[Construction and application of an artificial microRNA expression vector for inhibiting PAR4]
Na Han1, Li-sha Chu, Jiang Cao
1Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou 310016, China.
Aim:
To construct artificial microRNA expression vector targeting PAR4 and suppress the expression of PAR4 in human colorectal cancer SW620 cells with the artificial microRNA.
Methods:
Artificial microRNA was designed and amplified by two rounds of PCR and cloned into pMD-19T vector. The sequence of the cloned artificial microRNA was verified by DNA sequencing. Eight tandemly-repeated artificial microRNAs were subcloned into mammalian expression vector pcDNA3.1(+) to make the artificial microRNA- expressing vector pcDNA3.1(+)-8xPAR4-microRNA. The vector was transfected into human colorectal cancer SW620 cells, and stable transfectants were selected by G418. The expression of PAR4 was examined by Western blot.
Results:
DNA sequencing showed that the sequence of the cloned artificial microRNA targeting PAR4 was correct. Western blot result showed that the expression of PAR4 in SW620 cells stably transfected with pcDNA3.1(+)-8xPAR4-microRNA was markedly downregulated when compared to SW620 parental cells.
Conclusion:
Artificial microRNA expression vector targeting PAR4 is successfully constructed with significant suppression effect on PAR4 expression in SW620 cells. This provides the basis for future studies on the function of PAR4 and potential cancer gene therapy targeting PAR4.
Insights
An artificial microRNA expression vector targeting PAR4 was successfully constructed. This vector significantly suppressed PAR4 expression in human colorectal cancer SW620 cells, offering potential for gene therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Protease-activated receptor 4 (PAR4) plays a role in human colorectal cancer.
- Targeting specific genes like PAR4 is a strategy for cancer treatment.
Purpose of the Study:
- To construct an artificial microRNA (miRNA) expression vector targeting PAR4.
- To suppress PAR4 expression in human colorectal cancer SW620 cells using the artificial miRNA.
Main Methods:
- Artificial miRNA targeting PAR4 was designed, amplified, and cloned.
- Multiple copies of the artificial miRNA were inserted into a mammalian expression vector (pcDNA3.1(+)).
- The resulting vector was transfected into SW620 cells, and PAR4 expression was assessed via Western blot.
Main Results:
- DNA sequencing confirmed the correct sequence of the artificial miRNA targeting PAR4.
- Western blot analysis revealed a significant downregulation of PAR4 expression in SW620 cells transfected with the artificial miRNA vector compared to controls.
Conclusions:
- A functional artificial miRNA expression vector targeting PAR4 was successfully constructed.
- The vector demonstrated significant suppression of PAR4 expression in colorectal cancer cells.
- This study lays the groundwork for investigating PAR4's function and developing PAR4-targeted cancer gene therapy.
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