The Expression And Clinical Significance Of CrkⅡ In Laryngeal Squamous Cell Carcinoma

Objective: The purpose of this study is to investigate the expression of Crk II protein in laryngeal squamous cell carcinoma(LSCC),to explore the relationship between Crk II protein and the clinicopathological features of LSCC as well as to study the effect of down-regulation of Crk II expression on proliferation,migration and invasion of LSCC Hep-2 cells by knock downing the expression of Crk II protein with the application of RNA interference method.Methods: The expression of Crk II protein in LSCC tissues was detected by immunohistochemical method(SP method)in 54 LSCC patients who had undergone surgical resection.The relationship between the expression of Crk II protein and the clinical stage,pathological grade,lymph node metastasis and age of LSCC was analyzed.According to the coding sequence of human Crk II gene in Gen Bank database(series number: NM 016823.3),si RNA interference sequences for Crk II gene was designed by using the online design software(Invitrogen company),and unrelated sequences were designed as negative control(NC).The constructed RNA interference plasmid and NC plasmid was transfected into Hep2 cells by liposome transfection method.After 24 h,the transfection efficiency was observed under an inverted fluorescence microscope and then photographed.The experiment will be assigned into three groups,namely the experimental group(transfected with Crk II RNA interference plasmid),the NC group(transfected with NC plasmid)and the blank control group(without any transfection).After successful transfection,the down-regulated m RNA expression of Crk II was detected by real-time PCR,and the protein expression of Crk II was detected by western blot analysis.Cell proliferation,migration and invasion were detected by MTT assay,scratch test and transwell invasion assay,respectively.Results: 1.No or little expression of Crk II was found in 22 cases of adjacent normal tissues away from LSCC tissues,and the positive rate of Crk II expression was 9.1%;37 cases of LSCC tissues were found with expression of Crk II in 54 cases of LSCC tissues,and the positive rate of Crk II expression was 68.5%.The positive expression rate of Crk II in LSCC tissues was significantly higher than that in adjacent normal tissues(P<0.05).The positive expression rate of Crk II was significantly different in clinical stage,pathological grade and lymph node metastasis of LSCC(χ2 = 9.643,χ2= 4.987,χ2= 3.833,all P< 0.05).2.Crk II RNA interference plasmid was successfully constructed and transfected into Hep2 cells.The expression of endogenous Crk II m RNA and protein in LSCC Hep-2 cells was significantly inhibited after transfection(P<0.01).3.MTT assay results demonstrated that after transfection,the OD value of Hep-2 cells in the experimental group was lower than that in the blank control group and the NC group at all time points(P<0.01).The OD value increased gradually with time,but there was no significant difference in OD value between the NC group and the blank control group(P>0.05).4.The findings of scratch test suggested that the cell migration distance of the experimental group was significantly lower than that of the blank control group and the NC group at 48 h(t=8.591,t=19.666,P<0.01).and the migration distance of the blank control group was not significantly different from that of the NC group(t=1.403,P>0.05).5.Transwell invasion assay results showed that there was no significant difference between NC group and blank control group in the number of cells passing through the matrix membrane to the basolateral chamber(t=0.256,P>0.05).The average number of cells passing through the matrix membrane to the basolateral chamber in the experimental group was significantly lower than that in the blank control group and the NC group(t=10.226,t=9.707,P<0.01).Conclusion: 1.Crk II protein may play an important role in the development of LSCC by promoting the invasion and metastasis of LSCC cells.2.Successful construction of Crk II RNA interference plasmid can effectively down-regulate the expression of Crk II in Hep-2 cells.3.After down-regulating the expression of Crk II in LSCC Hep-2 cells,the cell proliferation,migration and invasion ability were significantly decreased,suggesting that Crk II protein may become a new target for the treatment of LSCC.