Until now it was held that miRNAs—noncoding snippets of RNA, only a few nucleotides long—work exclusively by suppressing protein expression in dividing cells, including cancer cells. A study published in ACS Central Science, overhauls this notion, and shows some miRNAs can boost protein expression as well.

Lara Mahal
Lara Mahal, PhD, Canada Excellence Research Chair in Glycomics and professor of chemistry at the University of Alberta, is the senior author of the study.

“Our study upends common assumptions on how miRNAs work. Right now, we think of microRNA as down-regulators of protein expression, stemming from their discovery as natural analogs of silencing RNA (siRNA)…There is evidence that upregulation has been seen in other common cancer genes, but was ignored by scientists because it did not fit their expectations,” said senior author of the study, Lara Mahal, PhD, Canada Excellence Research Chair in Glycomics and professor of chemistry at the University of Alberta, Canada. “We clearly show miRNAs can directly enhance and repress the expression of proteins in cancer cells.”

A few scattered reports, including a 2007 study led by Joan Steitz, PhD, professor of biophysics and biochemistry at the Yale School of Medicine, had reported the upregulation of protein synthesis by miRNAs in nondividing cells and mitochondria. However, such occurrences were deemed outliers.

Contrary to expectations, Mahal and her team observed that the majority of miRNAs upregulated an enzyme (ST6GAL1, β-Galactoside α2,6-sialyltransferase) that catalyzes the conjugation of a sugar (sialic acid) through a glycosylation mechanism called α-2,6-sialylation in a variety of actively dividing cancer cells. Micro RNAs are known to play a role in the development of cancer.

One of the established functions of miRNAs is to act as regulators of glycosylation, one of the most diverse and abundant post-translational modifications that attaches a wide range of carbohydrates (sugars, like sialic acid) to proteins. These sugars act as lamps on a lighthouse, signaling a cell’s physiological or pathological status. Abnormal sugar signals on cell surfaces throw off detection of cells by the immune system and may lead to cancer.

In earlier work, Mahal and her team developed a fluorescence assay (miRFluR) that measures the interaction and activity of miRNAs, which can be used to detect the expression level of proteins regulated by miRNAs.

“We used our high-throughput miRNA validation system to examine the comprehensive regulation of α-2,6-sialylation by miRNA by looking at the regulation via the 3′-untranslated region (3’-UTR) of the genes ST6GAL1 and ST6GAL2 (a minor enzyme we still don’t understand),” said Mahal.

The researchers found that most miRNAs directly attached to the 3′-UTR of the gene and upregulated ST6GAL1 catalyzed α-2,6-sialylation in noncancerous human cells and several types of cancer cells.

“We found a surprising result. The majority of miRNA targeting ST6GAL1 upregulated protein expression in our assay,” said Mahal. “The assay was run in HEK-293-T cells, a common, actively dividing cell line. We then validated the results on regulation of ST6GAL1 and associated glycosylation in four different cancer cell lines and found that upregulation was real.”

miRNA
Direct miRNA−mRNA interactions upregulate protein expression and α-2,6-sialylation in a variety of actively dividing normal and cancer cells. [Adapted from Jame-Chenarboo F, ACS Central Science, 2022]
Mahal’s team also found that the direct binding of miRNA to the 3’UTR of ST6GAL1 used the same proteins that Steitz’s team proposed in their 2007 paper.

This finding contradicts the current understanding that miRNAs only downregulate protein synthesis. By expanding and correcting our understanding of how miRNAs work, the current study opens doors for the development of novel miRNA-based therapeutics.

Earlier work by Mahal’s team and others showed that ST6GAL1 drives the development of cancer and metastasis in the pancreas and other organs. In the current study, Mahal’s team validated two miRNAs (miR-221 and miR-212) as enhancers of ST6GAL1 protein expression. Both these miRNAs are upregulated in pancreatic cancer.

“We believe that these miRNAs help maintain high levels of ST6GAL1 and 2,6-sialylation in pancreatic cancer. We show that using anti-miRs to inhibit endogenous miRNA in pancreatic cancer cells lowers both ST6GAL1 levels and concomitant sialylation,” said Mahal.

Mahal added, “It makes sense that the tuning of glycosylation enzymes would require direct upregulation by miRNA as well as downregulation, as, in general, these are low-abundance transcripts. Many of the most important drug targets such as GPCRs, receptor tyrosine kinases, and other membrane proteins are also low-abundance.”

At present, there are several clinical trials underway that exploit miRNA-based approaches for cancer treatment. It is therefore crucial that the mechanism of action of miRNAs is clearly understood. Mahal believes the current findings could lead to identification of new miRNA-based drug targets that focus on the upregulation of protein synthesis.