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miR-106a Is Downregulated in Peripheral Blood Mononuclear Cells of Chronic Hepatitis B and Associated with Enhanced Levels of Interleukin-8.

Hong Z, Hong H, Liu J, Zheng X, Huang M, Li C, Xia J - Mediators Inflamm. (2015)

Bottom Line: The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients.Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects.This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong 519000, China.

ABSTRACT

Aims: This study aimed to investigate miR-106a expression in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients and to analyze the function of miR-106a.

Materials and methods: miR-106a expression levels in PBMCs from 40 healthy controls and 56 CHB patients were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The luciferase activity assays were used to determine whether miR-106a binds to 3'UTR of IL-8. miR-106a mimics and inhibitors were transfected into healthy PBMCs. IL-8 mRNA and protein levels were detected and determined by qRT-PCR and ELISA, respectively.

Results: The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients. IL-8 was augmented in CHB patients and was inversely correlated with miR-106a levels. The luciferase activity assays indicated that IL-8 is a target of miR-106a. Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects.

Conclusions: This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8.

No MeSH data available.


Related in: MedlinePlus

IL-8 is a direct target of miR-106a. (a) Up: potential sites in IL-8 3′UTR targeted by miR-106a. 3′UTR of IL-8 was cloned into a luciferase reporter vector. Mutated sequences were generated in the seed regions to abolish binding of the corresponding miRNAs. Down: HEK293 cells were cotransfected with miR-106a mimics or inhibitor and the luciferase reporter constructs harboring IL-8 or mutant IL-8 3′UTR fragments. The luciferase reporter assays were performed 48 h after transfection. The luciferase activities were measured and normalized to a Renilla luciferase activity. (b) miR-106a expression level was determined after transfection with miR-106a mimics or inhibitor. (c) IL-8 mRNA and protein levels after transfection with miR-106a mimics were assessed by qRT-PCR and ELISA. (d) IL-8 mRNA and protein levels after transfection with miR-106a inhibitor were assessed by qRT-PCR and ELISA. ∗p < 0.05.
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fig3: IL-8 is a direct target of miR-106a. (a) Up: potential sites in IL-8 3′UTR targeted by miR-106a. 3′UTR of IL-8 was cloned into a luciferase reporter vector. Mutated sequences were generated in the seed regions to abolish binding of the corresponding miRNAs. Down: HEK293 cells were cotransfected with miR-106a mimics or inhibitor and the luciferase reporter constructs harboring IL-8 or mutant IL-8 3′UTR fragments. The luciferase reporter assays were performed 48 h after transfection. The luciferase activities were measured and normalized to a Renilla luciferase activity. (b) miR-106a expression level was determined after transfection with miR-106a mimics or inhibitor. (c) IL-8 mRNA and protein levels after transfection with miR-106a mimics were assessed by qRT-PCR and ELISA. (d) IL-8 mRNA and protein levels after transfection with miR-106a inhibitor were assessed by qRT-PCR and ELISA. ∗p < 0.05.

Mentions: Using the Targetscan 6.0, we found that IL-8 was one of the possible target genes for miR-106a. To demonstrate it, a luciferase assay was employed for detailed analysis. HEK293 cells were cotransfected with the IL-8 3′-UTR construct (WT) or its mutant (MUT) and miR-106a mimics or miR-106a inhibitor, followed by a measurement with the luciferase reporter assay (Figure 3(a)). As expected, miR-106a mimics decreased IL-8 translation, whereas miR-106a inhibitor increased the activity of IL-8 translation (Figure 3(a)). However, the luciferase activity of the IL-8 mutant was not influenced by miR-106a mimics or miR-106a inhibitor (Figure 3(a)). Next, we determined the effect of miR-106a on IL-8 mRNA and protein expression levels by qRT-PCR and ELISA. We firstly determined the efficiency of the transfection of miR-106a mimics and inhibitor. As is shown in Figure 2(b), transfection of miR-106a mimics into PBMCs can significantly increase its expression, while miR-106a inhibitor downregulated its expression level. We next assessed the mRNA and protein expression levels of IL-8 in the cells. IL-8 mRNA and protein levels were significantly downregulated in healthy PBMCs transfected with miR-106a mimics compared with the cells transfected with control mimics, whereas the expressions of IL-8 were significantly upregulated in cells transfected with miR-106a inhibitors compared with cells transfected with control inhibitors (Figures 3(c) and 3(d)).


miR-106a Is Downregulated in Peripheral Blood Mononuclear Cells of Chronic Hepatitis B and Associated with Enhanced Levels of Interleukin-8.

Hong Z, Hong H, Liu J, Zheng X, Huang M, Li C, Xia J - Mediators Inflamm. (2015)

IL-8 is a direct target of miR-106a. (a) Up: potential sites in IL-8 3′UTR targeted by miR-106a. 3′UTR of IL-8 was cloned into a luciferase reporter vector. Mutated sequences were generated in the seed regions to abolish binding of the corresponding miRNAs. Down: HEK293 cells were cotransfected with miR-106a mimics or inhibitor and the luciferase reporter constructs harboring IL-8 or mutant IL-8 3′UTR fragments. The luciferase reporter assays were performed 48 h after transfection. The luciferase activities were measured and normalized to a Renilla luciferase activity. (b) miR-106a expression level was determined after transfection with miR-106a mimics or inhibitor. (c) IL-8 mRNA and protein levels after transfection with miR-106a mimics were assessed by qRT-PCR and ELISA. (d) IL-8 mRNA and protein levels after transfection with miR-106a inhibitor were assessed by qRT-PCR and ELISA. ∗p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525765&req=5

fig3: IL-8 is a direct target of miR-106a. (a) Up: potential sites in IL-8 3′UTR targeted by miR-106a. 3′UTR of IL-8 was cloned into a luciferase reporter vector. Mutated sequences were generated in the seed regions to abolish binding of the corresponding miRNAs. Down: HEK293 cells were cotransfected with miR-106a mimics or inhibitor and the luciferase reporter constructs harboring IL-8 or mutant IL-8 3′UTR fragments. The luciferase reporter assays were performed 48 h after transfection. The luciferase activities were measured and normalized to a Renilla luciferase activity. (b) miR-106a expression level was determined after transfection with miR-106a mimics or inhibitor. (c) IL-8 mRNA and protein levels after transfection with miR-106a mimics were assessed by qRT-PCR and ELISA. (d) IL-8 mRNA and protein levels after transfection with miR-106a inhibitor were assessed by qRT-PCR and ELISA. ∗p < 0.05.
Mentions: Using the Targetscan 6.0, we found that IL-8 was one of the possible target genes for miR-106a. To demonstrate it, a luciferase assay was employed for detailed analysis. HEK293 cells were cotransfected with the IL-8 3′-UTR construct (WT) or its mutant (MUT) and miR-106a mimics or miR-106a inhibitor, followed by a measurement with the luciferase reporter assay (Figure 3(a)). As expected, miR-106a mimics decreased IL-8 translation, whereas miR-106a inhibitor increased the activity of IL-8 translation (Figure 3(a)). However, the luciferase activity of the IL-8 mutant was not influenced by miR-106a mimics or miR-106a inhibitor (Figure 3(a)). Next, we determined the effect of miR-106a on IL-8 mRNA and protein expression levels by qRT-PCR and ELISA. We firstly determined the efficiency of the transfection of miR-106a mimics and inhibitor. As is shown in Figure 2(b), transfection of miR-106a mimics into PBMCs can significantly increase its expression, while miR-106a inhibitor downregulated its expression level. We next assessed the mRNA and protein expression levels of IL-8 in the cells. IL-8 mRNA and protein levels were significantly downregulated in healthy PBMCs transfected with miR-106a mimics compared with the cells transfected with control mimics, whereas the expressions of IL-8 were significantly upregulated in cells transfected with miR-106a inhibitors compared with cells transfected with control inhibitors (Figures 3(c) and 3(d)).

Bottom Line: The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients.Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects.This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong 519000, China.

ABSTRACT

Aims: This study aimed to investigate miR-106a expression in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients and to analyze the function of miR-106a.

Materials and methods: miR-106a expression levels in PBMCs from 40 healthy controls and 56 CHB patients were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The luciferase activity assays were used to determine whether miR-106a binds to 3'UTR of IL-8. miR-106a mimics and inhibitors were transfected into healthy PBMCs. IL-8 mRNA and protein levels were detected and determined by qRT-PCR and ELISA, respectively.

Results: The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients. IL-8 was augmented in CHB patients and was inversely correlated with miR-106a levels. The luciferase activity assays indicated that IL-8 is a target of miR-106a. Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects.

Conclusions: This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8.

No MeSH data available.


Related in: MedlinePlus