Fig. 3
LncRNA ROR promotes H3K4m3 on TIMP3 by recruiting MLL1, thus further promoting the transcription of TIMP3. MCF-7 cells were treated with sh-TIMP3/oe-TIMP3 and sh-lncRNA ROR/oe-lncRNA ROR. a The mRNA expression of TIMP3 in breast cancer tissues and adjacent normal tissues determined by RT-qPCR. *p < 0.05 vs. the adjacent normal tissues. b The protein expression of TIMP3 after MCF-7 cells were transfected with sh-TIMP3 or oe-TIMP3 determined by Western blot analysis. c The protein levels of Vimentin, N-cadherin, MMP-2, MMP-9, Ki67, PCNA, Bcl-2, E-cadherin, Bax and Cleaved caspase-3 in MCF-7 cells transfected with sh-TIMP3 or oe-TIMP3 determined by Western blot analysis. d Apoptosis of MCF-7 cells transfected with sh-TIMP3 or oe-TIMP3 measured by flow cytometry. e Invasion of MCF-7 cells transfected with sh-TIMP3 or oe-TIMP3 measured by Transwell assay. f Proliferation of MCF-7 cells transfected with sh-TIMP3 or oe-TIMP3 measured by EdU assay. b–f *p < 0.05 vs. that of the cells transfected with sh-NC, #p < 0.05 vs. that of the cells transfected with oe-NC. g The protein expression of TIMP3 after MCF-7 cells were transfected with sh-lncRNA ROR or oe-lncRNA ROR determined by Western blot analysis. h The localization of lncRNA ROR in MCF-7 cells detected by FISH assay. i The MLL1 enrichment after lncRNA ROR overexpression and silencing detected by RNA-pull-down assay. j The level of lncRNA ROR pulled-down with MLL1 determined by RIP assay. k The expression of TIMP3 in MCF-7 cells transfected with oe-MLL1 or sh-MLL1 measured by RT-qPCR. l The expression of TIMP3 in MCF-7 cells transfected with oe-MLL1 or sh-MLL1 measured by Western blot assay. m The expression of TIMP3 in MCF-7 cells transfected with oe-MLL1 or sh-MLL1 measured by qPCR after ChIP assay using antibody to H3K4me3. n The expression of TIMP3 in MCF-7 cells transfected with oe-lncRNA ROR or sh-lncRNA ROR measured by qPCR after ChIP assay using antibody to H3K4me3. In j–p *p < 0.05 vs. that of the cells transfected with sh-NC, #p < 0.05 vs. that of the cells transfected with oe-NC. The above data are measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups are analyzed by independent sample t test. The experiment is repeated 3 times. RT-qPCR, reverse transcription quantitative polymerase chain reaction; TIMP3, tissue inhibitors of metalloproteinase 3; EdU, 5-Ethynyl-2′-deoxyuridine; MMP, matrix metalloproteinase; ChIP, Chromatin immunoprecipitation; FISH, Fluorescent in Situ Hybridization; PCNA, proliferating cell nuclear antigen; Bax, Bcl-2-associated protein x; Bcl-2, B cell lymphoma 2; NC negative control