RESUMO
Objective: To investigate the effect of silencing rapamycin insensitive companion of mammalian target of rapamycin (RICTOR) gene expression on the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and to explore its possible molecular mechanism. Methods: The expression level of RICTOR protein in esophageal squamous cell carcinoma TE1, ECa109, EC9706, KYSE450 and KYSE790 cells were detected by Western blotting. RICTOR-shRNA or the Control-shRNA was transfected into ECa109 cells by LipofectAMINE, and the ECa109 cells stably expressing RICTOR-shRNA or the Control-shRNA were screened and named as ECa 109-RICTOR-shRNA or ECa109-control-shRNA cells. The effect of everolimus on the proliferation of ECa109-RICTOR-shRNA cells was detected by CCK-8 assay. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt)/mammalian target of rapamycin (mTOR) signal pathwayrelated RICTOR, Akt, phospho-Akt (p-Akt) (Ser473), ribosome protein subunit 6 kinase of 70 kDa (p70S6K), phospho-p70S6K (p-p70S6K), proline-rich Akt substrate of 40 kDa (PRAS40) and phospho-PRAS40 (p-PRAS40) (Thr246) proteins in everolimus-treated ECa109-control-shRNA and ECa109-RICTOR-shRNA cells were detected by Western blotting. The nude mouse xenograft tumor models of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells were established and treated with everolimus, then the effect of everolimus on tumor growth in nude mice was evaluated. Results: RICTOR protein was expressed in five esophageal squamous cell carcinoma cell lines, especially in ECa1 09 cells. Compared with the Control-shRNA, RICTOR-shRNA inhibited the proliferation of ECa1 09 cells. Everolimus inhibited the proliferation of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells, especially to the former; the values of half maximal inhibitory concentration (IC50) were (17.68± 1.25) μmol/L and (36.84±1.57) μmol/L, respectively. The RICTOR-shRNA decreased the expression levels of p-Akt (Ser473) and p-PRAS40 (Thr246) (both P 0.05), which indicated that RICTOR-shRNA inhibited the phosphorylated activation of Akt and PRAS40 induced by everolimus. Both RICTOR-shRNA and everolimus inhibited the growth of ECa109 cell xenografts in nude mice (all P < 0.05), while the inhibitory effect was strongest in RICTOR-shRNA+everolimus group (P < 0.001). Conclusion: Silencing RICTOR gene can improve the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and the molecular mechanism may be associated with the down-regulation of RICTOR expression to inhibit the phosphorylated activation of Akt and PRAS40 induced by everolimus.
RESUMO
Objective To construct recombinant lentivirus silence vector aiming at rictor gene in mTORC2 specific protein, and to investigate its regulation on mTORC2/SGK1 signal pathway and the effect on pulmonary alveolar epithelial sodium ion chan-nel,as well as the role in acute respiratory distress syndrome(ARDS)and acute lung injury.Methods The interfering vector plas-mid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293T cells.The viral supernatant was collected,centrifuged,concentrated and purified for obtaining recombinant lentivirus.The virus titer was detected and the virus was infected to A549 cells.Stable cell lines were screened.RT-PCR was used to confirm the silencing situation of target gene rictor.The expression situation of various signal indexes in this pathway was detected by PCR and Western blot.Results The recombinant lentivirus of silence gene rictor was successfully constructed and transfected to A549 cell for obtaining stable cell lines.Compared with blank and control groups,the mRNA levels of rictor,downstream SGK1 andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased (P <0.05 ).Meanwhile,the protein levels of rictor,downstream SGK1,P-SGK andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased compared with the other two groups(P<0.05).Conclusion Silence rictor gene has the obvious regulation effect on mTORC2/SGK1 signal pathway,meanwhile affects the expression of pulmonary alveolar epithelial cellular α-,β-and γ-ENaC at gene and protein level.It is speculated that mTORC2/SGK1 may be an important signal pathway for regulating the clearance capacity of pulmonary alveolar epithelial cells on pulmonary alveolar fluid and simultaneously affecting the pulmonary edema formation.