ABSTRACT
Objective To explore the effect and mechanism of IncRNA EVADR on the proliferation and migration in human colorectal cancer cell lines HCT116 and LOVO. Methods HCT116 and LOVO cell lines were transfected with IncRNA EVADR by overexpressing lentivirus system. CCK8 assay was performed to measure the growth of HCT116 and LOVO cells after overexpression of EVADR. Transwell migration was performed to determine if EVADR promote HCT116 and LOVO cells migration. Finally, the expression of Ecadherin and transcription factor Snail, Slug, ZEB1 and ZEB2 were detected by Western blot and realtime quantitative PCR respectively. Results We successfully established colorectal cancer cells strains HCT116, LOVO which can stably overexpress IncRNA EVADR and the capacity of proliferation and migration in overexpression group was significantly improved (P＜0.05). The expression of Ecadherin was decreased while mesenchymal markers Snail, Slug, ZEB1 and ZEB2 were increased in EVADR overexpression HCT116 and LOVO cells. Conclusions Overexpression of IncRNA EVADR in HCT116 and LOVO cells can significantly promote the proliferation and migration of HCT116 and LOVO cells which may play an important role in regulating epithelial-mesenchymal transition in colorectal cancer cells.
ABSTRACT
<p><b>OBJECTIVE</b>To clone and express Streptococcus suis serotype 2 (S. suis 2) sly gene for constructing an foundation on identification of S. suis 2 protective antigen.</p><p><b>METHODS</b>The sly gene was amplified from S. suis 2 clinical isolate strain 05ZYH33 genome DNA by PCR. The gene fragment was inserted into the expression vector pET-30b(+) to build pET30b-sly. When recombinant vector pET30b-sly was identified by restriction enzyme cutting and DNA sequencing as a correct one, subsequently it was transformed to E. coli Rosetta for expression under IPTG induction. The obtained fusion protein was purified by Ni-NTA affinity chromatography. The immunologic and hemolysis activity of the purified protein was proved through Western blot and hemolysis assay respectively.</p><p><b>RESULTS</b>The PCR product was around 1500 bp. The gene segment inserted into the recombinant vector was proven to be completely identical with the sly gene sequence in the total genome sequence of S. suis 2. The target protein expressed was up to 30% of the total somatic protein under IPTG induction. The protein purity reached above 80% after purification. The protein could be recognized by human serum infected with S. suis 2 and could dissolve swine erythrocytes with the Hemolytic titer as 256.</p><p><b>CONCLUSION</b>The expression vector pET30b-sly was successfully constructed. The target protein could be over-expressed in E. coli and possessed its biological activity after purification.</p>