As researchers begin to appreciate the broad role of microRNAs (miRNAs) in diverse cellular processes, miRNA detection and profiling have become increasingly active areas of research. Applied Biosystems offers a complete portfolio of products and protocols for the discovery and profiling of miRNAs. Here, miRNA profiling tools are used to investigate miRNA differences in primary human hepatocytes compared to liver cancer cell lines.

Powerful Tools for miRNA Profiling

The resources you need for miRNA studies, from isolation through discovery, profiling, quantitation, and validation, are now available from a single source. Applied Biosystems offers innovative solutions to streamline miRNA research, enabling scientists to address the fundamental questions about miRNA function. The workflow described in this study demonstrates a highly streamlined and sensitive solution for miRNA profiling in cultured cells.

The TaqMan® MicroRNA Cells-to-CT™ Kit features a breakthrough method for lysing cultured cells while removing genomic DNA and preserving RNA integrity, making it possible to perform miRNA expression profiling directly in cultured cell lysates without RNA purification. The Megaplex™ PreAmp Primer preamplification step leads to increased sensitivity such that even miRNAs with low expression levels can be detected by real-time PCR. Megaplex Primer Pools, in conjunction with TaqMan MicroRNA Arrays, enable the rapid generation of an expression profile for up to 667 human miRNAs from as little as 1 ng of total RNA in a single working day.

When used together in a single workflow, these tools enable faster, easier, and higher throughput miRNA expression profiling. To demonstrate the utility of this workflow, miRNA profiles from primary human hepatocytes were examined and compared to those from two cell lines derived from hepatocarcinomas, HepG2 and Huh-7.

Experimental Approach

Figure 1 shows an overview of the miRNA workflow used in this study which focused on 377 human miRNAs represented in the Megaplex Primer Pool A. Primary human hepatocytes were obtained from a male donor with normal liver tissue; liver cancer cell lines were also derived from males. Aliquots of 5,000 HepG2 cells, Huh-7 cells, and primary human hepatocytes were processed using the TaqMan MicroRNA Cells-to-CT Kit. The resulting cell lysates were then subjected to reverse transcription using the TaqMan MicroRNA Reverse Transcription Kit in combination with the Megaplex RT Primers for Human Pool A. The cDNA product was then preamplified using the corresponding Megaplex PreAmp Primers for Human Pool A and TaqMan PreAmp Master Mix. Following preamplification, the products were diluted and mixed with TaqMan Universal PCR Master Mix and added into the TaqMan Human MicroRNA A Arrays for quantitation of miRNA expression. Since the completion of this study, additional Megaplex Primers have been released, enabling the rapid profiling of 667 human miRNAs using the workflow described here.

Figure 1. miRNA Profiling Workflow. Triplicate 5,000 cell aliquots of each cell type were processed for 8 minutes in TaqMan® Cells-to-CT™ Lysis Solution, Stop Solution was added, and the samples were incubated for 2 minutes. Lysates were then reverse transcribed using the TaqMan® MicroRNA Reverse Transcription Kit and Megaplex™ RT Primers. They were then preamplified for 12 cycles using the corresponding Megaplex PreAmp Primers and TaqMan PreAmp Master Mix. Finally, each sample was diluted and transferred into a TaqMan Array with Universal PCR Master Mix, No AmpErase® UNG and the real-time PCR was carried out on an Applied Biosystems 7900HT Fast Real-Time PCR System.

Sensitive miRNA Profiling from Liver Cancer Cell Lines

In this study, several miRNAs were found to vary in abundance in the HepG2 and Huh-7 liver cancer cell lines compared to primary human hepatocytes (Figures 2 and 3). There was greater miRNA variation between primary and cancer cell lines than between the two cancer cell lines, as shown by the R2 values (Figure 2). In addition, more miRNAs were differentially expressed (|ΔCT| >3.5) between primary and cancer cells than between the two cancer cell lines (Figure 3A). The miRNAs with CT values of <32 and |ΔCT| >3.5 for both cell lines are shown in Figure 3B, and are similarly expressed in both cancer cell lines. Eight miRNAs were expressed at higher levels in the cancer cell lines compared to primary hepatocytes, and one miRNA was expressed at lower levels.

Figure 2. HepG2, Huh-7, and Primary Hepatocyte miRNA Comparison. CT values obtained from biological triplicate TaqMan® Arrays for primary human hepatocytes, and HepG2 and Huh-7 cell lines are plotted. Samples that were undetermined were set at 40. miRNAs that had CT values of >32 for both cell types were omitted. R2 values were calculated without the non-detected samples.

Figure 3. miRNA Comparison Between Primary Hepatocytes and Cancer Cell Lines. Panel A. For the comparison between primary hepatocytes and cancer cell lines, ΔCT values for each miRNA target were calculated by subtracting the CT of the cancer cell line from the CT of the primary human hepatocytes. For the comparison between the two cancer cell lines, the HepG2 CT was subtracted from the Huh-7 CT. The second column indicates the number of miRNAs in each comparison that had CT values less than 32 in both cell lines and a ΔCT greater than 3.5. Panel B. The miRNAs with a ΔCT value greater than 3.5 and a CT value less than 32 in both cell lines are shown. Eight miRNAs were shown to have greater abundance in both cancer cell lines compared to primary hepatocytes, one had lower abundance. Let-7e had no significant change compared to primary human hepatocytes.

These results are consistent with published studies on the role of miRNAs in cancer. miR-106a and the miR-17-92 complex, which includes miR-20a [1], are known to be oncogenic [2], and miR-20a has been demonstrated to be anti-apoptotic [1]. The miR-17-92 complex regulates the E2F family of transcription factors for cell cycle and apotosis [1]. miR-222 has been shown to promote cancer cell proliferation by suppressing p27 (Kip1), a cell cycle regulator and tumor suppressor. Certain cancer cell lines require high amounts of miR-222 and miR-221 to suppress p27 (Kip1) activity [3]. miR-19a may also be involved in cell growth; when cells were transfected with an antisense oligonucleotide of the miRNA, cell growth was greatly reduced [4]. Downregulation of miR-126 has been demonstrated in colon tumors compared to normal colon tissue [5]. Also, miR-126 reconstitution into a colon cancer cell line resulted in growth inhibition. All of these observations correlate well with the data presented here (Figure 3B).

No previous studies were found on miR-125a-5p, miR-19b, miR-454, or miR-483-5p, all of which were found to be more highly expressed in the oncogenic cells. These miRNAs were not identified as potentially relevant in cancer using less sensitive platforms. Their future study may provide additional insight into the role of miRNAs in cancer and gene regulation.

A Complete and Streamlined Solution for miRNA Profiling

The streamlined miRNA profiling approach used in this study provides a simple and efficient means to detect and analyze the miRNA profiles from cancer cell lines. The TaqMan MicroRNA Cells-to-CT Kit, Megaplex Primer Pools, and TaqMan MicroRNA Arrays offer the benefits of simple, rapid sample processing, and the sensitivity, specificity, and dynamic range of TaqMan real-time PCR.

Scientific Contributors

Laura M Chapman, Richard Fekete, and Alexis Lennart, Yulei Wang, David Keys • Applied Biosystems