Prp19 Arrests Cell Cycle Via Cdc5l in Hepatocellular Carcinoma Cells

International Journal of Molecular Sciences

Article Prp19 Arrests Cell Cycle via Cdc5L in Hepatocellular Carcinoma Cells

Renzheng Huang 1, Ruyi Xue 1, Di Qu 2, Jie Yin 1,* and Xi-Zhong Shen 1,2,3,*

1 Department of Gastroenterology, Zhongshan Hospital of Fudan University, Shanghai 200032, China; [email protected] (R.H.); [email protected] (R.X.) 2 Key Laboratory of Medical Molecular Virology, Shanghai Medical College of Fudan University, Shanghai 200032, China; [email protected] 3 Shanghai Institute of Liver Diseases, Zhongshan Hospital of Fudan University, Shanghai 200032, China * Correspondence: [email protected] (J.Y.); [email protected] (X.-Z.S.); Tel.: +86-21-6404-1990 (ext. 2070) (J.Y. & X.-Z.S.); Fax: +86-21-6403-8038 (J.Y. & X.-Z.S.)

Academic Editor: Akihiro Tamori Received: 16 January 2017; Accepted: 31 March 2017; Published: 7 April 2017

Abstract: Pre-mRNA processing factor 19 (Prp19) is involved in many cellular events including pre-mRNA processing and DNA damage response. Recently, it has been identiﬁed as a candidate oncogene in hepatocellular carcinoma (HCC). However, the role of Prp19 in tumor biology is still elusive. Here, we reported that Prp19 arrested cell cycle in HCC cells via regulating G2/M transition. Mechanistic insights revealed that silencing Prp19 inhibited the expression of cell division cycle 5-like (Cdc5L) via repressing the translation of Cdc5L mRNA and facilitating lysosome-mediated degradation of Cdc5L in HCC cells. Furthermore, we found that silencing Prp19 induced cell cycle arrest could be partially resumed by overexpressing Cdc5L. This work implied that Prp19 participated in mitotic progression and thus could be a promising therapeutic target of HCC.

Keywords: pre-mRNA processing factor 19; cell division cycle 5-like; cell cycle; translation; repressing the translation of mRNA; lysosome-mediated degradation

1. Introduction Liver cancer, about 70% to 90% of cases of which are hepatocellular carcinoma (HCC), is the ﬁfth most common cancer in men and the seventh in women, and represents the second leading cause of cancer death among men and the sixth leading cause of cancer death among women worldwide [1]. Although surveillance in high-risk population allows diagnosis of HCC at an early stage, most cases still present at advanced and unresectable stage, and limited therapeutic options are available [2]. The development of HCC is a multistep process driving the progressive transformation of normal hepatocytes into highly malignant derivatives [3]. Among them, cell cycle deregulation is a common feature of human cancer [4]. Therefore, targeting the mitotic stage of the cell cycle may be an effective approach for cancer therapy [5]. The highly conserved Pre-mRNA processing factor 19 (Prp19)-associated complex (Prp19C) is a large multi-faceted protein complex that functions in diverse cellular processes, and novel roles of Prp19C are emerging [6]. Prp19C is a critical component of DNA repair and DNA damage checkpoint complexes, which is linked to deleterious consequences-cancer [7]. Prp19C is mainly composed of Prp19, cell division cycle 5-like (Cdc5L), pleiotropic regulator 1 and breast carcinoma ampliﬁed sequence 2 [8,9]. Among them, Prp19 forms a tetramer as a key architectural component for Prp19C stability [10]. Prp19 possesses E3 activity [11,12], and plays diverse roles in pre-mRNA splicing [13] and DNA damage response [14,15]. Despite disorders of these cellular events being linked to tumorigenesis, the potential role of Prp19 in HCC is still elusive. Cdc5L has been reported to play

Int. J. Mol. Sci. 2017, 18, 778; doi:10.3390/ijms18040778 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 778 2 of 9

Int. J. Mol. Sci. 2017, 18, 778 2 of 9 a crucial role in mitotic progression. Overexpression of Cdc5L in mammalian cells causes shorter G2phase phase and and reduced reduced cell cell size size [16] [ 16and] and knockdown knockdown of Cdc5L of Cdc5L results results in dramatic in dramatic mitotic mitotic arrest arrest [17] and [17 ] andchromosome chromosome misalignments misalignments [18]. [Recently,18]. Recently, both Prp19 both Prp19and Cdc5L and Cdc5L have been have characterized been characterized to be tooverexpressed be overexpressed in HCC in tissues HCC tissuesand their and overexpressi their overexpressionon are positively are positively correlated correlatedwith poor prognosis with poor prognosis[17,19]. Considering [17,19]. Considering the importance the importance of Prp19 of Prp19and Cdc5L and Cdc5L within within this this complex, complex, the the relationship relationship betweenbetween them them deserves deserves toto bebe investigatedinvestigated inin HCC.HCC. InIn thisthis study,study, wewe demonstratedemonstrate thatthat thethe levelslevels of Prp19 and Cdc5L Cdc5L are are overexpressed overexpressed in in HCC HCC specimens,specimens, andand positivelypositively correlated correlated with with each each ot other.her. In In HCC HCC cells, cells, Prp19 Prp19 binds binds with with Cdc5L Cdc5L and andits itsdownregulation downregulation results results in inreduction reduction of of Cdc5L. Cdc5L. Mechanistic Mechanistic insights insights reveal reveal that that silencing silencing Prp19 Prp19 compromises translation activity of Cdc5L and facilitates lysosome-induced degradation of Cdc5L. compromises translation activity of Cdc5L and facilitates lysosome-induced degradation of Cdc5L. Furthermore, Prp19 knockdown arrests cell cycle at G2/M stage and targeting Prp19-induced mitotic Furthermore, Prp19 knockdown arrests cell cycle at G2/M stage and targeting Prp19-induced mitotic arrest is partially reversed by overexpressing Cdc5L in HCC cells. Taken together, the above findings arrest is partially reversed by overexpressing Cdc5L in HCC cells. Taken together, the above ﬁndings indicate that Prp19 takes part in the cell cycle regulation, rendering the potential of Prp19 as a indicate that Prp19 takes part in the cell cycle regulation, rendering the potential of Prp19 as a diagnotherapeutic target in HCC. diagnotherapeutic target in HCC.

2.2. Results Results

2.1.2.1. The The PositivePositive CorrelationCorrelation ofof Prp19Prp19 andand Cdc5LCdc5L in HCC ToTo investigate investigate thethe rolesroles ofof Prp19Prp19 andand Cdc5LCdc5L inin HCCHCC tissues, IHC staining was was performed performed with with tissuestissues from from 69 69 HCC HCC samples.samples. OurOur resultsresults demonstrateddemonstrated that, in in cancerous cancerous tissue, tissue, Prp19 Prp19 and and Cdc5L Cdc5L werewere both both mainlymainly locatedlocated at the the nucleus, nucleus, while while in in paracancerous paracancerous tissue tissue they they were were mainly mainly located located at atthe the cytoplasm, cytoplasm, and and this this phenomenon phenomenon was was confirmed conﬁrmed by confocal by confocal laser laserscanning scanning (Figure (Figure S1). In S1). HCC In HCCtissues, tissues, Prp19 Prp19 and Cdc5L and Cdc5L were weremore moreabundant abundant (64/69 (64/69 for Prp19; for Prp19;63/69 for 63/69 Cdc5L) for Cdc5L)compared compared to paired to pairedparatumor paratumor tissue tissue(Figure (Figure 1A), which1A), which was further was further confirmed conﬁrmed by western by western blot (13/14) blot (13/14) (Figure(Figure 1C). Sixty-1C). Sixty-ninenine HCC HCC clinical clinical specimens specimens were were subjected subjected to analyze to analyze the the correlation correlation between between Prp19 Prp19 and and Cdc5L. Cdc5L. Positive correlation between Prp19 level and Cdc5L expression was observed (R2 = 0.9146, p < 0.001; Positive correlation between Prp19 level and Cdc5L expression was observed (R2 = 0.9146, p < 0.001; Figure 1B). Figure1B).

FigureFigure 1.1. TheThe Positive Positive Correlation Correlation of ofpre-mRNA pre-mRNA processi processingng factor factor 19 (Prp19) 19 (Prp19) and cell and division cell division cycle cycle5-like 5-like (Cdc5L) (Cdc5L) in hepatocellular in hepatocellular carcinoma carcinoma (HCC). (HCC).(A) Paracancerous (A) Paracancerous and Cancerous and Cancerous tissue sections tissue sectionsfrom HCC from patients HCC patients were marked were marked with antibodies with antibodies against againstPrp19 and Prp19 Cdc5L and Cdc5L(upper (upperpanel, panel,scale bar scale = bar100 = μ 100m); µLowerm); Lower panel panelindicated indicated the amplified the ampliﬁed view of view upper of panel upper (scale panel bar (scale = 20 μ barm); = (B 20) Theµm); positive (B) The positivecorrelation correlation of Prp19 of Prp19and Cdc5L and Cdc5L immu immunoactivitynoactivity score score in HCC in HCC tissues; tissues; (C) ( CPrp19) Prp19 and and Cdc5L Cdc5L expressionexpression inin tumortumor tissuetissue (C)(C) andand pairedpaired paratumorparatumor tissue (P) from HCC patient patient specimens specimens using using westernwestern blot. blot.

Int. J. Mol. Sci. 2017, 18, 778 3 of 9 Int. J. Mol. Sci. 2017, 18, 778 3 of 9

2.2. Prp192.2. Prp19 Binds Binds with with Cdc5L Cdc5L and and Modulates Modulates Cdc5L Cdc5L ExpressionExpression in in HCC HCC Cells Cells BecauseBecause of the of the relationship relationship between between Prp19 Prp19 andand Cdc5L described described as asabove, above, we weproposed proposed that that Prp19 may infect the expression of Cdc5L. In vivo binding between Prp19 and Cdc5L was observed Prp19 may infect the expression of Cdc5L. In vivo binding between Prp19 and Cdc5L was observed in in Huh7 and SMCC-7721 cells. In contrast to normal mouse IgG, Cdc5L could be detected in the Huh7proteins and SMCC-7721 precipitated cells. by Inanti-Prp19 contrast (Figure to normal 2A), mouse and a IgG,similar Cdc5L phenomenon could be could detected be detected in the proteins in precipitatedanother byHCC anti-Prp19 cell line HCCLM3 (Figure2 andA), HEK293T and a similar cell li phenomenonne (Figure S2). couldHuh7 and be detectedSMCC-7721 in anothercells were HCC cell linetransfected HCCLM3 with and siPrp19-1, HEK293T siPrp19-2 cell line or control (Figure siRN S2).A for Huh7 48 h. and Contrary SMCC-7721 to the cells cells transfected were transfected with with siPrp19-1,control siRNA, siPrp19-2 silencing or Prp19 control obviously siRNA inhibited for 48 h. Cdc5L Contrary expression to the in cellsboth HCC transfected cell lines with (Figure control siRNA,2B–D). silencing Prp19 obviously inhibited Cdc5L expression in both HCC cell lines (Figure2B–D).

FigureFigure 2. Prp19 2. Prp19 binds binds with with Cdc5L Cdc5L and and modulates modulates Cdc5LCdc5L expression expression in in HCC HCC Cells. Cells. (A) (EndogenousA) Endogenous interactioninteraction between between Prp19 Prp19 and and Cdc5L Cdc5L waswas detected in in Huh7 Huh7 and and SMMC-7721 SMMC-7721 cells; cells;(B) Reduction (B) Reduction of of Cdc5LCdc5L expression expression inin HCCHCC cellscells transfectedtransfected with with siRNAs siRNAs targeting targeting Prp19; Prp19; (C,D (C) ,TheD) Thebar barcharts charts demonstratedemonstrate the densitometrythe densitometry ratio ratio of of Prp19 Prp19 or or Cdc5LCdc5L protein to to GAPDH GAPDH (loading (loading control) control) (* p < (* 0.05).p < 0.05). The dataThe aredata mean are mean± SD ± SD of of three three independent independent experiments.experiments. NC, NC, control control siRNA. siRNA.

2.3. Prp19 Modulates Cdc5L Expression Via Inhibiting mRNA Translation and Facilitating Lysosome-Mediated Degradation in HCC Cells The decreased expression of Cdc5L protein in siRNA mediated Prp19 knockdown in HCC cells Int.was J. Mol.due Sci. to2017 either, 18, 778decreased synthesis or increased degradation. Firstly, qPCR was performed.4 of 9 Inhibiting Prp19 had no significant effect on mRNA level of Cdc5L compared with control siRNA (Figure 3A). Then the post-transcriptional modulation needs to be investigated. Huh7 cells were transfectedtransfected withwith indicatedindicated siPrp19siPrp19 oror controlcontrol siRNAsiRNA forfor 4848 h,h, andand thenthen culturedcultured withwith cyclohexamidecyclohexamide inin the absence absence or or presence presence of of the the proteasome proteasome inhi inhibitorbitor MG132. MG132. Endogenous Endogenous expression expression of Cdc5L of Cdc5L was waselevated elevated by treating by treating with cyclohexamide, with cyclohexamide, but the but inhibition the inhibition of the proteasome of the proteasome with MG132 with does MG132 not doesprevent not the prevent loss of theCdc5L, loss indicating of Cdc5L, that indicating proteasome-mediated that proteasome-mediated degradation was degradation not responsible was notfor responsiblePrp19-induced for Prp19-inducedmodulation of modulationCdc5L (Figure of Cdc5L 3B). Furthermore, (Figure3B). Furthermore, we analyzed we whether analyzed lysosome- whether lysosome-mediatedmediated degradation degradation worked in worked this setting. in this Compar setting.ed Compared with cells withtransfected cells transfected with control with siRNA, control a siRNA,decrease a of decrease Cdc5L ofin Cdc5LHCC cells in HCC treated cells with treated siPrp19 with was siPrp19 partially was partiallyreversed reversedby lysosome by lysosome inhibitor inhibitorchloroquine. chloroquine. This suggested This suggested that Prp19 that facilitated Prp19 facilitated lysosomal lysosomal degradation degradation of Cdc5L of Cdc5L in HCC in HCCcells 0 cells(Figure (Figure 3C).3 C).Since Since thethe translational translational activity activity ofof 5′ 5-UTR-UTR is isextremely extremely important important for for growth growth factorsfactors 0 controllingcontrolling cellcell cyclecycle [[20],20], thethe effecteffect ofof Prp19Prp19 onon thethe translationaltranslational activityactivity ofof 55′-UTR-UTR ofof Cdc5LCdc5L inin HCCHCC cellscells waswas alsoalso detecteddetected byby luciferaseluciferase activityactivity analysis.analysis. We found thatthat thethe luciferaseluciferase activityactivity ofof cellscells treatedtreated withwith siPrp19 siPrp19 was was decreased decreased by by about about 30% 30% compared compared to that to ofthat cells of treatedcells treated with control with control siRNA (FiguresiRNA 3(FigureD), indicating 3D), indicating that Prp19 that modulated Prp19 modulated the translational the translational activity of activity Cdc5L. of These Cdc5L. ﬁndings These revealedfindings revealed that Prp19 that knockdown Prp19 knockdown inhibited inhibited Cdc5L expression Cdc5L expression via facilitating via facilitating lysosome-mediated lysosome- degradationmediated degradation and inhibiting and inhibiting mRNA translation mRNA translation in HCC cells. in HCC cells.

Figure 3.3. Prp19Prp19 modulates Cdc5L expression via inhibitinginhibiting mRNA translationtranslation andand facilitatingfacilitating lysosome-mediatedlysosome-mediated degradation degradation in in HCC HCC cells. cells. (A )(A The) The mRNA mRNA levels levels of Cdc5L of Cdc5L were were measured measured by qPCR by inqPCR HCC in cells HCC treated cells withtreated siPrp19 with orsiPrp19 control or siRNA;control (BsiRNA;) Huh7 (B cells) Huh7 were cells transfected were transfected with indicated with siRNAs.indicated After siRNAs. 48 h After transfection, 48 h transfection, Huh7 cells Huh7 were cells treated were sequentially treated sequentially with 100 withµg/mL 100 CHXμg/mL for CHX 1 h, 20forµ 1M h,MG132 20 μM MG132 for another for another 6 h, and 6 followedh, and followed by western by western blot; (C blot;) Huh7 (C) andHuh7 SMMC-7721 and SMMC-7721 cells were cells transfectedwere transfected with siPrp19. with siPrp19. After 48 hAfter transfection, 48 h transfection, these cells werethese treated cells withwere 25 treatedµM Chloroquine with 25 μ forM anotherChloroquine 12 h, for and another followed 12 byh, westernand followed blot; (byD) western Huh7 and blot; SMMC-7721 (D) Huh7 and cells SMMC-7721 were transfected cells were with indicatedtransfected siRNAs. with indicated After 24 h siRNAs. transfection, After they 24 wereh tran co-transfectedsfection, they with were control co-transfected reporter pRL-TK with control vector andreporter pGL3-5 pRL-TK0UTR-Cdc5L vector and for anotherpGL3-5′ 48UTR-Cdc5L h, and then for luciferase another 48 activity h, and was then measured luciferase (*** activityp < 0.001). was Transfectionsmeasured (*** werep < 0.001). performed Transfections in triplicate, were and performed mean ± inSD tr wereiplicate, calculated and mean for ± each SD were condition. calculated NC, controlfor each siRNA. condition. CHX, NC, cycloheximide. control siRNA. CHX, cycloheximide.

2.4. Prp19 Regulates Mitotic Progression via Cdc5L in HCC Cells Considering that Cdc5L is a cell cycle regulator of the G2/M transition, we wondered whether Prp19 arrested the cell cycle via Cdc5L. Flow cytometric analysis using cells transfected with targeting siRNAs and plasmids was performed. In Huh7 cells, siRNA targeting Prp19 efﬁciently inhibited Int. J. Mol. Sci. 2017, 18, 778 5 of 9

2.4. Prp19 Regulates Mitotic Progression via Cdc5L in HCC Cells Considering that Cdc5L is a cell cycle regulator of the G2/M transition, we wondered whether Prp19Int. J. Mol. arrested Sci. 2017 ,the18, 778cell cycle via Cdc5L. Flow cytometric analysis using cells transfected with5 of 9 targeting siRNAs and plasmids was performed. In Huh7 cells, siRNA targeting Prp19 efficiently inhibited the expression of the Prp19 and Cdc5L, and Cdc5L expressing vector remarkably rescued the expression of the Prp19 and Cdc5L, and Cdc5L expressing vector remarkably rescued Cdc5L Cdc5L expression (Figure 4A). In contrast to cells treated with control siRNA, the percentage of G2/M expression (Figure4A). In contrast to cells treated with control siRNA, the percentage of G2/M phase phase increased from 12.25% to 20.89% in the presence of siPrp19. Prp19 knockdown-induced increased from 12.25% to 20.89% in the presence of siPrp19. Prp19 knockdown-induced elevation elevation of the percentage of G2/M phase was inhibited by exogenous expression of Cdc5L of the percentage of G2/M phase was inhibited by exogenous expression of Cdc5L (decreased from (decreased from 22.92% to 11.07%) (Figure 4B,C). A similar phenomenon was observed in SMMC- 22.92% to 11.07%) (Figure4B,C). A similar phenomenon was observed in SMMC-7721 cells (Figure S3). 7721 cells (Figure S3). These results indicated that Prp19 participated in mitotic progression via Cdc5L inThese HCC results cells. indicated that Prp19 participated in mitotic progression via Cdc5L in HCC cells.

FigureFigure 4. 4. Prp19Prp19 regulates regulates mitotic mitotic progression progression via via Cdc5L Cdc5L in in HCC HCC cells. cells. ( (AA)) Huh7 Huh7 cells cells were were transfected transfected withwith indicated indicated siRNAs. After 2424 hh transfection,transfection, these these cells cells were were transfected transfected with with indicated indicated plasmids plasmids for foranother another 48 48 h, andh, and followed followed by by western western blot; blot; (B )(B Flow) Flow cytometry cytometry analysis analysis of of cell cell cycle cycle distribution distribution of ofcells cells transfected transfected with with indicated indicated siRNAs siRNAs and and plasmids plasmids inin ((AA);); ((CC)) QuantiﬁcationQuantification of cell cycle cycle shown shown inin ( (BB).). Data Data were were representative representative of of three three in independentdependent experiments. NC, NC, control siRNA.

3.3. Discussion Discussion AsAs one one of of the the leading leading causes causes of of cancer-related cancer-related de deathsaths in in the the world, world, the the survival survival rate rate of of HCC HCC is is veryvery low low [1,2], [1,2], which which is is mainly mainly due due to to the the complicated complicated molecular molecular mechanism mechanism of of HCC. HCC. Lately, Lately, aberrant aberrant expressionexpression of of Prp19 Prp19 was was observed observed in in most most HCC HCC tissu tissueses and and was was positively positively correlated correlated with with vascular vascular invasion,invasion, tumor tumor capsule capsule breakthrou breakthroughgh and and poor poor prognosis prognosis [19]. [19]. Prp1 Prp199 may may inhibit inhibit chemotherapeutic chemotherapeutic drugs-induceddrugs-induced apoptosis apoptosis in in HCC HCC cells cells [15]. [15 ].These These findings findings indicate indicate the theimportant important role role of Prp19 of Prp19 in the in developmentthe development of HCC. of HCC. Cdc5L, Cdc5L, another another core core compon componentent of ofthe the Prp19C, Prp19C, is isfound found to to modulate modulate the the expressionexpression of aa setset ofof genes genes involved involved in in the the mitosis mitosis and and facilitate facilitate mitotic mitotic processing processing [21]. [21]. In an In in-depth an in- depthstudy, study, it was it shown was shown that Prp19 that Prp19 exerts exerts bioactivities bioactivities alone oralone as aor part as a of part a protein of a protein complex complex in tumor in tumorformation formation [22]. This [22]. study This demonstratedstudy demonstrated the positive the positive correlation correlation of Prp19 of andPrp19 Cdc5L and Cdc5L in HCC in tissues HCC tissuesand identified and identified the interaction the interaction between between them in them HCC in cells. HCC Considering cells. Considering that Cdc5L that Cdc5L is involved is involved in cell incycle cell processing, cycle processing, we also foundwe also that found Prp19 that facilitated Prp19 G2/Mfacilitated transition G2/M in transition HCC cells. in Furthermore,HCC cells. Furthermore,Prp19 knockdown-induced Prp19 knockdown-induced cell cycle arrest cell was cycle partially arrestreserved was partially by overexpression reserved by overexpression of Cdc5L. of Cdc5L.Recently, Mu et al. reported the mutual modulation of components within Prp19C and hypothesizedRecently, thatMu thiset effectal. reported might be the due mutual to the requirement modulation for of each components component within to maintain Prp19C complex and hypothesizedstability [21]. Ourthat studythis effect firstly might proved be that due Prp19 to the modulated requirement Cdc5L for expression each component via inhibiting to maintain mRNA translation and facilitating lysosome-mediated degradation in HCC cells, which further verified the hypothesis proposed elsewhere [21]. It has been reported that the Trp-Asp forty-amino-acid repeat (WD-40 repeat) domain of Prp19 is essential for protein binding [23] and Prp19 forms tetramers to Int. J. Mol. Sci. 2017, 18, 778 6 of 9 provide a scaffold for Prp19C organization [10]; therefore, Prp19 may provide an interaction platform to maintain complex stability and avoid lysosome-mediated degradation. Depletion of Prp19 may impair mRNA translation of components within this complex. This study firstly revealed that both Prp19 and Cdc5L were overexpressed and positively correlated in HCC tissues. Furthermore Prp19 bound with Cdc5L and participated in mitotic processing by modulating Cdc5L. Recently, therapeutic modalities such as anti-mitotic drugs are booming [4], and some pharmaceutical products exert antitumor effects by inducing cell cycle arrest at the G2/M phase [24–26],suggesting that the inhibition of Prp19C could be a targeted anti-mitotic cancer therapy. This may provide theoretical basis to elucidate the role of Prp19C during the pathogenesis of HCC.

4. Materials and Methods

4.1. Patients and Samples All 69 HCC samples were randomly retrieved from patients who underwent curative resection and veriﬁed by post-operational histopathology at Liver Cancer Institute, Zhongshan Hospital of Fudan University from 1 December 2006 to 31 December 2008. The clinic pathologic characteristics of 69 patients were summarized in Table S1. The protocol was approved by the Institutional Ethics Committee of Zhongshan Hospital of Fudan University (Shanghai, China) at 25 February 2016 (Code, Y2016-042), and written informed consent was obtained from all study participants.

4.2. Immunochemistry and Immunofluorescence Slides of paraffin-embedded primary HCC tissues were used for immunochemistry and stained with antibodies against Prp19 and Cdc5L. The exact procedure and the semi-quantitative method were reported elsewhere [27]. Images were processed with a Nikon microscope with NIS Element F3.2 software (Nikon, Melville, NY, USA). When subjected to immunofluorescence assay, unfixed frozen sections were generally processed as described below, briefly. First, sections were incubated with rabbit anti-human Prp19 polyclonal antibody (1:250) and mouse anti-human Cdc5L Monoclonal antibody (1:20) overnight at 4 ◦C after blocked by Phosphate buffered saline (PBS) containing 5% Bovine serum albumin (BSA). They were then washed by PBS carefully. These sections were incubated with Alexa Fluor® 647-conjugated AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor® 488-conjugated AffiniPure Donkey Anti-Mouse IgG (both 1:500) at room temperature for 2 h (Jackson ImmunoResearch Inc., West Grove, PA, USA). Finally, sections were added with Mounting Medium for Flurescence with DAPI and detected by Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

4.3. Cell Lines, Plasmids, siRNAs, Reagents and Chemical Agents Huh7, SMMC-7721, HCCLM3 and HEK293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) where they were characterized by mycoplasma detection, DNA-Fingerprinting, isozyme detection and cell vitality detection. All cell lines were cultured in DMEM with 10% fetal bovine serum. All these cells were cultured in a humidiﬁed incubator at 37 ◦C in the presence of 5% CO2. Human Cdc5L in pcDNA3.1/myc-His(−) vector was generated according to the standard PCR protocol. All primers and siRNAs sequences were listed in Table S2. Transfection of plasmids and siRNAs were performed with Lipofectamine® 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Antibodies recognizing Prp19 and Cdc5L were purchased from Abcam Ltd. (Cambridge, UK). Antibodies against GAPDH was from Cell Signaling Technology (Beverly, MA, USA). Chloroquine was dissolved in sterile nuclease-free water (Cell Signaling Technology, Beverly, MA, USA). Cycloheximide was dissolved in sterile nuclease-free water and used at 100 µg/mL (Sigma-Aldrich, St. Louis, MO, USA). MG132 was dissolved in dimethyl sulfoxide (DMSO) and used at 20 µM (Merck KGaA, Darmstadt, Germany). Int. J. Mol. Sci. 2017, 18, 778 7 of 9

4.4. Real-Time PCR (qPCR) Total RNA was exacted by TRIzol (Invitrogen, Carlsbad, NY, USA). The ﬁrst strand cDNA synthesis was carried out with AMV RNA PCR kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Subsequent qPCR was performed using a SYBR Green Premix Ex Taq (TaKaRa) on ABI StepOne Plus system (Applied Biosystems, Foster City, CA, USA). The relative mRNA level of speciﬁc genes was calculated using the ddCt method after normalization against GAPDH. The primers of interested genes were as follows: Prp19: 50-GTGCC AAGTT CCCAA CCAAG TGTT-30 (forward) and 50-AGCAC AGTGG CTTTG TCTTG AAGC-30 (reverse); GAPDH: 50-TCGAC AGTCA GCCGC ATCTT CTTT-30 (forward) and 50-GCCCA ATACG ACCAA ATCCG TTGA-30 (reverse); Cdc5L: 50-TCTCT GAAG CTCC TCTC GGC-30 (forward) and 50-CATC CTCG GTAT TCCT CCAT ACG-30 (reverse).

4.5. Western Blot Assay For western blot, cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 with protease inhibitor), and their extracts were clarified via centrifugation. The cell lysate proteins were separated on 10% SDS-PAGE gel and then transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked in blocking solution (50 mM Tris-HCl, 150 mM NaCl, 5% (w/v) non-fat dry milk and 0.1% Tween-20) at room temperature for 2 h, followed by incubation with appropriate primary antibodies at 4 ◦C overnight. After incubating with appropriate secondary antibodies at room temperature for 1 h, gels were scanned and quantified using ImageQuant LAS 4000 mini (GE Healthcare, New York, NY, USA).

4.6. Immunoprecipitation Analysis Briefly, Huh7 and SMCC-7721 cells under corresponding treatments were harvested in immunoprecipitation lysis buffer supplemented with a complete protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Mannheim, Germany). The cell lysates were immunoprecipitated with corresponding antibodies and detected by western blot.

4.7. Dual Luciferase Analysis First, pGL3-50UTR-Cdc5L, which contains a 299 bp promoter fragment of mRNA of Cdc5L inserted upstream of firefly luciferase coding sequences, was constructed from pGL3-Basic Vector, which lacks eukaryotic promoter and enhancer sequences and allows inserted upstream of the promoter. After the treatment of siPrp19 or control siRNA, Huh7 and SMCC-7721 cells were co-transfected with control reporter pRL-TK Vector and pGL3-50UTR-Cdc5L. Luciferase activities were measured 48 h after transfection. For each condition, the ratio of firefly to Renilla luciferase was determined and normalized to the value obtained for HCC cells transfected with control siRNA (Relative Expression). The value of the sample transfected with control siRNA was set at 100%. Data are presented as the means ± SEM of three independent experiments (in triplicate).

4.8. Flow Cytometric Analysis For ﬂow cytometric analysis, cells were collected in phosphate-buffered saline (PBS) containing 2 mmol/L ethylenediaminetetraacetic acid and ﬁxed in cold ethanol (70–80%) for at least 18 h. Fixed cells were washed three times in PBS and then permeabilized with 1% TritonX-100 for 20 min and incubated with 1 mg/mL RNase A in PBS for 20 min. Subsequently, the cells were stained with 50 µg/mL propidium iodide (PI) (Becton–Dickinson, San Jose, CA, USA) in PBS-TritonX-100 for an additional 20 min at 4 ◦C. At last, the cells were analyzed for cell cycle distribution using a BD Int. J. Mol. Sci. 2017, 18, 778 8 of 9

FACSCalibur (Beckton Dickinson, Franklin Lakes, NJ, USA) ﬂow cytometer and analyzed using Cell Quest software supplied by the manufacturer.

4.9. Statistical Analysis Statistical analyses were conducted on SPSS 19.0 for Windows. Student’s t-test was used to compare differences between different groups (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/4/778/s1. Acknowledgments: This work was supported by grants from Shanghai Science and Technology Commission (No. 16ZR1406100), National Nature Science Foundation of China (No. 81301820, No.81472673, No.81672720, No.81672334, No. 81502696), and National Clinical Key Special Subject of China. Author Contributions: Xi-Zhong Shen and Jie Yin conceived of and designed the experiments; Renzheng Huang, and Ruyi Xue performed the experiments; Di Qu provided material support; Renzheng Huang, Ruyi Xue and Jie Yin analyzed the data and wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Torre, L.A.; Bray, F.; Siegel, R.L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. Global cancer statistics, 2012. CA Cancer J. Clin. 2015, 65, 87–108. [CrossRef][PubMed] 2. El-Serag, H.B. Hepatocellular carcinoma. N. Engl. J. Med. 2011, 365, 1118–1127. [CrossRef][PubMed] 3. Merle, P.; Trepo, C. Molecular mechanisms underlying hepatocellular carcinoma. Viruses 2009, 1, 852–872. [CrossRef][PubMed] 4. Manchado, E.; Guillamot, M.; Malumbres, M. Killing cells by targeting mitosis. Cell Death Differ. 2012, 19, 369–377. [CrossRef][PubMed] 5. Asghar, U.; Witkiewicz, A.K.; Turner, N.C.; Knudsen, E.S. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat. Rev. Drug Discov. 2015, 14, 130–146. [CrossRef][PubMed] 6. Chanarat, S.; Strasser, K. Splicing and beyond: The many faces of the Prp19 complex. Biochim. Biophys. Acta 2013, 1833, 2126–2134. [CrossRef][PubMed] 7. Mahajan, K. hPso4/hPrp19: A critical component of DNA repair and DNA damage checkpoint complexes. Oncogene 2015, 2279–2286. [CrossRef][PubMed] 8. Wan, L.; Huang, J. The PSO4 protein complex associates with replication protein A (RPA) and modulates the activation of ataxia telangiectasia-mutated and Rad3-related (ATR). J. Biol. Chem. 2014, 289, 6619–6626. [CrossRef][PubMed] 9. Jurica, M.S.; Moore, M.J. Pre-mRNA splicing: Awash in a sea of proteins. Mol. Cell 2003, 12, 5–14. [CrossRef] 10. Ohi, M.D.; Kooi, C.W.V.; Rosenberg, J.A.; Ren, L.P.; Hirsch, J.P.; Chazin, W.J.; Walz, T.; Gould, K.L. Structural and functional analysis of essential pre-mRNA splicing factor Prp19p. Mol. Cell. Biol. 2005, 25, 451–460. [CrossRef][PubMed] 11. Ohi, M.D.; Vander Kooi, C.W.; Rosenberg, J.A.; Chazin, W.J.; Gould, K.L. Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat. Struct. Biol. 2003, 10, 250–255. [CrossRef][PubMed] 12. Vander Kooi, C.W.; Ohi, M.D.; Rosenberg, J.A.; Oldham, M.L.; Newcomer, M.E.; Gould, K.L.; Chazin, W.J. The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases. Biochemistry 2006, 45, 121–130. [CrossRef][PubMed] 13. Wahl, M.C.; Will, C.L.; Luehrmann, R. The Spliceosome: Design Principles of a Dynamic RNP Machine. Cell 2009, 136, 701–718. [CrossRef][PubMed] 14. Zhang, N.; Kaur, R.; Lu, X.; Shen, X.; Li, L.; Legerski, R.J. The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links. J. Biol. Chem. 2005, 280, 40559–40567. [CrossRef][PubMed] 15. Yin, J.; Zhang, Y.A.; Liu, T.T.; Zhu, J.M.; Shen, X.Z. DNA damage induces down-regulation of Prp19 via impairing Prp19 stability in hepatocellular carcinoma cells. PLoS ONE 2014, 9, e89976. [CrossRef][PubMed] 16. Ajuh, P.; Lamond, A.I. Identiﬁcation of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1. Nucleic Acids Res. 2003, 31, 6104–6116. [CrossRef][PubMed] Int. J. Mol. Sci. 2017, 18, 778 9 of 9

17. Qiu, H.; Zhang, X.; Ni, W.; Shi, W.; Fan, H.; Xu, J.; Chen, Y.; Ni, R.; Tao, T. Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma. Dig. Dis. Sci. 2016, 61, 795–805. [CrossRef][PubMed] 18. Bernstein, H.S.; Coughlin, S.R. A mammalian homolog of ﬁssion yeast Cdc5 regulates G2 progression and mitotic entry. J. Biol. Chem. 1998, 273, 4666–4671. [CrossRef][PubMed] 19. Yin, J.; Wang, L.; Zhu, J.M.; Yu, Q.; Xue, R.Y.; Fang, Y.; Zhang, Y.A.; Chen, Y.J.; Liu, T.T.; Dong, L.; et al. Prp19 facilitates invasion of hepatocellular carcinoma via p38 mitogen-activated protein kinase/twist1 pathway. Oncotarget 2016, 7, 21939–21951. [PubMed] 20. De Benedetti, A.; Graff, J.R. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004, 23, 3189–3199. [CrossRef][PubMed] 21. Mu, R.; Wang, Y.B.; Wu, M.; Yang, Y.; Song, W.; Li, T.; Zhang, W.N.; Tan, B.; Li, A.L.; Wang, N.; et al. Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe. Cell Death Dis. 2014, 5, e1151. [CrossRef][PubMed] 22. Yin, J.; Zhu, J.M.; Shen, X.Z. New insights into pre-mRNA processing factor 19: A multi-faceted protein in humans. Biol. Cell 2012, 104, 695–705. [CrossRef][PubMed] 23. Gotzmann, J.; Gerner, C.; Meissner, M.; Holzmann, K.; Grimm, R.; Mikulits, W.; Sauermann, G. hNMP 200: A novel human common nuclear matrix protein combining structural and regulatory functions. Exp. Cell Res. 2000, 261, 166–179. [CrossRef][PubMed] 24. Wang, W.; Cheng, J.; Zhu, Y. The JNK Signaling pathway is a novel molecular target for S-propargyl-L-cysteine, a naturally-occurring garlic derivatives: Link to its anticancer activity in pancreatic cancer in vitro and in vivo. Curr. Cancer Drug Targets 2015, 15, 613–623. [CrossRef][PubMed] 25. Jung, C.; Hong, J.Y.; Bae, S.Y.; Kang, S.S.; Park, H.J.; Lee, S.K. Antitumor activity of americanin a isolated from the seeds of phytolacca americana by regulating the ATM/ATR signaling pathway and the Skp2-p27 axis in human colon cancer cells. J. Nat. Prod. 2015, 78, 2983–2993. [CrossRef][PubMed] 26. Chelsky, Z.L.; Yue, P.; Kondratyuk, T.P.; Paladino, D.; Pezzuto, J.M.; Cushman, M.; Turkson, J. A resveratrol analogue promotes ERKMAPK-dependent STAT3 serine and tyrosine phosphorylation alterations and antitumor effects in vitro against human tumor cells. Mol. Pharmacol. 2015, 88, 524–533. [CrossRef] [PubMed] 27. Ruan, Y.; Sun, L.; Hao, Y.; Wang, L.; Xu, J.; Zhang, W.; Xie, J.; Guo, L.; Zhou, L.; Yun, X.; et al. Ribosomal RACK1 promotes chemoresistance and growth in human hepatocellular carcinoma. J. Clin. Investig. 2012, 122, 2554–2566. [CrossRef][PubMed]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).