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Published on: March 9, 2022
[Establishment of a cellular model with human NADH-cytochrome b5 reductase deficiency via RNA interference]
Yuepeng Zhuang1, Shuiliang Wang, Fenghua Lan
1Center for Laboratory Medicine, the Second Minitary Medical University, Fuzhou General Hospital, Fuzhou, Fujian, 350025 People's Republic of China.
Objective:
To establish a cell line with human NADH-cytochrome b5 reductase (b5R) deficiency via RNA interference (RNAi).
Methods:
Two siRNA expressing vectors targeting the b5R mRNA were designed and constructed. Hepatocellular carcinoma BEL-7402 cells were transiently transfected with the two recombinants by lipofectamine (TM) 2000, and semi-quantitative RT-PCR was carried out to analyze the suppression of b5R mRNA; BEL-7402 cells stably transfected with the two siRNA expressing vectors were selected in the media with G418. By analyses of the mRNA, enzymatic activity and protein level of b5R, several cell clones with deficiency of b5R were established. The cell growth curve of BEL-7402 cells with b5R deficiency was detected by MTT assay.
Results:
Two siRNA expressing vectors targeting b5R mRNA were obtained, namely pSib5R-1 and pSib5R-2. When BEL-7402 cells were transfected transiently with pSib5R-2, the expression of b5R mRNA was significantly suppressed with a suppression ratio of 68.3%, indicating that pSib5R-2 could trigger the degradation of b5R mRNA effectively. Eighteen clones stably integrated exogenous plasmids were obtained. In two clones from pSib5R-2 transfection, the expression of b5R mRNA was suppressed by up to 48.2% and 56.2%, and the enzymatic activity was inhibited by up to 54.6% and 63.5%, respectively. The protein levels also decreased significantly. The defect of b5R did not change the cell growth rate.
Conclusion:
The expression of b5R in BEL-7402 could be suppressed by vector-based RNA interference effectively. We established a cellular model with defect of b5R successfully, which can be used as a tool in investigation of the biological function of b5R and molecular mechanism of type II recessive congenital methemoglobinemia.
Insights
Researchers created a cell line deficient in human NADH-cytochrome b5 reductase (b5R) using RNA interference. This new cellular model effectively studies b5R function and congenital methemoglobinemia.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Context:
- NADH-cytochrome b5 reductase (b5R) is crucial for various cellular redox reactions.
- Deficiency in b5R is linked to type II recessive congenital methemoglobinemia.
- Establishing a cellular model is essential for studying b5R's biological functions and disease mechanisms.
Purpose:
- To establish a human cell line with deficient NADH-cytochrome b5 reductase (b5R) expression.
- To utilize RNA interference (RNAi) technology for targeted gene silencing of b5R.
- To create a valuable tool for investigating b5R's role in cellular processes and disease pathogenesis.
Summary:
- Two siRNA-expressing vectors targeting b5R mRNA were constructed and transfected into BEL-7402 cells.
- Stable cell clones with significantly suppressed b5R mRNA, protein levels, and enzymatic activity were successfully established.
- MTT assays indicated that b5R deficiency did not alter the cell growth rate in the established BEL-7402 cell line.
Impact:
- Successfully generated a b5R-deficient cellular model using vector-based RNA interference.
- The established cell line serves as a critical tool for exploring the biological functions of b5R.
- This model will aid in elucidating the molecular mechanisms underlying type II recessive congenital methemoglobinemia.
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