CONCLUSIONS: This study demonstrated the presence of D2 and A2A receptors in human RPE at both mRNA and protein levels. D2 and A2A receptors may play an important role in the maintenance of the function of human retinal pigment epithelium. Furthermore, their cooperation appeared to be important in the development of axial myopia and retinal and choroidal neovascularization.

[Expression of muscarinic acetylcholine receptor-1 in human retinal pigment epithelium].

OBJECTIVE: The aim of this study was to identify the presence of muscarinic acetylcholine receptors-I (M1 receptor) in human retinal pigment epithelium (RPE) in order to determine the role of M1 receptor in the maintenance of function of RPE and its role in the occurrence and development of myopia. METHODS: The 3rd-5th passages of RPE cells established in our laboratory were used in the present study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression of M1 receptor in cultured RPE. Immunocytochemistry was used to detect M1 receptor protein in the RPE cells. RESULTS: Cultured RPE demonstrated mRNA expression of M1 receptor in RT-PCR. Protein of M1 receptor was presented in the RPE under immunocytochemistry. CONCLUSIONS: This study demonstrated the presence of M1 receptor in human RPE at both mRNA and protein levels. M1 receptor plays an important role in the maintenance of function of RPE. Injection of M1 receptor antagonist into the vitreous can delay the occurrence and inhibit the development of myopia, which is possibly related to the inhibition of RPE cells function.

[Placement of double cannula using trocar puncture for abdominal abscess drainage].

OBJECTIVE: To study the effects of placement of double cannula using trocar puncture for intra-abdominal abscess drainage. METHODS: A retrospective study was performed to investigate the clinical data of 32 patients undergoing intra-abdominal abscess drainage with double cannula placed using trocar puncture between June 2010 and December 2010. TECHNIQUES: the location and size of the abscess was evaluated by ultrasound and CT. Placement of double cannula using trocar puncture was performed under CT or ultrasound guidance. RESULTS: Trocar puncture was successful in all the patients. One patient died of liver metastasis and multiple organ failure after surgery for pancreatic cancer. One patient required laparotomy and drainage because non-localization of sepsis from intestinal fistula. The remaining 30 patients experienced alleviation of septic symptoms after drainage and eventually cured. The mean healing time was(7±3) days. Two patients developed subcutaneous bleeding and were management by local compression. CONCLUSIONS: Placement of double cannula using trocar puncture for intra- abdominal abscess drainage results in satisfactory outcomes. This technique is especially suitable for abscesses with viscous drainage, those with the presence of phlegmon or necrotic debris, and those with multiple large cavities.