Discussion About Effects And Mechanism Of Rad On Ovarian Cancer Cells Skov3Migration And Invasion

The incidence of ovarian cancer in gynecologic malignancies was in third place.Due to theincidence of occult, when most patients has already diagnosed,it has occurred with metastasis,which is the reason for the high mortality rate, and therefore the recurrence rate was as high as50%-80%, resulting in5-year survival rate was only25%-30%.Existing treatments can notcontrol the recurrence of ovarian cancer,and it is difficult to prolong survival of patients.Inrecent years, gene therapy for cancer treatment is becoming the research focus, especially theemergence and development of RNA interference technology, and clinical drug development ofRNA interference, which make it possible for gene therapy on ovarian cancerMetastasis and invasive is the most important biological characteristics of ovariancancer,cell migration is started by the formation of pseudopodia and the adhesion with thematrix, it was found that the small G protein Rho GTPases superfamily, especially Rac1andCdc42, take participate in this process regulation, thus affect tumor cell migration andinvasion.In this article Rac1was selected as a target, to interfere with cell pseudopodiaformation, and its mechanism was studied,which would provided a new method for ovariancancer treatment.ObjectiveTo construct the Rac1RNA interference vectors and expression vectors, to observe itseffect on the migration and invasion of ovarian cancer cell Skov3, and to explore its mechanism,to explore new avenues for ovarian cancer treatment.MethodAccording to GenBank accession number of Rac1NM₀06908and shRNA designprinciples, Rac1was selected as the target and4shRNA oligonucleotides was designed.Andapplying Primer Premier5.0and the NCBI's "Make specific primers with Primer-BLAST",geneprimers for amplify Rac1CDS region was designed.Application of recombinant DNAtechnology, Rac1RNA interference vectors containing GFP reporter gene was constructed and,TA recombinant vector of Rac1was constructed according to TA cloning. Application of Rac1RNA interference vectors was identified by applying restriction enzymes BamH â…  or Pstâ… single enzyme digestion, Rac1TA cloning recombinant vector was identified by BamH â… andHind â…¢double enzyme digestion. The cloned gene sequence of positive recombinant vector was detected through sequencing test and the correct of the sequence was analyzedthrough"nucleotide blast" in the NCBI.Rac1of TA cloning recombinant vector was subcloned into the eukaryotic expressionvector pEGFP-C1containing the reporter gene EGFP and was identified by BamHâ…  and Hindâ…¢double restriction enzyme digestion.RNA interference vectors and expression vectors mediated Liposome Lipofectamine2000-n was transfected into human ovarian cancer Skov3, the transfection efficiency wasinitially determined under the fluorescence microscope in48h.Cells were collected and thetransfection efficiency was determined through flow cytometry. The effective RNA interferencevectors and expression vectors was screened and identified by RT-PCR and immunoh-istochemistry.After the effective Rac1RNA interference vector and Rac1expression vectors weretransfected into Skov3cells, and migration and invasion of Skov3cell on the silence andincrease of Rac1was obversed under laser confocal microscope, ultrastructural changes of thetransfected cells was obversed under SEM.Skov3cells transfected Rac1RNA interference and expression vector were respectivelycollected.ThemRNA expression of factors such on MMP-2and TIMP2, IRSp53, WAVE1,WAVE2and Arp2/3weredetected by RT-PCR.Result1. After Rac1RNA interference vector was carried through restriction enzyme Pst â…  orBamH â… single digestion and Rac1TA cloning recombinant vector was carried through BamHâ…  and Hind â…¢double single digestion, agarose gel electrophoresis showed the cloned DNAfragment size was in line with the theoretical value.When the selected recombinant vectors weresequenced, oligonucleotide sequence of four cloned Rac1RNA interference is fully consistentwith the theoretical sequence, the sequence of TA cloning of Rac1was the same as landingsequence with the GenBank.2. Rac1recombinant expression vector pEGFP-C1-Rac1digested by BamH â…  and Hindâ…¢ double restriction enzyme, agarose gel electrophoresis showed there was inserts in it, positiveexpression vector was detected for subsequent screening experiments.3. After Rac1RNA interference vector was transfected into Skov3cell lines in48h,transfection efficiency was preliminary observated under fluorescence microscope, and thetransfection efficiency was29.6%by flow cytometry.When Rac1mRNA expression of Skov3 cells transfected Rac1RNA interference vector was detected by RT-PCR, Rac1mRNAexpression was reduced in Skov3cells transfected pGPU6/GFP/Rac1-524; Rac1proteinexpression of transfected Skov3cells was carried by Immunohistochemical detection, theexpression of Rac1decreased in group transfected pGPU6/GFP/Rac1-524.These showed thatpGPU6/GFP/Rac1-524was valid Rac1RNA interference vectors, so it was chosed forfollow-up experiments.4.Similarly, transfection efficiency was preliminary observated under fluorescencemicroscope after Skov3cells were transfected Rac1expression vector pEGFP-C1-Rac1, and thetransfection efficiency was28%by flow cytometry. Rac1mRNA expression was increased incells transfected pEGFP-C1-Rac1by RT-PCR analysis. Rac1protein expression was increasedin cells transfected with pEGFP-C1group relative to non-transfected group.These prompted thatexogenous Rac1cells can increase Skov3Rac1expression.5.Through the observing, measuring under laser confocal microscope to and scanningelectron microscope,the results showed that in each transfection group, cells transfectedpGPU6/GFP/Rac1-524migrated the shortest distance, disappeared plate-like pseudopodia,decreased invasion capacity; cells transfected pEGFP-C1-Rac1migrated the longerdistance,grew plate-like pseudopodia,increased invasion capacity relative to non-transfectedgroup. These showed that silence of Rac1can block Skov3cell migration, invasion, increasedexpression of Rac1can enhance Skov3cell migration and invasion.6.The expression of MMP2and TIMP2, IRSp53, WAVE1, WAVE2and Arp2/3participating in Rac1signaling pathways were decreased in Skov3cells transfected thepGPU6/GFP/Rac1-524by RT-PCR detection,but these factors expression was increased in cellstransfected pEGFP-C1-Rac1.Conclusion1.Four Rac1RNA interference vector and Rac1expression vectors containing reportergene was successfully constructed;2. Rac1RNA interference reduces the Rac1mRNA and protein expression in transfectedSkov3cell, thus an effective Rac1RNA interference vector was screened out;3. Rac1expression vector can increase the Rac1mRNA and protein expression intransfected Skov3cell;4. silence of Rac1can block Skov3cell invasion, disappeared plate-like pseudopodia.Theblocking mechanism may be that it could reduce the expression of MMP2and TIMP2, IRSp53,WAVE1, WAVE2and Arp2/3变异体1⁵. 5. Increased expression of Rac1can enhance the Skov3invasion ability, increase cell platepseudopodia, which may achieve through increasing the expression of MMP2and TIMP2,IRSp53, WAVE1, WAVE2and Arp2/3变异体1⁵.