The role and mechanisms of action of microRNAs in cancer drug resistance.

MicroRNAs (miRNAs) are small non-coding RNAs with a length of about 19-25 nt, which can regulate various target genes and are thus involved in the regulation of a variety of biological and pathological processes, including the formation and development of cancer. Drug resistance in cancer chemotherapy is one of the main obstacles to curing this malignant disease. Statistical data indicate that over 90% of the mortality of patients with cancer is related to drug resistance. Drug resistance of cancer chemotherapy can be caused by many mechanisms, such as decreased antitumor drug uptake, modified drug targets, altered cell cycle checkpoints, or increased DNA damage repair, among others. In recent years, many studies have shown that miRNAs are involved in the drug resistance of tumor cells by targeting drug-resistance-related genes or influencing genes related to cell proliferation, cell cycle, and apoptosis. A single miRNA often targets a number of genes, and its regulatory effect is tissue-specific. In this review, we emphasize the miRNAs that are involved in the regulation of drug resistance among different cancers and probe the mechanisms of the deregulated expression of miRNAs. The molecular targets of miRNAs and their underlying signaling pathways are also explored comprehensively. A holistic understanding of the functions of miRNAs in drug resistance will help us develop better strategies to regulate them efficiently and will finally pave the way toward better translation of miRNAs into clinics, developing them into a promising approach in cancer therapy.

miR-27b-3p inhibits proliferation and potentially reverses multi-chemoresistance by targeting CBLB/GRB2 in breast cancer cells.

Drug resistance remains a major problem in the treatment of conventional chemotherapeutic agents in breast cancers. Owing to heterogeneity and complexity of chemoresistance mechanisms, most efforts that focus on a single pathway were unsuccessful, and exploring novel personalized therapeutics becomes urgent. By a system approach, we identified that microRNA-27b-3p (miR-27b), a miRNA deleted in breast cancer tissues and cell lines, has a master role in sensitizing breast cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo. Mechanistic analysis indicated that miR-27b enhanced responses to PTX by directly targeting CBLB and GRB2 to inactivate both PI3K/Akt and MAPK/Erk signaling pathways. Further, miR-27b was identified as a promising molecular biomarker in chemoresistance, clinicopathological features, and prognosis for breast cancer patients. In conclusion, we propose that combinational use of miR-27b and chemotherapeutic agents might be a promising therapeutic strategy to increase long-term drug responses in breast cancers.

A miR-20a/MAPK1/c-Myc regulatory feedback loop regulates breast carcinogenesis and chemoresistance.

Chemoresistance often leads to the failure of breast cancer treatment. MicroRNAs (miRNAs) play an important role in the progression and chemoresistance of cancer. However, because of the complexity of the mechanisms of chemoresistance and the specificity of miRNA regulation in different cell types, the function of miR-20a in breast cancer chemoresistance is still unclear. Here, by using miRNA microarray and high-content screening techniques, we found that miR-20a/b were significantly downregulated in breast cancer tissues compared with normal breast tissues, and low miR-20a/b expression was correlated with poor survival in breast cancer patients. Ectopic overexpression of miR-20a sensitized breast cancer cells to a broad spectrum of chemotherapy drugs and suppress their proliferation both in vitro and in vivo. Further study demonstrated that miR-20a directly targeted the 3'untranslated region of MAPK1, and thus downregulated the expression of P-gp and c-Myc by inhibiting the MAPK/ERK signaling pathway, whereas c-Myc can bind to the promoter region of the miR-20a gene to promote the expression of miR-20a. Together, our study identified a novel miR-20a/MAPK1/c-Myc feedback loop that regulates breast cancer growth and chemoresistance. These findings suggest that miR-20a synergizing with anticancer drugs will be a promising treatment strategy, especially for chemoresistant patients.

Resveratrol promotes MICA/B expression and natural killer cell lysis of breast cancer cells by suppressing c-Myc/miR-17 pathway.

Major histocompatibility complex class I chain-related proteins A and B (MICA and MICB) are important ligands for recognition of tumor cells by immune effector cells. Here, we report that resveratrol upregulated the protein and mRNA expression of MICA and MICB in breast cancer cells, which in turn promoted breast cancer cell lysis by natural killer (NK) cells in vitro and in vivo. Antibodies against NK group 2 member D blocked this effect. The 3'-untranslated regions of MICA and MICB were found to be direct binding targets of miR-17. MICA and MICB expression increased or decreased in breast cancer cells transfected with a miR-17 inhibitor or mimic, respectively. C-Myc overexpression/knockdown increased/decreased transcription of the miR-17-92 cluster host gene. Resveratrol suppressed c-Myc expression, which inhibited the transcription of miR-17-92 cluster, thereby downregulating miR-17. MiR-17 expression correlated inversely with MICA and MICB expression and overall survival in two sets of breast cancer specimens. Resveratrol thus upregulates MICA and MICB by suppressing the c-Myc/miR-17 pathway in breast cancer cells, and increases the cytolysis of breast cancer cells by NK cells. This suggests resveratrol has the potential to promote antitumor immune responses in breast cancer patients.

Silencing NKG2D ligand-targeting miRNAs enhances natural killer cell-mediated cytotoxicity in breast cancer.

NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DLs). NKG2DLs are expressed on malignant cells and sensitize them to early elimination by cytotoxic lymphocytes. We investigated the clinical importance of NKG2DLs and the mechanism of NKG2DL regulation in breast cancer (BC). Among the NKG2DLs MICA/B and ULBP1/2/3, the expression levels of MICA/B in BC tissues were inversely associated with the Tumor Node Metastasis stage. We first found that the high expression of MICB, but not MICA, was an independent prognostic factor for overall survival in patients with BC. Investigation into the mechanism revealed that a group of microRNAs (miRNAs) belonging to the miR-17-92 cluster, especially miR-20a, decreased the expression of ULBP2 and MICA/B. These miRNAs downregulated the expression of MICA/B by targeting the MICA/B 3'-untranslated region and downregulated ULBP2 by inhibiting the MAPK/ERK signaling pathway. Functional analysis showed that the silencing of NKG2DL-targeting miRNAs in BC cells increased NK cell-mediated cytotoxicity in vitro and inhibited immune escape in vivo. In addition, histone deacetylase inhibitors (HDACis) increased NKG2DL expression in BC cells by inhibiting members of the miR-17-92 cluster. Thus, targeting miRNAs with antisense inhibitors or HDACis may represent a novel approach for increasing the immunogenicity of BC.

[Isolation of heterotrophic nitrifiers which can tolerate high concentration of ammonia-nitrogen and the optimization of their nitrogen removal efficiency in wastewater].

The removal capabilities and tolerance of high concentration of ammonia-nitrogen of heterotrophic nitrifiers were studied. Methods included multi-point sampling, domestication, gradient dilution of domestication liquid, color indicator as rapid nitrification detection and isolation from streaking plate were conducted to screen heterotrophic nitrifiers. The strains were identified according to the sequence analysis of 16S rDNA. After inoculating the strains into ammonia-nitrogen wastewater, changes of nitrogen compounds were measured in order to understand their denitrification characteristics. The denitrification efficiency was optimized by improving the C/N ratio, changing the compatibility of the strains and mixing the compatible strains with the domesticated bacterial suspension. Finally 8 high-efficiency heterotrohic nitrifiers were obtained, and named as N1-N8 respectively. Phylogenetic analysis showed that 8 strains belonged to Comamonas genus, Rhodococcus genus, Pseudomonos genus, Arthrobacter genus and Paracoccus genus, respectively. When the initial concentration of ammonia nitrogen was 256.9 mg x L(-1) and the C/N was 5.5 of the artificial wastewater, the removal rates of ammonia nitrogen by the strains were about 65%-80%, and the stain N4 was the best. When the C/N ratio of the wastewater increased to 8.0, the ammonia nitrogen removal rates of the strains correspondingly increased to about 80% -90%. As the strains compatibility, the denitrification rate of N4 + N5 + N6 was 88.2% in the artificial wastewater with initial ammonia nitrogen concentration was 261.1 mg x L(-1) and initial C/N ratio was 5.5, which was higher than that of any single strain. The ammonia nitrogen removal rate could reach to 99.8% when N4 + N5 + N6 were combined with the domesticated bacterial suspension. In the artificial wastewater, when the initial ammonia nitrogen increased to 446.9 mg x L(-1) and the C/N ratio decreased to 3.2, the ammonia nitrogen removal rate of the mixed strains which composed of N4 + N5 + N6 and domesticated bacterial suspension was 99.9%. There was almost no nitrite and nitrate nitrogen accumulated in eventually, and the total nitrogen removal rate was 66.5%. The nitrogen which was assimilated by the strains was only 33% of the lost ammonia nitrogen. It shows that the strains which could not be isolated in the domesticated bacterial suspension had significant synergies effects on ammonia nitrogen removal of the isolating strains.

[Isolation, identification and characterization of a high-efficient heterotrophic nitrifier].

OBJECTIVE: In order to improve the rate of the heterotrophic nitrification, we screened and identified a high-efficient heterotrophic nitrifier, as well as studied its nitrification characteristics and nitrification conditions. METHODS: We obtained activated sludge samples from sewage and chemical fertilizer factories and farmland. We then utilized sodium citrate and ammonium chloride as carbon and nitrogen source. We used methods including domestication, gradient dilution of domestication liquid, isolation from streaking plate and color indicator as rapid nitrification detection. Finally a high-efficient heterotrophic nitrifier was obtained. We identified this strain according to its physiological, biochemical properties and the sequence analysis of 16S rDNA. After inoculating the strain into artificial ammonia-nitrogen wastewater, changes of nitrogen compounds were measured in order to understand the nitrification characteristics. Nitrification condition was also optimized by changing the carbon source, dissolved oxygen, C/N ratio, temperature and pH of the medium. RESULTS: The heterotrophic nitrifier was a gram-negative bacilli. It neither fermented glucose, nor produced indole. Oxidase and catalase tests were positive. It could produce alkali if organic salt was provided. The strain shared 99.7% sequence identity of its 16S rDNA with ES-SDK-3 of Alcaligenes sp. In the artificial wastewater with 182.30 mg/L ammonia nitrogen as initial concentration, the removal efficiency by the strain was 99. 8% after 30h cultivation. The average nitrogen removal rate was 9. 61 mg-N/L/h in its exponential phase. It produced almost no NO(2-)-N and NO(3-)-N in the entire nitrification process. The optimal carbon source is sodium citrate. Higher dissolved oxygen and C/N ratio favor its nitrification. When temperature is ranged from 30 degrees C to 35 degrees C and pH is ranged from 5.0 to 9.0, it can completely remove ammonia nitrogen. CONCLUSIONS: The strain was identified as Alcaligenes genus, and named as Alcaligenes sp. HN-S. Our research confirmed that the Alcaligenes sp. HN-S had significant advantages over heterotrophic nitrifiers that were screened previously with aspect of ammonia nitrogen removal rate. The research of its nitrification condition definitely provided necessary theory support for a new biology process to remove nitrogen with high efficiency.