Study Of The Influence And Mechanism On The Apoptosis Of The Orthotopic Implantation Tumor Of Human Gastric Cancer In Nude Mice By Inhibition To ZNF139with SiRNA

Objective: Gastric cancer is very common and has been a serious threatto human health. In recent years, a large number of researches have shown that,many members of the Krüppel family can regulate the transcription andexpression of the gene which is related to the apoptosis to cause theoccurrence and development of the tumor. ZNF139is one of the members ofthe Krüppel family. The previous research showed that:the expression ofZNF139gene is high in most of human gastric cancer cells, which is closelyrelated to the occurrence and development of the gastric cancer.However,there is no report in whether the expression of ZNF139gene isrelated to the apoptosis of gastic cancer cells and the expression of Bax andBcl-2gene,which needs to be confirmed by experiments.The siRNA-ZNF139was applied to silence the ZNF139gene expression of orthotopic implantationtumor of human gastric cancer cell line SGC-7901in nude mice in thisexperiment. The purpose is to observe and investigate the effect ofsiRNA-ZNF139on the cell apoptosis of orthotopic implantation tumor ofhuman gastric cancer in nude mice,exploring the relationship between theZNF139gene and the apoptosis of human gastric cancer cells and themechanism of its effect.Methods:1The recombinant plasmid of siRNA-ZNF139and negative controlplasmid of control-siRNA was designed and synthetized by the limitedcompany of TIANGEN Biochemical Technology. The recombinant plasmidwas extracted from the Endotoxin-free Kit.After the extraction,theconcentration and purity of the extracted recombinant plasmid was examinedby Ultraviolet spectrophotometer. The silencing efficiency of the recombinant plasmid of siRNA-ZNF139was examined by means of RT-PCR.2Human gastric cancer cell line SGC-7901was repeatedly implantedunder the skin of nude mice of BALB/C-(nu/nu). The human gastric cancertissue blocks which were passaged six times were taken to the stomach wall ofnude mice.The life status of nude mice and the growth of the tumor wereobserved regularly. The orthotopic implantation model of human gastriccancer in nude mouse was built.3When the tumor diameter was about5mm, the nude mice wererandomly divided into3groups, the blank control group, the negative controlgroup and the experimental group.There were5nude mice in each group. Thefirst day, the third day, the fifth day, the seventh day, the ninth day,thesiRNA-ZNF139of20μg, control-siRNA of20μg, and the normal saline of50μl were respectively injected into orthotopic implantation tumors in nudemice in corresponding group. The growth of orthotopic implantation tumorswas observed every day.4Before each tumor of the nude mice was injected and after theorthotopic implantation tumors were taken out of the nude mice, the longestdiameter (A) and shortest path (B) were mearsured. According to theformula, the volume (mm3) of the tumor=1/2×A×B2. The approximatevolume of the tumor was calculated and the curve of the tumor growth wasdrawed.5After the intervention on the nude mice for5times, all of the nude micewere sacrified by cervical dislocating on the eleventh day. The whole of thetumor was taken out. The tumor was measured by weight and the inhibitoryrate was calculated.The inhibition rate of the tumor=(the tumor weight of thecontrol group-the tumor weight of the experimental group)/the tumorweight of the control group×100%.6The nude mice were sacrificed. The whole of the tumor was taken out.A small amount of the specimens of the fresh tumor was taken out to producethe suspension of the single cell. The livability of gastric cancer cells in threegroups was determined by means of MTT. 7The apoptosis rate of gastric cancer cells in nude mice in three groupswas detected by means of the flow cytometry.8The expression of ZNF139, Bax and Bcl-2mRNA of gastric cancer cellin nude mice was determined and compared by means of RT-PCR.Results:1. Detection of ZNF139’s silencing efficiencyThe expression of ZNF139of gastric cancer tissue in the blank controlgroup (0.3586±0.0361) was significantly higher than that in the experimentalgroup (0.1237±0.0131), the difference was statistically significant (P=0.00).The expression of ZNF139of gastric cancer tissue in the negative controlgroup (0.3601±0.0296) was significantly higher than that in the experimentalgroup (0.1237±0.0131), the difference was statistically significant(P=0.00).The silencing efficiency was65.7%.2.The establishment of the orthotopic implantation model of human gastriccancer cell line SGC-7901in nude miceWe built an orthotopic implantation model of human gastric cancer cell lineSGC-7901in nude mice successfully. The tumor formation rate was100%(15/15).3.The effect of recombinant plasmid siRNA-ZNF139on the orthotopicimplantation tumor of human gastric cancer cell in nude mice3.1The growth of the orthotopic implantation tumorThe tumor volume of nude mice in the blank control group（361.21±15.09）mm3was significantly bigger than that in the experimental group(153.14±5.78)mm3,the difference was statistically significant (P=0.00).Thetumor volume of nude mice in the negative control group （354.09±9.79）mm3was significantly bigger than that in the experimental group（153.14±5.78）mm3, the difference was statistically significant (P=0.00). There was nostatistically significant difference between the blank control group and thenegative control group (P=0.32).Compared with the blank control group andthe negative control group, the tumor growth of nude mice in the experimentalgroup was significantly slower（Fig.5）.But the tumor growth of nude mice in the negative control group was slower than that in the blank control group.3.2The effect of recombinant plasmid siRNA-ZNF139on the inhibition oftumorThe tumor weight of nude mice in the blank control group（2.41±0.13）g was significantly more than that in the experimental group（1.11±0.06）g,the difference was statistically significant (P=0.00). The tumor weight of nudemice in the negative control group（2.30±0.10）g was significantly more thanthat in the experimental group（1.11±0.06）g, the difference was statisticallysignificant (P=0.00). There was no statistically significant difference betweenthe blank control group and the negative control group (P=0.11). Comparedwith the blank control group,the tumor inhibition rate in the experimentalgroup was53.94%and the tumor inhibition rate in the negative control groupwas4.56%.4.The effect of recombinant plasmid siRNA-ZNF139on the cell livability ofthe orthotopic implantation tumor of human gastric cancer cell in nude miceThe absorbance of gastric cancer cells in the blank control group(0.5014±0.0487) was significantly higher than that in the experimentalgroup(0.2768±0.0048),the difference was statistically significant (P=0.00).The absorbance of gastric cancer cells in the negative control group(0.4926±0.0715) was significantly higher than that in the experimentalgroup(0.2768±0.0048),the difference was statistically significant (P=0.01).There was no statistically significant difference between the blank controlgroup and the negative control group (P=0.97).5. The effect of recombinant plasmid siRNA-ZNF139on the apoptosis of theorthotopic implantation tumor of human gastric cancer cell in nude miceIn the blank control group the apoptosis of gastric cancer cells in nudemice (18.3800±4.6855)%was significantly lower than that in the experimentalgroup （33.3900±2.2393）%,the difference was statistically significant(P=0.00).In the negative control group the apoptosis of gastric cancer cells innude mice (16.9160±2.6913)%was significantly lower than that in theexperimental group（33.3900±2.2393）%,the difference was statistically significant (P=0.00).There was no statistically significant difference betweenthe blank control group and the negative control group (P=0.51).6.The expression of the ZNF139, Bax and Bcl-2/mRNA of nude mice gastriccancer tissues in three groups.6.1The expression of the ZNF139mRNAThe expression of ZNF139of gastric cancer tissue in the experimentalgroup (0.1237±0.0131) was significantly lower than that in the blank controlgroup (0.3586±0.0361), the difference was statistically significant (P=0.00).The expression of ZNF139of gastric cancer tissue in the experimental group(0.1237±0.0131) was significantly lower than that in the negative controlgroup (0.3601±0.0296), the difference was statistically significant(P=0.00).There was no statistically significant difference between the blankcontrol group and the negative control group (P=0.93).6.2The expression of the Bax mRNAThe expression of Bax of gastric cancer tissue in the experimental group(0.4981±0.0704) was significantly higher than that in the blank control group(0.2036±0.0531),the difference was statistically significant (P=0.00).Theexpression of Bax of gastric cancer tissue in the experimental group(0.4981±0.0704) was significantly higher than that in the negative controlgroup (0.1981±0.0547), the difference was statistically significant (P=0.00).There was no statistically significant difference between the blank controlgroup and the negative control group (P=0.89).6.3The expression of the Bcl-2mRNAThe expression of Bcl-2of gastric cancer tissue in the experimental group(0.0357±0.0039) was significantly lower than that in the blank control group(0.1379±0.0366), the difference was statistically significant (P=0.00).Theexpression of Bcl-2of gastric cancer tissue in the experimental group(0.0357±0.0039) was significantly lower than that in the negative controlgroup (0.1437±0.0379), the difference was statistically significant (P=0.00).There was no statistically significant difference between the blank controlgroup and the negative control group (P=0.77). Conclusion:1The orthotopic implantation model of human gastric cancer cell lineSGC-7901in nude mice was successfully built.2The application of siRNA-ZNF139inhibited ZNF139gene could makethe orthotopic implantation tumor of nude mice smaller in size and growthrate.3The recombinant plasmid of siRNA-ZNF139can effectively reduce thelivability of the gastric cancer cell in nude mice and promote the apoptosis ofthe gastric cancer cells.4The recombinant plasmid of siRNA-ZNF139can effectively decreasethe expression of ZNF139mRNA of orthotopic implantation tumor cell innude mice.Silencing the expression of ZNF139can increase the expression ofBax and decrease the the expression of Bcl-2, thereby inducing cell apoptosisand inhibiting the growth of the orthotopic implantation tumor of humangastric cancer in nude mice.