• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1508-1518
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• 2021

Hsa_circ_0001947 is associated with multiple cancers, but its function in non-small cell lung cancer (NSCLC) is ambiguous and needs further research. The targeting relationship among circ_0001947, miR-661, and downstream of tyrosine kinase 7 (DOK7) was predicted by database and further verified by dual-luciferase reporter assay, while their expressions in cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). After transfection, cell biological behaviors and expressions of miRNAs, miR-661 and DOK7 were determined by cell function experiments and qRT-PCR, respectively. Circ_0001947 was low-expressed in NSCLC tissues and cells. Circ_0001947 knockdown intensified cell viability and proliferation, induced cell cycle arrest at S phase, suppressed apoptosis and evidently enhanced miR-510, miR-587, miR-661 and miR-942 levels, while circ_0001947 overexpression did the opposite. MiR-661 was a target gene of circ_0001947 that participated in the regulation of circ_0001947 on cell biological behaviors. Furthermore, DOK7, the target gene of miR-661, partly participated in the regulation of miR-661 on cell viability. Hsa_circ_0001947 acts as a sponge of miR-661 to repress NSCLC development by elevating the expression of DOK7.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3767-3772
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• 2014

Introduction: MicroRNAs have emerged as post-transcriptional regulators that are critically involved in tumorigenesis. This study was designed to explore the effect of miRNA 133b on the proliferation and expression of TBPL1 in colon cancer cells. Methods: Human colon cancer SW-620 cells and human colon adenocarcinoma HT-29 cells were cultured. MiRNA 133b mimcs, miRNA 133b inhibitors, siRNA for TBPL1 and scrambled control were synthesized and transfected into cells. MiR-133b levels in cells and CRC tumor tissue was measured by real-time PCR. TBPL1 mRNA was detected by RT-PCR. Cell proliferation was studied with MTT assay. Western blotting was applied to detect TBPL1 protein levels. Luciferase assays were conducted using a pGL3-promoter vector cloned with full length of 3'UTR of human TBPL1 or 3'UTR with mutant sequence of miR-133b target site in order to confirm if the putative binding site is responsible for the negative regulation of TBPL1 by miR-133b. Results: Real time PCR results showed that miRNA 133b was lower in CRC tissue than that in adjacent tissue. After miR-133b transfection, its level was elevated till 48h, accompanied by lower proliferation in both SW-620 and HT-29 cells. According to that listed in http://www.targetscan.org, the 3'-UTR of TBPL1 mRNA (NM_004865) contains one putative binding site of miR-133b. This site was confirmed to be responsible for the negative regulation by miR-133b with luciferase assay. Further, Western blotting and immunohistochemistry both indicated a higher TBPL1 protein expression level in CRC tissue. Finally, a siRNA for TBPL1 transfection obviously slowed down the cell proliferation in both SW-620 and HT-29 cells. Conclusion: MiR-133b might act as a tumor suppressor and negatively regulate TBPL1 in CRC.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10181-10185
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• 2015

Background: MicroRNAs, small noncoding RNA molecules, can regulate mammalian cell growth, apoptosis and differentiation by controlling the expression of target genes. The aim of this study was to investigate the function of miR-323-5p in the glioma cell line, U251. Materials and Methods: After over-expression of miR-323-5p using miR-323-5p mimics, cell growth, apoptosis and migration were tested by MTT, flow cytometry and cell wound healing assay, respectively. We also assessed the influence of miR-323-5p on the mRNA expression of IGF-1R by quantitative real-time reverse transcriptase PCR (qRT-PCR), and on the protein levels by Western blot analysi. In addition, dual-luciferase reporter assays were performed to determine the target site of miR-323-5p to IGF-1R 3'UTR. Results: Our findings showed that over-expression of miR-323-5p could promote apoptosis of U251 and inhibit the proliferation and migration of the glioma cells. Conclusions: This study demonstrated that increased expression of miR-323-5p might be related to glioma progression, which indicates a potential role of miR-323-5p for clinical therapy.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.134-139
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• 2010

Mycobacterium tuberculosis (Mtb) is one of the most successful pathogens to infect one third of world population. Th1-mediated immunity against Mtb infection is known as critical to express mycobacteriostatic function but it is not sufficient to resolve the infection. In this study, to verify the possibility Mtb itself change the gene expression to survive against host immune response, expression pattern of selected H37Rv genes, 16S rRNA, acr, fbpA, aceA, and ahpC, during the course of infection was measured with absolute quantitation method using real-time RT-PCR. The total number of transcripts of 16S rRNA increased during the course of infection, which was coincide with the increasing CFU. The total number of fbpA transcripts per CFU, which encode typical secreted Mtb antigen, Ag85A, increased for 10 days of infection before decreasing. The number of transcripts of acr per CFU, which encode heat shock protein, ${\alpha}$-crystallin, increased during the infection, and ahpC and aceA, they both are enzymes produced in oxidative stressful condition, increased for 20 days and then slightly decreased on day 30. These findings are one of survival strategy of pathogen evading host immune response lead to persistent infection inside host cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.15
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• pp.6269-6273
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• 2014

Growing evidence suggests that miR-150-5p has an important role in regulating genesis of various types of cancer. However, the roles and the underlying mechanisms of miR-150-5p in development of colorectal cancer (CRC) remain largely unknown. Transwell chambers were used to analyze effects on cell migration and invasion by miR-150-5p. Quantitative real-time PCR (qRT-PCR), Western blotting and dual-luciferase 3' UTR reporter assay were carried out to identify the target genes of miR-150-5p. In our research, miR-150-5p suppressed CRC cell migration and invasion, and MUC4 was identified as a direct target gene. Its effects were partly blocked by re-expression of MUC4. In conclusiomn, miR-150-5p may suppress CRC metastasis through directly targeting MUC4, highlighting its potential as a novel agent for the treatment of CRC metastasis.

• Nutrition Research and Practice
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• v.13 no.1
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• pp.58-63
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• 2019

BACKGROUND/OBJECTIVES: Telomeres are located at the chromosomal ends and progressively shortened during each cell cycle. Telomerase, which is regulated by hTERT and c-MYC, maintains telomeric DNA sequences. Especially, telomerase is active in cancer and stem cells to maintain telomere length for replicative immortality. Recently we reported that walnut phenolic extract (WPE) can reduce cell viability in a colon cancer stem cell (CSC) model. We, therefore, investigated the effect of WPE on telomere maintenance in the same model. MATERIALS AND METHODS: $CD133^+CD44^+$ cells from HCT116, a human colon cancer cell line, were sorted by Fluorescence-activated cell sorting (FACS) and treated with WPE at the concentrations of 0, 10, 20, and $40{\mu}g/mL$ for 6 days. Telomere lengths were assessed by quantitative real-time PCR (qRT-PCR) using telomere specific primers and DNA extracted from the cells, which was further adjusted with single-copy gene and reference DNA ($ddC_t$). Telomerase activity was also measured by qRT-PCR after incubating the PCR mixture with cell protein extracts, which was adjusted with reference DNA ($dC_t$). Transcriptions of hTERT and c-MYC were determined using conventional RT-PCR. RESULTS: Telomere length of WPE-treated cells was significantly decreased in a dose-dependent manner ($5.16{\pm}0.13$ at $0{\mu}g/mL$, $4.79{\pm}0.12$ at $10{\mu}g/mL$, $3.24{\pm}0.08$ at $20{\mu}g/mL$ and $3.99{\pm}0.09$ at $40{\mu}g/mL$; P = 0.0276). Telomerase activities concurrently decreased with telomere length ($1.47{\pm}0.04$, $1.09{\pm}0.01$, $0.76{\pm}0.08$, and $0.88{\pm}0.06$; P = 0.0067). There was a positive correlation between telomere length and telomerase activity (r = 0.9090; P < 0.0001). Transcriptions of both hTERT and c-MYC were also significantly decreased in the same manner. CONCLUSION: In the present cell culture model, WPE reduced telomere maintenance, which may provide a mechanistic link to the effect of walnuts on the viability of colon CSCs.

• The Korean Journal of Food And Nutrition
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• v.27 no.2
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• pp.267-273
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• 2014

This study found antibacterial activity of $DMfree^{(R)}$ [green tea extract] on facultative bacteria by direct petri dish method and gene array of obligatory M. leprae infected mesenchymal stem cells (MSC). While DMfree showed DPPH radical scavenging effect and high contents of polyphenol, it did not inhibit growth of facultative bacteria such as E. coli and S. aureus on the petri dish. The result does not exclude a possible antibacterial effect of organic solvent extract of green tea rather than DMfree which comes from the water extract of green tea. Pre-treatment of DMfree appeared to have no effect on copy number of 14 genes compared with control MSC by real-time RT-PCR. However pre-treatment of DMfree on M. leprae infected MSC revealed a significant decrease of anti-inflammatory cytokine (IL-6), (P<0.038) and sharp down-regulation of pro-inflammatory cytokine (IL-1). Enhanced expression of VEGFR-1 mRNA was noted in DMfree pretreated M. leprae infected MSC group (P<0.003). These results show that DMfree would stabilize M. leprae infected MSC from further inflammation by down-regulating anti-inflammatory cytokine (IL-6) and pro-inflammatory cytokine (IL-$1{\beta}$). This is the first report on DMfree inhibition of IL-6 and IL-$1{\beta}$ expression in M. leprae infected MSC. Further experiments that detect protein levels of IL-$1{\beta}$ and IL-6 may support the result of this gene array.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.7
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• pp.954-960
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• 2008

MiRNAs (microRNAs) are a class of small non-coding RNA molecules of ~21 nucleotides that down- regulate the expression of target genes at post-transcriptional level. In this study, we first accomplished a preliminary scan of miRNA expression using 65 and 90 day fetal pig skeletal muscle samples by microarray hybridization, and 34 miRNAs showed strong positive signals. Five of these miRNAs were selected for further investigation by real-time RT-PCR. The statistical analyses indicated that three miRNAs exhibited significant differential expression (p<0.05) during porcine muscle development from 65 to 90 days of gestation, e.g., miR-24 and miR-424 were down-regulated while miR-133a was up-regulated. Multi-tissue RT-PCR was performed to detect the expression patterns of the five miRNA precursors. The results showed that most of these precursor miRNAs were ubiquitously expressed in different porcine tissues.

• Journal of Life Science
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• v.30 no.9
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• pp.772-782
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• 2020

Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.993-1003
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• 2013

Methanotrophs are the most important sink of $CH_4$, which is a more highly potent greenhouse gas than $CO_2$. Methanotrophic abundance and community diversity in cover soils from two typical semi-aerobic landfills (SALs) in China were detected using real-time polymerase chain reaction (real-time-PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA genes, respectively. Real time-PCR showed that Type I methanotrophs ranged from $1.07{\times}10^6$ to $2.34{\times}10^7$ copies/g soil and that of Type II methanotrophs from $1.51{\times}10^7$ to $1.83{\times}10^8$ copies/g soil. The ratio of Type II to Type I methanotrophic copy numbers ranged from 5.61 to 21.89, indicating that Type II methanotrophs dominated in SAL. DGGE revealed that Type I methanotrophs responded more sensitively to the environment, changing as the community structure varied with different soil types and locations. Methylobacter, Methylosarcina, and Methylomicrobium for Type I, and Methylocystis for Type II were most prevalent in the SAL cover layer. Abundant interflow $O_2$ with high $CH_4$ concentration in SALs is the reason for the higher population density of methanotrophs and the higher enrichment of Type II methanotrophs compared with anaerobic landfills and other ecosystems, which proved a conclusion that increasing the oxygen supply in a landfill cover layer would greatly improve $CH_4$ mitigation.