The Role Of YY1in BRL-3A And IEC-6Cells Under Mimic Hypoxia

【Background】Now, the fact is the data from the animal study can not represent the actual clinicalsituation, new view on the knowing data on trauma hemorrhagic shock was needed and theeffective targeting therapy on hemorrhagic shock needs to be developed. Previously, wedeveloped a left lateral lobotomy and2.5ml/100g body weight hemorrhagic shock ratmodel, in the model, the mortality24h after the trauma hemorrhage treatment close to50%, further microarray gene chip analysis between the death rats and the survivals foundthat about100genes may contribute to the survival benefit, EPHX2and AOC3wereamong these regulation gene networks, putative regulation factor of EPHX2and AOC3were analyzed, YY1is one of these regulation factors.Zinc finger protein YY1was a ubiquitous,conservative,multifunction regulation genes,was thought bind to more than7-10%binding site of human genes. Over expression ofYY1was found involved in the development,progression,infiltration of various cancers,and potential to be another therapy target of cancers. But we still don't know therelationship between YY1and the liver cells and small intestinal mucosa epithelial cellsunder hemorrhagic shock and hypoxia.【Objective】First, the expression of YY1and the apoptosis of cells under hypoxia was detected, andthen, the YY1miR vector was introduced in BRL-3A and IEC-6cells under hypoxia, theexpression of YY1and the apoptosis/proliferation of these cells was evaluated. By theseprotocol, to evaluate the relationship between transcription factor YY1and the function ofBRL-3A,IEC-6cells in vitro.【Methods】1. The expression of YY1in BRL-3A and IEC-6cells under hypoxia by gradient concen-tration (0-500μM)of CoCl2were detected by western blotting and q RT-PCR assay.2. Effective RNAi vector was designed,synthetized and screened, then pLenti6.3-EFPG-YY1-miR vector was packaged by lenti virus,293T cells were infected byvector, the efficiency of infection was evaluated, and optimized multiple of infectionwas confirmed.3. The expression of YY1in BRL-3A/IEC-6cells under hypoxia by100μM CoCl2andnormoxic condition after vector infection were detected by western blotting,immunofluorescence and q RT-PCR assay. 4. The apoptosis and the proliferation of BRL-3A/IEC-6cells under hypoxia by100μMCoCl2and normoxic condition after vector infection were assessed by CCK-8andFlow cytometry assay.【Results】1. The expression of YY1decreased gradually as the increasing of CoCl2on protein andmRNA level24h and72h after treatment on mRNA level, the differential expressionof YY1occurred at the concentration of CoCl2increased to250μM on protein level inIEC-6cells and which occurs under500μM CoCl2treatment in BRL-3A cells.2. From4designed targeting site were evaluated, the effective vector was screened andpackaged by lenti virus, the construction of pLenti-EGFP-YY1-miR vector wassuccessful.3. pLenti6.3-EGFP-YY1-miR down regulated the expression of YY1in BRL-3A andIEC-6cells on protein and mRNA level under normoxic condition. The futherdownregulation of of YY1in BRL-3A and IEC-6cells only on mNRA level whenhypoxia treatment introduced, but not happened on protein level in two cells.4. pLenti6.3-EGFP-YY1-miR induced the apoptosis of BRL-3A cells under normoxiccondition, and aggregated the apoptosis of BRL-3A and IEC-6cells under hypoxia by100μM CoCl2treatment when compared with which under normoxic condition. Theproliferation were slow down in two cells by pLenti6.3-EGFP-YY1-miR vectorintrouduction under both hypoxia and normoxic condition.【Conclusion】1. The expression of YY1decreased and the function of BRL-3A cells attenuated with theincreasing concentration of CoCl2。pLenti-EGFP-YY1-miR vector knockdown theexpression of YY1in BRL-3A and IEC-6cells on protein and mRNA level.pLenti6.3-EGFP-YY1-miR increased the apoptosis and slowed down the proliferationof BRL-3A cells under normoxic condition So, we deduced that the down regulation ofYY1involved in the apoptosis and proliferation change in BRL-3A cells underhypoxia.2. The efficacy of pLenti-EGFP-YY1-miR knowndown assay under hypoxia need to bereassessed for it's ATP dependent feature.