Arch Gynecol Obstet DOI 10.1007/s00404-017-4308-x

GYNECOLOGIC ONCOLOGY

The role of the SDF-1/ CXCR7 axis on the growth and invasion ability of endometrial cancer cells Hong‑qin Gu1 · Zhen‑bo Zhang1 · Jia‑wen Zhang1 · Qian‑qian Wang1 · Xiao‑wei Xi1 · Yin‑yan He1 

Received: 5 September 2016 / Accepted: 27 January 2017 © Springer-Verlag Berlin Heidelberg 2017

Abstract  Purpose  Stroma-derived factor-1 (SDF-1) and its receptor C-X-C chemokine receptor-4 (CXCR4) are involved in human endometrial carcinoma (EC) progression. CXCR7 is another important receptor of SDF-1 and has a higher affinity with SDF-1 compared with that of CXCR4. This paper aims to study the effects of the SDF-1/CXCR7 axis on the growth and invasion ability of EC cells. Methods  CXCR7 expression was evaluated by quantitative RT-PCR, immunohistochemistry, immunocytochemistry and Western blotting in EC cell lines and 30 cases of primary EC tissue from patients. EC cell line proliferation and migration were assessed following knockdown of CXCR7 by MTT and transwell assays. Results  The results showed that CXCR7 was highly expressed at both mRNA and protein levels in the EC cells and tissue. siCXCR7 effectively silenced CXCR7 in Ishikawa and AN3CA cells. Treatment with 17β-oestradiol (17β-E2) significantly increased the levels of CXCR7 and SDF-1 in Con, siCon and siCXCR7 treated Ishikawa. siCXCR7 persistently inhibited CXCR7 expression, even in cells treated with 17β-E2. Moreover, in vitro functional analyses, silencing CXCR7 resulted in decreased proliferation in Ishikawa and AN3CA cells. Treatment with 17β-E2 and SDF-1 significantly promoted the growth and migration in siCon treated Ishikawa and AN3CA. Interestingly, * Xiao‑wei Xi [email protected] * Yin‑yan He [email protected] 1



Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

in response to 17β-E2 and SDF-1 stimulation, siCXCR7 continuously inhibited the growth and invasion of Ishikawa and AN3CA cells. Conclusion  Our results indicate that SDF-1/CXCR7 plays a positive role in the proliferation and invasion of EC cells. CXCR7 inhibition treatment may provide a promising strategy for anti-tumour therapy for EC. Keywords  SDF-1 · CXCR7 · CXCR4 · Oestrogen · Endometrial carcinoma

Introduction Endometrial carcinoma (EC), which originates from the uterine epithelium, is the most common gynaecological malignancy. Every year, approximately 288,000 new cases and 50,327 deaths occur worldwide [1]. EC has two major subtypes, Type I and Type II, which are distinguished based on different aetiologies and prognosis. The 5-year survival rates of patients with high grade Type I tumours are between 45 and 77%. Type II EC is characterized by invasive disease spread at the time of diagnosis, with survival rates below 60% [2, 3]. Stromal-derived factor-1 (SDF-1), known as C-X-C motif chemokine 12 (CXCL12), is a member of the CXC chemokine family [4, 5]. C-X-C chemokine receptor-7 (CXCR7) is a receptor for SDF-1. The binding of SDF-1 to CXCR7 leads to the activation of several downstream pathways that regulate cell chemotaxis, survival, proliferation and migration [6, 7]. Of note, the roles of SDF-1 and CXCR7 have been well described in breast, lung, prostate, and bladder cancers and colorectal carcinoma [8–13]. For example, the expression of SDF-1 in bladder cancer tissues was higher than in normal bladder tissues and is related to

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the differentiation degree and invasive depth of cancer tissues [14]. Expression of SDF-1 in primary breast cancer is associated with the presence of circulating tumour cells in peripheral blood [15]. Moreover, CXCR7 levels are significantly higher in colorectal carcinoma tissues than in normal tissues [16] and positively correlate with disease severity in nasopharyngeal carcinoma tissues [17]. In 2014, Walentowicz-Sadlecka et al. [18] first reported the important role of SDF-1 as a predictor of negative clinicopathological characteristics of EC patients. They found that higher SDF-1 expression was associated with a higher risk of recurrence. However, the effects of SDF-1/CXCR7 on the functions of EC cells are poorly understood. Therefore, this study aims to investigate the effects of SDF-1/ CXCR7 on the proliferation and migration of EC cells.

Arch Gynecol Obstet

Immunohistochemistry and immunocytochemistry

Materials and methods

CXCR7, SDF-1 and I-TAC detection was performed by incubating Ishikawa, AN3CA and tissue sections with the following primary antibodies: rabbit-anti-human CXCR7 (1:50; Abcam, Cambridge, MA,USA), rabbit-anti-human SDF-1 (1:50; Cell Signaling, USA) and rabbit-anti-human I-TAC (1:100; Novus, USA). Following incubation (overnight, 4 °C), the sections were washed with PBS, and a secondary anti-mouse biotinylated antibody was applied. Finally, the sections were incubated with an avidin–biotin complex, and the reaction products were visualized by incubation with diaminobenzidine chromogen. Sections were then counterstained with haematoxylin, dehydrated and mounted. The cells were fixed in 4% paraformaldehyde for 15  min, and they were washed three times with PBS. The cells were incubated with CXCR7 (1:50), SDF-1 (1:50) and I-TAC (1:50).

Tissue specimens and cell lines

Cell culture and transfection

Thirty cases of tissue specimens that originated from EC patients at the Department of Gynecology of Shanghai First People’s Hospital (Shanghai, China) between March 2003 and June 2011 were utilized, among which there were 30 women with an average age of 58.4 years (range from 27 to 83 years old). The patients were ranked according to the International Federation of Gynecology and Obstetrics (FIGO) staging standard. None of the patients received hormonal drugs, radiation or chemotherapy before surgery. Prior to the study, the patients’ consent and approval from the Institute Research Ethics Committee of Shanghai first people’s hospital was obtained. Human EC cell lines (Ishikawa and AN3CA) were kindly provided by Prof. Feng you ji (Dept of Gynaecology and Obstetrics, Shanghai First People’s Hospital, Shanghai, China). Ishikawa is an oestrogen nuclear receptor (oestrogen receptor alpha, ER-a) positive expression cell while the oestrogen nuclear receptor is not expressed in AN3CA cells.

Ishikawa and AN3CA were cultured in DMEM-F12 medium (Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS) (Grand Island, NY, USA). The cells were maintained in a humidified 37 °C incubator with 5% ­CO2. For knockdown of CXCR7 expression, 1 × 105 cells were seeded into a six-well plate and cultured overnight (90–95% confluence) before transfection with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The sequences of CXCR7 siRNA (siCXCR7) and control siRNA (siCon) were synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China) and are as follows: siCXCR7: Sense: 5′-CAC​CGC​CGG​AAG​ATC​ATC​TTC​TCC​TAT​TCA​AGA​ GAT​AGG​AGA​AGA​TGA​TCT​TCC​GGT​TTT​TTG-3′; Antisense: 5′-GAT​CCA​AAA​AAC​CGG​AAG​AT CATCT TCT​ CCT​ ATC​ T CT​ T GA​ ATA​ G GA​ G AA​ G AT​ G AT​ C TT​ C CG​ GC-3′; siCon: 5′-CAC​CGT​TCT​CCG​AAC​GTG​TCA​CGT​ CAA​GAG​ATT​ACG​TGA​CAC​GTT​CGG​AGA​ATT​TTTTG3′; Antisense: 5′-GAT​CCA​AAA​AAT​TCT​CCG​AAC​ GTG​TCA​CGT​AAT​CTC​TTG​ACG​TGA​CAC​G TTC​GGA​ GAAC-3′. The efficiencies of siCXCR7 and siCon were tested by Western blotting.

Real‑time quantitative PCR Trizol (Invitrogen, Carlsbad, CA) was applied to total RNA extract from Ishikawa and AN3CA. A reverse transcription kit (Takara Biotech, Dalian, LTD) was used to reverse transcribe the RNA into cDNA, and the primers for targeted molecules are listed in Table  1. Quantitative RT-PCR was performed using SYBR Master Mix (Takara Biotech, Dalian, LTD) on a PTC-200 (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The relative fold changes of the tested genes were analysed using the ­2−△△Ct method with GAPDH as the reference gene (Table 2).

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Western blotting At 48  h after transfection, the cells in each group were lysed with RIPA buffer (Fermentas, USA). The levels of proteins in the lysate were measured with a BCA protein assay kit (Roche). Then, equal amounts of proteins were separated on a 10% SDS–PAGE and transferred to PVDF membranes (Millipore Inc., MA, USA). The membrane was blocked in 5% non-fat milk for 1  h and then incubated with anti-CXCR7 (1:250, Abcam Inc., Cambridge,

Arch Gynecol Obstet Table 1  The primer sequences of GAPDH and chemokine receptors mRNA

Size (bp)

Primer sequences

Position

CCR1

NM_001295

327

CCR2

HUMMCP1R

255

CCR3

AF026535

315

CCR4

NM_005508

351

CCR5

NM_000579

280

CCR6

AY242126

404

CCR7

NM_001838

353

CCR8

NM_005201

299

CCR9

HSA132337

292

CCR10

AF215981

303

CCR11

AF193507.1

310

CXCR1

NM_000634

297

CXCR2

NM_001557

967

CXCR3

NM_001504

426

CXCR4

AF025375

302

CXCR5

NM_001716

321

CXCR6

NM_006564

295

CXCR7

N_M020211.2

285

XCR1

NM_005283

397

CX3CR1

NM_001337

653

GAPDH

BC029618

235

Sense: 5′-ACCT GCAG CCTT CACT TTCC TCA-3′ Antisense: 5′-GGCG ATCA CCTC CGTC ACTTG-3′ Sense: 5′-CCAA CTCC TGCC TCCG CTCTA-3′ Antisense: 5′-CCG​CCA​AAA​TAA​CCGA TGTG ATAC-3′ Sense: 5′-TGGC GGTG TTTT TCAT TTTC-3′ Antisense: 5′-CCGG CTCT GCTG TGGAT-3′ Sense: 5′-GAAG AAGA ACAA GGCG GTGA AGAT-3′ Antisense: 5′-ATG​GTG​GAC​TGC​GTGT AAGA TGAG-3′ Sense: 5′-TGCT ACTC GGGA ATCC TAAA AACT-3′ Antisense: 5′-TTCT GAAC TTCT CCCC GACA AA-3′ Sense: 5′ –CCTG GGGA ATAT TCTG GTGG TGA-3′ Antisense: 5′-CATC GCTG CCTT GGGT GTTG TAT-3′ Sense: 5′-GTGC CCGC GTCC TTCT CATC AG-3′ Antisense: 5′-GGCC AGGA CCAC CCCA TTGT AG-3′ Sense: 5′-GCCG TGTA TGCC CTAA AGGT-3′ Antisense: 5′-ATGG CCTT GGTC TTGT TGTG-3′ Sense: 5′-CACT GTCC TGAC CGTC TTTG TCT-3′ Antisense: 5′-CTTC AAGC TTCC CTCT CTCC TTG-3′ Sense: 5′-TGCT GGAT ACTG CCGA TCTA CTG-3′ Antisense: 5′-TCTA GATT CGCA GCC​CTA​GTT​GTC​-3′ Sense: 5′-TCCT CCCT GTAT TCCT CAC​AAT​AG-3′ Antisense: 5′-CTGG GGAC TTTA GTTA CTGC CAC-3′ Sense: 5′-CAGA TCCA CAGA TGTG GGAT-3′ Antisense: 5′-TCCA GCCA TTCA CCTT GGAG-3′ Sense: 5′-CTTT TCTA CTAG ATGC CGC-3′ Antisense: 5′-GAAG AGAG CCAA CAAA GG-3′ Sense: 5′-ACCT AGCT GTAG CAGA CACG-3′ Antisense: 5′-CATA GCAG TAGG CCAT GACC-3′ Sense: 5′-GAAC TTCC TATG CAAG GCAG TCC-3′ Antisense: 5′-CCAT GATG TGCT GAAA CTGG AAC-3′ Sense: 5′-AAC​TAC​CCG​CTA​ACG​CTG​GAAAT GGAC-3′ Antisense: 5′-CAC​GGC​AA AGG​GCA​AGA​TGA​AGAC C-3′ Sense: 5′-ATGG CAAT GTCT TTAA TCTC GACA A-3′ Antisense: 5′-TGA​AAG​CT GGTC ATG​GCA​TAG​TAT​ T-3′ Sense: 5′-AAG​AAG​ATG​GTA​CGC​CGT​GTC​GTC​TGC​ATC​C TGGTG-3′ Antisense: 5′-CTC​GGC​GTC​CAG​TGAC CAG​GAG​AAG​CAC​AGC​AGC​CGGA-3′ Sense: 5′-TGAC CATC CACC GCTA CC-3′ Antisense: 5′-ATCT GGG​TCC​GAA​ACA​GC-3′ Sense: 5′-TTGA GTA​CGA​TGA​TTT​GGC​TGA​-3′ Antisense: 5′-GGCT TTGG CTTT CTTG TGG-3′ Sense: 5′-GGG​GAG​CCA​AAA​GGG​TCA​TCA​TCT​-3′ Antisense: 5′-GAG GGG CCA TCC ACA GTC TTCT-3′

606–628 912–932 126–146 357–380 734–753 1032–1048 758–781 1085–1108 994–1017 1252–1273 183–205 564–586 566–587 897–918 532–551 811–830 830–852 1099–1121 807–829 1085–1108 391–414 678–701 133–152 410–429 500–518 1448–1466 349–368 755–774 315–337 594–616 88–114 384–408 587–611 857–881 683–718 931–968 368–385 747–764 122–143 756–774 395–418 608–629

MA, UK), anti-SDF-1 (1:250; Cell Signaling, USA) and anti-GAPDH monoclonal antibody (1:3000, sigma) overnight at 4 °C. Subsequently, the membrane was washed with TBST three times and incubated with secondary antibody for 1  h at room temperature. Finally, the blots

were developed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA) [19]. Moreover, at 24 h after transfection, the cells were incubated with ­10− 9  mol/L 17β-E2 for 24  h, and CXCR7 expression was measured as described above.

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Table 2  Characteristics of endometrial cancer patients N Type of EC  Endometrioid adenocarcinoma  Undifferentiated carcinoma  Clear cell carcinoma  Squamous cell carcinoma  Others FIGO stage  IA  IB  IIA  IIB  IIIB  IVA Surgery applied  Radical hysterectomy  Total abdominal hysterectomy  Subradical hysterectomy  Subradical hysterectomy + pelvic lymph node dissection  Others

%

8 5 8 4 5

26.7 16.7 26.7 13.3 16.7

11 9 5 2 2 1

36.7 30.0 16.7 6.7 6.7 3.3

6 4 14 3

20.0 13.3 46.7 10.0

3

10.0

Cell proliferation assay Cell growth activities were examined using an MTT assay. Briefly, the cells were seeded in 96-well plates with a density of 2 × 104/ml and cultured for 48 h. Four hours before the end of the study, MTT (5 mg/L, TAKARA) was added to each well and incubated for 4  h at 37 °C. The optical density value of each well was measured using a microplate reader with a test wavelength of 450 nm. A total of eighteen groups for Ishikawa and AN3CA were included: Control (Con), siCon, siCXCR7, 17β-E2 treated Con, 17β-E2 treated siCon, 17β-E2 treated siCXCR7, SDF-1 (Peprotech, USA) treated Con, SDF-1 treated siCon, and SDF-1 treated siCXCR7. Each group had six parallel control samples. The concentration of SDF-1 and 17β-E2 used were 1­ 0− 9 mol/L and 100 ng/ml, respectively. Cell migration assay For the migration assay, 5 × 105 cells were washed, resuspended in serum-free DMEM, and placed in the top portion of a transwell chamber (COSTAR, USA). The lower compartment of the chamber contained 500 μl of complete culture solution as a chemoattractant. SDF-1 and 17β-E2 were added into the lower compartment of the chamber. The cells attached on the upper surface of the filter were removed by wiping with a cotton swab. Finally, the cells located on the lower surface were fixed by formaldehyde

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for 20 min and then stained in 0.1% crystal violet dye colour overnight. The cells that migrated to the lower surface were quantified by counting under a light microscope with magnification of ×200 in five different predetermined fields. Statistical analysis The results were expressed as the mean ± standard deviation. The data were analysed by one-way analysis of variance (SPPS 17.0 software) and the least-significant difference (LSD) test, and a P < 0.05 was considered statistically significant.

Results Chemokine receptor expression in endometrial cancer cells As seen in Fig. 1, the expression of a series of chemokine receptors was investigated in human EC cell lines (Ishikawa and AN3CA) using semi-quantitative RT-PCR analysis. The results indicated that CXCR7 mRNA was highly expressed in both Ishikawa and AN3CA (Fig. 1). CXCR7, SDF‑1 and I‑TAC expression in EC cells and tissue CXCR7 mRNA is highly expressed in EC cells, and therefore, we further investigated whether CXCR7 and its ligands (SDF-1 and I-TAC) were expressed at the protein level in EC cells and tissue. The results of immunohistochemistry (Fig.  2) showed that CXCR7 and SDF-1 were highly expressed in Ishikawa, AN3CA and EC tissue by positive staining (brown granules). However, we also noted that I-TAC had a very low expression in Ishikawa and AN3CA compared to that of SDF-1. Interestingly, I-TAC was not expressed in the EC tissue. siCXCR7 persistently inhibited CXCR7 and SDF‑1 expression of EC cells in response to 17β‑E2 As shown in Fig. 3a, b, there is no difference in expression of CXCR7 between the Con and siCon groups. However, compared with siCon, siCXCR7 pretreated Ishikawa and AN3CA showed a decreased level of CXCR7 (P < 0.05). The transfection efficiency was approximately 70%. Moreover, ­10− 9 mol/L 17β-E2 was applied to stimulate the cells. As shown in Fig.  3c, d, in Ishikawa, 17β-E2 pretreatment significantly increased the levels of CXCR7 and SDF-1 in Con, siCon and siCXCR7 (P < 0.05, Fig. 3c, d); compared with 17β-E2 pretreated siCon, the expression of CXCR7

Arch Gynecol Obstet

Fig. 1  mRNA expression of chemokine receptors in Ishikawa and AN3CA. The tested genes were analysed using the 2­ −△△Ct method, and the results were presented as the relative fold changes of the reference gene GAPDH

and SDF-1 in 7β-E2 pretreated siCXCR7 was obviously reduced (P < 0.05, Fig.  3c, d). In AN3CA cells, we found 17β-E2 treatment had no effect on the expression of CXCR7 and SDF-1 in all of the groups. These data indicate that siCXCR7 effectively inhibited CXCR7 and SDF-1 expression even in the cells treated with 17β-E2. Knockdown of CXCR7 inhibited the proliferation of Ishikawa and AN3CA cells

Fig. 2  Expression of CXCR7, SDF-1 and I-TAC in Ishikawa, AN3CA and EC tissue. Original magnifications: ×200. Immunocytochemistry analysis was performed in three independent trials, and immunohistochemistry analysis was conducted on the 30 clinical specimens

As presented in Fig. 4a, in Ishikawa cells, there was no difference in the proliferation level between Con and siCon. However, the growth of the siCXCR7 treated group was significantly decreased compared to the siCon group (P < 0.05). Moreover, pretreatment of ­10− 9 mol/L 17β-E2 (or 100  ng/ml SDF-1) resulted in the increased proliferation of Con and siCon (P < 0.05) and had no simulative effects on the proliferation of the siCXCR7 group. In AN3CA cells, there was no difference in the proliferation between Con and siCon. Pretreatment with siCXCR7 significantly lowered the growth compared

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Fig. 3  Protein expression of CXCR7 and SDF-1 in siCXCR7 pretreated Ishikawa and AN3CA. a, b The expression of CXCR7 in siCXCR7 pretreated Ishikawa and AN3CA, *P < 0.05 compared to siCon. c, d The effects of 17β-E2 on CXCR7 and SDF-1 levels in siCXCR7 pretreated Ishikawa, *P < 0.05 compared to siCon, # P < 0.05, compared to the corresponding group without E2 (M) treat-

ment. e, f: The effects of 17β-E2 on the CXCR7 and SDF-1 levels in siCXCR7 pretreated AN3CA, *P < 0.05 compared to siCon. The assays were performed in three independent trials. The expression of the targeted proteins was represented as the fold change over control (GAPDH)

to Con and siCon (P < 0.05). However, pretreatment with 100  ng/ml SDF-1 in Con and siCon resulted in increased levels of proliferation (P < 0.05) while treatment with 17β-E2 did not. As expected, pretreatment with ­10− 9  mol/L 17β-E2 (or 100  ng/ml SDF-1) had no simulative effects on the siCXCR7 group.

Knockdown of CXCR7 inhibited 17β‑E2 and SDF‑1 induced invasion in EC cells

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To determine the role of the SDF-1/CXCR7 axis in EC cell invasiveness, we next evaluated the impact of SDF-1 and CXCR7 on EC cell migration by transwell assay. As shown

Arch Gynecol Obstet

Fig. 4  Knockdown of CXCR7 significantly inhibited proliferation and invasion of EC cells. a, b The down-regulation of CXCR7 by siCXCR7 suppressed the growth of Ishikawa and AN3CA. The growth of EC cells were detected by MTT assay. The assays were performed in three independent trials. *P < 0.05 compared to siCon, # P < 0.05, as compared to the corresponding group without E2 (M) and SDF-1 treatment. c, d Silencing CXCR7 by siCXCR7 reduced the invasion ability in E2 (M) and SDF-1 treated Ishikawa. Transwell assays were performed in three independent trials to evaluate the

invasion ability of Ishikawa. #P < 0.05 compared to siCon, *P < 0.05, compared to the corresponding group without E2 (M) and SDF-1 treatment. e, f Silencing CXCR7 by siCXCR7 reduced the invasion ability in E2 (M) and SDF-1 treated AN3CA cells. Transwell assays were performed in three independent trials to evaluate the invasion ability of AN3CA cells. #P < 0.05 compared to siCon, *P < 0.05, compared to the corresponding group without E2 (M) and SDF-1 treatment

in Fig. 4c–e, no difference in invasion ability was observed between the siCXCR7 and siCon treated groups. The average numbers of cells that migrated through the membrane in the Ishikawa (siCon and siCXCR7) and AN3CA (siCon and siCXCR7) groups were 56, 60, 48 and 51, respectively. In Ishikawa cells, pretreatment with ­10− 9 mol/L 17β-E2 (or 100 ng/ml SDF-1) resulted in enhanced invasion ability in the siCon group. Compared with the 17β-E2 (or 100  ng/ ml SDF-1) treated siCon group, the numbers of migrated cells in the 17β-E2 (or 100 ng/ml SDF-1) treated siCXCR7 group were significantly reduced (P < 0.05). In AN3CA cells, pretreatment with SDF-1, but not 17β-E2, could increase the invasion ability in the siCon group. However, compared with the 17β-E2 (or SDF-1) treated siCon group, the number of migrated cells in the 17β-E2 (or SDF-1) treated siCXCR7 group was significantly decreased (P < 0.05).

Discussion SDF-1 is a chemokine and is ubiquitously expressed in both embryonic and adult tissues, with the highest levels detected in the liver, pancreas, spleen and heart [20]. CXCR4 and CXCR7 are receptors of SDF-1. The binding of SDF-1 with its receptors can induce the migration of cells expressing CXCR4 and CXCR7 [20]. Interestingly, as reported previously, SDF-1 has approximately ten times higher affinity to CXCR7 compared to CXCR4 [21]. The expression of CXCR4 in EC tissue was higher compared to the different controls [22, 23]. SDF-1 in combination with CXCR4 also functionally promotes proliferation, migration and invasion of EC cells, whereas treatment with an anti-CXCR4 monoclonal antibody and CXCR4 antagonist (AMD3100) resulted in decreased levels of EC cell migration [12, 22, 23]. In 2014, Walentowicz et al. [18] reported

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that higher SDF-1 expression was associated with a higher risk of recurrence. CXCR7 expression did not reveal any significant differences between the proteins expressed in the primary tumour cells and the clinicopathological features. However, whether the SDF-1/CXCR7 axis affects the functions of EC cells is still unknown. To achieve this goal, we used Ishikawa and AN3CA cells, which are positive and negative for ER, respectively. Ishikawa cells originate from a well-differentiated adenocarcinoma of human endometrial epithelium and maintain steroid receptors for androgen, progesterone and oestrogen. AN3CA typically arises in atrophic endometrium via a mechanism unrelated to oestrogen exposure. These cell lines are regarded as ideal models to study the response of EC with or without oestrogen exposure [24–26]. Our data showed that CXCR4 and CXCR7 are highly expressed in Ishikawa and AN3CA. Interestingly, we observed negative CCR2 expression. CCL2 and CCR2 gene variants have been reported to be associated with EC in Turkish women [27]. Therefore, CXCR7 may play an important role in the development of EC. As SDF-1 and I-TAC are ligands of CXCR7, further study was conducted to detect the expression of SDF-1, I-TAC and CXCR7 in EC cells and EC tissue. Our results showed that SDF-1 and CXCR7 are highly expressed, and I-TAC is lowly expressed, which is consistent with previously published papers. To investigate the effects of CXCR7 on the proliferation and apoptosis of EC cells, we employed siRNA to knock down CXCR7 in Ishikawa and AN3CA cells. In Ishikawa, 17β-E2 significantly increased the levels of CXCR7 and SDF-1 in the Con, siCon and siCXCR7 groups. However, siCXCR7 persistently inhibited CXCR7 and SDF-1 expression even in cells treated with 17β-E2. In AN3CA, 17β-E2 had no effect on CXCR7 and SDF-1 expression in any of the groups. These data support the hypothesis that 17β-E2 could activate the SDF-1/CXCR7 axis in ER positive cells. Additionally, depletion of CXCR7 markedly attenuated the proliferation of Ishikawa and AN3CA cells. Both 17βE2 and SDF-1 increased the proliferation and invasion of siCon treated Ishikawa. However, SDF-1 could increase the proliferative and invasive ability of AN3CA while 17β-E2 had no effect. The activation of ER by oestrogen induces SDF-1 gene expression, and SDF-1/CXCR4 signalling also promotes ER transcriptional activation [28]. In this study, CXCR4 in ER negative AN3CA could not be activated by 17β-E2. An unexpected finding was that 17β-E2 and SDF-1 ineffectively increased the growth and invasion of siCXCR7 treated cells. Theoretically, CXCR4 signalling could be activated in siCXCR7 treated Ishikawa (by 17β-E2 or SDF-1) and AN3CA (by SDF-1). The current results indicate that CXCR4 activation induced by 17β-E2 or SDF-1 has no effect on the proliferation and invasion of CXCR7-deficient cells. Yan et  al. [29] concluded that

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blocking CXCR7 with its antagonist CCX733 impaired SDF-1/CXCR4 induced EPC adhesion to active HUVECs and trans-endothelial migration. Zabel BA et  al. reported that SDF-1 greatly potentiated trans-endothelial migration (TEM) and that EC cells were more effectively inhibited by CXCR7-specific small molecule antagonists and less effectively inhibited by a CXCR4 antagonist [30]. Based on these results, we suspect that the biological functions of EC cells are mainly controlled by SDF-1/CXCR7 rather than SDF-1/CXCR4 signalling, and further clarification is needed. In conclusion, our data strongly suggest that CXCR7 serves as a potential therapeutic target in EC. Our research indicates that CXCR7 plays an important role in the regulation of proliferation and invasion in human EC, and CXCR7 inhibition may provide a promising strategy for anti-tumour therapy of EC. Acknowledgements  This work was supported by the National Natural Sciences Foundation of China (No. 81001154). Compliance with ethical standards  Conflict of interest  The authors declared that there is no conflict of interest. Research involving human participants  This article contains tissue from human participants. All of the procedures performed in studies involving human participants were in accordance with the ethical standards of the Institute Research Ethics Committee of Shanghai first people’s hospital and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent  Informed consent was obtained from all of the individual participants included in the study. Authors’ contributions  Hong-qin Gu: Project development, manuscript writing. Zhen-bo Zhang, Jia-wen Zhang: Data collection or management. Qian–qian Wang: Data analysis. Xiao-wei Xi, Yin-yan He: Protocol/project development.

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