Preliminary Study On The Effect Of Silencing RhoA Gene On Proliferation,migration And Invasion Of Gallbladder Cancer Cells

Objectives:1.To investigate the expression of RhoA protein in human gallbladder carcinoma.2.To study the effect of RhoA gene interference on the proliferation,migration and invasion of gallbladder cancer cells in vitro.3.The effect of EIF5A2 gene interference on the expression of RhoA and ROCKI protein in gallbladder cancer cells was confirmed by cell experiment in vitro.Methods:1.24 pairs of paraffin sections of gallbladder carcinoma and cholecystitis used in this experiment were all from the Department of Hepatobiliary surgery of the second affiliated Hospital of Kunming Medical University.The expression of RhoA protein in gallbladder cancer tissue and cholecystitis tissue was detected by average MOD.The MOD values of the two groups conformed to normal distribution.Independent sample t-test of SPSS23 software was used to analyze whether there was difference between them.2.Culture gallbladder cancer cell line,adjust the cell state,synthesize three designed siRNA-RhoA and screen one with excellent effect.The experiment was carried out by transient transfection method,which was divided into the following groups:siRNA-NC group and siRNA-RhoA group.The changes of mRNA and protein expression of RhoA and ROCK I were detected by qPCR and Western Blot techniques.The proliferation of cells in siRNA-NC group and siRNA-RhoA group was detected by CCK-8 method at 0 h,12 h,24 h,48 h and 72 h after treatment.The migration ability of cells in siRNA-NC group and siRNA-RhoA group was detected by fine cell scratch method,and the invasive ability of cells in siRNA-NC group and siRNA-RhoA group was detected by Transwell method.Use GraphPad prism8 to analyze data and draw drawings.3.Culture gallbladder cancer cell line,adjust the cell state,synthesize the designed siRNA-EIF5A2(the corresponding sequence has been verified and provided by our research group),using the method of transient transfection,divided into the following groups:3 siRNA-NC group,siRNA-EIF5A2 group,Western Blot was used to detect the changes of RhoA and ROCK I protein expression after 48h treatment,and GraphPad prism 8 was used to analyze the data and map.Results:1.It was found that RhoA protein was expressed in human gallbladder carcinoma and cholecystitis by microscope.The MOD of MOD,gallbladder carcinoma and cholecystitis were 24771±18373 and 7674±4275,respectively.The difference was statistically significant(P<0.003).This experiment proved that the expression of RhoA protein in human gallbladder carcinoma tissue was significantly increased.2.The expression of RhoA gene was detected by qPCR.With GAPDH as the internal reference,the results showed that the mRNA expression level of RhoA in siRNA-RhoA group(0.067±0.006)was significantly lower than that in siRNA-NC group(1.467±0.428).There was significant difference in the relative expression of RhoA gene between the two groups.The results showed that the pair of siRNA designed in this experiment could inhibit the expression of RhoA gene at RNA level.According to the results of Western Blot,the protein bands of RhoA and ROCKI in siRNA-RhoA group were weaker than those in siRNA-NC group,and the expression levels of RhoA and ROCKI in siRNA-RhoA group were significantly lower than those in siRNA-NC group(P<0.001,P<0.001),which indicated that the pair of siRNA designed in this experiment could also inhibit the RhoA gene of gallbladder cancer cells at the protein level and reduce the protein encoded by ROCKI.According to the results of CCK-8,there were significant differences in A450 absorbance values of GBC-SD cells between siRNA-NC group and siRNA-RhoA group at 24 h,48 h and 72 h after transfection(P=0.004;P=0.004;P=0.001),and the difference was the most significant(P<0.05).It is suggested that interfering with RhoA gene can inhibit the proliferation of human gallbladder cancer cells.According to the results of scratch experiment:through the scratch area,the percentage of wound healing was calculated,the ability of cell migration in siRNA-RhoA group was weaker than that in siRNA-NC group,and the difference was statistically significant(P=0.007).Transwell invasion experiment;three chambers were randomly selected for cell count under × 100 microscope,and siRNA-RhoA group(98.The invasiveness of GBC-SD was decreased in siRNA-NC group(162.333 ±17.785),siRNA-RhoA group(667±11.504),and there was significant difference between the two groups(P=0.006).3.The results of Western Blot showed that the bands of RhoA protein and ROCK I protein in siRNA-EIF5A2 group were weaker than those in control group,and those in siRNA-EIF5A2 group were weaker than those in control group.Conclusions:1.RhoA protein is expressed in human gallbladder tissue and highly expressed in human gallbladder carcinoma tissue.2.RhoA-SiRNAused in this experiment has a certain inhibitory effect on the expression of RhoA gene in gallbladder cancer cells in vitro,and silencing RhoA gene can inhibit the proliferation,migration and invasion of gallbladder cancer cells in vitro.3.Reducing the expression of EIF5A2 gene in gallbladder cancer cells in vitro can decrease the expression of RhoA and ROCKI proteins.