In Vitro Effects Of Rac1 Silencing On The Proliferation,Migration And Invasion Of Tongue Squamous Cancer Cells

Objective: Squamous cell carcinoma of the tongue is one of the most common malignant tumors in the head and neck.Despite the surgery,radiotherapy and chemotherapy of tongue cancer has been improved continuously in recent decades,the survival rates of five years did not raised significantly.Therefore,it is particularly important to explore the molecular mechanism of tongue squamous cell carcinoma.Rac1(Ras-related C3 botulinum toxin substrate 1),as an important member of Rho GTPase family,has been proved to play an important role in regulating tumor proliferation,motility and apoptosis.It has become a research hotspot of the molecule-targeted therapy for malignant tumors.At present,the research mostly focused on the correlation of Rac1 with gastric cancer,breast cancer,colon cancer and other parts of the tumor,there were few study about the effects of Rac1 on the growth and development of tongue cancer.In this study,we constructed small interfering RNA(small interfering RNA,si RNA)to silence Rac1 gene,and explored the molecular mechanism of Rac1 on proliferation,invasion and migration of tongue squamous cell carcinoma.Methods: Login Genebank and identify the gene sequence of human Rac1,design and synthesize three si RNA sequences of Rac1,then transfect three si RNAs through liposome into three groups of CAL27 cells.At the same time,negative control group was transfected independent nucleotide sequence and blank control group was only added transfection reagent,without si RNA.(1)The expression of Rac1 m RNA in CAL27 cells after 48 h transfection was detected by real-time quantitative real-time polymerase chain reaction(q PCR).(2)The expression of Rac1,PAK1,LIMK1,Cyclin D1,p21 and p27 protein in CAL27 cells after 48 h transfection were detected by Western blot.(3)The proliferation and cell cycle of CAL27 cells after 48 h transfected were detected by CCK-8 proliferation assay and flow cytometry.(4)The changes of migrative and invasive ability of CAL27 cells after transfection were detected by wound healing assay and Transwell test.The data was analyzed by SPSS 18.0.T test was used for the comparison among groups,and a single factor analysis of variance was used for the comparison among the various numbers.P<0.05 had statistical significance.Result:(1)The expression of Rac1 m RNA in three groups of Rac1 si RNA cells decreased after 48 h transfection(P < 0.05).(2)The expression of,Rac1,PAK1,LIMK1,Cyclin,D1 proteins in the experimental group significantly decreased in comparison with negative control group and blank control group(P<0.05);the expression of p21 and p27 proteins increased significantly(P<0.05).(3)cell proliferation activity of si RNA group was significantly decreased after 48 h transfection,and the difference was statistically significant(P<0.05).(4)The wound healings in the experimental group were slower than those in negative control group and blank control group(P<0.05).(5)The number of cells passing through the filter membrane in negative control group and blank group was more than that in Rac1-si RNA group,the difference was statistically significant(P<0.05).Conclusion: The specific suppression of Rac1 gene can inhibit the proliferation,migration and invasion ability of tongue cancer?CAL27 cells,and the potential mechanism may be related to down-regulation of PAK1,LIMK1 and Cyclin D1 expression,up-regulation of p21 and p27 protein.Rac1 may be an effective target for the gene treatment of tongue cancer.