The Role Of CXCL16/CXCR6 Chemokines In The Growth Of Lung Cancer And Its Correlation With NF-?B

BackgroundsLung cancer is one of the common malignant tumors clinically,with the highest mortality of cancer in the world.In China,lung cancer incidence increased gradually in the last few years.NSCLC is the major type of lung cancer,accounting for 80?85%.Despite the advanced treatments clinically,the patients with advanced lung cancer are not candidates for curative surgery,leading to remaining high mortality.There was not enough and clear evidence on the mechanisms for the progression of lung cancer metastasis,though many investigations focused on the pathogenesis and metastasis of lung cancer.Therefore,new biomarkers are needed to use in prognosis and to treat lung cancer.In the first part,we collected tumorous and adjacent tissues from 40 NSCLC patients.By measuring CXCL16 and CXCR6 expressions and detecting inflammatory factors' level,we analyzed the association of CXCL16/CXCR6 with inflammatory factors and clinical characteristics.In the second part,we silenced CXCR6 or inhibited NF-?B for confirming the co-interaction between CXCL16/CXCR6 and NF-?B on the biological characteristics of lung cancer cells,in order to investigate the influences of CXCL16/CXCR6 on lung tumor pathogenesis and metastasis.Part ?.Association of CXCL16/CXCR6 with clinical characteristics1.1 ObjectivesBy detecting CXCL16/CXCR6 expression and serum inflammatory factors' level,as well as analyzing the clinical data from 40 NSCLC patients,investigated the association of CXCL16/CXCR6 with inflammation and clinical characteristics.1.2 Methods1.2.1 Clinical data collectionThe clinical data of 40 patients who lived in NSCLC in Inner Mongolia were collected.27 males and 13 females were included,including 25 patients lower than 65 years old and 15 patients higher than 65 years old,average age 60.43±10.86 years old.1.2.2 Tumor samples collection40 NSCLC patients were without any radiotherapy or chemotherapy.All the patients received surgical resection,and the tumorous and adjacent tissues were sent to Pathology for confirming pathological stage,including 5 in stage ?,7 in stage ?,19 in stage ? and 9 in stage ?,as well as 12 squamous cell carcinoma(SC),20 adenocarcinoma(AC)and 8 adenosquamous carcinoma(ASC).27 patients were with lymphatic metastasis,while 13 were without.4 patients were with pleural invasion,while 36 were without.1.2.3 IHCCXCL16 and CXCR6 positive rates were measured by IHC.1.2.4 Hematoxylin and eosin(HE)stainingHE staining was carried on with HE staining kit.1.2.5 Blood samples collection and inflammatory factors detectionBefore and after surgery,venous blood samples were obtained from 40 NSCLC patients.Serum was separated and enzyme-linked immunosorbent assay(ELISA)was for detecting serum inflammatory factors' level,including high-mobility group box 1(HMGB1),interleukin-6(IL-6),interleukin-10(IL-10)and interleukin-1?(IL-1?).1.3 Results1.3.1 CXCL16/CXCR6 expression in tumorous and adjacent tissues from NSCLC patientsCXCL16 expression in NSCLC tumor tissues:20(50.0%)of strong positive(+++),10(25.0%)of moderate positive,8(20.0%)of weak positive,and 2(5.0%)of negative.CXCL16 expression in adjacent tissues:7(17.5%)of weak positive(+++),and 33(82.5%)of negative.CXCL16 expression in tumor tissues was higher than that in adjacent tissues(95.0%v.s.17.5%),significantly(t=14.859,P<0.05)CXCR6 expression in NSCLC tumor tissues:14(35.0%)of strong positive(+++),12(30.0%)of moderate positive,8(20.0%)of weak positive,and 6(15.0%)of negative.CXCL16 expression in adjacent tissues:4(10.0%)of weak positive(+++),and 36(90.0%)of negative.CXCL16 expression in tumor tissues was higher than that in adjacent tissues(85.0%v.s.10.0%),significantly(t=10.729,P<0.05)CXCL16 expression in NSCLC tumor tissues was higher than CXCR6(t=4.583,P<0.05).CXCL16 expression was positive with CXCR6 in NSCLC tumor tissues(r=0.894,95%CI=0.843?0.934,P<0.05)1.3.2 Correlation of tumor CXCL16/CXCR6 with serum inflammatory factors in NSCLC patientsAfter surgery,serum HMGB1(t=27.189,P<0.05),IL-1?(t=27.121,P<0.05),IL-6(t=13.966,P<0.05)and IL-10(t=12.599,P<0.05)levels fromg 40 NSCLC patients decreased significantly.Serum HMGB1 in NSCLC patients with strong positive(+++)and moderate positive(++)CXCL16 was higher than that with weak positive(+),significantly(F=4.476,P=0.009).Serum HMGB1 in NSCLC patients with strong positive(+++)and moderate positive(++)CXCR6 was higher than that with weak positive(+)and negative(-),significantly(F=4.476,P=0,009).Serum HMGB1 in NSCLC patients was positively correlated with tumor CXCL16(r=0.479,95%CI=0.261?0.678,P<0.05)and CXCR6(r=0.539,95%CI=0.308?0.715,P<0.05)levels.Serum IL-10 in NSCLC patients with strong positive(+++)CXCL16 was lower than that with moderate positive(++),significantly(P<0.05).1.3.3 Correlation of tumor CXCL16/CXCR6 with clinical characteristics from NSCLC patientsFor patients with different age,there was difference on CXCL16(F=4.775,P=0.035)distributions in NSCLC tumor,significantly.Tumor CXCL16 was positive correlated with the age of NSCLC patients(r=0.334,95%CI=0.036?0.585,P=0.035).For patients with or without smocking history,there was difference on CXCL16(F=13.096,P=0.001)and CXCR6(F=17.136,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.506,95%CI=0.217?0.738,P<0.001)and CXCR6(r=0.557,95%CI=0.281?0.751,P<0.001)were positively correlated with smocking history of NSCLC patients.For patients in different TNM stages,there was difference on CXCL16(F=9.129,P=0.001)and CXCR6(F=8.187,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.483,95%CI=0.245?0.679,P=0.002)and CXCR6(r=0.553,95%CI=0.346?0.733,P<0.001)were positively correlated with TNM stages of NSCLC patients.For patients with different tumor pathological types,there was difference on CXCL16(F=8.735,P<0.001)and CXCR6(F=9.815,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.531,95%CI=0.293?0.699,P<0.001)and CXCR6(r=0.552,95%CI=0.309?0.749,P<0.001)were positively correlated with SC,AC and ASC.For patients with or without lymphatic metastasis,there was difference on CXCL16(F=22.531,P<0.001)and CXCR6(F=22.022,P<0.001)distributions in NSCLC tumor,significantly.Tumor CXCL16(r=0.610,95%CI=0.355?0.816,P<0.001)and CXCR6(r=0.606,95%CI=0.338?0.807,P<0.001)were positively correlated with lymphatic metastasis.For patients with or without pleural infiltration,there was difference on CXCR6(F=5.695,P=0.022)distributions in NSCLC tumor,significantly.Tumor CXCR6(r=0.361,95%CI=0.180?0.530,P=0.022)was positively correlated with pleural infiltration.1.3.4 Correlations among clinical characteristics of NSCLC patientsGender of NSCLC patients was negatively correlated with smocking history(r=-0.414,95%CI=-0.701?0.102,P=0.008),but positively correlated with TNM stages(r=0.322,95%CI=0.083?0.535,P=0.043),tumor pathological type(r=0.328,95%CI=0.029-0.582,P=0.039)and lymphatic metastasis(r=0.368,95%CI=0.097?0.568,P=0.020).TNM stage of NSCLC patients was positively correlated with tumor pathological type(r=0.855,95%CI=0.706-0.954,P<0.001),lymphatic metastasis(r=0.771,95%CI=0.564?0.891,P<0.001)and pleural infiltration(r=0.431,95%CI=0.225?0.594,P=0.005).Tumor pathological type of NSCLC patients was positively correlated lymphatic metastasis(r=0.704,95%CI=0.581?0.851,P<0.001)and pleural infiltration(r=0.524,95%CI=0.285?0.691,P=0.001).1.3.5 Correlation of clinical characteristics with serum inflammatory factors in NSCLC patientsSerum HMGB1 of NSCLC patients was positively correlated with age(r=0.371,95%CI=-0.004?0.094,P=0.018),TNM stages(r=0.893,95%CI=0.815?0.936,P<0.001),tumor pathological types(r=0.683,95%CI=0.465-0.862,P<0.001),lymphatic metastasis(r=0.800,95%CI=0.703?0.892,P<0.001)and pleural infiltration(r=0.343,95%CI=0.168?0.501,P=0.030).1.4 ConclusionsCXCL16/CXCR6 expressed highly in tumor tissues,which was correlated with age,smocking history,TNM stages,tumor pathological types,lymphatic metastasis,pleural infiltration and serum inflammatory factors of NSCLC patients.CXCL16/CXCR6 could be the biomarkers in progression and metastasis of NSCLC.Part ?.Influence of co-interaction between CXCL16/CXCR6 and NF-?B on biological characters of lung cancer cells2.1 ObjectivesInvestigated the Influence of co-interaction between CXCL16/CXCR6 and NF-?B on biological characters of lung cancer cells by silencing CXCR6 or inhibiting NF-?B.2.2 Methods2.2.1 Cell culturingLung AC A549 cell and lung SC NCI-H520 cell were cultured with RPMI-1640 containing 10%fetal bovine serum(FBS),100UI/ml penicillin and 100?g/ml streptomycin.2.2.2 Silencing CXCR6Small interfering RNA(siRNA)targeting CXCR6 was used to transfect A549 and NCI-H520 cells.2.2.3 Inhibiting NF-?BJSH-23 was used to inhibit NF-?B in A549 and NCI-H520 cells.2.2.4 Proteins detectionCXCL16,CXCR6,p105,c-Rel and Rel-B levels in A549 and NCI-H520 cells were measured by western blotting(WB).2.2.5 CXCL16 locationImmunofluorescence(IF)was used to locate CXCL16 in A549 and NCI-H520 cells.2.2.6 Secreting CXCL16 detectionCXCL16 secretion in supernatant of A549 and NCI-H520 cells was measured by ELISA kit for 5 days.2.2.7 Cell proliferation detectionProliferation of A549 and NCI-H520 cells was measured by cell counting kit-8(CCK-8)for 5 days.2.2.8 Cell apoptosis and cell cycle detectionsCell apoptosis and cell cycle kit was used to measured A549 and NCI-H520 cells.2.2.9 Cell invasion detectionTranswell assay was used to detected invasion of A549 and NCI-H520 cells.2.3 Results2.3.1 Effect of silencing CXCR6 or inhibiting NF-?B on CXCL16/CXCR6 and related proteins expressions in NF-?BSilencing CXCR6 not only decreased CXCR6 expression in A549 and NCI-H520 cells,but also suppressed CXCL16 and related proteins expressions in NF-?B(p105,c-Rel,Rel-B).Inhibiting NF-?B not only decreased p105,c-Rel and Rel-B in A549 and NCI-H520 cells,but also suppressed CXCL16 and CXCR6 expressions.2.3.2 Effect of silencing CXCR6 or inhibiting NF-?B on CXCL16 expression and locationIn Control group,CXCL16 accumulated on cytomembrane of A549 and NCI-H520 cells,indicating CXCL16 showed as tCXCL16.In si-CXCL16 and in-NF-KB groups,silencing CXCR6 and inhibiting NF-?B both decreased tCXCL16 expression in A549 and NCI-H520 cells.2.3.3 Effect of silencing CXCR6 or inhibiting NF-?B on CXCL16 secretionIn A549 cell,CXCL16 secretion in si-CXCR6(r=0.997,95%CI=0.995?0.999,P<0.05),in-NF-?B(r=0.995,95%CI=0.991?0.998,P<0.05)and Control(r=0.992,95%CI=0.986-0.997,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from 24h to 120h,CXCL 16 secretion in Control group was higher than that in si-CXCR6 and in-NF-?B groups,significantly(F=128.195,P<0.05).In NCI-H520 cell,CXCL16 secretion in si-CXCR6(r=0.997,95%CI=0.995?0.999,P<0.05),in-NF-?B(r=0.968,95%CI=0.956?0.989,P<0.05)and Control(r=0.991,95%CI=0.985-0.996,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from 24h to 120h,CXCL 16 secretion in Control group was higher than that in si-CXCR6 and in-NF-?B groups,significantly(F=73.201,P<0.05).2.3.4 Effect of silencing CXCR6 or inhibiting NF-?B on lung cell proliferationA549 cell proliferation in si-CXCR6(r=0.978,95%CI=0.964?0.992,P<0.05),in-NF-?B(r=0.975,95%CI=0.945?0.993,P<0.05)and Control(r=0.976,95%CI=0.969-0.991,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from the 1st day to the 5nd day,optical density(OD)value in Control group was higher than that in si-CXCR6 and in-NF-?B groups,significantly(F=36.993,P<0.05).NCI-H520 cell proliferation in si-CXCR6(r=0.970,95%CI=0.949?0.988,P<0.05),in-NF-?B(r=0.985,95%CI=0.972?0.995,P<0.05)and Control(r=0.987,95%CI=0.981?0.995,P<0.05)groups increased as time went by,gradually and significantly.Comparing between the 3 groups,from the 1st day to the 5nd day,optical density(OD)value in Control group was higher than that in si-CXCR6 and in-NF-?B groups,significantly(F=18.448,P<0.05).2.3.5 Effect of silencing CXCR6 or inhibiting NF-?B on lung cell invasionComparing between groups,no matter in A549 cell(F=241.422,P<0.05)or in NCI-H520 cell(F=74.581,P<0.05),there were significant differences among si-CXCR6,in-NF-?B and Control groups.Meanwhile,invasive cells in Control group were more than that in si-CXCR6 and in-NF-?B groups,significantly(P<0.05).Comparing between A549 and NCI-H520 cells,there were significant differences on invasive cells in si-CXCR6(F=22.050,P<0.05)and Control(F=70.225,P<0.05)groups.2.3.6 Effect of silencing CXCR6 or inhibiting NF-?B on lung cell apoptosis and cell cycle2.3.6.1 Effect of silencing CXCR6 or inhibiting NF-?B on lung cell apoptosisComparing between,no matter in A549 cell(F=59.242,P<0.05)or in NCI-H520 cell(F=40.721,P<0.05),there were significant differences among si-CXCR6,in-NF-?B and Control groups.Meanwhile,apoptosis rate in Control group was lower than that in si-CXCR6 and in-NF-?B groups,significantly(P<0.05).Comparing between A549 and NCI-H520 cells,there were significant differences on apoptosis rate in Control(F=8.863,P=0.041)group.2.3.6.2 Effect of silencing CXCR6 or inhibiting NF-?B on lung cell cycleA549 cells in G1(F=696.087,P<0.05)and S(F=230.266,P<0.05),and NCI-H520 cells in G1(F=427.251,P<0.05)and S(F=255.513,P<0.05)showed significant differences among si-CXCR6,in-NF-?B and Control groups.In Control group,A549 and NCI-H520 cells in G1 was lower than that in si-CXCR6 and in-NF-?B groups,while that in S was higher than that in si-CXCR6 and in-NF-?B groups,significantly(P<0.05)Comparing between A549 and NCI-H520 cells,there was significant difference on G1 in si-CXCR6(G1:F=23.287,P=0.008;S:F=0.056,P=0.825),in-NF-?B(G1:F-11.471,P=0.028;S:F=2.816,P=0.169)and Control(G1:F=44.518,P=0.003;S:F=15.249,P=0.017)groups,while on S only in Control group.2.4 ConclusionsCXCL16/CXCR6 co-interacted with NF-?B,which promoted progression and metastasis of lung cancer.