Effect Of RNA Interference-Mediated Silencing Of Maspin On Apoptosis Of Gastric Cancer Cell Line MKN-28

Objective: To construct and identify eukaryotic expression vector containing maspin shRNA(short hairpin RNA) and investigate the effect on apoptosis of gastric cancer cell line MKN-28 after transfected with recombinant plasmidMethods:1.ShRNA targeting maspin gene was conjugated into pGenesil-1.1 plasmid expressing green fluorescent protein. Recombinant plasmids were constructed including pGenesil-maspin1 and pGenesil-maspin2 positive plasmid and pGenesil-HK negative plasmid.2. RT-PCR and Western blot were used to detect maspin, Bcl-2 and Bax expression in transfected and control group, respectively.3.Flow cytometry was used to identify the cell cycle and cell apoptosis.Results:1. These plasmids were validated by gel electrophoresis and DNA sequence analysis.2. RT-PCR and Western blot analysis indicated that maspin expression was dramatically down-regulated in gastric cancer cell line MKN-28 after transfected with pGenesil-maspin positive plasmid.3.Bcl-2 expression identified by RT-PCR and Western blot was up-regulated (P<0.05) after positive transfection, with Bax expression down-regulated(P<0.05).4. Flow cytometric profiles indicated that positive plasmid transfection led to the decrease of the cell percentage of G0/G1phase (P<0.05) and the cell apoptosis percentage (P<0.05).Conclusions:1. The recombinant plasmid was constructed successfully.2. Transfection of maspin shRNA recombinant plasmid could inhibit expression of maspin effectively in human gastric cancer cell line MKN-28.3. Transfection of maspin shRNA could inhibit apoptosis of human gastric cancer cell lines. The molecular mechanism might be associated with altering cell cycle, Bcl-2 overexpression and Bax downexpression...