The Mechanism Of Apoptosis Of MG-63Cells Transfected With SiRNA-FANCD2through P53Signaling Pathway

Background:Fanconi anemia (FA) is a rare autosomal or X-linked recessive hereditarydisorder, which mainly shows aplastic anemia genetic syndrome in children. Thereare15subtypes of FA gene, each of which corresponds to subtype mutation ordeletion, and these FA gene subtypes and proteins interact each other to form the FApathway. FANCD2is the core protein of FA pathway, the critical event of FApathway is the FANCD2ubiquitination and de-ubiquitination. It has been shown thatFANCD2was involved in DNA damage repair, cell cycle regulation, chromatinremodeling, DNA methylation, apoptosis, etc., to maintain the normal function of thebody. Osteosarcoma (OS) is a primary malignant tumor from non-hematopoieticsystem, which serious harm to people’s health, especially the young people. Both FAand OS often occurred in adolescence, and the OS is a complication of FA; however,their interaction is still unclear. In the preliminary studies, we have found thatFANCD2was an important protein in OS, which played an important role in theoccurrence and progression of OS, but its mechanism still needs further study.Therefore, in this study, we will construct the siRNA-FANCD2to transfect MG-63cells; then detect the expression of FANCD2after gene silencing, to explore themechanism of MG-63apoptosis mediated by siRNA-FANCD2, thereby indicatingthe effect of FANCD2in the occurrence and progression of OS.Methods:1) Collect the OS tissues and surrounding normal tissues from Sino-JapaneseFriendship Hospital, extract the total RNA and proteins from the tissues, and detectthe expression of FANCD2gene and protein by RT-PCR and Western blot(WB).2)Construct siRNA-FANCD2plasmid and control siRNA in vitro, then the plasmids were transfected into MG-63cells with liposome, and the control group was set;24h and48h later, the expression of FANCD2protein was determined by WB toevaluate the effect of plasmid transfection.3) In group siRNA-FANCD2, groupcontrol siRNA and control group, after transfection for4,8,12,24and48h, the cellproliferation was detected by CCK8kits.4) In three groups, after transfection for24h and48h, the percentage of G0/G1, S, G2/M phase were detected by PI staining andflow cytometry; meanwhile, the cell apoptosis was detected by flow cytometryfollowed by AnnexinⅤ-FITC staining.5) After transfection of siRNA-FANCD2intoMG-63cells for24h and48h, the total RNA was extracted with TRIzol, and thenwas performed gene chip by Affymetrix GeneChip system to explore the geneexpression changes after silencing FANCD2. The gene chip result was validated byreal-time PCR (Q-PCR). Next, the related signaling pathway of apoptosis wasanalyzed combined with the WB results, to confirm the mechanism of apoptosismediated by FANCD2.Results:1) The FANCD2gene and protein were highly expressed in OS tissues,suggesting that FANCD2was closely related to the occurrence and regression of OS.2)24h and48h after siRNA-FANCD2interfere MG-63cells, the expression ofFANCD2protein was blocked, suggesting that siRNA-FANCD2had excellent genesilencing effect.3) CCK8results showed that cell proliferation wasn’t influenced inthe two control groups, but was inhibited in group siRNA-FANCD2within48h; cellcycle detection results showed that24h and48h after siRNA-FANCD2transfection,the percentage of G0/G1and G2/M phase were significantly increased, indicating thattargeted FANCD2RNAi could induce cell cycle block; AnnexinⅤ-FITC resultsshowed that24h and48h after siRNA-FANCD2transfection, the cell apoptosis wassignificantly increased, and the apoptosis was the main cell death methods, not thenecrosis. All three experimental results illustrated that after FANCD2gene silence,the cell proliferation would be inhibited and the apoptosis would be induced, but theapoptotic mechanism still needs further proof.4) To explore the apoptotic mechanism mediated by siRNA-FANCD2, the gene chip was performed on MG-63cells, we found that48h after siRNA-FANCD2transfection,103gene expressionswere up-regulated, and30genes were down-regulated, which were involved in54pathways in KEGG database. After analysis, we found that p53-caspase signalingpathway was the core event. Therefore, we detected the expression of TP53INP1,p53, p21, caspase-9and caspase-3mRNA with QPCR, and the results showed thatall the five gene expressions were increased; suggesting that targeted FANCD2RNAi could activate the upstream and downstream genes of p53pathway.Furthermore, we detected the expression of p53, phosphorylated-p53, p21,TP53INP1, cleaved caspase-9and cleaved caspase-3proteins, the results showedthat all proteins were increased except p53. The mechanism was that high expressionof TP53INP1could promote p53phosphorylation, phosphorylated-p53couldactivate p21, induce cell cycle block, thereby mediating cell apoptosis throughmitochondria; and the apoptosis was synergic with caspase pathway.Conclusion:The FANCD2gene and protein were highly expressed in OS tissues, suggestingthat FANCD2was closely related to the occurrence and regression of OS. AfterFANCD2gene silence in MG-63cells, the cell proliferation would be inhibited andthe apoptosis would be induced. The apoptosis was mediated by p53signalingpathway, that is, the high expression of TP53INP1could promote p53phosphorylation, phosphorylated-p53could activate p21, induce cell cycle block,and thereby cell apoptosis occurred. Our study provides a new thought for FAcompanied with OS, which has important biological significance.