| Part 1 Expression and biological behavior of USP15 in non-small cell lung cancer cell linesObjective To investigate the expression of ubiquitin specific peptidase 15(USP15)in cell line of non-small cell lung cancer(NSCLC)and normal lung epithelium,and its effects on biological behavior in vitro and in vivo.Methods USP15-shRNA was transfected into NSCLC cell lines A549 and H1299 by lentivirus infection.Stable cell line of USP15 knockdown were established,which were named as USP15KD A549 and USP15KD H1299.Western blot were used to detect the expression of USP15 in these cell lines.The proliferation of A549 and H1299 cells before and after interfering with USP15 was detected by CCK8,and the invasiveness of A549 and H1299 cells before and after USP15 were observed by Transwell test.In the tumorigenicity experiment,fresh lung tissues were taken to observe the quality and number of tumor nodules.The lung tissue was stained with paraffin section and HE staining to observe the tumorigenesis in nude mice.Results 1.The protein expression of USP15 in NSCLC cell lines A549 and H1299 were significantly higher than that in human lung epithelial cell BEAS-2B(P<0.05);2.The NSCLC cell model interfered with USP15 were successfully established,named as USP15KD A549 and USP15KD H1299.The protein expression of USP15 in USP15KD A549 was significantly lower than that in A549(P<0.05),and the protein expression of USP15 in USP15KD H1299 was also significantly lower than that in H1299(P<0.05);3.The proliferation ability of USP15KD A549 cells in 3 d,4 d and 5 d was significantly lower than that of A549 cells(P<0.05),and the proliferation ability of USP15KD H1299 cells was also significantly lower than that of H1299 cells when the cells were cultured after 3 d,4 d and 5 d(P<0.05).4.Transwell invasion assay showed that the invasion ability of USP15KD A549 cells was significantly lower than that of A549 cells(P<0.05);the invasion ability of USP15KD H1299 cells was also significantly lower than that of H1299 cells(P<0.05);5.Westernblot results showed that the relative expression of MMP3 and MMP9 in USP15KD A549 cells was significantly lower than that in A549(P<0.01),and the relative expression of metalloproteinases in USP15KD cells was significantly lower than that in A549(P<0.01);6.In vivo,results showed that USP15 interference inhibited the formation of tumor when injecting USP15KD through tail vein,and the number of lung nodules in nude mice of USP15KD H1299 group was significantly less than that of A549 group(P<0.05),and the total weight of lung tumor in nude mice of USP15KD H1299 group was also lower than that of H1299 group(P<0.05).Conclusions USP15 is overexpressed in NSCLC cell lines and its knockdown could inhibit the proliferation and invasiveness of NSCLC,and the growth of transplanted tumor in mouse,which suggests USP15 may be as a novel molecular target to use for targeting therapy of NSCLC.Part 2 Expression and clinical significance of USP15 in human non-small cell lung cancerObjective To investigate the expression of USP15 in cell line of non-small cell lung cancer(NSCLC)tissues and normal lung epithelium.Methods 30 NSCLC surgical patients in the Third People's Hospital of Yancheng were collected in this study.The carcinoma and adjacent tissue of lung primary tumor were fixed,embedded in paraffin,and immunofluorescence was applied to observe the expression of USP15 in these tissue.The expression of USP15 gene and proteins in carcinoma and adjacent tissue was detected by quantitative PCR,Western blot.Results 1.The results of immunofluorescence showed that the expression of USP15 in non-small cell lung cancer(NSCLC)was significantly higher than that in adjacent tissue,and the average fluorescence intensity of USP15 positive cells in squamous cell carcinoma,adenocarcinoma and adenosquamous carcinoma was significantly higher than that in adjacent tissue(P<0.05).The average number of USP15 positive cells in 40-fold field of squamous cell carcinoma,adenocarcinoma and adenosquamous carcinoma was significantly higher than that of adjacent tissue(P<0.05);2.The gene expression levels of USP15 in carcinoma tissue of patients with NSCLC was significantly higher than that in adjacent tissue(P<0.05);3.The relative protein expression of USP15 in carcinoma tissue of patients with NSCLC was significantly higher than that in adjacent tissue(P<0.05).Conclusions USP15 gene and proteins levels are overexpressed in NSCLC patients,which suggests USP15 may be correlated with pathogenesis and development of human NSCLC.Part 3 Expression of USP15 in the serum of non-small cell lung cancer patients and its clinical significanceObjective To investigate the expression of USP15 in the serum of non-small cell lung cancer(NSCLC)patients and its relationship with clinical pathological features;and to evaluate its clinical value as a marker alone,or in conjunction with other parameters in diagnosis of NSCLC.Methods Serum samples were collected from 73 NSCLC patients with definite pathological diagnosis,60 lung benign disease patients and 51 healthy subjects.Serum levels of USP15 were measured by enzyme-linked immunoabsorbent assay(ELISA),and the levels of carcinoma embryonic antigen(CEA),cytokeratin protein fragment 21-1(CYFRA21-1),and neuron specific enolase(NSE)were measured by electrochemiluminescence technique.The relationships of USP15 and related parameters with NSCLC were analysed by binary logistic regression.Receiver operating characteristic(ROC)curve was used to determine the clinical value of USP15 alone,or in conjunction with other parameters in diagnosis of NSCLC.The diagnostic efficiency of USP15 and other parameters was evaluated according to the area under the curve(AUC).Youden index was calculated and the best cut-off value,sensitivity and specificity according to its largest value.Results The serum levels of USP15 in NSCLC patients were higher than those in lung benign disease patients(P<0.01)and healthy subjects(P<0.01),and significant difference also was observed between lung benign disease patients and healthy subjects(P<0.01).Serum USP15 levels were significantly higher in NSCLC patients with TNM III-IV than those with TNM ?-?(P<0.01),but no difference was found in patients with different gender,age,pathological types,differentiation and whether tumor has spread to lymph nodes(P>0.05).Logistic regression analysis showed that there was a significantly positive correlation between USP15 and NSCLC(OR:1.004,95%CI:1.002-1.005,P<0.01).The AUC for USP15 was 0.796(95%CI:0.730-0.863)in diagnosis of NSCLC,and when the largest Youden index was taken as 0.52,the best cut-off value of USP15 was 1.24 ng/mL,and the corresponding sensitivity and specificity were 74%and 78%,respectively.The AUC for USP15 in conjunction with CYFRA21-1 was 0.865(95%CI:0.810-0.921)in diagnosis of NSCLC,and when the largest Youden index was taken as 0.66,the corresponding sensitivity and specificity were 86%and 79%,respectively;AUC for USP15 in conjunction with CEA was 0.830(95%CI:0.770-0.890)in diagnosis of NSCLC,and when the largest Youden index was taken as 0.56,the corresponding sensitivity and specificity were 80%and 77%,respectively;and the AUC for USP15 combined with CYFRA21-1 and CEA was 0.878(95%CI:0.826-0.930)in diagnosis of NSCLC,and when the largest Youden index was taken as 0.66,the corresponding sensitivity and specificity were 87%and 75%,respectively.Conclusions Serum USP15 levels are overexpressed in NSCLC patients and are risk factors for NSCLC,whose expression was correlated with clinical stages and differentiation of cancer.The combined detection of serum USP15 and CYFRA21-1 levels could improve the sensitivity and specificity of NSCLC diagnosis.These results suggest that USP15 is expected to be a biomarker for the diagnosis of lung cancer. |
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