| Objectives:Studies have shown that the abnormal changes of ubiquitin-proteasome system(UPS)are closely related to tumorigenesis.E3 ubiquitin ligase is a key component of UPS in eukaryotes,according to the structural characteristics,it can be divided into two categories: RING(really interested new gene)zinc finger domain class and HECT(homologous to E6 AP C-terminus)domain class.HECW1 is the least known molecule among the nine members of the NEDD4 family of HECT ubiquitin ligase,also known as NEDL1(Nedd4-Like Ubiquitin Protein Ligase 1).It has been reported that HECW1 can induce apoptosis by binding with p53 in cancer cells expressing wild-type p53.But HECW1 expression and its role in non-small cell lung cancer(NSCLC)remain unclear.Here,immunohistochemistry(IHC)was performed to assess HECW1 expression in NSCLC,and the relationship between HECW1 expression and the clinicopathological features ? overall survival(OS)and progression-free survival(PFS)of NSCLC patients were explored.Functional experiments were further performed to determine the effects of HECW1 on proliferation,migration and invasion of NSCLC cells and the underlying molecular mechanisms.Methods:1.160 NSCLC patients admitted to the First Affiliated Hospital of Anhui Medical University from January 2011 to December 2015 were selected as subjects,and the pathological data of all patients were collected.Immunohistochemistry(IHC)was performed to evaluate HECW1 expression.All NSCLC patients were divided into two groups: high expression of HECW1 group and low expression of HECW1 group,and the correlation between HECW1 expression and clinicopathological features of NSCLC patients was analyzed.All the patients were followed up for 5 years,and Kaplan-Meier method was used to analyze overall survival(OS)and progression-free survival(PFS).2.Quantitative real-time polymerase chain reaction(q RT-PCR)and western blot were performed to detect the m RNA and protein expression of HECW1 in paired NSCLC and non-tumor tissues from 34 NSCLC patients who received radical resection in the First Affiliated Hospital of Anhui Medical University from March2016 to July 2019.3.QRT-PCR and western blot were carried out to detect the m RNA and protein expression of HECW1 in NSCLC cell lines(H1299,A549,H460,H226)and normal human bronchial epithelial cells(BEAS-2B).4.HECW1 overexpression vector and Si-HECW1 were constructed and screened.NSCLC cells with low HECW1 expression(H1299 cells)were transfected with HECW1 overexpression vector,and NSCLC cells with high HECW1 expression(A549 cells)were transfected with Si-HECW1.QRT-PCR and western blot were performed to detect HECW1 expression to determine the overexpression and interference efficiency.CCK8 was selected to determine cell proliferation.Transwell assay was carried out to assess cell migration and invasion.5.QRT-PCR and western blot were performed to examine Smad4 m RNA and protein expression in H1299 cells transfected with HECW1 overexpressing vector and A549 cells transfected with si-HECW1.6.The proteasome inhibitor MG-132 was used in H1299 cells transfected with HECW1 overexpressing vector,and western blot was performed to detect the protein level of Smad4.7.Flag-HECW1 and Myc-Smad4 were co-transfected into H1299 cells,and western blot was performed to detect Smad4 ubiquitination via the HA-labelled ubiquitin protein.8.HECW1 overexpression vector and Smad4 overexpression vector were co-transfected into H1299 cells.CCK8 was selected to determine cell proliferation.Transwell assay was carried out to assess cell migration and invasion.Results:1.IHC results revealed that 104 patients exhibited the positive expression of HECW1 and 56 patients exhibited the negative expression of HECW1;among the 34 normal lung tissues,5 exhibited the positive expression of HECW1 and 29 exhibited the negative expression of HECW1.The positive expression rate of HECW1 in NSCLC tumor tissues was significantly up-regulated,compared with the normal lung tissues(65.000% vs.23.529%,P<0.001).HECW1 expression was significantly correlated with smoking,TNM stage,lymph node metastasis,and tumor size(P<0.05).There was no significance correlation with gender,sex,histology,and tumor location(P>0.05).Kaplan-Meier analysis revealed that NSCLC patients displayed significant differences in OS and PFS between high expression of HECW1 and low expression of HECW1(P<0.001),NSCLC patients with high expression of HECW1 showed poor OS and PFS.A multivariate analysis demonstrated that HECW1 expression was an independent prognostic factor for NSCLC(P<0.05).2.Compared to normal human bronchial epithelial cells(BEAS-2B),HECW1 expression were significantly up-regulated in NSCLC cell lines(H1299,A549,H460,H226)(P<0.01),both in m RNA and protein level.H1299(the lowest expression of HECW1)and A549(the highest expression of HECW1)cells were selected for the in vitro experiments.3.QRT-PCR and western blot results revealed that H1299 cells were successfully transfected with HECW1 overexpression vector(P<0.01).CCK-8 assay and transwell assay further showed that overexpressing HECW1 significantly enhanced the proliferation,migration,and invasion of H1299 cells(P<0.01).4.QRT-PCR and western blot results revealed that A549 cells were successfully transfected with si-HECW1(P<0.01).CCK-8 assay and transwell assay further showed that deleting HECW1 significantly reduced the proliferation,migration,and invasion of A549 cells(P<0.01).5.QRT-PCR and western blot results showed that overexpressing HECW1 significantly reduced Smad4 protein level(P<0.01),and the m RNA level of Smad4 was not affected in H1299 cells.QRT-PCR and western blot results also showed that deleting HECW1 significantly elevated Smad4 protein level(P<0.01),and the m RNA level of Smad4 was not affected in A549 cells.Western blot results further showed that proteasome inhibitor MG-132 significantly rescued the reduced protein level of Smad4 induced by HECW1 in H1299 cells(P<0.01).Flag-HECW1 and Myc-Smad4 were co-transfected into H1299 cells,and western blot results showed that overexpressing HECW1 significantly promoted Smad4 ubiquitination.6.H1299 cells were co-transfected with HECW1 and Smad4,western blot results showed that Smad4 significantly rescued the reduced protein level of Smad4 induced by HECW1(P<0.01).CCK-8 assay and transwell assay showed that overexpressing HECW1 significantly enhanced the proliferation,migration,and invasion of H1299 cells,but Smad4 dramatically reversed the increased proliferation,migration,and invasion of H1299 cells induced by HECW1(P< 0.01).Conclusion:1.HECW1 is highly expressed in NSCLC tumor tissues,and NSCLC patients with high expression of HECW1 have poor OS and PFS.HECW1 may be a prognosis marker of NSCLC.2.HECW1 promotes the proliferation,migration,and invasion of NSCLC cells via inducing the ubiquitination of Smad4. |
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