Background
Methods
Cell culture and treatment
Tissue extraction and grouping
Cell viability
Detection of cell apoptosis by flow cytometry
Hoechst staining
RNA isolation and real-time PCR
Microarray analysis
Western blot analysis
Fluorescence in situ hybridization (FISH)
Bioinformatics analysis of the differentially expressed microRNAs
Statistical analysis
Results
H2O2 treatment decreased the viability of HLE-B3 cells
H2O2 treatment induced apoptosis in HLE-B3 cells
Microarray screening for microRNAs associated with H2O2 induced oxidative stress in HLE-B3 cells
miRNA Name | Fold Change | Forward Primer for RT-PCR |
---|---|---|
miR-630 | 4.14 | GCGAGTATTCTGTACCAGGGAAGGT |
miR-222-5p | 3.84 | CGCTCAGTAGCCAGTGTAGATCCT |
miR-210-3p | 3.23 | CTGTGCGTGTGACAGCGG |
miR-34a-5p | 2.59 | CTGGCAGTGTCTTAGCTGGTTGT |
miR-34b-5p | 2.44 | GCGTAGGCAGTGTCATTAGCTGATTG |
miR-335-3p | 0.45 | CGGCGTTTTTCATTATTGCTCCTGACC |
miR-15b-3p | 0.33 | CGGGCGAATCATTATTTGCTGCTCTA |
The association between the oxidation-induced miRNAs and nuclear opacity
Validation of the levels of miR-34a-5p, miR-630 and miR-335-3p by FISH
Identification of target genes and analysis of the miRNA/target gene network
MiRNA | Clustered target genes |
---|---|
miR-34a-5p | ATG2A, CALCR, SYVN1, RAD9A, TP53INP2, MAPK14, MAPK1, NR4A2, BTBD11, HSPB6, NMUR1, WIPI2, CRHR1, MAP3K15, FIGN, PTK6, TRPV4, BCL6, RRAGD, ATG4B, MAP2K1, TNFRSF11B, PPARG, CCL22, GABARAPL2, ERLIN2, PVR, SUGT1, SRC, GPR111, NFKBIA, MAPK13, XPC, GLP1R, FRK, C11orf34, TLR4, TP53INP1, SGTA, ERLIN1, UBA1, TMX4, UCN2, UNC45A, APITD1, CDCA5, HEBP1, ATM, PSME1, CXCL11, ATG5, MAP3K3, RAD9B, PSME3, DDB1, ULBP2, FES, BCAS3, PRKAG1, PRKAG3, PLA2G6, PPP6C, RAD51, IRAK2, UBE2NL, MAP4K4, GPX3,CXCL10 |
miR-630 | WEE2, MAP3K2, MAP4K3, STK39, DEFB132, MAPK14, ANKRD6, RAD1, ATG12, SGTB, MAP3K1, YES1, RB1CC1, BCL2L2, EDNRB, WEE1, FER, PAK7, DCLRE1C,UBXN2A, RAD18, PARP3, SLK, ATG2B, TMX3, BTBD3, TMX1, GPR37, CDC7, PPP2CA, IRAK3, TP63, RHD, FRK, CXCL13, POLR2D, GPX8, ITPR1, TLR4, SLK, UBE2N, SRPK2, CUL4B, TMX4, NBEAL1, CD226, CCL11, PIK3CA, DDB1, NLK |
miR-335-3p | MAPK9, CADM1, MAP3K2, PRKAA2, MAP4K3, NR4A3, GRAP, MAPK14, EIF2AK4, BTBD11, NLK, ANKRD17, SLA, CCS, UBE2B, FIGN, PPP6C, TXK, ERCC4, PDGFA, GPR126, BCL2L2, SMG1, WEE1, CCL5, UBXN2A, PPARG, FAS, ERLIN2, RYR2, CDC7, SUGT1, MED1, AQP4, TDG, PTH2R, IRAK3, PMS1, UBA6, MICA, TP63, HMGA2, TNFAIP3, CUL4B, SMAP1, PAK3, GABARAPL1, UBE2V2, ATM, MSH3, MAPK10, RAD9B, TMEM161B,RAB23 |
Co-regulated miRNAs | Target genes |
---|---|
miR-34a-5p/miR-630 | DDB1, TMX4, FRK, TLR4 |
miR-34a-5p/miR-335-3p | PPARG, ERLIN2, RAD9B, BTBD11, PPP6C, ATM, FIGN, SUGT1 |
miR-630/miR-335-3p | UBXN2A, BCL2L2, MAP3K2, MAP4K3, NLK, TP63, WEE1, CUL4B, IRAK3, CDC7 |
miR-34a-5p/miR-630/miR-335-3p | MAPK14 |