Differential Regulation of miR206 and miR423-3p in Rapidly and Slowly Progressing Amyotrophic Lateral Sclerosis Patients

This article was published in the 2024 issue of “Neuromolecular Medicine,” titled “Differential Regulation of miR206 and 423-3p in Rapidly and Slowly Progressing Amyotrophic Lateral Sclerosis (ALS) Patients.” The authors include Antonio Musarò, Gabriella Dobrowolny, Chiara Cambieri, Laura Libonati, Federica Moret, Irene Casola, Gaia Laurenzi, Matteo Garibaldi, Maurizio Inghilleri, and Marco Ceccanti, primarily from Sapienza University of Rome and its affiliated institutions in Rome, Italy.

Research Background

Amyotrophic Lateral Sclerosis (ALS) is a rare neuromuscular disease characterized by neurodegenerative changes in upper and lower motor neurons, typically leading to death within 3 years of diagnosis, with a 5-year survival rate of only 20-25% and a 20-year survival rate of about 5%. Despite significant efforts in developing effective biomarkers, many aspects of ALS remain unclear. To date, there are no fully reliable and irreplaceable diagnostic and prognostic biomarkers in clinical practice. Unlike other neurodegenerative diseases, finding reliable biomarkers is more challenging due to the rapid progression of ALS and the lack of pre-symptomatic models (such as mild cognitive impairment in Alzheimer’s disease).

Explored Biomarkers

Compared to cerebrospinal fluid, although NFL levels in blood show good sensitivity, specificity issues still exist in many cases, especially in ALS-like conditions. Among other factors, research on microRNAs (miRNAs) has attracted widespread attention. miRNAs are conserved non-coding, single-stranded short RNA sequences that play important roles in post-transcriptional regulation of gene expression and are tissue-specific.

Research Objectives

The main objective of this study was to evaluate the relationship between specific miRNAs and clinical and neurophysiological scores, and to explore their role as independent or dependent predictors of ALS progression.

Research Methods

Sample Selection

The study selected 45 patients definitively diagnosed with ALS from the Rare Neuromuscular Diseases Center at Sapienza University of Rome, Italy. Patients were divided into 22 rapid progression and 23 slow progression groups based on their change rate in the ALS Functional Rating Scale-Revised (ALSFRS-R) within 6 months of admission (progression index), and were balanced in terms of baseline ALSFRS-R scores, age, and onset type (spinal or bulbar onset).

Clinical and Experimental Data Collection

At baseline and 6-month follow-up, data were recorded for each patient including time from disease onset to diagnosis, ALSFRS-R scores, Medical Research Council (MRC) scores for upper and lower limbs, Compound Muscle Action Potential (CMAP) of right plantar and left ulnar nerves, Forced Vital Capacity (FVC), and serum Creatine Kinase (CK) levels. Monthly changes in all these variables were assessed by calculating progression slopes.

miRNA Extraction and Real-time Analysis

Circulating miRNAs were extracted from 200μL of serum using Qiagen’s miRNeasy Serum/Plasma Kit, with 1.43μL of Spike-in Kit added during extraction for relative quantification. Subsequently, reverse transcription was performed using the Mircury® LNA® RT Kit, followed by quantitative PCR using the QuantStudio™ 7 Flex Real-Time PCR System. The relative levels of each miRNA were calculated and reported using the 2−ΔΔCT method of relative expression.

Data Analysis

Independent sample t-tests were used to assess differences in individual miRNA levels between rapid and slow progression groups. Pearson coefficients and curve estimation models were used for regression analysis, and ROC curves were used to calculate optimal miRNA cut-off values for distinguishing between rapid and slow progression groups. Data analysis was performed using IBM SPSS version 27.0, with statistical significance set at p < 0.05.

Research Results

The rapid and slow progression groups were balanced in terms of age, gender, baseline ALSFRS-R scores, etc. Significant differences were found between the two groups in monthly ALSFRS-R progression index, MRC score changes, FVC, etc. miR206 levels were significantly higher in the rapid progression group compared to the slow progression group, while miR423-3p showed a negative correlation with the MRC score progression index in the slow progression group.

miR206 Levels

In the rapid progression group, miR206 levels were significantly higher than in the slow progression group, showing certain sensitivity and specificity. After adjustment, it was found that high miR206 levels and early diagnosis were associated with rapid progression rates.

miR423-3p Levels

High miR423-3p levels were independently associated with slow MRC progression, indicating a better prognosis. miR423-3p was shown to downregulate BIM protein, an anti-apoptotic protein that was found to be downregulated in slow-progressing ALS mouse models, suggesting a potential protective role in inhibiting BIM-mediated motor neuron apoptosis.

Conclusion

These data suggest that specific miRNAs may serve as prognostic factors for ALS, providing some clues about pathogenic and adaptive mechanisms as well as potential molecular targets. Increased miR206 levels and decreased miR423-3p levels are associated with rapid progression in ALS patients. These miRNAs, as biomarkers for ALS, may provide important value for clinical practice.