| Stathmin 1 is a phosphoprotein that universally exists in cytoplasm.Stathmin 1 plays critically important roles in the regulation of the microtubule cytoskeleton by combining with α/β tubulin heterodimers to prevent polymerization.The other way is by combining with microtubule directly to promote depolymerization of microtubules.That’s why stathmin 1 is also called microtubule-destabilizing protein.Microtubule dynamics are essential for many cellular processes such as cell division,cell migration and cell differentiation.Stathmin 1 plays important roles in regulating cell proliferation,migration/invasion,differentiation as a microtubule regulation protein..This research explored Stathmin 1 expression during muscle differentiation,constructed Stathmin 1 shRNA interference vector,and detected cell migration after Stathmin 1 was downregulated by Stathmin 1 shRNA.Firstly,mouse muscle was separated from hind legs during mouse embryonic period at E6.5,E8.5,E12.5,E15.5,and after birth at P1,P7,W2,W6.Total protein and mRNA was extracted from the muscle to detect the expression of Stathmin 1.Then the mouse myoblast cell C2C12 was induced to differentiate for detecting the change of Stathmin 1 expression during differentiation.The result showed low expression in mouse muscle before E10 in embryonic period and high Stathmin 1 expression at E12.5 and E15.5.Then Stathmin 1 expression decreased after birth,and it was rarely detected in adult mouse muscle.During C2C12 differentiation,Stathmin 1 highly expressed in proliferated cells,and decrease as C2C12 was differentiated to myotubs.The mouse muscle development begins from embryo when myoblast cells forms to divide and differentiate.During this stage,Stathmin 1 expression began to increased.Then Stathmin 1 expression decreased when the myoblast cells stop dividing,which was consistent to the result showed during C2C12 differentiation.Next,Stathmin 1 shRNA interference vectors were constructed specific to mouse Stathmin 1 mRNA.Stathmin 1 shRNA was designed,synthesized and annealled to form double strands to insert into the pDC316-EGFP-U6.STMN1/shRNA-control,STMN1/shRNA 1,STMN1/shRNA2 were constructed by this way.Then these plasmids were transfected into mouse melanoma cell B16F10,Stathmin 1 expression was detected after 48h.In addition,migration of mouse melanoma cell B16F10 transfected by the interference plasmids was detected by wound healing assay.The result showed that cells transfected by STMN1/shRNA1,STMN1/shRNA2 expressed 81%and 70%lower Stathmin 1 in protein and 78%and 86%in mRNA compared to cells transfected by STMN1/shRNA-Control,which illustrated that Stathmin 1 shRNA interference vectors were contructed successfully.The wound healing assay result showed that the migration rate of cells transfected by STMN1/shRNA1 and STMNl/shRNA2 was slower than cells transfected by STMN1/shRNA-control,which indicated that decreased Stathmin 1 expression could suppress cell migration. |
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