Application Of Double-stranded RNA Of Sealing Unction On Identification Of Transcriptional Factors Ssociated With Drug Resistance Of Gastric Cancer

Gastric cancer is one of the most frequently encountered cancers in theworld for which surgeries and chemotherapy are the common and useful methods.Although survivial time may be increased, the expected efficacy cannot beachieved because of the generation of multiple drug resistance. Thusinvestigation of mechanism of drug resistance turns into a hotspot.Transcriptional factor is a kind of molecules with wide distribution andmultiform regulating modes. Currently just a few methods are applied on therelationship between transcriptional factors and drug resistance, which is abottleneck problem to explain mechanisms of MDR.We carried out an Oligonucleotide Array-based Transcription Factor Assayto gastric cancer cell lines SGC7901and SGC7901/ADR and31transcriptionalfactors with different expression between two cell lines were obtained. We foundthat several factors would share the same cis-element. This kind of phenomenon is called ‘redundancy’, which means that factors sharing same cis-element mayhave similar function on induction of drug resistance. According to this kind ofphenomenon we separated the31transcriptional factors into11groups: factorsassociated with AML, AP-2A, CEBP, AP-1, CUTL-1, E4BP4, FOXO, FXR, HSF,PEBP, and PITX2.RNA interfering cannot be applied in the identification of the transcriptionalfactors associated with resistance against Adriamycin for the sake of hard workand unstable phenotype siRNA bring. According to the same cis-elementsmentioned above, if there is some method which can deal with these elements,the difficulties would be settled. TFD strategy is the method fitting for the work.Transcriptional factor decoy strategy (TFD strategy) is a kind of strategywith function of competitively sealing functions of transcriptional factors viaexogenetic cis-elements. It is evident that this kind of strategy can be applied toachieve our objective. However some defects of TFD strategy block our way,such as the low efficiency of function molecules ODN (oligo-deoxynucleotide)into cell nucleus. Although several settlements are developed such asanti-degradation against lysoome or cross bonding of NLS (nuclear localizationsignal peptide), they cannot be widely applied for high cost or complicatedtechniques.The process of double-stranded RNA expression of hair-pin shRNA attractedour focus then. What we images was whether double-stranded RNA with similarsequences with ODN (T>U) could have similar sealing function with ODN. If ourimaging could be achived, not only the cost would be reduced, but the efficiencyof function molecules into cell nucleus could also be increased via viralpackaging.In the previous study we have demonstrated the expected function of this new kind of RNA (VB-ODN). However the specificity of this kind of RNA andits function on gastric cancer cells remain unknown. The present study willinvestigate this kind of vector for which we wish to apply on the identification oftranscriptional factors associated with drug resistance of gastric cancer.Objectives1. To obtain the direct evidence of the combination between this kind of RNAand nuclear protein via EMSA assay;2. To demonstrate the specificity of this kind of RNA;3. To verify the conclusion in gastric cancer cell lines;4. To identify transcriptional factors associated with drug resistance of gastriccancer via this kind of RNA.Methods1. Further investigation of mechanisms. EMSA assay was used to verify whetherprobes with same structure to target dsRNA bind NFκB protein indirectly ordirectly.2. Specificity investigation. The vectors constructed previously were applied totransferred into HEK293cell lines and Genome Gene ExpressionMicroarray were carried out to demonstrate the specificity of function ofVB-ODN.3. Verification of VB-ODN in adherent gastric cancer cell lines SGC7901andMKN45.4. Designed and synthesized VB-ODN targeted various cis-elements to identifytranscriptional factors associated with drug resistance of gastric cancer..Expression levels of MDR1and MRP1were detected to verify the results.Results1. Directive evidence was obtained via EMSA assay that probes with same structure to target dsRNA bind NFκB protein directly.2. Results of gene CHIP showed that functions and signal pathways of NFκBwere involved into that of screened genes, such as cell proliferation, apoptosis,and canaliculus generation. Cis-elements were also found at the regulatingregains of some of these genes. All of these partly demonstrate the specificityof function of VB-ODN.3. Decreased cell proliferation and increased apoptosis were also observed whenVB-ODN were transferred into gastric cancer cell lines SGC7901andMKN45. And in treated cells mRNA expression level of NFκB did not changemarkedly and levels of downstream genes of NFκB were down-regulated. Allof these mean that VB-ODN can display the similar functions in gastriccancer cells.4. Gastric cancer cell lines SGC7901and SGC7901/ADR were transferred withVB-ODNs targeted cis-elements of different transcriptional factors andtreated with Adriamycin. VB-ODN1(AML sub-group targeted) was theprimarily screened key sub-group associated with induction of drug resistanceof gastric cancer. VB-ODN4(AP-1associated factors) was the selectedVB-ODN which is associated with reversion of drug resistance of gastriccancer. Results of expression levels of MDR1and MRP1were similar as thatin previous studies.ConclusionsIn the present study we made further investigation of mechanism ofVB-ODN which can seal function to transcriptional factor of dsRNA with similarsequence to its cis-element (T>U) and supplied direct evidance. We preliminarilydemonstrated the specificity of this kind of function and the functions ofextending molecules associated with VB-ODN. We demonstrated the function of VB-ODN in gastric cancer cell lines and via this kind of RNA tool primarilyidentified the transcriptional factors associated with Adriamycin resistance ofgasctric cancer. These findings would contribute to develop our knowledge ofmodels of transcriptional factor action, and VB-ODN4has a big potential to be atarget to reverse drug resistance of gastric cancer.