Circular RNA Implicated in MG, Study Suggests
Altered production of circular RNA impaired the process of muscle cell growth and regeneration in people with myasthenia gravis (MG), a study has suggested.
More studies investigating the role of circular RNA in MG may identify new molecular markers to monitor disease progression and reveal therapeutic targets, the researchers noted.
The study, “Upregulation of circ-FBL promotes myogenic proliferation in myasthenia gravis by regulation of miR-133/PAX7,” was published in the journal Cell Biology International.
In MG, a mistaken immune response targets the connection between nerve endings and muscle cells at the neuromuscular junction, leading to muscle weakness and fatigue.
This altered immune attack impairs the function of muscle stem cells, called satellite cells (SCs), which normally grow to replenish damaged muscle tissue, a process known as myogenesis. Studies suggest altered SC function delays muscle regeneration in MG patients.
Circular RNA (circRNA) are small segments of RNA that form closed loops and are thought to block microRNAs, which bind to messenger RNA (mRNA) — the molecule that carries the instructions from genes to make proteins. Consequently, circRNA and microRNA work together to regulate protein production.
The abnormal expression (production) of circRNA has been associated with various diseases, but their role in MG has not been investigated thoroughly.
Now, researchers in China analyzed 35 MG patients and 11 healthy volunteers to detail circRNA in MG. Blood, cells, and muscle tissue were isolated from participants to analyze RNA, along with various immune-related molecules.
The first set of experiments confirmed the altered immune response in MG patients by demonstrating increased levels of pro-inflammatory signaling proteins (cytokines) in the blood of patients compared to healthy controls. The numbers of regulatory T-cells also increased in MG patients; T-cells play a role in suppressing altered immune responses.
A protein marker for SC activation and growth, known as Pax7, was increased in MG patients, as well as the number of Pax7-positive SCs. The increase also was seen in an experimental autoimmune MG (EAMG) mouse model.
RNA analysis found that many circRNA molecules were expressed abnormally in people with MG. In particular, one circRNA called circ-FBL increased significantly.
Computer-based analysis conducted to find the microRNA target of circ-FBL identified multiple targets, including miR-133, miR-144, miR-146, miR-153, miR-217, miR-513a, miR-370, and miR-627. Exploratory experiments in cells offered evidence that miR-133 was a circ-FBL target.
This finding was confirmed by showing that a mutated version of circ-FBL was unable to interact with miR-133 in cells compared to normal circ-FBL. Moreover, miR-133 levels in the blood of MG patients increased significantly.
Additional computer-based analyses suggested that the SC marker protein Pax7 then became a downstream target of miR-133.
To understand the regulatory functions between circ-FBL, miR-133, and PAX7, myoblast cells were used, which are precursor cells that become muscle cells via differentiation — the process that transforms different cell types within the muscle.
The overexpression of miR-133 or PAX7 in myoblasts did not impact circ-FBL expression, whereas the overexpression of circ-FBL decreased miR-133 in cells. Adding a miR-133 mimic (identical to miR-133) promoted miR-133 expression, but blocking PAX7 did not.
Additionally, circ-FBL overexpression increased PAX7 production in myoblasts, while miR-133 mimic partially suppressed PAX7 production.
Altogether, these experiments showed that “miR-133 and PAX7 are circ-FBL downstream targets and PAX7 is a miR-133 downstream target,” the researchers wrote.
Finally, overexpression of circ-FBL led to the proliferation of myoblasts, whereas the overproduction of miR-133 and a PAX7 blocker suppressed this proliferation ability even after excess circ-FBL expression.
“Taken together, our study showed that upregulation of circ-FBL promoted myogenic proliferation in patients with MG by regulating miR-133/PAX7,” the researchers concluded.
“It is essential to combine basic experiments with clinical data to further investigate circRNA and identify new molecular markers related to the prognosis, prediction, and therapeutic targets for clinical patients,” they added.