MCPIP1-mediated NFIC Alternative Splicing Inhibits Proliferation Of Triple-negative Breast Cancer Via Cyclin D1-Rb-E2F1 Axis

Part Ⅰ The expression and clinical relevance of MCPIP1 in triple-negative breast cancerObjective To explore the differential expression of monocyte chemotactic protein induced protein 1(MCPIP1)between triple-negative breast cancer(TNBC)tissues and corresponding adjacent normal mammary tissues as well as the differential expression between TNBC cell lines and immortalized mammary epithelial cell line.To analyze the association of MCPIP1 expression with survival and clinicopathological characteristics of TNBC patients.Methods The bioinformatics techniques was applied to analyze the differential expression of MCPIP1 between TNBC tissues and corresponding adjacent normal mammary tissues.The expression level of MCPIP1 was detected by immunohistochemistry(IHC)staining in TNBC microarray.Kaplan–Meier survival curve was performed to assess the relationship between MCPIP1 expression and overall survival(OS).The Chi-square test was performed to analyze the association between clinicopathological parameters of TNBC patients and MCPIP1 expression.Quantitative real-time PCR(q RT-PCR)and western blot were performed to detect MCPIP1 expression in TNBC cell lines and immortalized mammary epithelial cell line.Result MCPIP1 expression was markedly downregulated in TNBC tissues and cell lines compared to the corresponding normal samples and cell line.High MCPIP1 levels were correlated with prolonged OS of TNBC patients(hazard ratio,3.281;95% confidence interval,1.341–8.028;P = 0.043).However,MCPIP1 expression levels were not significantly associated with the clinicopathological characteristics,including age,grade,tumor size,lymph node status,and TNM staging.Conclusion MCPIP1 was downregulated in TNBC tissues and cell lines,and high MCPIP1 expression was highly predictive of better prognosis in TNBC patients.These results suggested that MCPIP1 might be a reliable prognostic factor of TNBC.Part Ⅱ The effect of MCPIP1 on the cell cycle and proliferation of TNBC cellsObjective To investigate the effect of MCPIP1 on TNBC cell proliferation and cell cycle.Methods CCK8,colony formation assay,Ed U assay and flow cytometry were performed to detect cell viability,cell proliferation and cell cycle of TNBC cells(MDAMB-231 and MDA-MB-157)in which MCPIP1 was overexpressed and silenced.Xenograft models were constructed to detect the effect of MCPIP1 on the growth of subcutaneous xenograft.Result CCK-8 assay showed that enforced expression of MCPIP1 significantly suppressed the viability of TNBC cells.Colony formation assay and Ed U assay showed that MCPIP1 overexpression suppressed TNBC cell proliferation.Cell cycle analysis showed that MCPIP1 over-expressing TNBC cells exhibited a higher proportion of cells within the G0/G1 stage and lower in the S stage.In contrast,silencing MCPIP1 significantly increased TNBC cell proliferation and promoted cell cycle transition from the G0/G1 stage to the S stage.Animal experiment showed that enforced expression of MCPIP1 hindered tumor growth.Conclusion MCPIP1 inhibited cell proliferation and prevents cell cycle progression of TNBC cells which functioned as a tumor suppressor in TNBC.Part Ⅲ Identification of MCPIP1-dependent AS events in TNBC cells and exploration of the mechanism of MCPIP1 regulation of NFIC ASObjective To identify MCPIP1-regulated ASEs(alternative splicing events)in TNBC cells and to explore the mechanism by which MCPIP1 regulated NFIC alternative Splicing(AS).Methods RNA-seq analysis was used to identify MCPIP1-regulated alternative splicing events(RASEs)in MDA-MB-231 cells after MCPIP1 overexpression.Further functional analysis was performed to investigate the potential function of MCPIP1-regulated alternative splicing genes(RASGs)in TNBC cells.q RT-PCR was performed to detect the ratio of the exons 9 and 10 skipping when MCPIP1 was overexpressed in MDA-MB-231 and MDA-MB-157 cells.q RT-PCR was performed to detect CTF5 expression level in MDA-MB-231 and MDA-MB-157 cells after MCPIP1 overexpression or knockdown as well as in xenografts.i RIP-seq was performed to obtain a transcriptome-wide binding profile of MCPIP1 in MDA-MB-231 cells.HOMER algorithm was applied to search for overrepresented motifs in MCPIP1-bounding peaks.Minigene experiment was conducted to verify that MCPIP1 regulates the skipping of exons 9 and 10 in the NFIC.Result RNA-seq data showed that a total of 762 ASEs were associated with MCPIP1 expression in MDA-MB-231 cells,including 153 p MXE,22 5p MXE,127 A3 SS,16 A3SS&ES,154 A5 SS,17 A5SS&ES,129 ES,152 IR,26 MXE,and 104 cassette exon.Functional analysis revealed that these RASGs were mainly enriched in the mitotic cell cycle,DNA replication,Erb B signaling pathway,and Notch signaling pathway.Further analyzing RNA-seq data,we found that the ratio of exons 9 and 10 skipping in NFIC which generates the 428-amino-acid-long CTF5(ENST00000341919.7)was increased after MCPIP1 overexpression.The results of q RT-PCR also showed that ratio of exons 9 and 10 skipping in NFIC was increased after MCPIP1 overexpression in MDA-MB-231 and MDA-MB-157 cells.Moreover,q RT-PCR assay showed that MCPIP1 overexpression increased the expression of CTF5,whereas MCPIP1 knockdown resulted in decreased expression of CTF5.We also confirmed that enforced expression of MCPIP1 upregulated CTF5 expression in xenografts by q RT-PCT.i RIP-seq data showed that MCPIP1 selectively bound to gene regions including the CDS,3′UTR,5′UTR,and intron regions.Integrated analysis showed that 154 genes overlapped between MCPIP1-RASGs and MCPIP1 binding peaks in two replicates and NFIC was one of them.In addition,we confirmed MCPIP1 binding to NFIC using quantitative RIP-PCR in two replicate i RIP samples.We found the over-presentation of CGGCCG motif in both replicated i RIP samples using HOMER algorithm.The results of minigene experiment showed that a significant increase in exon 9 and 10 skipping in constructed NFIC wide-type minigene in MCPIP1-overexpression cells compared with the vector group,whereas no significant change in exon 9 and 10 skipping was observed between vector and MCPIP1-overexpression groups in NFIC mutant minigene.Conclusion MCPIP1 extensively regulated ASEs in TNBC cells and MCPIP1-RASGs was significantly correlated with the regulation of cell cycle progression and proliferation in TNBC.MCPIP1 directly bound the pre-m RNA of NFIC to regulate exon 9 and 10 skipping,promoting the production of CTF5.Part Ⅳ The mechanism of MCPIP1 inhibition of TNBC cell proliferationObjective To investigate the role of CTF5 in TNBC cells and whether MCPIP1 inhibits TNBC cell proliferation via upregulating CTF5 to inhibit cyclin D1-Rb-E2F1 axis.Methods The bioinformatics techniques were applied to analyze the correlation of MCPIP1 expression with CTF5 expression as well as the differential expression of CTF5 between TNBC tissues and normal tissues.CCK8,colony formation assay,Ed U assay and flow cytometry were performed to detect cell viability,cell proliferation and cell cycle of MDA-MB-231 and MDA-MB-157 cells after CTF5 overexpression;meanwhile western blot was performed to detect the expression level of cyclin D1,pR,t-Rb,E2F1.MDA-MB-231 and MDA-MB-157 cells were co-transfected with CTF5 overexpression plasmid and si MCPIP1,colony formation assay and Ed U assay were performed to detect cell proliferation;flow cytometry was performed to detect cell cycle;western blotting was performed to detect the expression level of cyclin D1,p-R,t-Rb,E2F1.MDA-MB-231 and MDA-MB-157 cells were co-transfected with CTF5 overexpression plasmid and cyclin D1 overexpression plasmid,colony formation assay and Ed U assay were performed to detect cell proliferation;flow cytometry was performed to detect cell cycle;western blotting was performed to detect the expression level of CTF5,cyclin D1,p-R,t-Rb,E2F1.Western blotting was performed to detect the expression level of cyclin D1,p-R,t-Rb,E2F1 when MCPIP1 was overexpressed or silenced in MDA-MB-231 and MDA-MB-157 cells.IHC staining was performed to detect the expression level of MCPIP1,cyclin D1 and Ki67 in xenografts.Immunofluorescence assay was performed to determine CTF5 subcellular localization.Integrative Genomics Viewer(IGV)was used to predict CTF5 binding site in cyclin D1 promoter.Ch IP assay was performed to detect whether CTF5 binds to the promoter of cyclin D1 in MDA-MB-231 and MDA-MB-157 cells.Luciferase reporter assay was performed to detect whether CTF5 inhibits cyclin D1 transcription.Result Bioinformatics analysis showed that there was a positive correlation between MCPIP1 and CTF5 expression in clinical TNBC samples from TCGA.Although there was no significant difference in CTF5 expression between TNBC tissues and normal tissues,we observed a trend of reduction of CTF5 expression in TNBC tissues.CCK-8,colony formation,and Ed U assays showed that overexpression of CTF5 markedly hindered TNBC cell proliferation.Meanwhile,cell cycle analysis showed that elevated expression of CTF5 inhibited cell cycle transition from G0/G1 to S stage in MDA-MB-231 and MDA-MB-157 cells.The results of the rescue experiments displayed that the acceleration in cell proliferation and cell cycle preogression induced by MCPIP1 downregulation was inhibited by restoration of CTF5 expression.Cyclin D1 expression was notably decreased in CTF5-overexpressing cells,which was accompanied by the reduction in phosphorylated retinoblastoma(p-Rb)and E2F1 levels.In contrast,the expression level of total Rb(t-Rb)remained unchanged.Furthermore,overexpression of cyclin D1 restored the impaired proliferation,stagnant cell cycle,and the decreased p-Rb and E2F1 levels.Overexpression of MCPIP1 markedly downregulated the expression of cyclin D1,p-Rb and E2F1,while knockdown of MCPIP1 significantly upregulated the expression of cyclin D1,p-Rb and E2F1.The result of IHC showed that enforced expression of MCPIP1 suppressed the expression of cyclin D1 and Ki67 in xenografts.Western blot assay showed restoration of CTF5 resulted in reduced cyclin D1 accumulation induced by MCPIP1-knockdown.Meanwhile,the downstream signaling molecules p-Rb and E2F1 both showed a corresponding reduction.Immunofluorescence assay showed that CTF5 was mainly located in the nucleus of TNBC cells.The result of IGV showed that the cyclin D1 promoter harbored one potential CTF5 binding site(chr11:69455832-69456066).The Ch IP assay showed that CTF5 can directly bind to cyclin D1 promoter.Luciferase reporter assay showed that CTF5 inhibited cyclin D1 transcription.Conclusion CTF5 functions as a tumor suppressor in TNBC.CTF5 downregulated cyclin D1 expression by directly repressing cyclin D1 transcription.MCPIP1 inhibited TNBC proliferation by upregulating CTF5 to repress cyclin D1-Rb-E2F1 axis.