| PART Ⅰ Metabolic volume parameters based on different thresholds with baseline 18F-FDG PET/CT as prognostic factors for survival in stage III non-small cell lung cancerPurpose: Stage Ⅲ non-small-cell lung cancer (NSCLC) manifests in a range of heterogeneous patients, from those with potentially operable lesions to those with inoperable advanced lesions. Concurrent chemo-radiotherapy is recommended as the standard treatment of Stage Ⅲ non-small-cell lung cancer (NSCLC) patients.However, the 5-year survival has not significantly improved during the past decade.To improve the outcomes of patients, better and more detailed individualized treatments must be developed, and doing so depends on the identification of a more reliable prognostic factor. Therefore, it is essential to find prognostic factors for specific cohort of patients. Studies have indicated that the intratumoral heterogeneity can be described by the variability in the voxel intensity of Fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) within the tumor volume. Hence, we presumed that certain thresholds of metabolic volume parameters such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) might serve as prognostic factors of Stage Ⅲ NSCLC patients.Methods: A total of 78 cases with stage Ⅲ NSCLC were confirmed by histology or cytology and treated with concurrent chemo-radiotherapy. Before treatment, the patients were examined for staging, which included bronchoscopy, transthoracic biopsy, CT or MR of the brain, and contrast-enhanced CT of the chest and abdomen.We recorded the clinical variables, such as sex, age, stage, histology, ECOG performance status, EGFR mutations, tumor location. We computed MTV2.5 on the basis of an absolute SUV threshold of 2.5. We also evaluated MTV values on the basis of relative thresholds, i.e., MTV40%, MTV50%, MTV60%, MTV70%, MTV80%, and MTV90%, which were defined as volumes with an SUV greater than 40%, 50%, 60%,70%,80%,and 90% of SUVmax, respectively. TLG50% was calculated as MTV50%multiplied by the SUVmean, and TLG60% was calculated in a similar manner. The prognostic values of the PET parameters and clinical variables were assessed with Cox proportional hazards regression analysis of the overall survival (OS) and progression-free survival (PFS) for both univariate and multivariate analyses. The differences in the estimated factors among the subgroups were evaluated using the log-rank test.Results: The median follow-up time for all patients was 24.5 months (range 12-39 months). In the univariate analysis, MTV50%,MTV60%,MTV70%,TLG50%,TLG60%,ECOG and sex were significant prognostic factors of OS [P(MTV50%)=0.013,P(MTV60%)=0.002,P(MTV70%)=0.024.P(TLG50%)=0.013,P(Tl.G60%)=0.029,P(ECOG)=0.011,P(sex)=O.008], while MTV50%, MTV60%. MTV70%, TLG50%, TLG60%, and ECOG were significantly associated with PFS [P(MTV50%)=0.004,P(MTV60%)=0.007,P(MTV70%)=0.020, P(TLG50%)=0.006. P(TLG60%)=0.038, P(ECOG)=0.005]. In the multivariate analysis, TLG50% was significantly associated with OS (HR=0.423,P=0.023), and also significant prognostic factor of PFS [HR=0.457,P(TLG50%)=0.029].MTV60% was significantly associated with PFS (HR=0.402. P (MTV60%)=0.042). These PET/CT parameters were separately analyzed and were adjusted for age, sex, stage,histology, ECOG, and location.Conclusions: Volume-based PET pretreatment parameters have prognostic value in nonsurgical stage III NSCLC. A higher MTV60% predicts a worse PFS. TLG50% is negatively correlated with both OS and PFS. These would be helpful to identify proper cohorts for individualized therapeutic schedules.Part Ⅱ Inhibition of miR-150 suppresses the growth and metastasis of NSCLC by restoring APC expressionPurpose: Altered miRNA expression has been shown to be involved in non-small cell lung cancer (NSCLC) progression. However, the exact mechanism underlying miRNA alteration in NSCLC is still elusive. In this study, the associations of miR-150 with the pathological stage and lymph node (LN) metastasis of NSCLC as well as the roles of miR-150 in the aggressive biological behaviors of NSCLC cells were explored. Furthermore, APC was predicted to be a target of miR-150 in NSCLC by bioinformatics methods, which were verified by the loss- or/and gain-of-function experiments.Matierals and methods: we found elevated expression of miR-150 in the tumor tissues of 87 NSCLC patients. Quantitative PCR (qPCR) was conducted on an ABI 7900 Fast Real-Time PCR system (ABI, USA) according to the manufacturers’ instructions. U6 was used as an internal control for miR-150 expression and Glyceraldehyde-3-phosphate dehydrogenase (GADPH) as a control for APC. For wound-healing assays, cell layers were scratched using P200 pipette tips. The scratched cells were washed with PBS and cultured for 24 h. For invasion assays, the transwell migration chambers were coated with Matrigel, and the transwell assay was performed on H1299 cells as reported previously. For MTT assays, cells were transfected with miR-150 inhibitor or inhibitor NC. Cells were incubated with MTT for 1 h, and the absorbance at 490 nm (OD490) was measured. For colony formation assays,H1299 cells were transfected with miR-150 inhibitor or inhibitor NC, cultured and stained with crystal violet. Bioinformatics prediction of the potential target of miR-150 was performed with TargetScan (http://www.targetscan.org/). Statistical differences were determined by paired Student’s t test,Pearson’s chi-square analysis,one-way or two-way analysis of variance (ANOVA) and Bonferroni’s multiple comparisons tests and Pearson’s correlation analysis.P<0.05 was considered statistically significant.Results: Among 87 NSCLC patients. the frequencies of high miR-150 expression in tumor tissues were 7/25 (28%). 16/33 (49.43%) and 19/29 (65.52%) in patients with stage Ⅰ, Ⅱ and Ⅲ cancer (x2=7.569; P=0.0227 Fig. 1B), respectively, and were 10/39 (25.64%) and 32/48 (66.67%) in the absence and presence of LN metastasis(x2=14.5;P=0.001; Fig. 1C),respectively. Furthermore,we also found that miR-150 was increased in six human lung cancer cell lines in contrast to the normal lung cell line. MTT data showed that the proliferation of miR-150 inhibitor-transfected H1299 cells was decreased compared with that of cells transfected with inhibitor NC vector 24, 48 and 72 h after transfection (all P values<0.05). FACS analysis was further conducted to investigate the effects of miR-150 on the cell cycle. MiR-150 inhibition resulted in the accumulation of cells in G1 phase and a consequent reduced proportion of cells transitioning to S and G2/M phases (all P values<0.05). The miR-150 inhibitor-transfected cells showed delayed gap closure in the wound-healing assay(P<0.01) and decreased invasive activity through the matrigel in transwell assays compared with that of inhibitor NC-treated cells (P<0.01;). Western blotting analysis revealed that the miR-150 inhibitor-transfected cells showed up-regulated expression of E-cadherin as well as down-regulated expression of Vimentin and EMT-specific transcription factors (Snail and Slug) (all P values<0.05). The silenced expression of the miR-150-targeted anti-oncogene APC led to the augmented colony formation and invasion through matrigel in WT cells (both P values<0.01) and further rescued these impaired aggressive behaviors in cells transfected with the miR-150 inhibitor (all P values<0.01). The silenced expression of miR-150 up-regulated APC expression and decreased accumulation of the downstream signaling molecule (3-catenin (both P values<0.001), both of which were nearly reversed by the siRNA-induced suppression of APC expression (both P values<0.001).Conclusion: miR-150 expression was up-regulated in both NSCLC tissues and cell lines, and the level of miR-150 was positively correlated with the pathological stage and LN metastasis of NSCLC. In NSCLC cells, knockdown of miR-150 could suppress NSCLC cell growth, block cell cycle progression, attenuate migration and invasion and inhibit EMT. APC was predicted to be a target of miR-150 using bioinformatics methods, which was further confirmed by luciferase reporter assays and loss- and gain-of-function experiments. Our data indicated that the negative effects of knockdown of miR-150 expression on the proliferation and invasion of NSCLC cells was mediated by the restored APC level and repressed intracellular Wnt signaling. |
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