RESEARCH ARTICLE

Macrophage immigration inhibitory factor promotes cellproliferation and inhibits apoptosis of cervical adenocarcinoma

Peng Guo & Jing Wang & Junxiu Liu & Meng Xia & Wen Li &Mian He

Received: 25 November 2014 /Accepted: 26 January 2015 /Published online: 26 February 2015# International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract As a multifunctional cytokine, macrophage mi-gration inhibitory factor (MIF) is associated with inflam-mation and tumorigenesis; however, the role of MIF incervical adenocarcinoma (ADC) is not fully understood.In this study, we aimed to examine the expression ofMIF in ADC and explore the mechanism of MIF inADC progression. MIF expression was positively relatedto ADC clinicopathological features of carcinoma diam-eter and lymph node metastasis. MIF knockdown in-duced cell cycle arrest of G1/S transition in ADC cells,upregulation of the expressions of p21 and p27, anddownregulation of the expressions of Cdk4, CyclinD2,and CyclinE2. In MIF knockdown cells, the expressionsof proapoptotic proteins of Bax, caspase-3, cleaved cas-pase-3, and cleaved-PARP were upregulated, and the ex-pressions of antiapoptotic proteins of Bcl-2, pAkt, andp53 were downregulated. It indicated that MIF knock-down inhibited cell proliferation and induced apoptosisin ADC cells. MIF might be a novel molecular marker indiagnosis and therapy of ADC.

Keywords Macrophagemigration inhibitory factor . Cervicaladenocarcinoma . Cell proliferation . Apoptosis

Introduction

Cervical cancer is the third most common cancer and thefourth leading cause of cancer deaths among women inthe world. More than 500,000 new cases and around 275,000 deaths occur each year [1]. The incidence of cervicalcancer decreases due to the implementation of cervicalcytology and HPV detection, especially for squamous cellcarcinoma (SCC) [2]. However, the incidence of cervicaladenocarcinoma (ADC) rises, especially in young women[3]. Compared with SCC, ADC is more likely to have aworse outcome and escapes from being detected [4]. Theetiology of cervical cancer is absolutely related to persis-tent infection of HPV [5]. While majority of the HPVinfections remain asymptomatic and be cleared by im-mune system within 12 months [6], only few casesprogressed to cervical cancer [7]. It indicates that HPVinfection alone is insufficient for cervical cancer, and theabnormal host genes are critical in the development ofcervical cancer [8]. Therefore, in addition to the HPVDNA testing, the discovery of vital diagnostic and thera-peutic markers against ADC should be prioritized.

Macrophage migration inhibitory factor (MIF) is firstidentified as a T cell-derived lymphokine with 12.5-kDamonomeric molecular weight [9]. It has been reportedthat MIF played an important role in various autoim-mune and inflammatory disorders [10]. Moreover, sever-al studies have indicated that MIF was involved incancerogenesis and progression [11]. MIF might promoteproliferation by stimulating MAPK/PI3K/Akt pathwaysin lung ADC cell [12] and inhibit apoptosis by bindingto tumor suppressor p53 [13]. In addition, coexpressionlevel of MIF and its receptor CD74 could be a surrogatemarker to test the efficacy of antiangiogenic drugs inbreast cancer [14]. The finding of Rong jie Cheng sug-gested that MIF and CD74 were overexpressed in SCC

Peng Guo and Jing Wang have an equal contribution to this study work.

P. Guo : J. Wang : J. Liu :M. Xia :M. He (*)Department of Obstetrics and Gynecology, The First AffiliatedHospital of Sun Yat-sen University, #58 Zhongshan er Road,Guangzhou, Guangdong 510080, People’s Republic of Chinae-mail: mianhe64@163.com

W. Li (*)Laboratory of General Surgery, The First Affiliated Hospital of SunYat-sen University, #58 Zhongshan er Road,Guangzhou, Guangdong 510080, People’s Republic of Chinae-mail: wenli28@163.com

Tumor Biol. (2015) 36:5095–5102DOI 10.1007/s13277-015-3161-4

and might play critical roles in the pathogenesis andangiogenesis of cervical cancer [15]. In this study, ouraims are to examine the expression of MIF in ADC tis-sues and explore the possible mechanism of MIF in pro-gression of ADC cells.

Patients and methods

Patients and specimens

Seventy-five fresh-frozen tissue samples were collected fromADC patients with radical hysterectomy (The first affiliatedhospital of Sun Yat-sen university, Guangzhou, China), includ-ing 10 patients at stage IA, 45 patients at stage IB, 13 patients atstage IIA, and 7 patients at stage IIB. All of the patients re-ceived neither preoperative radiotherapy nor chemotherapy.The normal cervical tissues of ten patients with hysteromyomawho received hysterectomy were taken as the control group.Informed consent from each patient was obtained. The proce-dures have been approved by the ethics review committee ofthe First Affiliated Hospital of Sun Yat-sen University and arein accordance with the Helsinki Declaration of 1975.

Immunohistochemical staining

Immunohistochemistry was performed to detect the expres-sion of MIF in ADC and normal cervical tissues. Paraffin-embedded cervical tissue samples were dewaxed in xylene,rehydrated in graded ethanol. Antigen recovery was per-formed in 10-mmol/L boiling sodium citrate buffer at pH 6.0for 10 min at 92~98 °C, and specimens were incubated with0.3 % H2O2 for 15 min. Nonspecific binding was blockedwith normal horse serum for 20 min at room temperature.The sections were incubated with monoclonal mouse anti-human MIF antibody (Santa Cruz) that was 1:100 at 4 °Covernight. The sections were washed with PBS and incubatedwith biotinylated secondary antibody for 30 min (diluted1:1000, DAKO EvisionTM). Sections were then treated withABC solution at 37 °C for 30 min and incubated with 3,3′-diaminobenzidine (DAB) for 5 min. Sections were counterstained using Harris hematoxylin. Cells with brown stainingto membranes or cytoplasm were considered positive.

MIF expression was evaluated according to the ratio ofpositive cells per specimen and stained intensity. The ratio ofpositive cells per specimen was evaluated quantitatively andscored 0 for staining of ≤1 %, 1 for staining of 2 to 25 %, 2 forstaining of 26 to 50 %, 3 for staining of 51 to 75 %, and 4 forstaining ≥75 % of the cells examined. Intensity was graded asfollows: 0, no signal; 1, weak; 2, moderate; and 3, strongstaining. A total score of 0 to 12 was finally calculated andgraded as negative (−; score 0–1), weak (+; 2–4), moderate(++; 5–8), and strong (+++; 9–12).

Cell culture and transfection

An established Human Hela 229 cervical carcinoma cells(TCHu187, Shanghai Cell Bank of Science Academy of Chi-na, Shanghai, China) were used for this study. Hela 229 cellswere incubated in RPMI1640 medium (GBICO) containing10 % fetal calf serum (GBICO), under a 5 % CO2 atmosphereat 37 °C.

The effective siRNA (5′GGG TCT ACA TCA TCGATT A-3′) targeting MIF gene was used to construct therecombinant plasmid, which was transfected to Hela 229cells by lipofectamine 2000 according to the manufac-turer’s instructions (Invitrogen). Then, the stably MIF si-lenced cell lines were screened by using G418. RT-PCRand Western blotting analysis were used to examine MIFmRNA and protein expression. The Hela 229 cell linewith MIFshRNA was successfully established, in whichMIF was stably silenced and was used in the followingexperiments. The Hela 229 cells with null vector transfec-tion were used as the control group.

Cell growth assay using CCK-8 method

Hela 229 cells of MIF shRNA group and control group werecultured in 96-well plates, at a density of 1.0×104 cells perwell. Six hours later, 10 μL CCK-8 (Dojindo, MolecularTechnologies, Inc) was distributed into each well, and theabsorbance at 450 nm was detected after 1-h culture; theCCK-8 assay was tested at 0, 24, 48, 72, and 96 h after trans-fection. All experiments were repeated at least three timeswith similar results.

Cell cycle and cell apoptosis analysis by flow cytometry

Cultured cells of MIF shRNA group and control group at24 and 48 h were collected. The cells were fixed with70 % cold ethanol at 4 °C overnight, and then washedwith ice-cold PBS twice. The cells were stained withpropiduim iodide (KEYGEN, Nanjing China) (50 ng)containing RNase (100 ng) for 30 min at 37 °C. Thepercentage of cells in each phase of the cell cycle wascounted by flow cytometer (FACS Calibur, BD, USA).All analysis was performed in triplicate.

Cultured cells of MIF shRNA group and control group at24 and 48 h were collected for apoptotic analysis by flowcytometry analysis. The cells were washed with cold PBStwice and were stained with Anannexin V-FITC and 10 μlpropiduim iodide. The percentage of apoptotic cells was de-tected using flow cytometer (FACS Calibur, BD, USA). Foreach analysis, 1×105 cells were scanned. All analysis wasperformed in triplicate.

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Western blotting analysis

Cultured cells of MIF shRNA group and control group at 48 hwere collected to extract proteins. Equal amounts of proteinwere separated on 12 % SDS-PAGE and transferred topolyvinylidene fluoride membrane (Millipore Corparation,Bedford, MA, USA). The membranes were blocked inblocking buffer (10 % nonfat milk in PBST) at room temper-ature for 1 h, and then incubated with antibodies at 4 °C over-night. The antibodies included MIF (Santa Cruz), p21 (SantaCruz), p27 (Santa Cruz), CyclinE2 (Santa Cruz), CyclinD2(Santa Cruz), GAPDH (Santa Cruz), Cdk4 (Cell SignalingTechnology, CST), Bcl-2 (CST), Bax (CST), caspase-3(CST), cleaved caspase-3 (CST), cleaved-PARP (CST),phosphorylation-Akt (CST), PARP (CST), Akt (CST), andp53 (CST). Immunoblots were developed by using anti-mouse-HRP or anti-rabbit-HRP secondary antibodies (SantaCruz). GAPDH was used as a referenced gene. All experi-ments were repeated at least three times with similar results.

Statistical analysis

All data were expressed as the mean±SD (standard deviation)and compared using ANOVA or t test analysis. The χ2 testwas used for comparisons between immunohistochemical andclinicopathological parameters. Statistical significance was

assumed as p<0.05. All statistical analyses were processedusing SPSS software program version 13.0.

Results

MIF expression and its clinicopathological significancein ADC

MIF expression was increased in ADC tissues compared withcontrol group. We found that MIF was overexpressed in68.0 % (51/75) of ADC samples. MIF protein was mainlyexpressed in the cytoplasm of ADC cells (Fig. 1). In addition,MIF expression is positively correlated with carcinoma diam-eter and lymph node metastasis (Table 1).

Effect of MIF knockdown on ADC cell cycle and cellproliferation

To investigate the effect of MIF knockdown on cell cycle,flow cytometry analysis was used to examine the DNA con-tent in each phase of cell cycle. In MIF knockdown cells, anincrease of DNA content in G1 phase and a decrease in Sphase were found at 24 and 48 h after culturing (Fig. 2a–c).MIF knockdown leaded to an arrest of cell cycle transitionfrom G0/G1 phase into S phase in ADC cells.

Fig. 1 The expression of MIF was markly increased in ADC tissues and positively correlated with clinical pathological stage. MIF protein mainlylocates in cytoplasm of ADC cells

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To investigate the effect of MIF knockdown on cell prolif-eration, CCK8 assays were used to examine cell proliferationof Hela 229 cells. As shown in Fig. 2d, at 24, 48, 72, and 96 hafter culturing, proportion of MIF shRNA cells was decreasedthan that of control group. MIF knockdown suppressed theproliferation of ADC cells.

Western blot was used to explore the expressions of pro-teins related to cell cycle progression and proliferation. InMIFknockdown cells, the expressions of Cdk4, Cyclin D2, andCyclinE2 were downregulated, while the expressions of p21and p27 were increased (Fig. 4c). The densitometric analysisof these proteins is shown in Fig. 5b.

Effect of MIF knockdown on cell apoptosis in ADC cells

The effect of MIF knockdown on Hela 229 cell apoptosis wasexamined by flow cytometry. MIF knockdown promoted cellapoptosis in the early stage and late stage (Fig. 3a and b). Our

results suggested that MIF would inhibit apoptosis in Hela229 cells.

In MIF knockdown cells, the expressions of proapoptoticproteins of Bax, caspase-3, cleaved caspase-3, and cleaved-PARP were upregulated, and the expressions of antiapoptoticprotein Bcl-2, pAkt, and p53 were downregulated (Fig. 4b–e).The densitometric analysis of these proteins is shown inFig. 5c–e. There were no changes on the expressions of Aktand PARP. Moreover, the ratio of Bcl-2/Bax was decreased inMIF knockdown cells (Fig. 5f).

Discussion

Several studies have indicated that MIF was involved incancerogenesis and progression of various tumors. However,the role ofMIF in ADC progression is not fully understood. Inthis study, we demonstrated that MIF was overexpressed inADC and was positively correlated with clinicopathologicalcharacters of carcinoma diameter and lymph node metastasis.MIF knockdown inhibited cell cycle progression and prolifer-ation, as well as promoted cell apoptosis.

MIF expression was increased in the ADC samples of tu-mor size over than 4 cm and in the patients with lymph nodemetastasis. It indicated that MIF promoted the progression ofADC. The cell cycle progression, particularly the G1 to Sphase transition, is controlled by a series of protein complexescomposed of cyclins and cyclin-dependent kinases (CDKs).The activity of CDKs is controlled by CDK inhibitors (CKIs),including p21 (Cip10) and p27 (Kip1), which are able to in-hibit cell cycle progression and induce cell cycle arrest [16].The cyclinD and cyclin E regulate the transition from G1 intoS phase. CyclinD2/Cdk4 complexes and Cyclin E2/Cdk2complexes are inhibited by both p21 and p27 [17, 18]. Inour study, MIF knockdown induced cell cycle arrest of G1/Stransition in ADC cells and upregulated the expressions ofp21 and p27, as well as downregulated the expressions ofCdk4, CyclinD2, and CyclinE2. It suggested that MIF knock-down inhibited cell cycle progression and proliferation ofADC cells through Cdk/Cyclin pathways.

In MIF knockdown ADC cells, the expressions ofproapoptotic proteins of Bax, caspase-3, cleaved caspase-3,and cleaved-PARP were upregulated, and the expressions ofantiapoptotic proteins of Bcl-2, pAkt, and p53 were downreg-ulated. Apoptosis induction is arguably the most potent de-fense against cancer growth [19]. Apoptosis is normally abalanced system tightly regulated by antiapoptotic andproapoptotic effectors, including proteins of the Bcl-2 family[20]. It has been demonstrated that MIF delayed cleavage ofthe proapoptotic members of the Bcl-2 family Bid and Bax inthe neutrophils, suggesting that MIF inhibits apoptosis path-ways proximal to mitochondria activation. The Bcl-2 and Bcl-xL, as the antiapototic proteins, promote cell survival, while

Table 1 Correlation betweenMIF expression and the clinicopathologicalcharacteristics of the ADC patients

Variable feature n MIF p

+ −Age

≥40 46 30 16 0.515

<40 29 21 8

Differention

well 23 15 8 0.088

moderate 34 27 7

poor 18 9 9

Tumor size (cm)

≥4.0 32 26 6 0.034

<4.0 43 25 18

Lymphatic metastasis

Yes 13 12 1 0.029

No 62 39 23

Cervical invasion

≥1/2 21 12 9 0.272

<1/2 54 39 15

FIGO staging

Ia 10 4 6 0.054

Ib 45 35 10

IIa 13 9 4

IIb 7 3 4

Surgical margin

Yes 7 4 3 0.673

No 68 47 21

Parametrial extension

Yes 15 10 5 0.034

No 60 41 19

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the proapototic protein Bax promotes programmed cell death.The ratio of Bcl-2/Bax regulates cell apoptosis and determineswhether cells undergo programmed cell death [21]. Therefore,substances that target Bcl-2 family signals are effective drugsin cancer treatment [22]. Poly-ADP-ribose polymerase(PARP) is a nuclear protein with an ADP-ribosyl transferasecatalytic domain with multiple cellular functions includingtranscriptional regulation, DNA damage repair, cell prolifera-tion, and apoptosis [23, 24]. It has been reported that theincrease of the Bax/Bcl-2 ratio may be responsible for theconcomitant apoptosis due to the disruption of mitochondrialmembrane potential and the inactivation of key cellular pro-teins such as PARP-1 [25]. The caspase-3 cascade would beactivated by cytochrome c, which is released from mitochon-dria [26]. Cytochrome c is an important mitochondrial proteinthat induces apoptosis when accumulated in the cytosol inresponse to diverse stress stimuli [27]. MIF also preventedrelease of cytochrome c and Smac from the mitochondriaand subsequent activation of caspase-3 [28]. Moreover,BAD, as one of the Bdeath-promoting^ members of the Bcl-2 family, is an important regulator of the cell death machinery,which has been reported to contribute to tumorigenesis inseveral cancers [29]. However, the effect of BAD on apoptosis

is still controversial. Several studies showed that overexpres-sion of BAD alone had no effect on apoptosis, the effect ofBAD on apoptosis in nonsmall cell lung cancer (NSCLC) wasa result of regulation and interaction with other Bcl-2 familymembers, including Bcl-xl, Bcl-2, and Bax [30, 31]. Anotherstudy suggested that BAD overexpression induced apoptosisin NSCLC cells, in which process expressions of mitochon-drial cytochrom c and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax, and caspase 8 expressions did not change. Itindicated that a mitochondrial pathway, in which processcytochrom c was released from mitochondrial to activate cas-pase 3, was involved in BAD overexpression-mediated apo-ptosis [32]. In the further study, the function of MIF siRNAcombination with Bcl-2 antigonist BAD on ADC apoptosiswould be explored.

Depending on the cellular context, binding of MIF to itsknown cell surface receptors can lead to activation of twofundamental signaling axes, namely, the mitogen-activatedprotein kinase (MAPK) pathway and PI3K/Akt pathway[33]. PI3K modulates signaling pathways implicated in cellgrowth, apoptosis, or both. Regulatory factors of this pathwayare frequently deregulated in an extensive number of tumortypes, making it an attractive target for tumor therapy [34].

Fig. 2 MIF knockdown induced cell cycle arrest and decreasedproliferation in Hela 229 cells. a DNA content in each phases of cellcycle of MIF shRNA cells and control group at 24 and 48 h afterculturing. b Proportion of Hela 229 cells in the two groups in G1 phaseat 24 h (***p<0.001) and 48 h (***p<0.001) after culturing. c Proportion

of Hela 229 cells in the two groups in S phase at 24 h (*p<0.05) and 48 h(***p<0.001) after culturing. d Proportion of proliferated Hela 229 cellsin the two groups at 24 h (*p<0.05), 48 h (*p<0.05), 72 h (***p<0.001),96 h (***p<0.001) after culturing

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Akt activity is modulated by PI3K, Akt activation induces cellcycle progression, survival, migration, and metabolismthrough phosphorylation of various physiological factors. Ac-tivation of PI3K and Akt is reported in several cancers [35, 36].It is indicated that MIF is an upstream regulator of the AKTpathway [37]. Phosphorylated and activated AKT leads apo-ptosis by inactivating proapoptotic proteins, including Bax andcaspase, and also by inducing the expression of antiapoptoticproteins of Bcl2 [38]. MIF was identified as a negative regu-lator of p53, and MIF was an important endogenous regulatorof p53 expression [39, 40]. Our results showed that the

phosphorylation level of Akt and p53 was dramatically lowerin MIF knockdown cells than in control cells.

On the cellular level, MIF is stored in the cytoplasmiccompartment and is released in response to several stimuli.The primary MIF receptor is CD74, and CD 74 can bind toCD44 to form a receptor complex and mediate the transduc-tion of MIF signaling [41]. CD74 can also form complexedwith the CXCR2 and CXCR4 to transmit MIF signals tointegrins in inflammatory cells [42, 43]. It has been reportedthat MIF-related signaling is triggered by its receptor CD 74and then channeled directly via the AKT pathway [33]. In

Fig. 3 MIF knockdown induced apoptosis in Hela 229 cells. aApoptosisanalyzed by flow cytometry at 24 and 48 h after culturing. b Proportion ofapoptotic cells in early stages at 24 h (***p<0.001) and 48 h (*p<0.05)

after culturing, as well as in late stages at 24 h (*p<0.05) and 48 h(*p<0.05) after culturing

Fig. 4 MIF knockdown induced apoptosis and inhibited proliferation ofHela 229 cell through Bcl-2/bax and cyclinD/E pathways. a Expressionsof MIF in MIF knockdown group and control group. b Expressions ofBcl-2, Bax in MIF shRNA group and control group. c Expressions of

Cdk4, CyclinD2, CyclinE2, P21, and P27 in MIF shRNA group andcontrol group. d Expressions of caspase-3, cleaved caspase-3, cleavedPARP, PARP in MIF shRNA group and control group. e Expressions ofpAkt, Akt, and p53 in MIF shRNA group and control group

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platelet, MIF induced CXCR7-Akt-dependent phosphoryla-tion of Bcl-2 antagonist of cell death (BAD) both in vitroand in vivo and limited activation-induced apoptosis viaCXCR-7 depending Akt signaling [44]. In our further study,inmmunofluorescence would be conducted to demonstrate thecolocalizate of MIF and its receptors.

This study demonstrated that MIF was overexpressedin ADC tissues. MIF knockdown inhibited ADC cellproliferation and promoted apoptosis. The mechanisminvolved inhibition of cell cycle proteins and inductionof apoptotic proteins. The present results suggest thatMIF may be an important diagnostic and therapeuticmarker against ADC. However, further investigation isneeded.

Acknowledgments The National Natural Science Foundation of China(No. 81172337, 30973395); Natural Science Foundation of GuangdongProvince of China (S2011010003516, S2011010004793)

Conflicts of interest None

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