| Objective: To observe the effects of LIMK1 silencing and ROS on intraperitoneal transplantation tumor of gastric carcinoma nude mice by constructing the LIMK1-silenced MGC803 cell line.Methods: Use Lentivirus-mediated RNAi to construct LIMK1 targeting silenced expression vectors to infect gastric carcinoma MGC803 cells,establish LIMK1-silenced expression of MGC803 cell line,set up interference group,empty vector(EV)group and control group,and use Western Blot technology to detect the expression of LIMK1 protein of the transfected gastric carcinoma cells in each group.MGC803 cells of the three groups were collected.Nude mice were injected intraperitoneally with gastric cell suspensions to construct enterocoelia transplantation tumor model.The tumorigenesis was observed,and after enterocoelia transplantation tumors were formed,each group was divided into two groups randomly,the experimental group and control group were gavaged with ROS and saline respectively.42 days later,nude mice were dissected,tumor formation and tumor metastasis in abdominal cavity of nude mice from each group were observed,the number of tumors in nude mice from each group was calculated,the longest diameter and total weight of tumors was measured,and calculate the tumor inhibition rate,observe the effects of ROS and LIMK1 silenceing on the growth and metastasis of enterocoelia transplantation tumor of nude mice;HE staining was used to observe the histomorphological changes of tumors in nude mice,immunohistochemistry and Western Blot were used to detect the expression of LIMK1 protein in enterocoelia metastatic tumor tissues from each group before and after ROS treatment.Results: 1.Successfully constructed the LIMK1-silenced gastric carcinoma MGC803 cell line.We commissioned Shanghai Genechem Co.,Ltd.to construct LIMK1 interference lentiviral recombinant vector.The gastric carcinoma MGC803 cells were infected with interference vector and empty vector respectively,and 72 h later a mass of green fluorescence was visible under the fluorescence microscope,which showed that the Lv-LIMK1-shRNAMGC803 cell line was successfully constructed.Western-Blot was used to detect the expression level of LIMK1,and the results indicated that,compared with the empty vector group and control group,the interference group had narrower LIMK1 protein bands with lighter color,the expression level was obviously decreased,and the differences were statistically significant(p<0.05),while there was no statistically significant difference of LIMK1 protein expression between the empty vector groups and control groups(p>0.05).2.Successfully constructed enterocoelia transplantation tumor model in gastric cancer nude mice42 days after inoculation,the nude mice were executed,the bloody ascites was visual observed in the abdominal cavity in some of the nude mice,several unevenly distributed miliary nodular tumors with different size could be seen on intestinal,mesenteric,greater omentum,abdominal wall,celiac lymph node and surface of liver,the tumorigenesis of enterocoelia organs in nude mice from each group was calculated,and the result manifest that,compared with the empty vector groups(B and E groups))and control groups(D and F groups),the LIMK1 interference groups(A and D groups)had significantly decreased tumorigenesis rate of celiac visceral organs and the differences were statistically significant(p<0.05);there is no significant difference between the empty vector groups and the control groups(p>0.05).3.Effect of LIMK1 silencing on ROS inhibition of enterocoelia transplantation tumor of gastric carcinoma MGC803 cells in nude mice(1)Specimens were dissected,the number,maximum diameter and total weight of metastatic tumors in enterocoelia of each visceral organ of nude mice from each group were measured,and the results indicate that the number,maximum diameter and total weight of tumors in LIMK1 interference groups were significantly lower than those in the EV groups and control groups,the differences were statistically significant(p<0.05).And after ROS treatment the number,maximum diameter and total weight of tumors from each group were significantly different from those before ROS treatment(p<0.05).Compared with control group(F group),the tumor inhibition rates in tumor weight of LIMK1 interference group(D group)and EV group(E group)were 35.54% and 3.85%,respectively,and compared with the corresponding groups before ROS treatment,tumor inhibition rates in tumor weight of LIMK1 interference group,EV group and blank control group after ROS treatment were 46.48%,19.57% and 20.90%,respectively.(2)The results of HE staining showed that compared with the groups without ROS gavage(D,E and F groups),the groups gavaged with ROS(A,B and C groups)had decreased cellular atypia,reduced mitotic figures,lighter nuclear staining and decreased nucleus-to-cytoplasm ratio,LIMK1 interference group had the minimal cellular atypia.(3)The results of immunohistochemical method showed that tumor tissues from each group visible LIMK1 expression,but compared with EV groups and control groups,LIMK1 in interference groups weakly expressed,and compared with the groups without ROS gavage,groups gavaged with ROS had lower LIMK1 expression.(4)Western Blot was used to detect the expression of LIMK1 protein of enterocoelia transplantation tumor tissue from each group.Compared with EV groups and control groups,interference groups had lower LIMK1 expression and the differences were statistically significant(p<0.05),while no statistically significant difference(p>0.05)found between EV groups and control groups.Compared with the groups without ROS gavage,groups gavaged with ROS had obviously decreased LIMK1 expression and the differences were significant(p<0.05).Conclusions:1.LIMK1 silencing can inhibit the growth and metastasis of enterocoelia tumor of gastric carcinoma MGC803 cells in nude mice.2.Rosiglitazone can inhibit the growth and metastasis of enterocoelia transplantation tumor of gastric carcinoma MGC803 cells in nude mice.3.LIMK1 may be the target of rosiglitazone to inhibit the growth and metastasis of celiac transplantation tumor of gastric carcinoma MGC803 cells in nude mice. |
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