Construction Of Twist Gene Plasmid Vector And Its Effect On Invasion Of Colon Carcinoma Cells

Objective:To construct steadily and highly expressing Twist gene eukaryotic expression plasmid vector, study the role of Twist gene on invasion of malignant cancer cell.Methods:①Twist gene was amplified by PCR from pCDNA4/myc-His A-Twist,and subcloned into pTracer-CMV/BSD through double enzyme digestion and oriented cloning, to construct pTracer-CMV/BSD-Twist.②The recombinant plasmid confirmed by DNA sequencing was transfected into SW480 cell line through Lipofectamine 2000,including pTracer-CMV/BSD-Twist, pTracer-CMV/BSD and negative control group.③The mRNA expression of Twist gene of three group cells were detected by fluorescence real-time quantitative PCR and the protein expressions were detected by Western bolt:④The ability of invasion of three group cells were examined by Transwell method.Results:①Double enzyme digestion and DNA sequence analysis identified that Twist gene eukaryotic expression vector pTracer-CMV/BSD-Twist was constructed successfully.②After transfected for 48hr,the brightness and concentrated gree fluorescence protein of pTracer-CMV/BSD-Twist and pTracer-CMV/BSD was shown by fluorescence microscope.③The expression of Twist gene at mRNA level in the pTracer-CMV/BSD-Twist was higher than that of the pTracer-CMV/BSD and the negative control group respectively,the difference had statistically significant (P<0.01). The difference between pTracer-CMV/BSD and negative control group had no statistically significant(P>0.05).④The anysis of Western bolt indicated that the pTracer-CMV/BSD-Twist showed higher expression of Twist protein than that of the pTracer-CMV/BSD and the negative control group respectively,the difference had statistically significant (P<0.01). There was no statistically significant between pTracer-CMV/BSD and negative control group (P>0.05).⑤The Transwell method showed that there was stronger invasive ability in the pTracer-CMV/BSD-Twist rather than that of the pTracer-CMV/BSD and the negative control group respectively,the difference had statistically significant (P<0.01). The difference between pTracer-CMV/BSD group and negative control group had no statistically significant(P>0.05).Conclusions:①The Steadily expressing high Twist gene eukaryotic expression plasmid vector pTracer-CMV/BSD-Twist is constructed successfully,and confirm that it can express Twist gene mRNA and protein after transfected into SW480 cell line.②Twist gene plays an important promoting role in invasion of malignant cancer cell.The high expressing of Twist gene can enhance the ability of invasion of malignant cancer cell significently.