Effects And Mechanisms Of LRP16Gene On The Radiosensitivity And Chemosensitivity Of HeLa Cell

DNA damage is a major risk to cellular homeostasis and genomic integrity. Cellularresponses to DNA damage are crucial for cell survival and for preventing thedevelopment of cancer. DNA damage initiates multiple cellular responses, including theDNA repair pathways, the cell cycle checkpoints, and apoptotic pathways,. Of all DNAlesions, DNA double-strand breaks(DSB) are extremely cytotoxic and can be inducedby IR, radiomimetic chemicals, topoisomerase I and II poisons, oxygen radicals formedin the course of normal metabolism. Being a special member of Macro domainproteins,LRP16previously appeared to be involved in the resistance of tumor cellstowards radiotherapy and chemical drugs. Weidong Han et al.implicated that exogenousexpressed CPNE5interact with NF-κB(P65) directly. Knockdown of LRP16markedlyattenuates NF-κB transcriptional activity with TNFα-induced.We investigated the regulatory role of LRP16on resistance to chemo-and radiationtherapruic agent(IR and ETO).Method: The extent of DNA damage was detected by single cell gel electrophoresis(SCGE).The expression of p-γH2AX was assessed to further test the effect of LRP16toDNA damage.The changes of cell viability and apoptosis of were tested by the methodof CCK-8assay and amphochromophil AnnexinⅤ-PI flow cytometry so as to test theeffect of IR or ETO–resistance. The mRNA levels of target genes was detected NF-kBtranscriptional activity.Results: Overexpression of LRP16desensitized HeLa cells to DNA damage induced byIR or ETO. The CCK-8assay revealed that knockdown of LRP16promoted the cell viability of cells treated with IR or ETO. In contrast to LRP16knockdown,overexpression of LPR16significantly inhibited cell viability. In addition，knockdownof LRP16also sensitizes cells to apoptosis. However,the flow cytometry result showsthat effect of overexpression LRP16to apoptosis depend on DNA damage agents typesand cells types.Knockdown of LRP16markedly attenuates NF-kB transcriptionalactivity with IR or ETO-induced.Conclusions: Our findings indicated that LRP16significantly enhanced the resistance oftumor cells towards radiotherapy and chemical drugs,which is achieved throughupregulating the transcriptional activity of NF-κB. Object： To verify that the association of expression of LRP16and DNA damage levelinduced by etoposide and ionizing radiation.Method：HeLa cells were transientlytransfected with s plasmid pcDNA3.1and pcDNA3.1-LRP16,then were treated withionizing radiation or etoposide which induces DNA damage. Single cell gelelectrophoresis(SCGE) was used test the DNA damage,and tail moment and Olive tailmoment was calculated by CASP software. Effect of overexpression of LRP16onphosphorylation of histone H2AX was examined by flow cytometer.Results: Westernblot demonstrated that the expression of LRP16transiently transfected withpcDNA3.l-LRP16significiently increased compared with the controls. CASP softwareresults indicated that the tail moment and Olive tail moment of LRP16transfected withpCDNA3.l-LRP16was significantly higher than the controls. In response to ETO or IRtreatment, higher levels of damage were observed in cells transfected with pCDNA3.l: the phosphorylation of histone H2AX increased.Conclusion: LRP16played specificrole in DNA damage etoposide and ionizing radiation-induced. Object： To verify that the association of expression of LRP16and apoptosis inducedby etoposide and ionizing radiation. Method： HeLa, H1299，MCF-7cells weretransiently transfected with siRNA （control siRNA,siRNA-374,siRNA-668） ofLRP16gene and then were treated with etoposide and ionizing radiation which inducesDNA damage at indicated time.CCK-8assay was used to detect cell viability, Effect ofthe expression of LRP16on cell apoptosis was examined by Annexin V-FITC apoptosisdetection kit, RT-PCR was used to detect the downstream gene level of NF-κB followedby etoposide and ionizing radiation treating when the expression of LRP16wasinhibited or overexpressed. Results:The cell viability of Hela cells tranfected wassignificiently inhibited(p<0.05).After treated with etoposide and ionizingradiation,apoptotic cells in the group with suppression of the LRP16significientlydecreased compared with the controls(p<0.05).However,LRP16does not alwaysfunction as an anti-apoptotic gene.In the overpression of LRP16group, it appears to apro-apoptotic gene in H1299cells.Conclusion: LRP16played specific role in cellviability and apoptosis. Down-regulation of LRP16expression level could significantlyinhibit transactivation activity of NF-κB induced by etoposede and ionizing radiation,bywhich it may contribute to chemo-and radio-therapeutic resistance.