Environmental arsenic pollution induced liver oxidative stress injury by regulating miR-155 through inhibition of AUF1.

Arsenic (As), a common environmental pollutant, has become a hot topic in recent years due to its potentially harmful effects. Liver damage being a central clinical feature of chronic arsenic poisoning. However, the underlying mechanisms remain unclear. We demonstrated that arsenic can lead to oxidative stress in the liver and result in structural and functional liver damage, significantly correlated with the expression of AUF1, Dicer1, and miR-155 in the liver. Interestingly, knockdown AUF1 promoted the up-regulatory effects of arsenic on Dicer1 and miR-155 and the inhibitory effects on SOD1, which exacerbated oxidative damage in rat liver. However, overexpression of AUF1 reversed the up-regulatory effects of arsenic on Dicer1 and miR-155, restored arsenic-induced SOD1 depletion, and attenuated liver oxidative stress injury. Further, we verified the mechanism and targets of miR-155 in regulating SOD1 by knockdown/overexpression of miR-155 and nonsense mutant SOD1 3'UTR experiments. In conclusion, these results powerfully demonstrate that arsenic inhibits AUF1 protein expression, which in turn reduces the inhibitory effect on Dicer1 expression, which promotes miR-155 to act on the SOD1 3'UTR region after high expression, thus inhibiting SOD1 protein expression and enzyme activity, and inducing liver injury. This finding provides a new perspective for the mechanism research and targeted prevention of arsenic poisoning, as well as scientific evidence for formulating strategies to prevent and control environmental arsenic pollution.

Enterococcus faecalis rnc gene modulates its susceptibility to disinfection agents: a novel approach against biofilm.

BACKGROUND: Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan. METHODS: E. faecalis rnc gene overexpression (rnc+) and low-expression (rnc-) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc- strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays. RESULTS: The results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc- biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents' groups. CONCLUSIONS: This study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm.

Dicer-mediated upregulation of BCRP confers tamoxifen resistance in human breast cancer cells.

PURPOSE: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. EXPERIMENTAL DESIGN: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. RESULTS: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. CONCLUSION: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity.

In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.

The combination of emergent RNA interference (RNAi) technology with in ovo electroporation in the chick embryo has the potential to provide a powerful and rapid means for functional analyses of novel genes in vivo. In this study, we show that electroporation of short 21-bp RNA duplexes (siRNAs) is a quick and simple method for silencing exogenous and endogenous gene expression in vivo. Quantitative comparisons with two other RNAi protocols that use long double-stranded RNA duplexes and endonuclease-digested duplexes (esiRNAs) demonstrate that siRNAs are significantly more effective at reducing gene expression. Furthermore, we also find that much higher amounts of siRNA are required for silencing of endogenous gene expression relative to plasmid-borne reporter constructs. In short, these results demonstrate that siRNAs are the most effective type of double-stranded RNA duplex for silencing gene expression and suggest that there might be important differences between silencing endogenous and exogenous genes. Finally, we review the parameters for each of these RNA-based methods of RNAi and the controls required to analyze RNAi data in the context of the developing vertebrate embryo.

Resistance of human hepatitis delta virus RNAs to dicer activity.

The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release approximately 21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no approximately 21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.