Study On Sensitivity To Chemotherapy Drugs Of SiRNA-ZNF139Gastric Cancer Cells And ZNF139in Proteomics

Gastric cancer is one of the major diseases considered a serious threat tohuman life and health, and it has become a major global public healthproblem.In China gastric carcinoma is the first place of the incidence andmortality rate in malignant tumors of the digestive system. The earlysymptoms of stomach cancer are atypical; therefore, when patients exhibit theapparent symptoms, the disease has already developed into the late stage.Therefore, the opportunity for radical surgery for these patients is lost, andchemotherapy becomes the primary means of their treatment. In recent years,new chemotherapy drugs and improved combined chemotherapy have beendeveloped. However, the effects of chemotherapy on gastric cancer patientsare still not satisfactory. The multidrug resistance (MDR) of gastric cancer isone of the main causes that result in the ill effects of chemotherapy treatment.Mechanisms of MDR are more complex, and it also influence by multiplefactors. Researching the effective ways of reversing MDR has become the keyto improving the efficacy of chemotherapy. Therefore, how to reverse MDR ingastric carcinoma and improve chemotherapy of gastric carcinoma become thecurrent hot spots in the study of gastric carcinoma and the pressing problemsin the clinical treatment of gastric carcinoma.Proteomics focuses on studies of the all proteins encoded by genes, andmakes complete analysis of the protein composition, expression levels andmodification state of the intracellular proteins with dynamic changes, andunderstands the interactions and associations among proteins, thus revealingthe protein function and its relationship with cell life activity Proteomicstechnological system is protein separation techniques based ontwo-dimensional polyacrylamide gel electrophoresis (2-DE) and proteinidentification techniques based on mass spectrometry (MS) and bioinformatics.The experiment was divided into four parts. In the first part, it was usedsiRNA to inhibit the expression of ZNF139in human gastric carcinoma cellSGC7901and researched its effect on chemotherapeutic sensitivity. In thesecond part, it was used siRNA to inhibit the expression of ZNF139inorthotopic transplantation tumor of human gastric cancer in nude mice andresearched its effect on chemotherapeutic sensitivity. From the research of thefirst and second part, it concluded that using siRNA can inhibit the expressionof ZNF139in human gastric carcinoma cell SGC7901and orthotopictransplantation tumor of human gastric cancer in nude mice, and sensitivity ofgastric carcinoma cell to chemotherapy drugs can be improve after inhibitingthe expression of ZNF139. In the third part, it was used the proteomicstechnology to separate and identify difference protein from human gastriccarcinoma cells SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice, and it was purpose to search the proteins whichrelated with ZNF139participation occurrence, development and multidrugresistance of gastric carcinoma. From the research of the third part, itconcluded that ZNF139possible participate occurrence, development andmultidrug resistance of gastric carcinoma through regulating the expression ofPDXK, ANXA2and Fascin. In the fourth part, it was used the qRT-PCR andWestern blot to detect the expression of PDXK, ANXA2and Fascin in humangastric carcinoma cell SGC7901and orthotopic transplantation tumor ofhuman gastric cancer in nude mice which ZNF139inhibited by siRNA. Fromthe research of the fourth part, it concluded that ZNF139possible participateoccurrence, development and multidrug resistance of gastric carcinomathrough increasing the expression of ANXA2, Fascin and reducing theexpression of PDXK.The specific contents of the four parts in this experiment are as follows:Part I: Inhibiting ZNF139expression by siRNA in human gastriccarcinoma cell SGC7901and researching its effect onchemotherapeutic sensitivity. Objective: to object human gastric carcinoma cell SGC7901, to detectthe expression of ZNF139in human gastric carcinoma cell SGC7901beforeand after using siRNA, to research its effect on chemotherapeutic sensitivity.Methods:1The siRNA was designed and synthesized according to the ZNF139.The siRNA-ZNF139plasmids were constructed. Human gastric carcinomacell SGC7901was cultured. The siRNA-ZNF139plasmid was transfected intohuman gastric carcinoma cell SGC7901.2The qRT-PCR method was applied to detect the expression of ZNF139on the mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group.3The Western Blot method was applied to detect the expression ofZNF139on the protein level in human gastric carcinoma cell SGC7901ineach experimental group.4The MTT method was applied to detect sensitivity to chemotherapydrugs in human gastric carcinoma cell SGC7901in each experimental group.Results:1The expression of ZNF139on the mRNA level in human gastriccarcinoma cell SGC7901in each experimental group.It was used the3positive plasmids and the negative control plasmid withconcentration for0.8μg/μl that transfected human gastric carcinoma cellSGC7901for48hours. From the results, it can be found that the relativeexpression quantity of ZNF139mRNA were highest in both blank controlgroup and negative control group, and there was no statistically significantdifference within both group (P>0.05). The relative expression quantity ofZNF139mRNA was lowest in no.1plasmid group, and there was statisticallysignificant difference within blank control group and no.1plasmidgroup(P<0.05). The inhibition rate of ZNF139was lowest in no.1plasmidgroup,84.41%.It was used the no.1plasmids concentration for0.4μg/μl,0.6μg/μl,0.8μg/μl and1.0μg/μl that transfected human gastric carcinoma cell SGC7901 for48hours. From the results, it can be found that the relative expressionquantity of ZNF139mRNA were highest in blank control group. The relativeexpression quantity of ZNF139mRNA was lowest in0.8μg/μl group, andthere was statistically significant difference within blank control group and0.8μg/μl group (P<0.05). The inhibition rate of ZNF139was lowest in0.8μg/μl group,84.42%.It was used the no.1plasmids concentration for0.8μg/μl that transfectedhuman gastric carcinoma cell SGC7901for24hours,48hours and72hours.From the results, it can be found that the relative expression quantity ofZNF139mRNA were highest in blank control group. The relative expressionquantity of ZNF139mRNA was lowest in48hours group, and there wasstatistically significant difference within blank control group and48hoursgroup (P<0.05). The inhibition rate of ZNF139was lowest in0.8μg/μl group,84.36%.2The expression of ZNF139on the protein level in human gastriccarcinoma cell SGC7901in each experimental group.The Western Blot method was applied to detect the expression ofZNF139on the protein level. Western blot results were consistent withqRT-PCR results.3The sensitivity to chemotherapy drugs in human gastric carcinoma cellSGC7901in each experimental group.According to the previous results, it was used the no.1plasmidsconcentration for0.8μg/μl that transfected human gastric carcinoma cellSGC7901for48hours. The MTT method was applied to detect sensitivity to5-FU, and ADR, CDDP, L-OHP, MMC, MTX, VP-16in human gastriccarcinoma cell SGC7901in each experimental group. From the results, it canbe found that the cell inhibition rate between blank control group and negativecontrol group was no statistically significant difference within both group(P>0.05). The cell inhibition rate was obviously higher compared with blankcontrol group, and there was statistically significant difference within bothgroup (P<0.05). Conclusions:1The expression of ZNF139on the mRNA level can be inhibited inhuman gastric carcinoma cell SGC7901by siRNA.2The expression of ZNF139on the protein level can be inhibited inhuman gastric carcinoma cell SGC7901by siRNA.3The sensitivity of gastric carcinoma cell to chemotherapy drugs5-FU,ADR, CDDP, L-OHP, MMC, MTX, VP-16can be improve after inhibiting theexpression of ZNF139in human gastric carcinoma cell SGC7901by siRNA.Part II: Inhibiting ZNF139expression by siRNA in orthotopictransplantation tumor of human gastric cancer in nude miceand researching its effect on chemotherapeutic sensitivity.Objective: to object orthotopic transplantation tumor of human gastriccancer in nude mice, to detect the expression of ZNF139in orthotopictransplantation tumor of human gastric cancer in nude mice before and afterusing siRNA, to research its effect on chemotherapeutic sensitivity.Methods:1Established orthotopic transplantation nude mice model of humangastric cancer and interventionHuman gastric carcinoma cell SGC7901cultured in vitro were collected.It was established subcutaneous transplantation tumor model in nude mice,and passage5times. It was get the vigorous growth of the6th generation ofsubcutaneous transplantation tumors. It was used the OB biological glue pastemethod transplanted the6th generation subcutaneously to orthotopictransplantation. It was observed the nude mice abdominal incision eating andceliac orthotopic tumor growth after surgery everyday. When diameter of theorthotopic transplantation tumor was10mm or so, it was begin to intervention.According to the blank control group, siRNA-ZNF139group, siRNA-ZNF139+oxaliplatin group, oxaliplatin and negative control groups intervene nudemice in random. During intervention every other day, it was measured the sizeof tumors, and calculated the volume of orthotopic transplantation tumoraccording to V=ab2/2(a, length diameter of tumor; b, short diameter of tumor). It was take out orthotopic transplantation tumor after two weeks.2The qRT-PCR method was applied to detect the expression of ZNF139on the mRNA level in orthotopic transplantation tumor of human gastriccancer in nude mice in each experimental group.3The Western Blot method was applied to detect the expression ofZNF139on the protein level in orthotopic transplantation tumor of humangastric cancer in nude mice in each experimental group.4The MTT method was applied to detect sensitivity to chemotherapydrugs in orthotopic transplantation tumor of human gastric cancer in nudemice in each experimental group.Results:1Morphological observation of orthotopic transplantation nude micemodel of human gastric cancer after interventionAfter two weeks during intervention, it was statistical volume oforthotopic transplantation tumor (V=ab2/2; a, length diameter of tumor; b,short diameter of tumor). From the results, it can be found that the volume oforthotopic transplantation tumor were biggest in both blank control group andnegative control group, and there was no statistically significant differencewithin both group (P>0.05). The volume of orthotopic transplantation tumorwas smallest in siRNA-ZNF139+oxaliplatin group, and there was statisticallysignificant difference within blank control group and siRNA-ZNF139+oxaliplatin group (P<0.05). There was statistically significant differencewithin siRNA-ZNF139group and blank control group (P<0.05). There wasstatistically significant difference within siRNA-ZNF139group andsiRNA-ZNF139+oxaliplatin group (P<0.05).There was statistically significantdifference within oxaliplatin group and blank control group (P<0.05). Therewas statistically significant difference within oxaliplatin group andsiRNA-ZNF139+oxaliplatin group (P<0.05). There was no statisticallysignificant difference within siRNA-ZNF139group and oxaliplatin group(P>0.05).2The expression of ZNF139on the mRNA level in orthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group.From the results, it can be found that the relative expression quantity ofZNF139mRNA were highest in both blank control group and negative controlgroup, and there was no statistically significant difference within both group(P>0.05). The relative expression quantity of ZNF139mRNA was lowest insiRNA-ZNF139group, and there was statistically significant difference withinblank control group and siRNA-ZNF139group(P<0.05). The inhibition rate ofZNF139was81.05%in siRNA-ZNF139group.3The expression of ZNF139on the protein level in orthotopictransplantation tumor of human gastric cancer in nude mice in eachexperimental group.The Western Blot method was applied to detect the expression ofZNF139on the protein level. Western blot results were consistent withqRT-PCR results.4The sensitivity to chemotherapy drugs in orthotopic transplantationtumor of human gastric cancer in nude mice in each experimental group.The MTT method was applied to detect sensitivity to5-FU, and ADR,CDDP, L-OHP, MMC, MTX, VP-16in orthotopic transplantation tumor ofhuman gastric cancer in nude mice in each experimental group. From theresults, it can be found that the cell inhibition rate between blank controlgroup and negative control group was no statistically significant differencewithin both group (P>0.05). The cell inhibition rate was obviously highercompared with blank control group, and there was statistically significantdifference within both group (P<0.05).Conclusions:1The growth of orthotopic transplantation tumor of human gastric cancerin nude mice by siRNA-ZNF139.2The expression of ZNF139on the mRNA level can be inhibited inorthotopic transplantation tumor of human gastric cancer in nude mice bysiRNA. 3The expression of ZNF139on the protein level can be inhibited inorthotopic transplantation tumor of human gastric cancer in nude mice bysiRNA.4The sensitivity of gastric carcinoma cell to chemotherapy drugs5-FU,ADR, CDDP, L-OHP, MMC, MTX, VP-16can be improve after inhibiting theexpression of ZNF139in orthotopic transplantation tumor of human gastriccancer in nude mice by siRNA.Part III: Proteomics study on the expression of ZNF139in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice inhibited by siRNAObjective: to object human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice, todetect the differences proteins in human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group by2D-DIGE and LC-MS.Methods:The differences proteins in human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group were separated by2D-DIGE.Application DeCyderDifferential Analysis Software selected difference obvious proteins and usedEttan spot picker to dig the points. The difference proteins were identified byLC-MS after in-gel digest. The SEQUEST computing method in BioWorkssoftware was applied for database retrieval.Results:About1958±67proteins are separated by2D-DIGE and the matchingrate is90.5%in human gastric carcinoma cell SGC7901. About5227±59proteins are separated by2D-DIGE and the matching rate is88.7%inorthotopic transplantation tumor of human gastric cancer in nude mice.It was select eight differences proteins in human gastric carcinoma cellSGC7901. Six kinds of proteins are identified by LC-MS: Pyridoxal kinase(PDXK)，Desmin, Nucleophosmin (NPM), Heat shock protein70(HSP70), Annexin A2(ANXA2) and Fascin. PDXK, Desmin and NPM wereup-regulating while HSP70, ANXA2and Fascin were down-regulate insiRNA-ZNF139group.It was select six differences proteins in orthotopic transplantation tumorof human gastric cancer in nude mice. Five kinds of proteins are identified byLC-MS: ANXA1, PDXK, Fascin, ANXA2and Heterogeneous nuclearribonucleoprotein (hnRNP). ANXA1, PDXK were up-regulating while Fascin,ANXA2and hnRNP were down-regulate in siRNA-ZNF139group.Conclusions:1PDXK, Desmin and NPM were up-regulating while HSP70, ANXA2and Fascin were down-regulate in human gastric carcinoma cell SGC7901after using siRNA-ZNF139.2ANXA1, PDXK were up-regulating while Fascin, ANXA2and hnRNPwere down-regulate in siRNA-ZNF139group in orthotopic transplantationtumor of human gastric cancer in nude mice after using siRNA-ZNF139.3ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through regulating the expression of PDXK,ANXA2and Fascin.Part IV: Research on effects of siRNA-ZNF139on expression of PDXK,ANXA2and Fascin in human gastric carcinoma cell SGC7901and orthotopic transplantation tumor of human gastric cancerin nude miceObjective: to object human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice, todetect the expression of PDXK, ANXA2and Fascin in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice before and after using siRNA,Methods:1Human gastric carcinoma cell SGC7901was cultured. ThesiRNA-ZNF139plasmid was transfected into human gastric carcinoma cellSGC7901 2Established orthotopic transplantation nude mice model of humangastric cancer and intervention3The qRT-PCR method was applied to detect the expression of PDXK,ANXA2and Fascin on the mRNA level in human gastric carcinoma cellSGC7901and orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group.4The Western Blot method was applied to detect the expression ofPDXK, ANXA2and Fascin on the protein level in human gastric carcinomacell SGC7901and orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group.Results:1The expression of PDXK, ANXA2and Fascin on the mRNA level inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice in each experimental group.The qRT-PCR method was applied to detect the expression of PDXK onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of PDXK mRNA were lowest in both blank control groupand negative control group, and there was no statistically significant differencewithin both group (P>0.05). The relative expression quantity of PDXK mRNAwas highest in siRNA-ZNF139group, and there was statistically significantdifference within blank control group and siRNA-ZNF139group (P<0.05).The qRT-PCR method was applied to detect the expression of ANXA2onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of ANXA2mRNA were highest in both blank controlgroup and negative control group, and there was no statistically significantdifference within both group (P>0.05). The relative expression quantity ofANXA2mRNA was lowest in siRNA-ZNF139group, and there wasstatistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05). The qRT-PCR method was applied to detect the expression of Fascin onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of Fascin mRNA were highest in both blank control groupand negative control group, and there was no statistically significant differencewithin both group (P>0.05). The relative expression quantity of Fascin mRNAwas lowest in siRNA-ZNF139group, and there was statistically significantdifference within blank control group and siRNA-ZNF139group (P<0.05).The qRT-PCR method was applied to detect the expression of PDXK onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of PDXK mRNA were lowest in both blankcontrol group and negative control group, and there was no statisticallysignificant difference within both group (P>0.05). The relative expressionquantity of PDXK mRNA was highest in siRNA-ZNF139group, and therewas statistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).The qRT-PCR method was applied to detect the expression of ANXA2onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of ANXA2mRNA were highest in both blankcontrol group and negative control group, and there was no statisticallysignificant difference within both group (P>0.05). The relative expressionquantity of ANXA2mRNA was lowest in siRNA-ZNF139group, and therewas statistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).The qRT-PCR method was applied to detect the expression of Fascin onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of Fascin mRNA were highest in both blankcontrol group and negative control group, and there was no statistically significant difference within both group (P>0.05). The relative expressionquantity of Fascin mRNA was lowest in siRNA-ZNF139group, and there wasstatistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).2The expression of PDXK, ANXA2and Fascin on the protein level inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice in each experimental group.The Western Blot method was applied to detect the expression of PDXK,ANXA2and Fascin on the protein level. Western blot results were consistentwith qRT-PCR results.Conclusions:1The expression of PDXK on the mRNA level increased and theexpression ANXA2, Fascin of on the mRNA level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.2The expression of PDXK on the protein level increased and theexpression of ANXA2, Fascin on the protein level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.3ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through increasing the expression of ANXA2,Fascin and reducing the expression of PDXK.Based on the four parts of this research, we could draw conclusionsas follows:1The expression of ZNF139on the mRNA levelcan be inhibited inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice by siRNA.2The expression of ZNF139on the protein levelcan be inhibited inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice by siRNA.3The sensitivity of gastric carcinoma cell to chemotherapy drugs can be improve after inhibiting the expression of ZNF139in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice by siRNA.4The growth of orthotopic transplantation tumor of human gastric cancerin nude mice by siRNA-ZNF139.5PDXK, Desmin and NPM were up-regulating while HSP70, ANXA2and Fascin were down-regulate in human gastric carcinoma cell SGC7901after using siRNA-ZNF139. ANXA1, PDXK were up-regulating while Fascin,ANXA2and hnRNP were down-regulate in siRNA-ZNF139group inorthotopic transplantation tumor of human gastric cancer in nude mice afterusing siRNA-ZNF139.6The expression of PDXK on the mRNA level increased and theexpression ANXA2, Fascin of on the mRNA level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.7The expression of PDXK on the protein level increased and theexpression of ANXA2, Fascin on the protein level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.8ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through increasing the expression of ANXA2,Fascin and reducing the expression of PDXK.