Abstract
BackgroundMicroRNAs (miRNAs) are reportedly involved in pancreatic ductal adenocarcinoma (PDAC) development. Current methods do not allow us to reliably monitor miRNA function. Asensors are adeno-associated virus (AAV) vector miRNA sensors for real-time consecutive functional monitoring of miRNA profiling in living cells.MethodsmiR-200a, -200b, -21, -96, -146a, -10a, -155, and -221 in three PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma cells (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were monitored by Asensors. Subsequently, the real-time consecutive functional profile of all miRNAs was evaluated.ResultsSelected miRNAs were detectable in all cell lines with high sensitivity and reproducibility. In the three PDAC cell lines, BxPC-3, CFPAC-1, and SW1990, the calibrated signal unit of the eight miRNAs Asensors was significantly lower than that of the Asensor control. However, in PANC-1 cells, miR-200a and -155 showed upregulation of target gene expression at 24 hours after infection with the sensors; at 48 hours, miR-200b and -155 displayed upregulation of reporter expression; and at 72 hours, reporter gene expression was upregulated by miR-200a and -200b. The result that miRNA could upregulate gene expression was further confirmed in miR-155 of hTERT-HPNE cells. Furthermore, miRNA activity varied among cell/tissue types and time.ConclusionIt is possible that miRNA participates in the pathophysiology of pancreatic cancer, but the current popular methods do not accurately reveal the real-time miRNA function. Thus, this report provided a convenient, accurate, and sensitive approach to miRNA research.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer with increasing incidence and mortality worldwide
The Asensors were quantified by qPCR, and when the amount matched that of the Asensor-infected target cells, the activity of miRNAs in cells paired with the target sequences of the Asensor was revealed by Gaussia luciferase (Gluc) expediently, accurately, and sensitively
To ensure that all recombinant adeno-associated virus (rAAV) could act as an miRNA sensor, we first evaluated the Asensor control, and Gluc secreted into the supernatant was assayed at 24, 48, 72 hours after infection
Summary
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer with increasing incidence and mortality worldwide. It is one of the major leading causes of cancer-related mortality with a five-year survival rate of 6–7% [1]. MicroRNAs (miRNAs) are noncoding RNAs that are 18–25 nucleotides long. They have emerged as a critical class of negative regulators of gene expression through the modulation of post-transcriptional activity of multiple target mRNAs. Recently, they have emerged as a critical class of negative regulators of gene expression through the modulation of post-transcriptional activity of multiple target mRNAs They regulate gene expression via complementarity with the 39untranslated region (39-UTR) of their target mRNAs. miRNAs regulate gene expression either by target mRNA degradation, repression of its translation, or sometimes by upregulation of the target gene. Asensors are adeno-associated virus (AAV) vector miRNA sensors for real-time consecutive functional monitoring of miRNA profiling in living cells
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