The Regulation Study Of Survivin On Related Gene Expression In The Formation Process Of Autophagy-lysosome In Tumor Cells

Objective:To study the regulation effects of Survivin on the expression of genes involved in the formation process of autophagic lysosome in tumor cells.Methods: The autophagy induction model was established in the laboratory,IKKβ inhibitor TPCA-1 and over expression Survivin-plasmid were used to intervene autophagy model.The morphology of autophagy and the status of autophagy were observed by transmission electron microscope(SEM)andconfocal laser scanning microscopy;qPCR and Western blot were used to detect the expression changes of genes involved in the formation of autophagy,cell apoptosis was detected by flow cytometry.Results: After rapamycin induction in ECA109 cells and KYSE450 cells,increased autophagy activities could be seen under transmission electron microscope;The increased levelof LC3 expression in cells was observed by confocal laser scanning microscopy;Western blot detection showed that the expression of LC3-I and LC3-II protein increased gradually after RAPA induction.The results of laser confocal microscopy showed thatover-expression of Survivin in KYSE450 cells showed adecreased number of red fluorescent cells(P<0.05),the number of cells containing red fluorescent particles decreased(P<0.05).After the TPCA–1intervention,the number of red and green fluorescent particles and the number of cells containing red fluorescent particles decreased in ECA109 cell autophagy model(P<0.05);KYSE450 cell autophagy model showed thatthe number of red fluorescent cells,the number of cells containing red fluorescent particles were reduced(P<0.05).qPCR was used to detect genes expression levels involved in the formation of autophagic-lysosome,the results showed that when Survivin was over expressed,theexpression of Survivin was increased in the gene transcription and protein expression levels(P<0.05);The mRNA transcription levels of Rab7,TAK1 and LAMP1 decreased significantly(P<0.05).After TPCA-1 intervention,the mRNA transcription level of Rab7 in the ECA109 cells was significantly increased(P<0.05)while the LAMP1 mRNA transcription level was significantly decreased(P<0.05).The mRNA transcription level of IKKβ,Rab7 and TAK1 in the KYSE450 cells was significantly dereased(P<0.05),but there was no significant change in protein level.Flow cytometry showed that the apoptosis of ECA109 cells increased after TPCA-1 intervention within 48 hours.Conclusion:Autophagy model can be successfully established by rapamycin stimulation.Survivin and TPCA-1 can inhibit autophagy.During the formation of autophagic lysosome,overexpression of Survivin may inhibit the transcription of Rab7,TAK1 and LAMP1 genes.TPCA-1 intervention within 48 hours can promote apoptosis in ECA109 cells.