E2Fs co-participate in cadmium stress response through activation of MSHs during the cell cycle.

Cadmium is one of the most common heavy metal contaminants found in agricultural fields. MutSα, MutSß, and MutSγ are three different MutS-associated protein heterodimer complexes consisting of MSH2/MSH6, MSH2/MSH3, and MSH2/MSH7, respectively. These complexes have different mismatch recognition properties and abilities to support MMR. However, changes in mismatch repair genes (OsMSH2, OsMSH3, OsMSH6, and OsMSH7) of the MutS system in rice, one of the most important food crops, under cadmium stress and their association with E2Fs, the key transcription factors affecting cell cycles, are poorly evaluated. In this study, we systematically categorized six rice E2Fs and confirmed that OsMSHs were the downstream target genes of E2F using dual-luciferase reporter assays. In addition, we constructed four msh mutant rice varieties (msh2, msh3, msh6, and msh7) using the CRISPR-Cas9 technology, exposed these mutant rice seedlings to different concentrations of cadmium (0, 2, and 4 mg/L) and observed changes in their phenotype and transcriptomic profiles using RNA-Seq and qRT-PCR. We found that the difference in plant height before and after cadmium stress was more significant in mutant rice seedlings than in wild-type rice seedlings. Transcriptomic profiling and qRT-PCR quantification showed that cadmium stress specifically mobilized cell cycle-related genes ATR, CDKB2;1, MAD2, CycD5;2, CDKA;1, and OsRBR1. Furthermore, we expressed OsE2Fs in yeasts and found that heterologous E2F expression in yeast strains regulated cadmium tolerance by regulating MSHs expression. Further exploration of the underlying mechanisms revealed that cadmium stress may activate the CDKA/CYCD complex, which phosphorylates RBR proteins to release E2F, to regulate downstream MSHs expression and subsequent DNA damage repairment, thereby enhancing the response to cadmium stress.

Upregulated expression of S100A6 in human gastric cancer.

OBJECTIVE: The expression of S100A6 (calcyclin), a member of the S100 calcium binding protein family, is elevated in a number of malignant tumors, but there have been few reports about its expression in gastric cancer. The aim of this study was to investigate its expression regulations in human gastric cancer and noncancerous mucosa, and the response to chemotherapeutic drugs in the gastric cancer cell line. MATERIALS AND METHODS: In one matched gastric cancer sample pair, the serial analysis of gene expression (SAGE) experiment was conducted to compare the gene expression profiles between cancerous and adjacent tissues. To detect the expression regulations among more cancerous tissues, microarrays were carried out and real-time RT-PCR was conducted to validate the results. At the protein level, Western blot and tissue microarray (TMA) examination were further used to verify S100A6 expression. The regulation detection of S100A6 with flurouracil and doxorubicin at the mRNA and protein level was performed in the SGC7901 cell line. RESULTS: With the SAGE strategy, five times more S100A6 tags were identified in cancer tissues than in normal tissues. With the cDNA microarray, S100A6 was found to be significantly upregulated in 21 of 42 (50%) nonselective gastric cancers. In 10 other paired samples, the upregulation of S100A6 was consolidated with RT-PCR and Western blot analysis as well. A total of 14 endoscopy-sectioned gastric noncancerous lesions and corresponding normal gastric mucosa were also applied to profile the gene expression; both cDNA microarray and RT-PCR demonstrated no significant alterations of S100A6 at the mRNA level. TMA examination showed that 34 of 52 (65.4%) cancer samples were positively stained, while only 17 of 80 (21.3%) noncancerous lesions were positively detected and all nine normal mucosae were detected to be negative. An in vitro experiment showed that in the gastric cell line SGC-7901, S100A6 mRNA was detected to be upregulated from 24 to 72 h after treatment with 5 mg/L 5-flurouracil or 0.3 mg/L doxorubicin, and there were two wave upregulations of the S100A6 protein. CONCLUSION: The observed regulated expression of S100A6 suggests that it is associated with gastric cancer tumorigenesis and quantitation of S100A6 is a promising tool for diagnosis of gastric cancer.