EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05.

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and ß-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT-PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.

Circular RNA circCLK3 promotes the progression of tongue squamous cell carcinoma via miR-455-5p/PARVA axis.

A previous study has elucidated that circular RNA circCLK3 acts as an oncogenic gene in cervical cancer. However, the role and regulatory mechanism of circCLK3 in tongue squamous cell carcinoma (TSCC) remain unknown. Quantitative real-time PCR was used to examine targeted gene expression in different groups. Cell viability and proliferation were investigated by MTT and 5-ethynyl-2'-deoxyuridine assays. Cell migration and invasion were detected by Transwell assays, and cell apoptosis was measured by flow cytometry analysis. The interaction among genes was investigated using luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay. In the present study, our findings revealed the upregulated expression of circCLK3 in TSCC tissues and cell lines. CircCLK3 knockdown suppressed cell proliferation, migration invasion, and induced cell cycle arrest at G0/G1 phase in TSCC. Moreover, circCLK3 acted as a molecular sponge for miR-455-5p. PARVA was the target gene of miR-455-5p. Furthermore, the negative correlation between expression of miR-455-5p and circCLK3 or PARVA in TSCC tissues was discovered. Rescue assays indicated that PARVA overexpression reversed the circCLK3 knockdown-mediated inhibitory effects on the progression of TSCC. In summary, circCLK3 exerts its carcinogenic effects on TSCC progression via absorbing miR-455-5p to upregulate PARVA, which expands our knowledge on the underlying mechanism of TSCC.

LINC00460 facilitated tongue squamous cell carcinoma progression via the miR-320b/IGF2BP3 axis.

OBJECTIVE: We aimed to explore the role of long intergenic non-protein coding RNA 460 (LINC00460) in tongue squamous cell carcinoma (TSCC). METHODS: We enrolled 27 TSCC patients to explore LINC00460 expression in clinical TSCC samples. RT-qPCR measured expression of molecules in this research. Loss-of-function assays explored biological function of LINC00460 in TSCC cells. RNA pull-down assay, luciferase reporter assay, and RIP assay investigated mechanism of LINC00460 underlying TSCC cells. RESULTS: TSCC tissues and cell lines both showed high expression of LINC00460. Functionally, LINC00460 downregulation inhibited TSCC cell growth and promoted TSCC cell apoptosis. Additionally, LINC00460 silencing suppressed tumor growth in vivo. Mechanistically, LINC00460 bound with microRNA 320b (miR-320b) in TSCC cells. MiR-320b overexpression suppressed TSCC cell growth and promoted TSCC cell apoptosis. Moreover miR-320b targeted insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) 3'untranslated region in TSCC cells. Furthermore, IGF2BP3 silencing suppressed TSCC cell growth and promoted TSCC cell apoptosis. IGF2BP3 upregulation countervailed effects of silenced LINC00460 on TSCC cells. The LINC00460/miR-320b/IGF2BP3 axis was associated with lymph node metastasis of TSCC patients. CONCLUSION: Our research illustrated that LINC00460 facilitated TSCC progression via the miR-320b/IGF2BP3 axis, highlighting a potential insight for the treatment of TSCC.

Long noncoding RNA distal-less homeobox 2 antisense 1 restrains inflammatory response and apoptosis of periodontal ligament cells by binding with microRNA-330-3p to regulate Ro60, Y RNA binding protein.

OBJECTIVE: This study aims to investigate the role of long noncoding RNA distal-less homeobox 2 antisense 1 (DLX2-AS1) in lipopolysaccharide-induced inflammatory response and apoptosis of periodontal ligament cells (PDLCs). DESIGN: Lipopolysaccharide was used to induce inflammation response of PDLCs. The expression of DLX2-AS1, microRNA-330-3p and Ro60, Y RNA binding protein (RO60) in lipopolysaccharide-treated PDLCs was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Enzyme linked immunosorbent assay (ELISA) was performed to evaluate the concentration of inflammatory cytokines in PDLCs after DLX2-AS1 overexpression or RO60 downregulation. The apoptosis of PDLCs after lipopolysaccharide treatment or indicated transfection was analyzed by flow cytometry analysis. The level of apoptosis-related proteins, Bax and Bcl-2, were examined by western blotting. The binding capacity between microRNA-330-3p and DLX2-AS1 (or RO60) was verified by luciferase reporter assays. RESULTS: DLX2-AS1 was downregulated in PDLCs after lipopolysaccharide treatment. DLX2-AS1 overexpression decreased the production of inflammatory cytokines and inhibited cell apoptosis. microRNA-330-3p bound with DLX2-AS1 and displayed high expression in lipopolysaccharide-induced PDLCs. In addition, the downregulation of RO60, a target gene of microRNA-330-3p, reversed the suppressive influence of DLX2-AS1 overexpression on the inflammatory response and apoptosis of PDLCs. CONCLUSIONS: DLX2-AS1 restrains inflammatory response and apoptosis of PDLCs via the microRNA-330-3p/RO60 axis.

Exosomal lncRNA HEIH promotes cisplatin resistance in tongue squamous cell carcinoma via targeting miR-3619-5p/HDGF axis.

BACKGROUND: Accumulating evidence has suggested that long noncoding RNAs (lncRNAs) are involved in the progression of types of human cancers. It has been known that exosomes can mediate cell-cell crosstalk by transferring lncRNAs in tumor progression. This study aimed to investigate the role of exosomal lncRNA HEIH on cisplatin (DDP) resistance in tongue squamous cell carcinoma (TSCC). METHODS: The expression of HEIH in human oral keratinocytes cell line (HOK), DDP-sensitive TSCC cell line (SCC4/S) and DDP-resistant TSCC cell line (SCC4/DDP) was measured. SCC4/S and SCC4/DDP cells were transfected with sh-HEIH to examine TSCC cell proliferation and apoptosis. The DDP-resistant exosomes were extracted and identified. The expression of miR­3619-5p and TDGF in DDP-sensitive recipient cells was determined. The binding capacity between HEIH and miR­3619-5p, along with miR­3619-5p and TDGF was verified. RESULTS: HEIH expression was significantly upregulated in SCC4/DDP cells. Downregulation of HEIH inhibited DDP resistance and cell proliferation and promoted cell apoptosis. HEIH acted as a competing endogenous RNA (ceRNA) for miR­3619-5p to upregulate HDGF expression. Exosomal HEIH promoted cell proliferation and drug resistance and inhibited cell apoptosis by sponging miR­3169-5p and upregulating HDGF. CONCLUSION: Exosomal HEIH acted as a ceRNA for miR­3619-5p to upregulate HDGF, thereby promoting DDP resistance in TSCC cells.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa.

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa PAO1. In this work, when a TetR-family regulator gene, pip, was knocked out, we found that pyocyanin production was dramatically reduced, indicating that Pip positively regulates pyocyanin biosynthesis. With further phenazines quantification and ß-galactosidase assay, we confirmed that Pip positively regulates phz2 expression, but does not regulate phz1 expression. In addition, while the rpoS gene was deleted, expression of pip was down-regulated. Expression of rpoS in the wild-type PAO1 strain, however, was similar to that in the Pip-deficient mutant PAΔpip, suggesting that expression of pip could positively be regulated by RpoS, whereas rpoS could not be regulated by Pip. Taken together, we drew a conclusion that Pip might serve as an intermediate in RpoS-modulated expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa.

Overexpression of phzM contributes to much more production of pyocyanin converted from phenazine-1-carboxylic acid in the absence of RpoS in Pseudomonas aeruginosa.

Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT. To investigate this issue, we constructed phzM-lacZ fusion on a vector and on the chromosome. By quantifying ß-galactosidase activities, we confirmed that expression of the phzM was up-regulated when the rpoS gene was inactivated. However, no changes occurred in the expression of phzS and phzH when the rpoS was knocked out. Taken together, overproduction of the SAM-dependent methyltransferase (PhzM) might contribute to the increased pyocyanin in the absence of RpoS in P. aeruginosa.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

[Influence of Synonymous Codon Bias on the RNA Secondary Structure in Influenza-A Viruses].

Influenza-A viruses were selected as samples. Then, the relationship between synonymous codon bias and mRNA secondary structures was analyzed. Our data will provide a theoretical basis for further research on the synonymous codon and RNA properties of the influenza-A virus. Information on the nucleic-acid sequence of all influenza-A viruses in the National Center for Biotechnology Information was collected. The secondary structure of each nucleic-acid sequence was predicted. Then, the loop structure, stem structure, and free replicating energy of RNA were calculated. Based on this information, the specific flexibility of RNA was worked out. Simultaneously, the synonymous codon bias in each nucleic-acid sequence was counted. A database on the RNA secondary structure of the influenza-A virus was set up. Then, the relationship between the synonymous codon bias and the content of loop structure, stem structure, and flexibility were analyzed.Codon usage of 50% of amino acids was correlated significantly with the content of stem structure or the content of loop structure.Also,60% of amino acids were correlated significantly with average unit folding free energy. In addition, codon usage of 50% of amino acids was correlated significantly with the specific flexibility of RNA. For codons that were correlated significantly with stem structure and loop structure, the correlation between their usage with two types of structural content was completely contrary .Also, the correlation between specific flexibility with synonymous codon bias was better than that observed for other parameters. These results suggest that synonymous codon usage bias has important influences on RNA secondary structure.

Effect of citrus lemon oil on growth and adherence of Streptococcus mutans.

In order to exploit novel anticaries agents, we investigated the effects of citrus lemon oil (CLO), a type of natural product, on growth and adherence of the primary oral cariogenic bacteria Streptococcus mutans (S. mutans). The growth inhibitory effect was explored with a micro-dilution assay. Adherence was analyzed by colony counts on the respective surfaces and the adherence inhibition rate (AIR). Real time-PCR was used to investigate the effects of CLO on transcription of glucosyltransferase (Gtf) encoding genes, gtfB, C and D. Neson-Somogyi method was used to measure the effects of CLO on Gtf activity. The minimum inhibitory concentration of CLO against S. mutans was 4.5 mg/ml. The CLO effectively reduced the adherence of S. mutans on glass surface (the AIR were from 98.3 to 100 %, P > 0.05) and saliva-coated enamel surface (the AIR were from 54.8 to 79.2 %, P < 0.05). CLO effectively reduced the activity of Gtf and the transcription of gtfs in a dose dependent manner (P < 0.05). In conclusion, CLO can effectively inhibit the growth and the adherence to glass and saliva-coated enamel surfaces of S. mutans. It can also inhibit the transcription of gtfs, as well as the Gtf enzyme activity.

[Effects of lemon peel extracts on lactate dehydrogenase and sucrase activity of Streptococcus mutans].

OBJECTIVE: To investigate the effect of lemon peel extracts (LPE) on the activity of lactate dehydrogenase and sucrase of Streptococcus mutans (Sm). METHODS: After serial dilution with trypticase soy broth (TSB) medium containing 2% glucose, LPE was used as the experimental group, and TSB without LPE as the control group. Sm was added to each group, which was then cultured for 6, 18, 24 and 48 hours in the anaerobic tank. The activity of lactate dehydrogenase(LDH) was measured with the method of oxidation of reduction coenzymeIand the pH value of the culture solution was also detected. The activity of the sucrose was determined with the method of coloration of 3,5-dinitrosalicylic acid. RESULTS: The activity of LDH, sucrase and the changes of solution pH were decreased with the increase of the concentration of LPE (P < 0.01). The activity of LDH were declined from (0.8025 ± 0.0913) × 10(3) U/L to (0.2099 ± 0.0283) × 10(3) U/L; the activity of sucrase were declined from (-0.0107 ± 0.0003) × 10(3) U/L to (-0.0078 ± 0.0002) × 10(3) U/L; the ΔpH were declined from (2.8067 ± 0.0404) to (2.5033 ± 0.0416) (24 h results). The differences were significant between experimental groups and the control group (P < 0.01), and there were also significant differences among experimental groups with different LPE concentration (P < 0.01). The inhibitory effect of acid generation and lactate dehydrogenas' activity of Sm were positively correlated (P < 0.01). CONCLUSIONS: LPE can inhibit the activity of lactate dehydrogenase, sucrase and the acid production capacity of the Sm in a dose dependent manner. The inhibitory effects in logarithmic phase is stronger than that in other phases of growth cycle.