| Objective: Gastric cancer is one of the most common digestive tractcancers worldwide with high incidence, high mortality, and low earlydiagnosed or poor treatment effect in the advanced stage. In2008, theincidence of gastric cancer was nearly one million and740000people died.China is one of countries which have the highest incidence and mortality ofgastric cancer. At present, the treatment of gastric cancer is mainly byoperation and chemotherapy is subordinate. However, most of gastric cancershave been found in moderate and advanced stages with poor operation effect,so the conservative therapy can only be received. Because of low sensitivity tochemotherapy and high drug resistance, effect of conservative therapy ongastric cancer is poor. The survival rate of advanced gastric cancer in fiveyears is still hovering around30%-40%. With the further development ofcancer genetics and molecular biology, the pathogenesis of gastric cancer hasbeen understood profoundly, it is a disease which has involved in multi-step,multi-stage and multi-factor. In process of normal cell growth, because ofabove reasons are likely to cause disharmony of cell proliferation, resulting inoccurrence of gastric cancer. If we intervene the expression of certain genes inthis progress, it will play positive effect in the prognosis of gastric cancer. Inrecent years, the effect of Zinc Fingers protein has caused widespreadattention in tumor. Krüppel family of Zinc Fingers protein is one oftranscription factor of Zinc Fingers which can influence development ofcancer through regulation transcription and express of related gene. Forexample, ZNF-139presents high expression in osteosarcoma tissue as specificprotein marker in pathogenesis of osteosarcoma. ZNF-139is one of ZincFingers Krüppel family members, which presents high expression in gastriccancer tissue, estimated that it may participate regulation of development of gastric cancer and MDR. This research will study the tumor from gastriccancer orthotopic transplanted nude mouse established by SGC7901cells;explore the influence of silent transcription factor ZNF-139on tumorproliferation.Methods:1Synthesis small interfering RNA on ZNF-139, and transfect it intocultured human gastric cancer SGC-7901in different concentrations throughliposome LipofectamineTM2000mediating. Inhibition rate was detected byqRT-PCR.2Collect human gastric SGC-7901cells in logarithmic phase of growthand configure to cell suspension which was injected subcutaneously in nudemouse. Isolate gastric cancer tissue in1mm3when the diameter of tumor was1.5cm and inoculate into the other nude mouse’s subcutaeous in order topassage between rodents. The transplanted tumor was isolated5timesaccording to above methods and cut into scale of1mm3tissue. Prepare modelof gastric cancer orthotopic transplanted nude mouse through orthotopictransplanting cancer tissues into serous membrane of gastric wall.3SiRNA was transfected into tumor of nude mouse through liposomeLipofectamineTM2000mediating. Negative control group (transfected withempty plasmid group, hereinafter referred to negative group) and blank controlgroup (injection with saline group, hereinafter referred to blank group) wereset up. Kill mouse and remove tumor after transfection in suitable time, spare.4Three groups of different factors treated tumor were configured to cellsuspension in order to primarily culture. Proliferation was detected in0,24,48and72hours by MTT.5The tumors were fixed with70%alcohol after transfection of48hoursand configured into cell suspension. The changes of cell cycle between threegroups were compared by flow cytometry. The proliferation index of threegroups were calculated.6Detect expression of ZNF-139, PCNA, Cycling D in three groups oftumor by application of RT-PCR. Results:1Experiments in vitro indicated that the establishment of SiRNA-ZNF-139would silence specifically expression of ZNF-139in SGC-7901cell. Theresults of qRT-PCR showed that relative expression quantity of ZNF-139mRNA in SGC-7901cell were0.992±0.0637，0.662±0.701，0.346±0.0245，0.144±0.216，0.339±0.0658respectively after0μg/μl、0.4μg/μl、0.6μg/μl、0.8μg/μl、1.0μg/μl recombinant plasmid transfection. Statistical analysisshowed significance difference between each group (P<0.01) and the highestinhibition rate was85.58%in group of0.8μg/μl.2MTT assay results showed that: OD values of experimental group, negativegroup and control group in0hours were0.126±0.031、0.128±0.042、0.125±0.041respectively; the OD values in24hours were0.225±0.063、0.228±0.038、0.221±0.054respectively;the OD values in48hours were0.342±0.104、0.495±0.094、0.499±0.127and the72hours values were0.482±0.054、0.665±0.038、0.667±0.038. There is no obvious difference in thegroup of0hours and24hours (P>0.05); cells in experimental group wassignificantly less than the negative control group and blank group in48hoursand72hours (P<0.05), and there is no significant difference in negative groupand control group.(P>0.05).3The results of Flow cytometry showed: cells percentages of experimentalgroup, negative group and blank group in G0/G1phase were43.80±0.92、36.17±0.35、36.00±0.53respectively after transfected injection48hours finally;cells percentages in S phase were30.10±1.97、38.00±0.80、37.90±0.89cellspercentages of experimental group, negative group and blank group in G2/Mphase were25.73±0.643、25.37±0.551、26.1±0.65. The cells of experimentalgroup were significantly more than negative group and blank group in G0/G1phase, which had statistical significance (P<0,05); the number of cells ofnegative group and blank group in G0/G1phase had no difference (P>0.05);the cells of experimental group was significantly less than negative group andblank group in S phase, which had statistical significance (P<0.05); cellsbetween negative and blank group in S phase had no significant difference (P>0.05), the number of cells of three groups in G2/M phase had no difference(P>0.05) The proliferation index of experimental group, negative group andblank group were36.2%,63.83%,64%。4Expression of ZNF-139, PCNA and CyclinD1.4.1Expression of ZNF-139mRNA: RT-PCR results showed thatZNF-139mRNA had expression in the experimental group, negative group andblank group, the values of IOD were0.158±0.051、0.359±0.055、0.356±0.038;The expression in experimental group was significantly lower than in negativegroup and blank group (P<0.05), the difference was statistical significant.4.2Expression of PCNAmRNA: RT-PCR results showed that：PCNAmRNAhad expression in the experimental group, negative group and blank group, thevalues of IOD were0.311±0.069、0.650±0.117、0.651±0.127; The expression inexperimental group was significantly lower than in negative group and blankgroup (P<0.05), the difference was statistical significant.4.3Expression of CyclinD1mRNA: RT-PCR results showed that CyclinD1mRNA had expression in the experimental group, negative group and blankgroup, the values of IOD were0.154±0.042、0.507±0.045、0.508±0.078;Theexpression in experimental group was significantly lower than in negativegroup and blank group (P<0.05), the difference was statistical significant.Conclusion:1The plasmid of small interfering RNA against ZNF-139which caninhibit expression of ZNF-139effectively2Human gastric cancer orthotopic transplanted nude mouse canapproximately simulate biological behavior of gastric cancer in the body withadvantages of simple operation and high rate of tumor formation which canused as identical experimental tools for study of gastric cancer.3Tumor cells of gastric cancer orthotopic transplanted nude mouseproliferate slowly and most of cells stagnated in G0/G1phase aftertransfection small interfering RNA against ZNF-139into nude mouse4ZNF-139is gene regulated factor in transfection level, its mechanismof inhibition cell proliferation lies in si-RNA-ZNF-139may decline genetic expression of PCNA and CyclinD1and further influence the progressof cell cycle, slow down the tumor cell proliferation and play role inanti-tumor. |
PDF Full Text Request