MBNL2 Regulates DNA Damage Response Via Stabilizing P21

International Journal of Molecular Sciences

Article MBNL2 Regulates DNA Damage Response via Stabilizing p21

Jin Cai 1,2, Ningchao Wang 1,2, Guanglan Lin 1,2, Haowei Zhang 1,3, Weidong Xie 1,3 , Yaou Zhang 1,3 and Naihan Xu 1,3,*

1 State Key Laboratory of Chemical Oncogenomics, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China; [email protected] (J.C.); [email protected] (N.W.); [email protected] (G.L.); [email protected] (H.Z.); [email protected] (W.X.); [email protected] (Y.Z.) 2 Open FIESTA Center, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China 3 Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China * Correspondence: [email protected]

Abstract: RNA-binding proteins are frequently dysregulated in human cancer and able to modulate tumor cell proliferation as well as tumor metastasis through post-transcriptional regulation on target genes. Abnormal DNA damage response and repair mechanism are closely related to genome instability and cell transformation. Here, we explore the function of the RNA-binding protein muscleblind-like splicing regulator 2 (MBNL2) on tumor cell proliferation and DNA damage response. Transcriptome and gene expression analysis show that the PI3K/AKT pathway is enriched in MBNL2- depleted cells, and the expression of cyclin-dependent kinase inhibitor 1A (p21CDKN1A) is signiﬁcantly affected after MBNL2 depletion. MBNL2 modulates the mRNA and protein levels of p21, which is independent of its canonical transcription factor p53. Moreover, depletion of MBNL2 increases the phosphorylation levels of checkpoint kinase 1 (Chk1) serine 345 (S345) and DNA damage response, and the effect of MBNL2 on DNA damage response is p21-dependent. MBNL2 would further alter tumor cell fate after DNA damage, MBNL2 knockdown inhibiting DNA damage repair and DNA damage-induced senescence, but promoting DNA damage-induced apoptosis. Citation: Cai, J.; Wang, N.; Lin, G.; Zhang, H.; Xie, W.; Zhang, Y.; Xu, N. Keywords: MBNL2; RNA-binding protein; p21; DNA damage response; Chk1; cancer MBNL2 Regulates DNA Damage Response via Stabilizing p21. Int. J. Mol. Sci. 2021, 22, 783. https:// doi.org/10.3390/ijms22020783 1. Introduction CDKN1A Waf1/Cip1 Received: 20 November 2020 The cyclin-dependent kinase inhibitor p21 , also known as p21 , has Accepted: 12 January 2021 multiple functions in cell cycle, apoptosis, DNA replication/repair and transcriptional reg- Published: 14 January 2021 ulation in response to a variety of stimuli, including ionizing, UV radiation and genotoxic stress [1,2]. The tumor suppressor p53 acting as the main transcriptional factor of p21, Publisher’s Note: MDPI stays neu- various stresses upregulating p53 activity will subsequently induce p21 expression. G1/S tral with regard to jurisdictional clai- cell cycle progression is prevented by p21 primarily through its inhibition on CDK2 activity, ms in published maps and institutio- which is indispensable for the phosphorylation of Rb and the consequent activation of nal afﬁliations. E2F-dependent gene expression [3,4]. Thus, attributed to the role of cyclin-dependent kinase inhibitor, p21 promotes cell cycle arrest after DNA damage through transcriptional repression on a set of cell cycle-regulatory genes [1]. The expression of p21 is tightly regulated at both transcriptional and post-transcriptional Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. levels. RNA-binding proteins play a crucial role in post-transcriptional regulation of pre- This article is an open access article mRNAs, involved in capping, splicing, polyadenylation, export, translation, stability, and distributed under the terms and con- other mRNA maturation processes. It has been reported that many RNA-binding proteins ditions of the Creative Commons At- post-transcriptionally regulate p21 mRNA stability. For instance, RNPC1, Rbm24, HuR, 0 tribution (CC BY) license (https:// and HuD bind to the AU-rich elements (AREs) within the 3 untranslated region (UTR) of creativecommons.org/licenses/by/ p21 transcript and increase p21 mRNA stability [5–8]. In contrast, PCBP1, PCBP2, PCBP4, 0 4.0/). and hnRNP K bind to the CU-rich elements in the 3 UTR of p21 mRNA and negatively

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regulate its stability [9–11]. FXR1P muscle-specific isoforms directly interact with p21 mRNA through a conserved G-quadruplex located in its 30UTR and subsequently reduce its half-life [12]. Muscleblind-like (MBNL) proteins are a family of deeply conserved RNA-binding proteins that regulate developmentally programmed alternative splicing [13,14]. Both MBNL and CUGBP Elav-like family (CELF) proteins play predominant roles in the pathogene- sis of myotonic dystrophy (DM), in which they are sequestered away from their normal RNA targets, leading to splicing mutations. There are three MBNL paralogs in mammals, MBNL1, MBNL2 and MBNL3, containing highly conserved zinc finger (ZnF) domains which bind preferentially to specific pre-mRNAs to regulate alternative splicing [15,16]. MBNL1 and MBNL2 are broadly expressed across many tissues, whereas MBNL3 primarily exists in the placenta [14]. Except their well-studied functions in DM, recent research has indicated MBNL proteins also actively participate in tumorigenesis and tumor progression through post-transcriptional mechanisms. MBNL1 inhibits tumor metastasis by modulating the stability of DBNL, TACC1, and Snail mRNA [17,18]. MBNL3 activates alternative splicing of lncRNA-PXN-AS1, which increases the expression of PXN to inhibit apoptosis of hepatocellular carcinoma cells [19]. MBNL2 is reported to inhibit tumor growth and invasion in hepatocellular carcinoma [20]; however, the molecular mechanism beyond the anti-tumor effect of MBNL2 is mainly unknown. In this report, we performed transcriptome analysis in control and MBNL2 siRNA- transfected cells. Gene expression profiling and KEGG pathway enrichment analysis show that the PI3K/AKT pathway is enriched in MBNL2-depleted cells and p21 is among the most differentially expressed genes. MBNL2 modulates the mRNA and protein levels of p21 independent of p53 but through binding to and post-transcriptionally regulating the stability of p21 mRNA. MBNL2 also affects DNA damage response through controlling the phosphorylation of Chk1. MBNL2 knockdown increases Chk1 S345 phosphorylation and the effect can be rescued by concurrent overexpression of p21. Moreover, MBNL2 alters tumor cell fate upon DNA damage, depletion of MBNL2 inhibiting DNA damage repair and DNA damage-induced cellular senescence, but provoking DNA damage-induced apoptosis.

2. Results 2.1. MBNL2 Modulates the PI3K/AKT Signaling Pathway To study the function of MBNL2 on tumorigenesis and tumor progression, we performed transcriptome and gene expression analysis in the HCT116 cells transfected with control or MBNL2 siRNA. The differentially expressed genes were subjected to KEGG pathway enrichment analysis, the PI3K/AKT signaling pathway was signiﬁcantly enriched in MBNL2-depleted cells (Figure1A). The PI3K/AKT pathway is an intracellular signal transduction pathway that promotes cell survival and proliferation in response to various extracellular signals [21,22]. CDKN1A (p21), a predominant cell cycle inhibitor, is one of the most differentially expressed genes after MBNL2 depletion in the PI3K/AKT pathway. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 14

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FigureFigure 1. MBNL2 1. MBNL2 regulates regulates p21 p21 expression. expression. (A ()A KEGG) KEGG pathway pathway enrichment enrichment analysisanalysis of of RNA-seq RNA-seq data datain the in HCT116 the HCT116 cells cells transfected transfected with with control control or MBNL2 siRNAs siRNAs (left). (left). The The Rich Rich factor factor refers refers to to the ratiothe ratio of the of thenumber number of ofthe the differentially differentially ex expressedpressed genesgenes to to the the total total number number of annotatedof annotated genes. genes.Normalized Normalized gene gene enrichment enrichment score ofscore gene of sets gene in thesets PI3K/AKT in the PI3K/AKT pathway waspathway specially was analyzed specially in analyzeddetail in (right). detail (B(right).) HCT116 (B) cellsHCT116 were cells transfected were transfected with control with or MBNL2 control siRNA, or MBNL2 empty siRNA, vector, empty or vector,MBNL2-myc or MBNL2-myc plasmids plasmids and subjected and subjected to qRT-PCR to qRT-PCR and immunoblotting and immunoblotting analysis. (C) analysis. qRT-PCR ( andC) qRT-PCRimmunoblotting and immunoblotting analysis in HeLa analysis cells in transfected HeLa cells with transfected control or MBNL2with control siRNA, or empty MBNL2 vector, siRNA, or emptyMBNL2-myc vector, or plasmids.MBNL2-myc Abbreviations: plasmids. si,Abbreviations: siRNA; CON, control;si, siRNA; Vec, CON, vector. control; *** p < 0.001. Vec, vector. *** p < 0.001. We then performed qRT-PCR and immunoblotting to verify the regulation of MBNL2 onWe p21 then expression. performed qRT-PCR qRT-PCR analysis and immunoblotti conﬁrmed thatng the to p21 verify transcript the regulation was signiﬁcantly of MBNL2 reduced in MBNL2-depleted HCT116 cells. Immunoblotting showed that the p21 pro- on p21 expression. qRT-PCR analysis confirmed that the p21 transcript was significantly tein level was also markedly reduced after MBNL2 knockdown, whereas ectopic expres- reducedsion in of MBNL2-depleted MBNL2 had no obvious HCT116 effect cells. on Immunoblotting p21 protein expression showed (Figure that1 B).the MBNL2p21 protein levelexhibited was also the markedly same regulatory reduced effect after in MBNL2 HeLa cells, knockdown, both qRT-PCR whereas and immunoblottingectopic expression of MBNL2demonstrating had no thatobvious p21 expression effect on p21 was prot largelyein decreasedexpression in (Figure MBNL2-depleted 1B). MBNL2 HeLa exhibited cells the same regulatory effect in HeLa cells, both qRT-PCR and immunoblotting demonstrating that p21 expression was largely decreased in MBNL2-depleted HeLa cells (Figure 1C). Moreover, we found that CCND1 (cyclin D1), another important cell cycle regulator in the PI3K/AKT pathway, was also differentially expressed after MBNL2 depletion (Figure 1A) and verified by molecular experiments in both HCT116 (Figure 1B) and HeLa cells (Figure 1C).

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(Figure1C ). Moreover, we found that CCND1 (cyclin D1), another important cell cycle reg- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 14 ulator in the PI3K/AKT pathway, was also differentially expressed after MBNL2 depletion (Figure1A ) and veriﬁed by molecular experiments in both HCT116 (Figure1B) and HeLa cells (Figure1C). 2.2. MBNL2 Regulates p21 Expression in a p53-Independent Manner 2.2. MBNL2 Regulates p21 Expression in a p53-Independent Manner The cyclin-dependent kinase inhibitor p21 is one of the most crucial transcriptional targetsThe of cyclin-dependentp53 to induce cell cycle kinase arrest, inhibitor apopto p21sis, is and one cellular of the most senescence crucial transcriptional[1,2]. To deter- minetargets whether of p53 MBNL2 to induce regulates cell cycle p21 arrest,expression apoptosis, through and its canonical cellular senescence transcription [1, 2factor]. To p53,determine we knocked whether down MBNL2 MBNL2 regulates in p53 p21−/− HCT116 expression or HeLa through cells. its In canonical accordance transcription with the decreasefactor p53, in wide we knocked type cells, down p21 MBNL2 mRNA leve in p53l was−/ −significantlyHCT116 or reduced HeLa cells. upon In depletion accordance of MBNL2with the in decrease p53−/− cells in wide (Figure type 2A,B). cells, Similarly, p21 mRNA th levele p21 wasprotein signiﬁcantly level was reducedmarkedly upon re- − − duceddepletion in the of cells MBNL2 transfected in p53 with/ cells MBNL2 (Figure siRNA2A,B). in Similarly,p53-null cells the p21(Figure protein 2C). levelThese was re- sultsmarkedly indicate reduced the regulatory in the cells effect transfected of MBNL2 with on MBNL2 p21 expression siRNA in is p53-null p53-independent. cells (Figure 2C). These results indicate the regulatory effect of MBNL2 on p21 expression is p53-independent.

Figure 2. MBNL2 regulates p21 expression independently of p53. (A) The mRNA levels of MBNL2, p21, and cyclin D1 Figure 2. MBNL2 regulates p21 expression independently of p53. (A) The mRNA levels of MBNL2, p21, and cyclin D1 werewere determined determined in in the the HCT116 HCT116 p53 p53−/−− or/ −(Bor) HeLa (B) HeLap53−/− p53 cells−/ transfected− cells transfected with control with or controlMBNL2 or siRNAs. MBNL2 (C siRNAs.) Immunob- (C) lottingImmunoblotting of MBNL2, of p21, MBNL2, and p21,cyclin and D1 cyclin in the D1 HCT116 in the HCT116 p53−/− p53or HeLa−/− orp53 HeLa−/− cells p53 −transfected/− cells transfected with control with or control MBNL2 or siRNAs.MBNL2 siRNAs.** p < 0.01, ** ***p < p 0.01, < 0.001. *** p < 0.001.

2.3.2.3. MBNL2 MBNL2 Regulates Regulates p21 p21 mRNA mRNA St Stabilityability and and Tumor Tumor Cell Cell Proliferation AA significant significant post-transcriptional post-transcriptional function function of of RNA-binding RNA-binding protei proteinsns is is to to modulate mRNAmRNA stability. stability. To To study study whether whether RNA-binding RNA-binding protein protein MBNL2 MBNL2 is is involved involved in in regulation regulation ofof p21 p21 mRNA mRNA stability, stability, we performed RIP and qRT-PCR analysis. HCT116 HCT116 cells cells were were transfectedtransfected with with His-tagged His-tagged MBNL2 MBNL2 plasmids plasmids and and immunoprecipitated immunoprecipitated with with the the anti-His anti-His antibody.antibody. MBNL2-associated MBNL2-associated RNAs RNAs were were purifi purifieded for for cDNA cDNA synthesis synthesis and and quantified quantified by qRT-PCRby qRT-PCR with with specific specific p21 p21primers. primers. Interest Interestingly,ingly, we found we found that thatp21 p21mRNA mRNA was wasco-im- co- munoprecipitatedimmunoprecipitated with with the theHis-MBNL2 His-MBNL2 protein, protein, indicating indicating MBNL2 MBNL2 could could physically physically as- sociateassociate with with p21 p21 mRNA mRNA (Figure (Figure 3A).3A). Then, Then, we we overexpressed overexpressed or or knocked knocked down MBNL2 inin HCT116 HCT116 cells, cells, treated treated cells cells with with the the transcription transcription inhibitor inhibitor actinomycin actinomycin D D (ActD), (ActD), and and detecteddetected the the half-life half-life of of th thee existing existing p21 p21 mRNA. mRNA. Depletion Depletion of of MBNL2 MBNL2 accelerated accelerated p21 p21 mRNAmRNA decay, decay, whereas whereas ectopic ectopic expression expression of of MBNL2 MBNL2 slightly slightly delayed delayed p21 p21 mRNA mRNA decay, decay, provingproving MBNL2 MBNL2 could could regulate regulate the the stability stability of the p21 transcript (Figure 33B,C).B,C). G1/SG1/S transition transition is is prevented prevented by by p21 p21 due due to to its its function function of of the the cyclin-dependent cyclin-dependent kinase kinase inhibitor,inhibitor, hence, hence, depletion depletion of of p21 p21 can can promote promote cell cell cycle cycle progression progression and and cell cellproliferation prolifera- [1,2];tion [we1,2 ]then; we explore then explore the role the of role MBNL2 of MBNL2 in tumor in tumor cell proliferation. cell proliferation. HCT116 HCT116 cells were cells transfectedwere transfected with control with control or MBNL2 or MBNL2 siRNA siRNAand subjected and subjected to PI/BrdU to PI/BrdU flow cytometry flow cytometry to detect cellto detectcycle distribution. cell cycle distribution. Depletion Depletionof MBNL2 ofin MBNL2creased the increased percentage the percentage of S phase ofcells S phase (Fig- urecells 3D). (Figure We 3alsoD). Weperformed also performed colony colonyformation formation assay; assay;depletion depletion of MBNL2 of MBNL2 significantly signifi- increasedcantly increased the number the number of colonies of colonies whereas whereas ectopic ectopic expression expression of MBNL2 of MBNL2 only only mildly mildly re- ducedreduced cell cell proliferation proliferation (Figure (Figure 3E).3E). These These results results indicate indicate that that MBNL2 MBNL2 modulates modulates cell cellcy- clecycle progression progression and and tumor tumor cell cell proliferation proliferation th throughrough post-transcriptional post-transcriptional regulation regulation on on p21. p21.

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FigureFigure 3. 3.Depletion Depletion of of MBNL2 MBNL2 destabilizes destabilizes p21 p21 mRNA mRNA and and promotes promotes tumor tumor cell cell proliferation. proliferation. ( A(A)) HCT116 HCT116 cells cells transfected transfected withwith His-MBNL2 His-MBNL2 plasmids plasmids were were subjected subjected to to RIP, RIP, and and MBNL2-associated MBNL2-associated p21 p21 mRNA mRNA was was determined determined byby qRT-PCR.qRT-PCR. ( B(B)) HCT116HCT116 cells cells transfected transfected withwith controlcontrol oror MBNL2MBNL2 siRNA,siRNA, (C)) emptyempty vector, or MBNL2-myc plasmids plasmids were were treated treated with with 5 5mg/mL mg/mL ActD ActD for for the the indicated indicated time. time. Norm Normalizedalized expression expression (qRT-PCR) ofof thethe remainingremaining p21p21 mRNAmRNA afterafter ActD ActD treatment treatment was performed using linear regression analysis. (D) Flow cytometry analysis of the percentage for G1, S, and G2/M cells was performed using linear regression analysis. (D) Flow cytometry analysis of the percentage for G1, S, and G2/M cells in in the HCT116 cells transfected with the indicated siRNAs, pulse-labeled with 1 mg/mL BrdU for 30 min before harvesting, the HCT116 cells transfected with the indicated siRNAs, pulse-labeled with 1 mg/mL BrdU for 30 min before harvesting, and subjected to PI/anti-BrdU staining. (E) Representative images (left) and quantification (right) of the colony formation E andassay subjected in the HCT116 to PI/anti-BrdU cells infected staining. with ( the) Representative indicated lentiviral images constructs (left) and and quantification left for 8-day (right) culture. of the ** p colony < 0.01. formation assay in the HCT116 cells infected with the indicated lentiviral constructs and left for 8-day culture. ** p < 0.01. 2.4. MBNL2 Regulates Chk1 Activation and DNA Damage Response 2.4. MBNL2 Regulates Chk1 Activation and DNA Damage Response Functional deficiency of DNA damage response and repair mechanism is closely as- sociatedFunctional with genome deficiency instability of DNA and damage cell transformation. response and Several repair mechanismstudies indicate is closely RNA- associatedbinding proteins with genome also take instability part in and the cellregulation transformation. of DNA Severaldamage studies response, indicate then RNA- affect bindingtumorigenesis proteins and also tumor take partprogression. in the regulation We next ofexplore DNA functions damage response, of the RNA-binding then affect tumorigenesisprotein MBNL2 and in DNA tumor damage progression. response. We HCT116 next explore cells were functions transfected of the RNA-bindingwith control or proteinMBNL2 MBNL2 siRNA/shRNA in DNA damageand then response. treated with HCT116 DNA cells damage were reagent, transfected the topoisomerase with control or I MBNL2inhibitor siRNA/shRNA camptothecin and(CPT) then for treated4 h to in withduce DNA DNA damage damage reagent, response. the topoisomerasePhosphorylation I inhibitorof Chk1 camptothecinby ATM/ATR (CPT)at S345 for within 4 h to the induce C-terminal DNA damage regulatory response. domain Phosphorylation is in particular ofessential Chk1 by for ATM/ATR both the DNA at S345 damage within and the replic C-terminalation checkpoint regulatory responses domain is in in vertebrates. particular essential for both the DNA damage and replication checkpoint responses in vertebrates. Immunoblotting revealed that the phosphorylation levels of checkpoint kinases Chk1 Immunoblotting revealed that the phosphorylation levels of checkpoint kinases Chk1 S345 and ATM S1981 were strongly increased after MBNL2 depletion upon DNA damage S345 and ATM S1981 were strongly increased after MBNL2 depletion upon DNA damage (Figure 4A). On the contrary, ectopic expression of MBNL2 slightly reduced the phos- (Figure4A). On the contrary, ectopic expression of MBNL2 slightly reduced the phosphory- phorylation levels of both kinases (Figure 4B). Next, we performed immunofluorescence lation levels of both kinases (Figure4B). Next, we performed immunofluorescence staining staining to detect the phosphorylation level of Chk1 S345 in HCT116 cells and discovered to detect the phosphorylation level of Chk1 S345 in HCT116 cells and discovered that that depletion of MBNL2 by shRNA increased the fluorescence intensity of Chk1 pS345 depletion of MBNL2 by shRNA increased the fluorescence intensity of Chk1 pS345 staining staining (Figure 4C). Furthermore, we examined the effect of MBNL2 on Chk1 phosphor- (Figure4C). Furthermore, we examined the effect of MBNL2 on Chk1 phosphorylation in ylation in HeLa cells. Similarly, knockdown of MBNL2 enhanced the phosphorylation HeLa cells. Similarly, knockdown of MBNL2 enhanced the phosphorylation level of Chk1 level of Chk1 S345, whereas overexpression showed weak effect (Figure 4D,E). S345, whereas overexpression showed weak effect (Figure4D,E).

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Figure 4. DepletionFigure of4. Depletion MBNL2 upregulates of MBNL2 DNAupregulates damage DNA response. damage (A )response. HCT116 cells(A) HCT116 transfected cells with transfected control or MBNL2 siRNA (left) andwith shRNA control lentiviralor MBNL2 constructs siRNA (left) (right), and (shRNAB) empty lentiviral vector, constructs or His-MBNL2 (right), plasmids (B) empty were vector, treated or with 1 µM CPT for 4 h orHis-MBNL2 mock-treated, plasmids then subjected were treated to immunoblotting with 1 μM CPT analysis.for 4 h or (mock-treated,C) Representative then imagessubjected (left) to im- of Chk1 pS345 immunostainingmunoblotting in the HCT116 analysis. cells transfected(C) Representative with control images or MBNL2(left) of Chk1 shRNA pS345 lentiviral immunostaining constructs and in treatedthe with 1 µM HCT116 cells transfected with control or MBNL2 shRNA lentiviral constructs and treated with 1 CPT for 4 h or mock-treated. Relative fluorescence intensity was quantified (right). (D) Immunoblotting of Chk1 pS345 in μM CPT for 4 h or mock-treated. Relative fluorescence intensity was quantified (right). (D) Im- the HeLa cells transfected with control or MBNL2 siRNA (left) and shRNA lentiviral constructs (right), (E) empty vector, or munoblotting of Chk1 pS345 in the HeLa cells transfected with control or MBNL2 siRNA (left) and MBNL2-GFPshRNA plasmids lentiviral and treated constructs with 1 (right),µM CPT (E for) empty 4 h or vector, mock-treated. or MBNL2-GFP * p < 0.05. plasmids and treated with 1 μM CPT for 4 h or mock-treated. * p < 0.05. 2.5. MBNL2 Regulates DNA Damage Response Independently of p53 but Relying on p21 2.5. MBNL2 RegulatesNext, DNA we Damage investigated Response whether Independently MBNL2 regulatesof p53 but DNARelying damage on p21response in p53-null Next, wecells. investigated Both HCT116 whether and MBNL2 HeLa p53 regu−/−latescells DNA were damage transfected response with controlin p53-null or MBNL2 siRNA cells. Both HCT116and treated and HeLa with p53 CPT−/ for− cells 4 h were to induce transfected DNA damage with control response. or MBNL2 Similar siRNA to the activation in and treated withwide CPT type for cells, 4 h depletionto induce ofDNA MBNL2 damage significantly response. increased Similar to the the phosphorylation activation levels of in wide type cells,Chk1 depletion S345 and of ATM MBNL2 S1981 significantly in both p53 −increased/− cells the (Figure phosphorylation5A,B), indicating levels the regulatory of Chk1 S345 effectand ATM of MBNL2 S1981 in on both DNA p53 damage−/− cells response (Figure is5A,B), p53-independent. indicating the regulatory effect of MBNL2 on DNA damage response is p53-independent.

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− − FigureFigure 5.5. MBNL2 regulates the phosphorylation of Chk1 independently of of p53 p53 but but through through p21. p21. ( (AA)) HCT116 HCT116 p53 p53−/−/ or (orB) (HeLaB) HeLa p53 p53−/−− cells/− cellstransfected transfected with with control control or MBNL2 or MBNL2 siRNA siRNA were were treated treated with with 1 1μµMM CPT CPT for for 4 4 h h or mock-treated. TheThe samplessamples werewere subjectedsubjected toto immunoblottingimmunoblotting analysisanalysis ofof Chk1Chk1 pS345pS345 andand ATMATM pS1981.pS1981. ((CC)) HCT116HCT116 oror ((DD)) HeLaHeLa cellscells werewere transfectedtransfected withwith control,control, MBNL2,MBNL2, p21p21 siRNA,siRNA, respectively,respectively, andand treatedtreated withwith 11 µμMM CPTCPT forfor 44 hh oror mock-treated.mock-treated. SamplesSamples werewere collectedcollected andand subjectedsubjected toto immunoblottingimmunoblotting analysisanalysis ofof Chk1Chk1 pS345.pS345. ((EE)) HeLaHeLa cellscells werewere transfectedtransfected withwith controlcontrol oror MBNL2MBNL2 siRNAsiRNA with/withoutwith/without p21-pGL3 plasmids and treated with 1 µμM CPT forfor 44 hh oror mock-treated.mock-treated. TheThe samplessamples werewere subjectedsubjected toto immunoblottingimmunoblotting analysisanalysis ofof Chk1Chk1 pS345.pS345.

Earlier studies demonstrated that p21 can regulateregulate Chk1 activation both at transcrip- tionaltional and post-translational post-translational levels levels [23,24]. [23,24 ].Therefore, Therefore, wewe examined examined whether whether MBNL2 MBNL2 reg- regulatesulates the theactivation activation of Chk1 of Chk1 through through p21. We p21. transfected We transfected control, control, MBNL2, MBNL2, or p21 orsiRNA p21 siRNAin HCT116 in HCT116 or HeLa or cells HeLa and cells treated and treated cells with cells withCPT CPTfor 4 for h to 4 hinduce to induce DNA DNA damage damage re- response.sponse. As As expected, expected, depletion depletion of of MBNL2 MBNL2 or p21 signiﬁcantlysignificantly increased increased the the phosphoryla- phosphory- tionlation level level of Chk1of Chk1 S345 S345 in both in both HCT116 HCT116 and HeLaand HeLa cells (Figurecells (Figure5C,D). 5C,D). Moreover, Moreover, concurrent con- overexpressioncurrent overexpression of p21 rescued of p21 therescued increased the increased Chk1 phosphorylation Chk1 phosphorylation induced byinduced MBNL2 by knockdown,MBNL2 knockdown, proving thatproving MBNL2 that regulatesMBNL2 regulates Chk1 activation Chk1 activation and DNA and damage DNA response damage throughresponse p21 through (Figure p215E). (Figure 5E).

2.6. MBNL2 Regulates Tumor CellCell FateFate afterafter DNADNA DamageDamage DNA damage response halts cell cycle progression to allow DNA damage repair, whereaswhereas severesevere DNADNA damagedamage leadsleads toto apoptosisapoptosis andand cellularcellular senescence.senescence. SinceSince MBNL2MBNL2 showedshowed a regulatory regulatory effect effect on on DNA DNA damage damage re response,sponse, we we then then investigated investigated its itsimpact impact on ontumor tumor cell cell fate fate upon upon DNA DNA damage. damage. HCT116 HCT116 cells cells transfected transfected with with control or MBNL2MBNL2 shRNAshRNA were treated with CPT for 1212 hh andand thenthen releasedreleased fromfrom thethe drugdrug toto allowallow DNADNA γ damage repair.repair. WeWe used used γH2AX,H2AX, a a sensitive sensitive marker marker of of DNA DN double-strandA double-strand breaks, breaks, to assess to as- γ repairsess repair efficiency. efficiency.H2AX γH2AX disappeared disappeared at 6 h at after 6 h CPTafter releaseCPT release in control in control cells; however,cells; how- it was still detected at 9 h after CPT release in MBNL2-depleted cells, revealing knockdown ever, it was still detected at 9 h after CPT release in MBNL2-depleted cells, revealing of MBNL2 significantly delayed DNA damage repair (Figure6A). We also examined the knockdown of MBNL2 significantly delayed DNA damage repair (Figure 6A). We also effect of MBNL2 on repair of etoposide (Etop)-induced DNA damage. Similarly, depletion examined the effect of MBNL2 on repair of etoposide (Etop)-induced DNA damage. Sim- of MBNL2 delayed repair of Etop-induced DNA damage (Figure6B). Immunofluorescent ilarly, depletion of MBNL2 delayed repair of Etop-induced DNA damage (Figure 6B). Im- staining with the anti-γH2AX antibody in HCT116 cells showed that MBNL2 knockdown munofluorescent staining with the anti-γH2AX antibody in HCT116 cells showed that

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inhibitedMBNL2 knockdown DNA damage inhibited repair asDNA well, damage ﬂuorescence repair inas control well, fluorescence cells declining in control much faster cells afterdeclining CPT much release faster than after that inCPT MBNL2-depleted release than that cells in MBNL2-depleted (Figure6C). cells (Figure 6C).

Figure 6.6. DepletionDepletion ofof MBNL2MBNL2 impairsimpairs DNADNA damagedamage repairrepair andand DNADNA damage-induceddamage-induced senescencesenescence but promotespromotes DNADNA damage-induced apoptosis.apoptosis. ((AA)) HCT116HCT116 cellscells transfectedtransfected withwith control oror MBNL2MBNL2 shRNA lentiviral constructs were treated withwith 11 µμMM CPT,CPT, ((BB)) 1010 µμMM etoposideetoposide (Etop)(Etop) forfor 1212 hh andand releasedreleased intointo aa freshfresh mediummedium forfor thethe indicatedindicated time.time. TheThe samples were collected and subjected to immunoblotting analysis of γH2AX. (C) Representative images of γH2AX immunostain- were collected and subjected to immunoblotting analysis of γH2AX. (C) Representative images of γH2AX immunostaining ing in the HCT116 cells transfected with control or MBNL2 shRNA lentiviral constructs, treated with 1 μM CPT for 12 h, in the HCT116 cells transfected with control or MBNL2 shRNA lentiviral constructs, treated with 1 µM CPT for 12 h, and and released into a fresh medium for the indicated time. (D) HCT116 cells transfected with control or MBNL2 shRNA releasedlentiviral into constructs a fresh mediumwere treated for the with indicated 1 μM CPT time. for ( D0 h) HCT116or 24 h and cells the transfected Cell Counting with controlKit-8 assay or MBNL2 was performed shRNA lentiviralto detect constructspercentage were of cell treated survival. with 1(Eµ)M Immunoblotting CPT for 0 h or 24 of h cleaved and the CellPARP Counting and Casp Kit-8ase assay 3 in wasthe HCT116 performed ce tolls detecttransfected percentage with ofcontrol cell survival. or MBNL2 (E) ImmunoblottingshRNA lentiviral of constructs cleaved PARP (left), and empty Caspase vector, 3 in or the MBNL2-GFP HCT116 cells plasmids transfected (right) with and control treated or MBNL2 with 1 shRNAμM CPT lentiviral for 0, 12, constructs or 24 h. (F (left),) Representative empty vector, images or MBNL2-GFP (left) of SA- plasmidsβ-galactosidase (right) staining and treated in the with HCT116 1 µM CPTcells fortransfected 0, 12, or 24with h. (control,F) Representative MBNL2, p21 images siRNA (left) and of SA-treatedβ-galactosidase with 20 nM stainingCPT for in96 the h. HCT116The percentage cells transfected of SA-β-galactosidase-positive with control, MBNL2, p21cells siRNA was quantified and treated (right). with 20*** nMp < 0.001. CPT for 96 h. The percentage of SA-β-galactosidase-positive cells was quantiﬁed (right). *** p < 0.001.

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Next, we investigated the effect of MBNL2 on DNA damage-induced apoptosis. MBNL2 depletion inhibited cell survival upon DNA damage (Figure6D) and the hydrolytic cleavage of two signiﬁcant markers of apoptosis, Caspase 3 and PARP were increased after MBNL2 knockdown and decreased upon MBNL2 overexpression (Figure6E), indicating activation of DNA damage-induced apoptosis after MBNL2 depletion. DNA damage is a common driving factor of cellular senescence, so we lastly explored the effect of MBNL2 on DNA damage-induced senescence. HCT116 cells were transfected with control, MBNL2, or p21 siRNA and then treated with a low concentration of CPT for 96 h. Senescence- associated β-galactosidase (SA-β-galactosidase) has been widely used as a speciﬁc marker for senescent cells [25,26]. The SA-β-galactosidase assay showed that depletion of MBNL2 or p21 both strongly reduced the percentage of SA-β-galactosidase-positive cells, indicating an inhibitory effect of MBNL2 depletion on DNA damage-induced senescence (Figure6F ). Collectively, these results demonstrate that MBNL2 not only regulates DNA damage response, but also controls tumor cell fate after DNA damage.

3. Discussion The cyclin-dependent kinase inhibitor p21 is a well-known negative regulator of cell cycle and a predominant transcriptional target of p53. p21 plays multiple functions in cell cycle arrest, DNA damage response, apoptosis, and cellular senescence [2]. The expression of p21 is tightly controlled at both transcriptional and post-transcriptional levels [2,27–29]. Many RNA-binding proteins have been reported to regulate p21 mRNA stability through binding to its 30UTR [6–9,30–32]. In this research, we demonstrate that RNA-binding protein MBNL2 is a novel regulator of p21. The regulatory effect of MBNL2 on p21 is p53-independent; MBNL2 instead physically associates with p21 mRNA and modulates its stability. p21 is an active player in DNA damage response, and earlier studies have linked p21 induction to Chk1 inactivation. It is reported that p21 can transcriptionally repress Chk1 expression through the CDK2-pRb-E2F axis [24]. Deﬁcient p21 induction or p21 knockdown is accompanied by increased/prolonged Chk1 activation, which could contribute to G2 arrest maintenance [23]. Here, we show that MBNL2 depletion downregulates p21 expression and induces Chk1 hyperphosphorylation upon DNA damage. And we further prove that MBNL2 regulates Chk1 activation and DNA damage response independent of p53 but relying on p21, concurrent overexpression of p21 rescuing the MBNL2 knockdown- induced Chk1 hyperphosphorylation. Noticeably, the effect of MBNL2 knockdown on p21 expression and Chk1 activation is far greater than that of MBNL2 overexpression. DNA damage response results in checkpoint activation and cell cycle arrest. To protect genome integrity, cells have evolved extensive signaling cascade to coordinate DNA damage repair with cell cycle progression. Upon severe DNA damage, however, cells irreversibly exit the cell cycle and enter senescence or undergo apoptosis [2]. There are several lines of evidence suggesting that p21 protects numerous cell types from genotoxic stress-induced apoptosis by inhibition of CDKs which are essential for activation of Caspase cascade [33,34]. On the other hand, persistent DNA damage response drives cellular senescence, and p21 contributes to the induction of cellular senescence through CDK inactivation and cell cycle arrest [35–37]. p21 maintains the viability of DNA damage- induced senescent cells, treatment with a low concentration of CPT inducing senescence in wild type cells, but apoptosis in p21-null cells [38,39]. In this study, we demonstrate that knockdown of MBNL2 delays DNA damage repair, promotes DNA damage-induces apoptosis, but inhibits cellular senescence. Multiple evidences support the existence of crosstalk between the PI3K/AKT pathway and the DNA damage response [40–43]. We performed transcriptome and gene expression analysis and found that the PI3K/AKT pathway is enriched in MBNL2-depleted cells. Except p21 and cyclin D1, several members of the PI3K/AKT pathway are signiﬁcantly differentially expressed after MBNL2 depletion, hence, MBNL2 may regulate DNA damage Int. J. Mol. Sci. 2021, 22, 783 10 of 14

response and tumor cell proliferation through other unknown mechanisms, which needs further investigation.

4. Materials and Methods 4.1. Cell Culture and Treatment HCT116 (purchased from ATCC, Manassas, VA, USA) and HCT116 p53−/− (pro- vided by Prof. Zeping Hu, Tsinghua University), HeLa (purchased from ATCC) and HeLa p53−/− cells (purchased from EdiGene, Beijing, China) were maintained in RPMI 1640 and DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA), respectively, both supplemented with 10% FBS (Biowest, France) and 1% Antibiotic–Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). For the operation of transient transfection, cells at a density of 2.0 × 105 cells per well were transfected with 50 nM siRNA oligonucleotides (Santa Cruz Biotechnology, siMBNL2, sc-60990; sip21, sc-29427) or 1 µg/mL overexpression plasmids (synthetized from YouBio, Changsha, China) as indicated. All the transfections were performed in an opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA), facilitated by Lipofectamine reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Six hours after transfection, the cells were transferred to a fresh medium. And 48 h after transfection, the cells were harvested and lysed for further experiments. To generate stable MBNL2-depleted and MBNL2-overexpressed cell lines, shMBNL2 and MBNL2-myc lentiviral constructs were purchased from Genechem and infected HCT116 cells. The lentivirus-infected cell lines were cultured under the selection pressure of 2 µg/mL puromycin (Thermo Fisher Scientific, Waltham, MA, USA).

4.2. Transcriptome and Bioinformatics Analysis Transcriptome analysis was performed by Gene Denovo Biotechnology Co. (Guangzhou, China). Briefly, total RNAs of control or MBNL2 siRNA-transfected HCT116 cells were extracted using the RNAiso Plus Reagent (TaKaRa, Mountain View, CA, USA). Then, mRNA was enriched by Oligo (dT) beads fragmented into short fragments and reverse- transcribed with random primers. The cDNA fragments were purified, end repaired, poly(A) added, and ligated to sequencing adapters. The ligation products were size- selected, PCR-amplified, and sequenced. Raw reads were filtered, mapped to the reference genome, reconstructed to transcripts, and annotated. The gene expression level was quantified and differently expressed genes were analyzed using edgeR (version 3.12.1) (http://www.r-project.org/). Genes with a fold change ≥ 2 and a false discovery rate (FDR) < 0.05 were considered significant differentially expressed genes, which were then subjected to enrichment analysis of GO functions and KEGG pathways [32].

4.3. Immunoblotting Cells were rinsed twice with PBS, collected, and lysed in a protein lysis buffer (5 mL 1 M Tris-HCl (pH 8.0), 26 g urea, 1 mL Triton X-100, 2 pieces of protease inhibitor cocktail, extended to the total volume of 100 mL with ddH2O) on ice for 30 min. The cell lysates were centrifuged at 12,000 rpm for 15 min at 4 ◦C and protein concentration was determined using the Bradford assay (Beyotime, Shanghai, China). The cell lysates were then supplemented with a loading buffer (8% SDS, 0.5% bromophenol blue, 40% glycerol, 8% β-mercaptoethanol) and boiled at 100 ◦C for 10 min. Proteins were separated using an SDS-PAGE gel and electrotransferred onto nitrocellulose membrane. Membrane was successively blocked in TBST with 5% milk, incubated with primary antibodies (anti-MBNL2, ab171551, 1:750; anti-Chk1 pS345, #2348, 1:1000; anti-Chk1, sc-8408, 1:500; anti-ATM pS1981, ab81292, 1:500; anti-GAPDH, abs118936, 1:20000; anti-p21, #2947, 1:1000; anti-γH2AX, #9718, 1:1000; anti-p53, 10442-1-AP, 1:8000; anti-cyclin D1, #29787, 1:500; anti-β-actin, 60008-1-Ig, 1:1000; anti-Caspase 3, #9662, 1:1000; anti-cleaved PARP, #9541, 1:1000) overnight at 4 ◦C, and then conjugated with KPL peroxidase-labeled goat anti-rabbit/mouse secondary antibodies (1:5000, 52200336, SeraCare, Milford, MA, USA) at room temperature for 1 h. Membrane was ﬁnally supplemented with the Pierce ECL Int. J. Mol. Sci. 2021, 22, 783 11 of 14

Western Blotting Substrate (Thermo Fisher Scientiﬁc) and imaged with an autoradiography ﬁlm (FUJIFILM, Minato, Japan).

4.4. qRT–PCR The cells were homogenized using 1 mL RNAiso Plus Reagent (TaKaRa, Mountain View, CA, USA), supplemented with 200 µL chloroform, and vibrated brieﬂy. The mixture was then centrifuged (12,000 rpm, 4 ◦C, 15 min) into three layers. The clear top layer was carefully pipetted into a new tube. Equal volume of isopropanol was added, and total RNA was extracted after centrifugation (12,000 rpm, 4 ◦C, 10 min). The RNA pellet was then rinsed twice with 75% ethanol, dried, and dissolved in RNAase-free water. Complementary DNA was synthesized from 2 µg RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) and qPCR was performed with SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) and primer pairs on a qTOWER 2.2 (Jena, Germany). The relative expression of target genes was normalized to GAPDH by the 2−∆∆CT method. Primer sequences are listed as follows: MBNL2, forward primer 50-TCAAAGAGGAACATGCTCACG-30, reverse primer 50 -AACGGCCCTTTAGGGAATCAA-30; p21, forward primer 50-TGTCCGTCAGAACCCATGC-30, reverse primer 50-AAAGTC GAAGTTCCATCGCTC-30; cyclin D1, forward primer 50-ACAAACAGATCATCCGCAAACAC-30, reverse primer 50-TGTTGGGGCTCCTCAGGTTC-30; GAPDH, forward primer 50-AAGGCTGTGGGCAAGG-30, reverse primer 50-TGGAGG AGTGGGTGTCG-30.

4.5. Immunofluorescence The cells (2.0 × 105) were plated onto a 20 mm circle cover glass and treated as indicated. The cells were first fixed with 4% formaldehyde for 10 min, and permeabilized with 0.25% Triton X-100 for 15 min. The cells were then successively blocked in PBS with 3% BSA for 1 h, probed with primary antibodies (anti-Chk1 pS345, #2348, 1:50; anti-γH2AX, 05-636, 1:250) overnight at 4 ◦C, and incubated with Alexa Fluor 488 or 555- conjugated secondary antibodies (1:400, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. The cells were finally soaked in DAPI (Vector Laboratories, Burlingame, CA, USA), mounted on slides, sealed with nail polish, and dried in the dark. Images were acquired with a FLUOVIEW FV1000 (Olympus, Shinjuku, Japan) and quantified by ImageJ.

4.6. Flow Cytometry The cells were first incubated with BrdU (1 mg/mL) for 0.5 h before harvesting. Next, the cells were rinsed twice with PBS, trypsized to single cells, and resuspended in a growth medium. Cell pellets were then fixed with 4 ◦C 70% ethanol/PBS for at least 30 min, permeabilized with 4N HCl at room temperature for 15 min, and resuspended in PBT (PBS + 0.5% BSA + 0.1% Tween 20). Finally, cell pellets were successively incubated in 200 µL PBT containing 1/40 dilution of anti-BrdU antibody (#5292, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 30 min, 200 µL PBT containing 1/40 dilution of Alexa Fluor 488-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 30 min, 1 mL PBS containing 250 µg/mL RNase A and 25 µg/mL PI at room temperature for 30 min. The percentage of different phases of cell cycle were analyzed (10,000 events for each sample) using a BD Accuri C6 flow cytometer (San Jose, CA, USA).

4.7. RNA-Binding Protein Immunoprecipitation (RIP) The cells were transfected with empty vector or His-MBNL2 plasmids for 48 h and then the RIP assay was performed using a Magna RIP™ Kit (Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. In brief, the cells were harvested in the Int. J. Mol. Sci. 2021, 22, 783 12 of 14

RIP lysis buffer and incubated with rabbit IgG (ab172730) or anti-His-Tag (66005-1-Ig)- conjugated magnetic beads. RNAs bound by the immunoprecipitation magnetic complex were extracted and subjected to qRT-PCR analysis with the p21 primer.

4.8. Senescence-Associated β-Galactosidase (SA-β-Galactosidase) Assay The cells were transfected with indicated siRNAs for 48 h and then treated with 20 nM CPT for 96 h. The SA-β-galactosidase assay was performed using an SA-β-galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA, #9860) according to the manufacturer’s protocol.

4.9. Colony Formation Assay The cells (500–1000) were plated in 6 cm dishes and left for 8–10 days to allow colony formation. Then, colonies were rinsed with PBS, ﬁxed with 4% formaldehyde for 15 min, and stained with 4% crystal violet for 30 min. Images were acquired and the number of colonies was counted with ImageJ.

4.10. Cell Counting Kit-8 (CCK-8) Assay The cells (5 × 103) were plated into 96-well plates with four repeats and treated as indicated. Then, the cells were transferred to a fresh medium supplemented with 10% CCK-8 solution for additional 2 h. After a gentle shake, OD450 values of each well were detected and analyzed by GraphPad Prism 7.0.

4.11. Statistical Analysis The data were analyzed as the mean ± standard deviation (SD) of three independent experiments. All statistical analysis was performed using a two-tailed Student’s t-test. Statistical signiﬁcance was depicted as follows: * p < 0.05; ** p < 0.01, *** p < 0.001.

Author Contributions: Conceptualization, N.X.; methodology, J.C. and N.X.; validation, J.C. and N.X.; formal Analysis, H.Z.; investigation, J.C., N.W., and G.L.; writing—original draft preparation, J.C.; writing—review and editing, N.X.; visualization, J.C.; supervision, N.X.; project administration, W.X., Y.Z., and N.X.; funding Acquisition, N.X. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Natural Science Foundation of China (No. 31571418) and the Shenzhen Science and Technology Innovation Commission (JCYJ20170817161908969). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

PI3K Phosphatidylinositol 3-kinase MBNL2 Muscleblind-like splicing regulator 2 CPT Camptothecin GAPDH Glyceraldehyde 3-phosphate dehydrogenase CDK Cell cycle-dependent kinase ATM ataxia telangiectasia mutated ATR ataxia telangiectasia mutated and RAD3-related Chk1 Checkpoint kinase 1 siRNA Small interfering RNA shRNA Short hairpin RNA RIP RNA-binding protein immunoprecipitation qRT–PCR Quantitative reverse transcription PCR KEGG Kyoto Encyclopedia of Genes and Genomes Int. J. Mol. Sci. 2021, 22, 783 13 of 14

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