Abstract
Objective
The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell.
Methods
Total RNA was extracted from SGC-7901 cells to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction (PCR). The PCR product was separated by electrophoresis on 1% agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMPmiRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5α strain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5α via transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cells with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection efficiency was observed under fluorescence microscopy and cell counting kit-8 assay was conduced to evaluate the effect of miRNA-451 on SGC-7901 cell proliferation.
Results
Our results showed that pLMP-miRNA-451 expression vector was not only constructed successfully and effectively infected SGC-7901 cells, but also could repress the SGC-7901 cell proliferation.
Conclusion
The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cell lines.
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Supported by a grant from the Natural Science Foundation of China (No. 8100098).
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Chen, B., Xu, X. Construction of the human miRNA-451 expression vector and its expression in gastric carcinoma cell line SGC-7901. Chin. -Ger. J. Clin. Oncol. 12, 379–384 (2013). https://doi.org/10.1007/s10330-013-1176-0
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DOI: https://doi.org/10.1007/s10330-013-1176-0