The Mechanisms Of C3AR1 Gene Promoting HL60 Cells’ Migration And Drug Resistance

Part one.Identification of the Constructed HL60-C3AR1 cell lineObjective: To explore the function of Complement Component 3a Receptor1(C3AR1) in acute myeloid leukemia cell lines, the HL60-C3AR1 cell line was constructed.Methods: 1.The expression of GFP of HL60-C3AR1, HL60-Venus and HL60 was measured by FCM. 2.The m RNA expression level of C3AR1 on HL60-C3AR1,HL60-Venus and HL60 was detected with RT-PCR. 3.The protein expression levels of C3AR1 on HL60-C3AR1, HL60-Venus and HL60 were detected with Western Blot.Results: The GFP expression levels of HL60-C3AR1 and HL60-Venus was more than98% respectively, the m RNA and protein expression levesl were significantly increased in HL60-C3AR1 compared with HL60-Venus and HL60.Conclusion: C3AR1 gene has been successfully upregulated in HL60-C3AR1 cell line.Part two. Over-expression of C3AR1 in HL60 Promotes Cell Migrationand InvasionObjective:To explore the molecular mechanism of C3AR1 on the migration and invasion of AML cells, PCR ARRAY assay related was performed..Methods: The migration and invasion assay were performed used transwell system.PCR ARRAY assay combining with expression of CXCR4 were performed by either Real Time PCR or Flow Cytometry.Results:The ability of migration and invasion was significantly promoted in HL60-C3AR1 cells compared to negative control,without CXCR4 increasing. We found 16 genes including CCL2 were upregulated and 4 genes were downregulated by PCR array which were confirmed by real-time RT-PCR..Moreover,further study on CCL2 indicated its same action on migration and invasion in THP-1 cells.Conclusion: Over-expression of C3AR1 promoted the migration and invasion of HL60 cells, which may be mediated by a series of genes upregulated and downregulated..Part three. The effect of C3AR1 on HL60 cells’ resistance to TOPOⅡinhibitors: Etoposide(VP16),Doxorubicin and Mitoxantrone.Objective: To investigate the mechanism of C3AR1 genes promoting the effect of drug resistance to etoposide, doxorubicin, mitoxantrone in HL60 cells, PCR array related to apoptosis were performed.Methods: 1.HL60-C3AR1,HL60-Venus were treated with different concentrations of etoposide, doxorubicin, and mitoxantrone at 24 h, 48 h, and 72 h, cell toxicity of the two cells were done using CCK8. 2.The apoptosis levels were detected either by Flow cytometry or Hoechst 33342 dyeing test after treating cell with different concentrations of etoposide, doxorubicin, mitoxantrone for 24 hs 4.We used PCR ARRAY assay to explore the potential mechanism after treated cell with different concentrations of etoposide,doxorubicin, and mitoxantrone for 24 hs. 5. The different protein expressions of related genes were detected by Western blot.Results: HL60-C3AR1 showed obvious drug resistance to etoposide, doxorubicin,and mitoxantrone. After treated with etoposide,doxorubicin, mitoxantrone for 24 h,we detected much less apoptosis level in HL60-C3AR1 compared with HL60-Venus. Hoechst33342 staining showed that HL60-C3AR1 had less nucleus fragmentation than HL60-Venus. Through PCR ARRAY assay we found that the BIRC7、SOCS2、LCK gene were downregulated in HL60-C3AR1 and the protien levels of BIRC7、SOCS2、LCK were also downregulated by Western blot detection. Meanwhile, BCL-XL and BCL2 were upregulated and Bax and Bad were downregulated.Conclusion: C3AR1 promoted HL60 cells resistantance to TOPOⅡinhibitors such as Etoposide(VP16),Doxorubicin and Mitoxantrone by regulating BIRC7, LCK, SOCS2 gene through mitochondrial passway.