Rac1: The Important Role In Mediating Cell Invasion Of Squamous Cell Carcinoma Promoted By Rab23

Cutaneous squamous cell carcinoma(c SCC) is the second most common cancer overall among patients just after basal cell carcinoma(BCC). It is usually localized, but it can metastasize. Squamous cell carcinoma on head and neck is associated with severe disease and treatment related morbidity and has a 5-year survival rate of approximately 50%, mainly due to the strong invasive capacity. Recent years, the research about invasion, metastasis is the point of dermatology.As a member of the Rab family of small GTPase, Rab23 has involved in the transportation of vesicles. O ur previous study firstly observed that Rab23 was high expression in c SCC, which is negatively related to histological differentiation. Secondly, we found cell invasion by Transwell assay significantly enhanced by o verexpression of Rab23, and Rab23 could somehow regulate Rac1. However, the molecular mechanism by which Rab23 enhance cell invasion of c SCC still remains unclear. The aim of this study is to comprehensively clarify the mechanism underlying Rab23 promoting cell invasion of c SCC, from the view of clinical specimens, cell and animal experiments respectivel. Firstly, we are to detect the correlation of expression of Rab23 and Rac1 in clinical specimens of c SCC. Secondly, we are to confirm Rac1 as a central molecule in the regulation of c SCC cell invasion enhanced by Rab23, with stable cell lines of differe ntRab23 activity. Thirdly, we are to explore how Rab23 regulate Rac1 expression and activity through integrin. Lastly, we are to find Rac1’s downstream effectors, which play an important role in mediating cell invasion of c SCC promoted by Rab23.Methods: 1. Construct stably transfected cell lines infected by lentivirus construct stably transfected Sa3 cell lines with Rab23 different expression and detected Rab23、Rac1 expression by Western blot. 2. Rab23 promotes SCC cell invasion, and dependent on the activity of GTP enzyme. 1) cell experiment: ① SCC cell lines with Rab23 different expression invasion ability were detected by transwell assays. ② The migration ability were detected by cell scratch assay. 2) animal experiment:Investigate whether SCC cell invasion enhanced by Rab23 in nude mouse model. 3. Rac1 as a central molecule in the regulation of SCC cell invasion enhanced by Rab23 1) The expression of Rab23, Rac1 in c SCC, normal skin was detected by immunohistochemistry. Logistic regression analysis was used to analyse the relationship between Rab23 and Rac1 by SPSS 11.5. 2) The expression of Rab23, Rac1 in c SCC by laser confocal microscopy(LSCM). 3) Invasion and migration ability in SCC cell lines with Rab23 different expression were detected after inhibited Rac1. 4) The expression of Rab23, α5β1 integrin in c SCC by LSCM. Invasion and migration ability in SCC cell lines with Rab23 different expression were detected after inhibited integrin.results: 1. Rab23 expression in SCC cell lines with Rab23 different expression by western blotting:Rab23 high expressed in Sa3-Rab23-wt,Sa3-Rab23-Q68 L,weak expressed in Sa3- Rab23-S23 N,Sa3-Rab23-RNAi;Rac1 expression in SCC cell lines with Rab23 different expression by western blotting : Rac1 high expressed inSa3-Rab23-wt, Sa3-Rab23-Q68 L, weak expressed in Sa3-Rab23-S23 N,Sa3-Rab23-RNAi; 2. Sa3 SCC cell invasion ability by transwell assays : Cell invasion ability : Sa3-Rab23-Q68L>Sa3-Rab23-wt>Sa3-Rab23-S23N>Sa3-Rab23-RNAi. 3. Migration ability by cell scratch assay: Cell migration ability : Sa3-Rab23-Q68L>Sa3-Rab23-wt>Sa3-Rab23-S23N>Sa3-Rab23-RNAi. 4. Numor bearing nude mice : The size of 0.6cm3 nodules subcutaneous formed in Sa3-Rab23-Q68 L after 1 weeks but Sa3-Rab23-S23 N, Sa3-Rab23-RNAi. 5. Rab23, Rac1 expression in c SCC and normal skin by immunohistochemistry: Positively stained cel s were found in c SCC; Rab23, Rac1 protein was absent in normal epidermis or dermis. 6. LSCM results show: Sa3-Rab23-wt, Sa3-Rab23-Q68 L were high expression of Rab23 at cytomembrane as well as Rac1, α5β1 integrin co-localization with Rab23 at cytomembrane. 7. Invasion and migration ability in SCC cell lines Sa3 with Rab23 different expression were significantly decreased after inhibited Rac1. 8. Invasion and migration ability in SCC cell lines Sa3 with Rab23 different expression were significantly decreased after inhibited α5β1 integrin.Conclusions: 1. Rab23, Rac1 express in c SCC sections, and Rab23 expression in c SCC has obviously positive correlation with Rac1, suggesting Rab23 probably positive regulate Rac1. 2. Rab23 promotes SCC cell lines Sa3 invasion and migration, and probably through the regulation of Rac1 to promote invasion and migration. 3. Rab23 probably through the regulation of α5β1 integrin to control Rac1.