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Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor "CFIm25".

ABSTRACT: Cleavage factor "CFIm25", as a key repressor at proximal poly (A) site, negatively correlates to cell proliferation and tumorigenicity in various cancers. Hence, understanding CFIm25 mechanism of action in breast cancer would be a great benefit. To this aim four steps were designed. First, potential miRNAs that target 3'-UTR of CFIm25 mRNA, retrieved from Targetscan web server. Second, screened miRNAs were profiled in 100 breast cancer and 100 normal adjacent samples. Third, miRNAs that their expression was inversely correlated to the CFIm25, overexpressed in MDA-MB-231 cell line, and their effect on proliferation and migration monitored via MTT and wound healing assays, respectively. Fourth, interaction of miRNAs of interest with 3'-UTR of CFIm25 confirmed via luciferase assay and western blot. Our results indicate that CFIm25 considerably down-regulates in human breast cancer tissue. qRT-PCR assay, luciferase test, and western blotting confirm that CFIm25 itself could be directly regulated by oncomiRs such as miR-23, -24, -27, -135, -182 and -374. Besides, according to MTT and wound healing assays of cell lines, CFIm25 knockdown intensifies cell growth, proliferation and migration. Our results also confirm indirect impact of CFIm25 on regulation of mRNA's 3'-UTR length, which then control corresponding miRNAs' action. miRNAs directly control CFIm25 expression level, which then tunes expression of the oncogenes and tumor proliferation. Therefore, regulation of CFIm25 expression level via miRNAs is expected to improve treatment responses in breast cancer.

SUBMITTER: Tamaddon M

PROVIDER: S-EPMC7360588 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor "CFIm25".

Tamaddon Mona M Shokri Gelareh G Hosseini Rad Seyed Mohammad Ali SMA Rad Iman I Emami Razavi Àmirnader À Kouhkan Fatemeh F

Scientific reports 20200714 1

Cleavage factor "CFIm25", as a key repressor at proximal poly (A) site, negatively correlates to cell proliferation and tumorigenicity in various cancers. Hence, understanding CFIm25 mechanism of action in breast cancer would be a great benefit. To this aim four steps were designed. First, potential miRNAs that target 3'-UTR of CFIm25 mRNA, retrieved from Targetscan web server. Second, screened miRNAs were profiled in 100 breast cancer and 100 normal adjacent samples. Third, miRNAs that their ex ...[more]

PMID: 32665581

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Project description:Macrophages play a crucial role in our body’s development and tissue repair and protect against microbial attacks via cytokine secretion or phagocytosis. A previous study in our lab revealed a crucial role for regulation of mRNA 3' end cleavage and polyadenylation (C/P) in monocyte to macrophage differentiation. Among the various C/P factors involved, the subunit CFIm25 showed a striking increase at an early time point upon treatment of monocytes with the differentiating agent Phorbol Myristate Acetate (PMA), suggesting that CFIm25 may promote this process. To study its role in macrophage differentiation, CFIm25 was overexpressed in the HL-60 and THP-1 monocytic cells, followed by PMA-mediated differentiation. Both cell lines showed a significant increase in macrophagic characteristics and a decrease in monocytic features. Cell cycle was slowed down, accompanied by a greater decrease in the proliferation markers PCNA and cyclin D1 coupled with increased 3’UTR lengthening of cyclin D1 mRNA. Since alternative polyadenylation (APA) could be greatly affected by manipulating CFIm25, we performed mRNA 3' end-focused sequencing to reveal changes in the global APA landscape during differentiation and after CFIm25 overexpression. Among the genes whose 3’UTRs were shortened, TAB2 and TBL1XR1 which are known positive regulators of the NF-κβ signaling pathway, underwent shortening followed by increased protein expression compared to the control. Examination of other components of the NF-κβ pathway showed an increase at earlier time in NF-κβ-p50 and phosphorylated NF-κβ-p65 and in the NF-κβ, targets p21, Bcl-XL, ICAM1 and TNF-α upon CFIm25 overexpression There was also an increase in the shorter isoforms of NF-κB1 mRNA that encodes the NF-κβ p50 precursor. Depletion of CFIm25 suppressed the NF-κβ pathway, as evident by lengthening of TAB2, TBL1XR1, and NF-κB1 mRNAs accompanied by decreased expression of TAB2, TBLXR1, and the NF-κβ proteins. In conclusion, our study supports a model in which CFIm25 accelerates the monocyte to macrophage transition by promoting APA events which lead to activation of the NF-κβ pathway.

Project description:BackgroundMacrophages are required for development and tissue repair and protect against microbial attacks. In response to external signals, monocytes differentiate into macrophages, but our knowledge of changes that promote this transition at the level of mRNA processing, in particular mRNA polyadenylation, needs advancement if it is to inform new disease treatments. Here, we identify CFIm25, a well-documented regulator of poly(A) site choice, as a novel mediator of macrophage differentiation.MethodsCFIm25 expression was analyzed in differentiating primary human monocytes and monocytic cell lines. Overexpression and depletion experiments were performed to assess CFIm25's role in differentiation, NF-κB signaling, and alternative polyadenylation (APA). mRNA 3' end-focused sequencing was conducted to identify changes in poly(A) site use of genes involved in macrophage differentiation and function. Cell cycle markers, NF-κB pathway components, and their targets were examined. The role of CFIm25 in NF-κB signaling was further evaluated through chemical inhibition and knockdown of pathway regulators.ResultsCFIm25 showed a striking increase upon macrophage differentiation, suggesting it promotes this process. Indeed, CFIm25 overexpression during differentiation amplified the acquisition of macrophage characteristics and caused an earlier slowing of the cell cycle, a hallmark of this transition, along with APA-mediated downregulation of cyclin D1. The NF-κB signaling pathway plays a major role in maturation of monocytes to macrophages, and the mRNAs of null, TBL1XR1, and NFKB1, all positive regulators of NF-κB signaling, underwent 3'UTR shortening, coupled with an increase in the corresponding proteins. CFIm25 overexpression also elevated phosphorylation of the NF-κB-p65 transcription activator, produced an earlier increase in the NF-κB targets p21, Bcl-XL, ICAM1 and TNF-α, and resulted in greater resistance to NF-κB chemical inhibition. Knockdown of Tables 2 and TBL1XR1 in CFIm25-overexpressing cells attenuated these effects, reinforcing the mechanistic link between CFIm25-regulated APA and NF-κB activation. Conversely, depletion of CFIm25 hindered differentiation and led to lengthening of NFKB1, TAB2, and TBL1XR1 3' UTRs.ConclusionsOur study establishes CFIm25 as a key mediator of macrophage differentiation that operates through a coordinated control of cell cycle progression and NF-κB signaling. This linkage of mRNA processing and immune cell function also expands our understanding of the role of alternative polyadenylation in regulating cell signaling.

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Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor "CFIm25".

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Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor "CFIm25".

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