CXCL7 Promotes Proliferation And Invasion Of Cholangiocarcinoma Cells

BackgroundCholangiocarcinoma arises from the epithelial cells of the intrahepatic,perihilar and distal biliary tree.Despite significant improvements in surgery combined with chemotherapy using gemcitabine and cisplatin,the prognosis of cholangiocarcinoma patients still remains poor with a median survival of less than one year.In the recent decade,deregulations of various oncogenes or tumor suppressors have been implicated in malignant progression of human cancers including.Therefore,revealing the underlying mechanism of cholangiocarcinoma development and progression may help improve the development of effective therapeutic strategies.Chemokines,secreted by various cell types,play central roles in chemotaxis.According to the order of conserved cysteine residues,chemokines are classified into C,CC,CXC and C(X)3C,and CXC chemokines are further classified into ELR+CXC and ELR-CXC,according to the absence or presence of the amino-terminal ELR motif.Many studies have shown that chemokines are involved in the tumorigenesis and malignant progression of human cancers,and therefore may become potential therapeutic target for cancer treatment.For instance,Lung cancer cells were found to utilize the CXCL12/CXCR4 signaling to benefit growth and distant spread.CXCL5/CXCR2 axis can promote the migration and invasion of bladder cancer cells by activating PI3K/AKT-induced upregulation of MMP2/MMP9.CXCL7 is a platelet-derived growth factor that belongs to the CXC chemokine family,functioning as a potent chemoattractant and activator of neutrophils through binding to its receptor CXCR2.CXCL7 has been demonstrated to participate in a variety of cellular processes,such as DNA synthesis,glycolysis,mitosis,intracellular cAMP accumulation,prostaglandin E2 secretion,as well as the synthesis of hyaluronic acid and plasminogen activator.Moreover,it is also an antimicrobial protein with bactericidal and antifungal activity.Recently,CXCL7 has been found to be deregulated in human cancers,and plays a role in tumor growth.For instance,CXCL7 was found to promote the growth of clear cell renal cell carcinoma.Desurmont et al.reported that overexpression of CXCL7 and CXCR2 in liver metastases from colon cancer was correlated to shorter disease-free and overall survival.However,the role of CXCL7 in cholangiocarcinoma has never previously been studied.The present study aimed to investigate the expression of CXCL7 in cholangiocarcinoma tissues,as well as the regulatory role of CXCL7 in the malignant phenotypes of cholangiocarcinoma cells.MethodsPart I:The expression of CXCL7 is associated with disease progression and poor prognosis in cholangiocarcinoma patients1.Tissue Samples collectionThis study was approved by the legislation and ethical boards of Guangdong general Hospital,Guangdong Academy of Medical Sciences,Guangzhou,China.A total of 156 cholangiocarcinoma tissues and 35 adjacent non-tumor tissues were collected from our hospital.All written informed consents were obtained.Patients involved in this study received no preoperative chemotherapy or radiotherapy.All tissues were formalin fixed and paraffin-embedded.2.Immunohistochemical Staining AssayFour pm sections were deparaffinized and subjected to heat-induced antigen retrieval using citrate buffer for 22 minutes using a microwave oven,which were then incubated with primary antibodies,and then with secondary antibody for 1 h at room temperature.The reaction was developed using substrate diaminobenzidine and counterstained with hematoxylin.The protein expression was scored by 3 pathologists independently.The percentage of positively staining cells was graded as 0(no staining),,negative(-);>0 and?25%of cells positive,weak expression(+);>25 and<75%of cells positive,moderate expression(++):>75%of cells positive,strong expression(+++).3.Statistical MethodsCount data are expressed as rate and percentage.Statistical analysis was performed using SPSS 17.0(SPSS,Armonk,NY,USA).The differences between two groups were analyzed using chi-square test.P<0.05 indicated significant differences.Part II:Reduced CXCL7 expression inhibits the proliferation and invasion of cholangiocarcinoma cells1.Cell culturesHuman cholangiocarcinoma cell lines(HuCCT1,HuH28,QBC939,EGI-1,OZ and WITT)and human hepatic stellate cell line LX-1 were purchased from Cell Bank of Chinese Academic Institute,Shanghai,China.All cell lines were cultured in DMEM(Gibco,USA)added with 10%FBS(Gibco,USA)in a 37? incubator with 5%C02.2.Cells transfectionLipofectamine 2000 was used to conduct cell transfection,according to the manufacture's instruction.Briefly,cells were cultured to 70%confluence,and resuspended in serum-free DMEM medium.The blank pcDNA3.1 vector,pcDNA3.1-CXCL7 plasmid,non-specific siRNA,CXCL7 siRNA,and Lipofectamine 2000 were diluted with serum-free medium,respectively.The diluted Lipofectamine 2000 was then added into the diluted plasmid or siRNA,and incubated for 20 min at room temperature,and then added into the cell suspension.After incubation at 37?for 6h,the medium was replaced by the normal serum-containing medium.Then,cells were cultured for 48h before the following assays.3.RT-PCR analysisTotal RNA was extracted by using Trizol Reagent(Life Technologies),according to the manufacturer's instruction.A total of 800 ng RNA was converted into cDNA using Reverse Transcription Kit(Life Technologies),according to the manufacture's instruction.Real-time PCR was then performed by using Q-PCR Detection Kit(Life Technologies)on ABI 7500 thermocycler.The PCR steps were 95? for 10 min,and 40 cycles of denaturation at 95? for 15 sec and annealing/elongation step at 60? for 60 sec.GAPDH was used as an internal control.The relative expression was analyzed by the 2'??Ct method.4.Western blottingCells were lysed with ice-cold lysis buffer(50 mM Tris-HC1,pH 6.8,100 mM 2-ME,2%w/v SDS,10%glycerol).Protein was separated with 10%SDS-PAGE and then transferred onto a polyvinylidene difluoride(PVDF)membrane(Life Technologies).The PVDF membrane was incubated with PBS containing 5%milk overnight at 4?.After washed with PBS for 3 times,the PVDF membrane was incubated with primary antibodies(Abcam,Cambridge,MA,USA)at room temperature for 3 h.After washed with PBS for 3 times,the PVDF membrane was incubated with secondary antibody(Abcam)at room temperature for 1 h.Super Signal West Pico Chemiluminescent Substrate Kit(Pierce,Rockford,IL,USA)was then used to detect signals,according to the manufacture's instruction.The relative protein expression was analyzed by Image-Pro plus software 6.0,represented as the density ratio versus GAPDH.5.Enzyme-linked immunosorbent assay(ELISA)Cells in DMEM containing 10%FBS were seeded in a 6-well plate(1.5×105 cells/well)and cultured for 48 h.Then,the supernatant was collected,and centrifuged at 12000xg for 10 min.The secretion level of CXCL7 was detected using a human CXCL7 ELISA kit(Thermo Fisher,USA),according to the manufacturer's instructions.6.MTT assayMTT assay was used to examine cell proliferation-Briefly,cells were plated at a density of 10000 cells per well in 96-well plates.After cultured for 0,24,48 and 72 h,the cells were incubated with MTT at a final concentration of 0.5 mg/ml for 4 h at 37?.After the removal of the medium,150 mM DMSO solutions were added.The absorbance was read at 570 nm using a Bio-TekTM ELX-800TM Absorbance Microplate reader.7.Trans well assayTranswell assay was performed to examine the cell invasion using transwell chambers(BD,USA).Cell suspension containing 5×105 cells/ml was prepared in serum-free media,and 300?l of cell suspension was added into the upper chamber.Then,500?l of DMEM with 10%FBS was added into the lower chamber.Cells were incubated for 24 h.Then,we used a cotton-tipped swab to carefully wipe out the cells that did not migrate through the pores.The filters were fixed in 90%alcohol and stained by crystal violet,and observed under an inverted microscope(Olympus,Tokyo,Japan).8.Statistical MethodsAll data represent the results of above 3 times repeated the experiment,data are expressed as the mean ± SD.Statistical analysis was performed using SPSS 17.0(SPSS,Armonk,NY,USA).The differences between two groups were analyzed using student t test.Diverse variance of the comparison of the mean using the single factor analysis of variance(One-Way ANOVA),and F test,variance neat,two comparison using the LSD method;Variance not neat,two comparison using Dunnett's T3 method.P<0.05 indicated significant differences.ResultsPart ?:The expression of CXCL7 is associated with disease progression and poor prognosis in cholangiocarcinoma patients1.Upregulation of CXCL7 is associated with cholangiocarcinoma progressionIn the present study,our data indicated that the CXCL7 protein was mainly in the cytoplasm.The positive expression of CXCL7 was found in 66%(103/156)of cholangiocarcinoma cases,while only 23%(8/35)was detected in adjacent non-tumor tissues(P<0.05).The cholangiocarcinoma patients were divided into two groups,low CXCL7 expression group(negative and weak expression)and high CXCL7 expression group(moderate and strong expression).High CXCL7 expression was significantly associated with the poor differentiation,lymph node metastasis,vascular invasion,and advanced clinical stage of cholangiocarcinoma(P<0.05).However,the expression of CXCL7 was not associated with age,sex,and tumor size(P>0.05).After that,we further investigated the relationship between CXCL7 protein expression and the expression of Ki67,CA199,AFP,and P53 in cholangiocarcinoma.As indicated in Table 2,the expression of CXCL7 was significantly associated with the Ki67 expression(P<0.05).However,the CXCL7 expression was not associated with CA199,AFP,or P53 expression in cholangiocarcinoma(P>0.05).2.The increased CXCL7 expression is associated with poor prognosis of cholangiocarcinoma patientsThe cholangiocarcinoma patients with high CXCL7 expression had shorter overall survival time,when compared with those with low CXCL7 expression(P<0.05).Therefore,the increased expression of CXCL7 is associated with the advanced progression and poor prognosis of patients with cholangiocarcinoma.Part ?:Reduced CXCL7 expression inhibits the proliferation and invasion of cholangiocarcinoma cells1.Knockdown of CXCL7 reduces the proliferation and invasion of cholangiocarcinoma cellsWe examined the protein expression of CXCL7 and CXCR2 in several common cholangiocarcinoma cell lines including HuCCTl,HuH28,QBC939,EGI-1,OZ and WITT.As QBC939 cells showed the highest expression of CXCL7 and CXCR2,we used this cell line in the following experiments.To knockdown the expression of CXCL7,QBC939 cells were transfected with CXCL7-specific siRNA,or non-specific siRNA(NC siRNA),respectively.Our data showed that transfection with CXCL7-specific siRNA significantly decreased the mRNA and protein expression of CXCL7 compared to the control group(P<0.05).MTT assay and transwell assay further showed that knockdown of CXCL7 caused a significant decrease in the proliferation and invasion of QBC939 cells(P<0.05).These data suggest that CXCL7 plays a promoting role in the regulation of the malignant phenotypes of QBC939 cells.To further confirm these findings,QBC939 cells were transfected with pcDNA3.1-CXCL7 ORF plasmid,or blank vector as NC group,respectively.After transfection with pcDNA3.1-CXCL7 ORF plasmid,the mRNA and protein levels of CXCL7 were significantly increased(P<0.05),when compared to the control group,respectively.Moreover,overexpression of CXCL7 remarkably enhanced the proliferation and invasion of QBC939 cells(P<0.05).Taken together,we demonstrate that CXCL7 can promote the proliferation and invasion of cholangiocarcinoma cells.2.CXCL7 promotes the malignant phenotypes of cholangiocarcinoma cells in a paracrine-dependent mannerAs non-tumor cells in the tumor microenvironment can also secret CXCL7,we used 50 ng/ml of recombinant human CXCL7 to treat QBC939 cells for 24h.After treatment,the cell proliferation and invasion of QBC939 cells were examined.Treatment with CXCL7 significantly increased the proliferation and invasion of QBC939 cells,when compared to the control group,respectively(P<0.05).We further studied the effect of normal cell-derived CXCL7 on the malignant phenotypes of cholangiocarcinoma cells.Human hepatic stellate cell line LX-1 was transfected with CXCL7 ORF plasmid or blank vector,respectively.After transfection with CXCL7 ORF plasmid,the mRNA and protein expression of CXCL7 in LX-1 cells were significantly increased compared to the control group(P<0.05).ELISA data further indicated that the CXCL7 levels in the CM of CXCL7-overexpressing LX-1 cells were higher than those in the control group(P<0.05).The CM of LX-1 cells were further used to culture QBC939 cells,the proliferation and invasion of QBC939 cells cultured with the CM of CXCL7-overexpressing LX-1 cells were significantly increased,when compared to control group,respectively(P<0.05).These findings confirmed that CXCL7 may also play a promoting role in cholangiocarcinoma in a paracrine-dependent manner.3.The AKT signaling is activated by CXCL7 in cholangiocarcinoma QBC939 cellsIn the present study,western blotting data showed that knockdown of CXCL7 significantly decreased the activity of AKT signaling,when compared to the control group(P<0.05).On the contrary,overexpression of CXCL7 enhanced its activity,when compared to the control group in cholangiocarcinoma QBC939 cells(P<0.05).According to these data,we suggest that the activation of AKT signaling is involved in the CXCL7-induced proliferation and invasion of cholangiocarcinoma cells.ConclusionFrom what has been discussed above,we can draw the following conclusions:(1)CXCL7 plays promoting role in the proliferation and invasion of cholangiocarcinoma cells through activation of AKT signaling pathways.(2)Blockage of the connection between cholangiocarcinoma and adjacent tissue may be an effective strategy for the treatment of cholangiocarcinoma.