Anti-sense RNA inhibits the expression of synaptotagmin II in RBL-2H3 and enhances the exocytosis of lysosomes in RBL-2H3.
The expression of synaptotagmin II (Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8% and 10% of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.
Zhang
Jicheng
J
Department of Medical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Lu
Wenli
W
Li
Yirong
Y
Wu
Jianmin
J
Zhang
Chunguang
C
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D005089:Exocytosis; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism* / Q000473:pathology; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D050861:Synaptotagmin II / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D016372:RNA, Antisense; D012333:RNA, Messenger; D050861:Synaptotagmin II; D002402:Cathepsin D