<p>Circular RNA Hsa_circ_0004018 Inhibits Wnt/β-Catenin Signaling Pathway by Targeting microRNA-626/DKK3 in Hepatocellular Carcinoma</p>
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Dysexpression of circular RNAs has been identified in multiple types of cancer. Hsa_circ_0004018 was reported to be significantly downregulated in hepatocellular carcinoma (HCC) and to display HCC-stage-specific expression features. However, the role of hsa_circ_0004018 in HCC progression remains unclear.The expression of hsa_circ_0004018 or microRNA-626 (miR-626) was detected in tumor tissues and paired non-tumor tissues from HCC patients, as well as in one normal human liver cell line and 5 HCC cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, dye exclusion assay, clonogenic assay, scratch migration assay and transwell assay were used to measure cell proliferation and migration capacity, respectively. Luciferase report assay and RNA pull down assay were performed to explore the regulatory effect of certain molecules on the expression of target genes.We found that the expression of hsa_circ_0004018 was lower in tumor tissues than in their paired non-tumor tissues from 28 out of 41 HCC patients. The difference in the expression between tumor tissues and non-tumor tissues was statistically significant (p<0.001). Further analysis revealed that such lower expression in tumor tissues was much more common in bigger tumor size group (≥5cm) compared with the smaller tumor size group (<5cm) (85% vs 42%, p=0.0007). Similarly, hsa_circ_0004018 was downregulated in HCC cell lines. Additionally, a negative correlation between hsa_circ_0004018 and miR-626 expression was noticed in HCC tissues. Moreover, we observed that hsa_circ_0004018 interacted with miR-626/DKK3 and contributed to HCC cell proliferation and migration through inhibiting Wnt/β-catenin signaling pathway in vitro. Furthermore, hsa_circ_0004018 blocked xenograft tumor growth in vivo through inhibiting Wnt/β-catenin signaling pathway by targeting miR-626/DKK3.We revealed that hsa_circ_0004018/miR-626/DKK3 regulatory axis may be a possible novel therapeutic target for HCC.Keywords:
Clonogenic assay
Objectives To develop and evaluate a real-time fluorescent quantitative RT-PCR method for the detection of a novel CXC chemokine macrophage inflammatory protein-2γ(MIP-2γ)mRNA expression based on TaqMan technique.Method The expression of MIP-2γ mRNA was detected by TaqMan real time fluorscet quantitative RT-PCR. Results The dynamic range of the assay varied from 102 to 107 copies. The intra- and inter-assay coefficient variation (CV) were less than 122%. MIP-2γ mRNA is expressed in heart, liver and kidney of normal Balb/c mice, with the greatest expression in heart.Conclusion The detection of MIP-2γ mRNA expression by real time fluorescent quantitative RT-PCR based on TaqMan technique is more accurate,specific, sensitive and time-saved, which is suitable for use in clinical laboratory.
TaqMan
Coefficient of variation
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Quantitative Analysis
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Organoid
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Mononegavirales
Serial dilution
Standard curve
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Objective:To establish a real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR) for rubella virus(RV),a rubella virus specific PCR amplicon as external standard to analytically estimate this real time quantitative assay.Methods: Firstly,the primer and TaqMan probe concentrations,as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA.Next,and a RV specific PCR amplicon was made as external standard.Results: The RV specific PCR amplicon prepared for evaluation of the assay was 503bp,and its original concentration was 2.75×109 copies/μl.The real time quantitative assay was shown to have good linearity(r2=0.9986,P0.001).Conclusion: The established quantitative RT-PCR is a simple,rapid,quantitative and accurate assay for RV RNA.
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Rubella virus
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We recently established a method for quantitative determination of human catalytically active UDPglucuronosyltransferases (UGTs) other than UGT2A1 and UGT2B28 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and applied the method to an exhaustive analysis of localization in various human tissues. We report here an additional quantitative determination method targeting UGT2B28. To date, there have been no reports on the distribution of UGT2B28 mRNA expression in human tissues based on quantitative determination. Human UGT2B28 was clearly detected in the breast and adipose tissue. UGT2B28 expression in the breast was comparatively low, about 1.6% of GAPDH mRNA levels, and was less than 5% of normalized (against GAPDH) UGT2B7 and 2B10 mRNA expression levels in the liver. Although the UGT2B28 has 97% identity with UGT2B11 at the cDNA sequence level, the primers constructed for UGT2B28 did not detect UGT2B11. In addition, significant expression of UGT2B11 was detected in the liver, breast and kidney, and was clearly different from the distribution of UGT2B28. Therefore, we conclude that the real-time RT-PCR method established here is very specific for human UGT2B28 isozyme. Keywords: Human UGTs, mRNA expression, quantitative analysis, real-time RT-PCR, tissue distribution, UGT2B28, adrenal gland, skeletal muscle, primers, bilirubin
Glyceraldehyde 3-phosphate dehydrogenase
Quantitative Analysis
Tissue distribution
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Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion® MagMAX™, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5–3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.
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Application of Real Time Fluorescent quantitative reverse transcription polymerase chani reaction (rRT-PCR)to poliovirus identification in Chinese Poliomyelitis Laboratory Network and evaluation of the assay.According to Real-time RT-PCR recommended by WHO and developed by USA Centers for Disease Control and Prevention, 10 poliovirus isolates from laboratories of Chinese poliomyelitis network were tested for intratypic differentiation (ITD) and vaccine derived polioviruses (VDPVs) screening. The results of Real-time RT-PCR for 10 isolates were compared with those of VP, region sequencing.The Real-time RT-PCR results for 10 isolates did not completely consist with those of VP1 region sequencing. 5 Pre-VDPVs can not be identified by Real-time RT-PCR and type IVDPV from Shanxi province in 2009 was missed by the assay.The Real-time RT-PCR retrospective and prospective researches for large scale of polioviruses well be conducted to determine if the assay is applicable to Chinese Poliomyelitis Laboratory Network.
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