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Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations
Sara Missaglia1,2, Daniela Tavian1,2, Sandro Michelini3
1Laboratory of Cellular Biochemistry and Molecular Biology, CRIBENS, Università Cattolica del Sacro Cuore, 20145 Milan, Italy.
Abstract:
Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.
Insights
Forkhead-box C2 (FOXC2) and its lncRNA FOXC2-AS1 expression are similar in Lymphedema-distichiasis syndrome patients and controls. Frameshift mutations alter the FOXC2-AS1/FOXC2 ratio, potentially protecting cells from misfolded proteins.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Forkhead-box C2 (FOXC2) is crucial for lymphatic development, and its mutations cause Lymphedema-distichiasis syndrome (LD).
- A natural antisense long non-coding RNA, FOXC2-AS1, enhances FOXC2 mRNA stability.
- No prior studies have assessed FOXC2 and FOXC2-AS1 blood expression in LD patients.
Purpose of the Study:
- To investigate FOXC2 and FOXC2-AS1 expression levels in Lymphedema-distichiasis syndrome patients and healthy controls.
- To explore the correlation between FOXC2 and FOXC2-AS1 expression in different patient groups.
- To determine the functional impact of FOXC2-AS1 on FOXC2 protein levels and cellular behavior.
Main Methods:
- Quantitative real-time PCR to measure FOXC2 and FOXC2-AS1 expression.
- Correlation analysis to assess the relationship between RNA levels.
- Western blotting and confocal microscopy to evaluate protein levels and localization.
- Bioinformatic analysis to predict molecular interactions.
Main Results:
- FOXC2 and FOXC2-AS1 expression levels were comparable between LD patients and controls, with higher expression in females.
- A positive correlation between FOXC2 and FOXC2-AS1 was observed, except in patients with frameshift mutations.
- Patients with frameshift mutations exhibited a 1:1 FOXC2-AS1/FOXC2 ratio, unlike controls and other mutation types.
- Altering FOXC2-AS1 levels affected FOXC2 protein levels and induced nuclear protein aggregates, including DNA.
Conclusions:
- Patients with frameshift mutations show a reduced FOXC2-AS1/FOXC2 ratio due to decreased FOXC2-AS1 expression.
- The imbalance between FOXC2 mRNA and FOXC2-AS1 may serve as a protective mechanism against FOXC2 misfolded proteins.
- This study elucidates a novel molecular mechanism in Lymphedema-distichiasis syndrome pathogenesis.
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