Investigators at the Wistar Institute and North Carolina State University said that they have discovered, apparently for the first time, a molecule that inhibits microRNA.
Alexander Deiters, an assistant professor of chemistry at North Carolina State University, said that to identify the molecule, collaborator Qihong Huang developed a luciferase reporter gene assay to identify small-molecule inhibitors of the miRNA miR-21.
The researchers chose miR-21 because it’s “a pretty prominent” miRNA and is “highly upregulated in a variety of cancers, such as breast and brain cancers,” Deiters explained.
Huang, an assistant professor in the molecular and cellular oncogenesis program at the Wistar Institute, “used a luciferase reporter gene, and downstream from that reporter gene he put an miR-21 binding sequence,” Dieters told CBA News this week.
As a result, if miR-21 is present it will be loaded into the RISC complex in a manner similar to RNA interference, and that complex will sequence-specifically recognize that miR-21 target sequence downstream of luciferase, and inhibit the translation of the luciferase signal, Deiters added.
If miR-21 is present at high levels, no luciferase signal will be seen because the luciferase reporter gene is silenced by miR-21. “What we then did, when we screened for small molecules, is we exposed those cells to small molecules and then looked for an increase in the luciferase signal,” said Deiters. “We wanted to inhibit the inhibition of luciferase by miR-21, and bring the luciferase signal back up to normal and knock out the function of miR-21.”
The research, which Huang said represents “the first screen to identify miRNA function inhibitors,” was published in the Sept. 15 issue of Angewandte Chemie.
According to Deiters, an advantage of the assay over a knock down is its ability to provide a positive signal. “We do not knock something out with a small molecule; we basically get the luciferase signal back, so we get a positive readout, which means much less background,” he said.
The investigators put together the lentiviral reporter constructs to measure miRNA activity by introducing complementary sequences to those of mature miR-21, the specificity control miR-30, or a negative control linker sequence downstream of a luciferase reporter gene.
“We do not knock something out with a small molecule, we basically get the luciferase signal back, so we get a positive readout, which means much less background.”
These constructs were stably introduced into HeLa cells because they express high levels of oncogenic miR-21 and relatively low levels of miR-30, Huang told CBA News this week.
Identifying an Inhibitor
The researchers conducted a subsequent primary screen of more than 1,000 compounds from Wistar’s compound library and from Sigma-Aldrich’s Library of Pharmacologically Active Compounds at a compound concentration of 10.0 µM. The team plated 2,500 HeLa cells containing the reporter construct into each well of a 384-well plate, then incubated the plates overnight and added the compounds at a final concentration of 10.0 µM.
Next, the researchers used Promega’s Bright-Glo assay kit to measure luciferase signals 48 hours after treatment. An initial hit, diobenzene 1, produced a 251-percent increase in luciferase signal intensity. The team developed a preliminary structure-activity relationship following several rounds of screening and structural modification.
“We sort of did a medicinal chemistry project around that core structure and found another molecule that was about twice as active as our original hit,” said Deiters. That molecule, dubbed diazobenzene 2, had an EC50 of 2.0 µM, and it knocked down the level of miR-21 expression from 100 percent to 20 percent.
Deiters said that because miR-21 is linked to several malignancies, the investigators theorized that if they could down-regulate it with small molecules and bring it back to a normal level, “we can turn those cancer cells back into normal cells, or induce apoptosis in those cells.”
It has previously been demonstrated that when researchers used an antisense agent to knock out miR-21 in glioblastoma, a cancer in which it is highly upregulated, they induced cell death in those glioblastomas.
“We thought that could do the same with small molecules,” Deiters said.
The next step will be to make better compounds, Deiters said. “As you see in this paper, we still have 20 percent of miRNA function. And the compound that is reported in the paper does not induce apoptosis in glioblastoma cells.” This assay can also be used to screen for inhibitors of other miRNAs, he added.