Phylogenetic and ion-response analyses reveal a relationship between gene expansion and functional divergence in the Ca2+/cation antiporter family in Angiosperms.

KEY MESSAGE: Plant CaCA superfamily genes with higher tendency to retain after WGD are more gene expression and function differentiated in ion-response. Plants and animals face different environmental stresses but share conserved Ca2+ signaling pathways, such as Ca2+/Cation transport. The Ca2+/cation antiporters superfamily (CaCAs) is an ancient and widespread family of ion-coupled cation transporters found in all kingdoms of life. We analyzed the molecular evolution progress of the family through comparative genomics and phylogenetics of CaCAs genes from plants and animals, grouping these genes into several families and clades, and identified multiple gene duplication retention events, particularly in the CAX (H+/cation exchanger), CCX (cation/Ca2+ exchanger), and NCL (Na+/Ca2+ exchanger-like) families. The tendency of duplication retention differs between families and gene clades. The gene duplication events were probably the result of whole-genome duplication (WGD) in plants and might have led to functional divergence. Tissue and ion-response expression analyses revealed that CaCAs genes with more highly differentiated expression patterns are more likely to be retained as duplicates than those with more conserved expression profiles. Phenotype of Arabidopsis thaliana mutants showed that loss of genes with a greater tendency to be retained after duplication resulted in more severe growth deficiency. CaCAs genes in salt-tolerant species tended to inherit the expression characteristics of their most recent common ancestral genes, with conservative ion-response expression. This study indicates a possible evolutionary scheme for cation transport and illustrates distinct fates and a mechanism for the evolution of gene duplicates. The increased copy numbers of genes and divergences in expression might have contributed to the divergent functions of CaCAs protein, allowing plants to cope with environmental stresses and adapt to a larger number of ecological niches.

[Study on the Effect of Overexpression of miR-18a on Cellular Proliferation and Migration by Targeting ATM in Human Colorectal Cancer Cells].

OBJECTIVES: To study the regulation to colon cancer cellular biological properties through miR-18a targeting ataxia-telangiectasia mutated gene (ATM). METHODS: A target of miR-18a was predicted by using bioinformatics tools. The miR-18a mimics and inhibitors were designed and synthesized. The expression of endogenous miR-18a in colon cancer cell line HCT116 was up-regulated or down-regulated by transfection. The effect of overexpression of miR-18a on cellular proliferation, invasion and migration via regulation of ATM gene expression was confirmed in vitro by using qRT-PCR, Western blot, MTT assay, clone forming assay and Transwell method, respectively. RESULTS: ATM was identified as a potential target gene of miR-18a in the bioinformatics analysis. In addition, through transient transfection leading to the overexpression of miR-18a in HCT116 cell, the expression level of ATM was decreased. Down-regulation of HCT116 cell proliferation activity while significantly reducing HCT116 cell clone forming ability, lateral migration ability and longitudinal invasion ability were observed after transfected with miR-18a mimics. All of the changes were related to the overexpression of miR-18a. CONCLUSIONS: miR-18a inhibited the proliferation and migration of colon cance cell HCT116 through negative regulation of ATM expression.

[MiRNA224 Regulation Role on RKIP Gene Expression and Methylation of miRNA224 Gene Promoter Region in Esophageal Squamous Cell Carcinoma].

OBJECTIVES: To study the expression levels of tumor suppressor gene RIKP and miRNA224 in esophageal squamous cell carcinoma (ESCC) tissues. To determine whether miRNA224 targets RKIP and the methylation status of miRNA224 gene promoter region in esophageal carcinoma. METHODS: The expression levels of RKIP and miRNA224 in ESCC and normal tissue were detected by using immunohistochemistry and real-time qPCR, respectively. Luciferase assay was used to determine the targeting of miRNA224 to RKIP. The methylation status of miRNA224 promoter region was studied by bisulfite sequencing PCR (BSP). RESULTS: In 40 cases of ESCC, RKIP expression was significantly lower than that of normal tissue; miRNA224 expression was higher in ESCC than in paracancerous tissue. Luciferase assay showed that miRNA224 targets RKIP 3'UTR thus inhibit its expression. The miRNA224 gene promoter region was hypomethylated in ESCC. CONCLUSIONS: Compared with normal tissue, in ESCC, RKIP was downregulated, while miRNA224 was upregulated, and the promoter region of miRNA224 gene was hypomethylated. RKIP is the target of miRNA224, which may be closely related to esophageal squamous cell carcinoma.

[SIRT1 promote GTM cell DSBs repair and resist cellular senescence].

OBJECTIVE: To study the relationship between SIRT1 and glaucoma trabecular meshwork cell (GTM) cell on DNA double-strand breaks (DSBs) repair capability and resist cellular senescence. METHODS: The expressions of SIRT1 in GTM and normal trabecular meshwork (HTM) cell detected by RT-RCR and Western blot; HTM and GTM cells divided into four groups separately: Res group (treat cells with 0.5 micromol/L Resveratrol for 24 h), SIRT1-ShRNA group (cells infected with recombinant SIRT1-ShRNA), microRNA34a group (cells infected with recombinant microRNA34a) and control group. The expression level of SIRT1 in groups was detected by Western blot. SA-beta-Gal staining was applied to each group of cells at 10 h, 32 h, 3 d and 6 d to evaluate the senescence of the cells. DSBs and the expression of gamma-H2AX after treated with 1.33 mol/L H2O2 at 0 h, 1 h, 2 h were detected by comet electrophoresis and Western blot. RESULTS: The expression of SIRT1 were observed in both HTM and GTM cells, but the expression level in HTM was higher than that of GTM cells have the ability to express SIRT1, however the expression of SIRT1 was lower than HTM. Expression levels of SIRT1 presented following treads: Res > Control > microRNA34a > SIRT1-ShRNA. The dgree of senescence in GTM was higher than that in HTM cells when detected at the same time point with SA-beta-Gal staining. In the same cell line, the signs of senescence were appeared firstly and seriously in the cells treated with SIRT1-ShRNA in a time-dependent manner. Differently, after 24 h treatment with Res, the degree of senescence was decreased. The DSBs in GTM group was more than that of HTM group after treatment with oxidant when detected with Comet Electrophoresis and the the trends of the change was SIRT1-ShRNA > microRNA34a > Control > Res. The similar results also observed in the expression of gamma-H2AX. CONCLUSION: SIRT1 may be useful in predicting the development and prognosis of glaucoma; Res promotes the expression of SIRT1 significantly, and the SIRT1 may protects GTM from oxidative stress-induced DSBs, aging even apoptosis, and promotes cell cycle arrest, which may provide a new target to treat glaucoma.

[Determination of six metal elements in animal samples by flame atomic absorption spectrometry with wet digestion].

The contents of 6 metal elements (Ca, Mg, K, Na, Cu and Fe ) were determined in serum, urine, feces and different tissue and organs (heart, kidney, stomach, liver, spleen, intestine and lung) of Wistar rats and mice by flame atomic absorption spectrometry (FAAS) with wet digestion. The samples were digested by the mixture of HNO3 and HClO4 (4 : 1, V/V) at 120 degrees C, the correlation coefficient for the standard curves was 0.999 4-0.999 8, the relative standard deviation (RSD) was from 0.33% to 4.52%, and the recovery for the samples was from 97.7% to 104.2%, meeting the demand of elements content determination in biological samples. The assay method for the determination of the 6 metal elements in animal samples established in this study has the advantages of easy operation, high sensitivity, less sample requiring and accurate results, and the method can be widely used in the determination of various metal elements in biological samples.