Inhibition of mesangial cell proliferation by antisense oligodeoxynucleotide targeting preproendothelin-1 mRNA in
1Division of Nephrology, First Hospital, Beijing Medical University, Beijing 100034, China.
Objective:
To investigate the effects of endothelin-1 (ET-1) on mesangial cell proliferation in vitro.
Methods:
Antisense oligodeoxynucleotide (As-ODN) and its control sequences, sense (Se-ODN) and mismatch (Mis-ODN) oligodeoxynucleotides, targeting preproendothelin-1 (ppET-1) mRNA were delivered into cultured human mesangial cells (HMC) with lipofectin-mediated gene transfer method. The cytotoxicity of lipofectin was determined with lactate dehydrogenase (LDH) release assay. The efficiency of ODNs transfer into HMC was examined with biotinylated As-ODN staining. The effect of As-ODN on expression of ppET-1 mRNA was analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The action of As-ODN on HMC ET-1 secretion was tested by radioimmunoassay (RIA). The influence of As-ODN on HMC proliferation was evaluated with MTT method.
Results:
As-ODN was transferred into HMC with lipofectin without any impairment of cell viability. The ppET-1 mRNA expression, the ET-1 secretion and the cell proliferation were inhibited by As-ODN transferred into HMC, while Se-ODN and Mis-ODN transferred into HMC did not show any effects on all of these.
Conclusion:
Decrease of ET-1 secretion by HMC, caused by the down-regulation of ppET-1 mRNA expression after As-ODN transfer, led to inhibition of HMC proliferation. These results suggest ET-1 is one of the autocrine growth factors of HMC, and may play an important role in the pathogenesis of proliferative glomerulonephritis.
Insights
Endothelin-1 (ET-1) is an autocrine growth factor for human mesangial cells (HMC). Inhibiting ET-1 secretion via antisense oligodeoxynucleotides (As-ODN) reduces HMC proliferation, suggesting a role in glomerulonephritis.
Area of Science:
- Cell Biology
- Molecular Biology
- Nephrology
Background:
- Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogen.
- Its role in mesangial cell proliferation, a key process in glomerulonephritis, requires further elucidation.
Purpose of the Study:
- To investigate the effect of endothelin-1 (ET-1) on human mesangial cell (HMC) proliferation in vitro.
- To determine if ET-1 acts as an autocrine growth factor for HMC.
Main Methods:
- Lipofectin-mediated gene transfer of antisense oligodeoxynucleotides (As-ODN) targeting preproendothelin-1 (ppET-1) mRNA into cultured HMC.
- Assessed cytotoxicity using lactate dehydrogenase (LDH) release assay.
- Quantified ppET-1 mRNA expression via RT-PCR, ET-1 secretion by radioimmunoassay (RIA), and HMC proliferation using MTT assay.
Main Results:
- As-ODN effectively reduced ppET-1 mRNA expression and ET-1 secretion in HMC without affecting cell viability.
- As-ODN significantly inhibited HMC proliferation.
- Control sequences (Se-ODN and Mis-ODN) had no significant effects.
Conclusions:
- Down-regulation of ppET-1 mRNA expression and subsequent decrease in ET-1 secretion inhibit HMC proliferation.
- ET-1 functions as an autocrine growth factor for HMC.
- ET-1 may play a significant role in the pathogenesis of proliferative glomerulonephritis.
