| Background:As the primary pathological type of lung cancer,it has important clinical implications to explore regulatory mechanisms and therapeutic targets enabling NSCLC progression.Histone deacetylases 7(HDAC7),a member of class IIa HDACs,was first discovered in2000.Previous studies have confirmed that HDAC7 was a key endogenous molecular which promotes proliferation,metastasis and angiogenesis.The increased HDAC7 expression was identified in numerous malignancies,such as gastric cancer,lymphoma,glioma.Nevertheless,whether HDAC7 is involved in NSCLC progression and the specific regulatory mechanisms remain unknown.Objectives:1.To investigate the expression of HDAC7 in NSCLC and the impact on survival prognosis of NSCLC patients.2.To clarify the effects of high HDAC7 expression on promoting NSCLC proliferation and metastasis.3.To investigate whether FGF18 is involved in HDAC7 mediated NSCLC progression and the molecular mechanisms.4.To investigate whether USP10 regulates NSCLC progression via mediating HDAC7 ubiquitination.5.To investigate the expression of FGF18 in NSCLC and the impact on survival prognosis of NSCLC patients.To further comprehensively analyze the NSCLC prognosis via combining the expression HDAC7 and FGF18.Methods:1.To elucidate the impact of HDAC7 expression on survival of NSCLC patients,the tumor bioinformatics analysis was firstly applied.The HDAC7 expression of 319 NSCLC patients was detected by IHC staining.Then,the relationship between HDAC7 level and clinic data or survival prognosis of NSCLC patients were further analyzed.2.The HDAC7 OE or sh HDAC7 lentivirus were applied to establish the HDAC7 overexpression or knockdown NSCLC cell lines.The cells proliferation was evaluated by proliferation-related assays,such as CCK8 assay and Ed U assay.The cells metastasis was estimated by metastasis-related assays,such as scratch assay and nude mouse lung metastasis assay.The expression changes of cell cycle and EMT biomarkers were detected by western blot.3.The transcriptome changes after HDAC7 overexpression were screened by RNA-seq analysis and validated by western blot.By infecting FGF18 OE or sh FGF18 lentivirus,we downregulated FGF18 in HDAC7-overexpressing cells or upregulated FGF18 in HDAC7 knockdown cells.The colony formation assay,nude mouse tumor xenograft assay,scratch wound healing assay and nude mouse lung metastasis assay were used to further estimate whether FGF18 was involved in the NSCLC progression mediated by HDAC7.The interaction between proteins was screened using mass spectrometry analysis and co-immunoprecipitation(Co-IP)was applied to further validate whether HDAC7 binds with β-catenin.Moreover,western blot was used to assess the changes of deacetylation,phosphorylation and nuclear translocation of β-catenin,and estimate whether FGF18 expression was mediated by β-catenin/TCF4.4.The bioinformatics analysis was adopted to clarify the USP10 expression in NSCLC and its prognostic impact on survival of lung cancer.The mass spectrometry analysis and Co-IP was used to detect whether HDAC7 binds with USP10.The USP10 OE or sh USP10 lentivirus were applied to establish the USP10 overexpression or knockdown NSCLC cell lines,and the HDAC7 m RNA and protein expression changes were validated by q RT-PCR and western blot.Furthermore,IP experiment and western blot were used to analyze whether HDAC7 ubiquitination was affected by USP10.And proliferative and metastatic capacities of NSCLC cells were estimated by assays mentioned in methods part2.5.The bioinformatics analysis was applied to analyze the prognostic influence of FGF18 expression on survival of NSCLC patients.The FGF18 expression of 319 NSCLC patients was detected by IHC staining.Then,the relationship between FGF18 level and clinic data or survival prognosis of NSCLC patients were further analyzed.Later,the comprehensive analysis was conducted to evaluate the NSCLC prognosis via combining the HDAC7 and FGF18 expression.Results:1.The TCGA analysis suggested that NSCLC patients with high HDAC7 suffered dismal prognosis.The IHC indicated that 32.3%(103/319)NSCLC sections were categorized as HDAC7 positive while 16.9%(54/319)paired normal tissue sections were categorized as HDAC7 positive.The HDAC7 were dramatically upregulated in NSCLC tissues than that in paired normal tissue.The HDAC7 levels were positively linked to tumor size,lymph node metastases and distant dissemination of NSCLC patients.Furthermore,survival analysis suggested that NSCLC patients with high HDAC7 expression were linked to poor overall survival.And HDAC7 was the independent prognostic factor of NSCLC.2.Compared with control groups,HDAC7 overexpression remarkably increased the in vitro proliferative capability of NSCLC cells estimated by CCK8 assay,Ed U assay and colony formation assay.Conversely,knocking down HDAC7 suppressed cells proliferative capability and the weight of subcutaneous xenografts tumors.Moreover,HDAC7 overexpression remarkably enhanced cells migration ability verified by transwell migration assay and scratch wound healing assay.And knocking down HDAC7 weakened cells metastasis ability and the number of lung metastasis tumor nodules of nude mice.Overexpression of HDAC7 could upregulate CDK2 and Cyclin A2 expression to promote cell proliferation,and increase Snail and decreased E-cadherin thus enhancing EMT of NSCLC cells.3.The RNA-seq and western blot showed that FGF18 was strongly upregulated after HDAC7 overexpression.And FGF18 knockdown partially rescued the enhanced proliferative and metastatic ability of HDAC7 overexpression cells.The western blot also indicated that FGF18 knockdown could partially reverse the HDAC7-induced CDK2,Cyclin A2,Snail upregulation,and E-cadherin downregulation.Moreover,Co-IP elucidated that HDAC7 and β-catenin has the interaction in NSCLC cells.And the immunofluorescence(IF)indicated that there is the subcellular colocalization between HDAC7 and β-catenin.Western blot showed the acetylation of β-catenin at Lys49 and phosphorylation of β-catenin at Ser45 were significantly decreased in HDAC7up-regulating cells.The nuclear-cytosolic protein isolation assay revealed that HDAC7 overexpression promoted β-catenin nuclear import thus enhancing FGF18 expression.4.The Oncomine and TCGA analysis suggested that USP10 was significantly upregulated in NSCLC,and lung cancer patients with high USP10 suffered shortened overall survival.The mass spectrometry analysis and Co-IP indicated that HDAC7 and USP10 has the interaction in NSCLC cells.Although HDAC7 m RNA was not affected by the change of USP10,USP10-overexpressing could significantly increase HDAC7 protein content.Furthermore,IP and western blot also indicated that downregulation or inhibition of USP10 could dramatically increase the ubiquitination of HDAC7.And then,inhibiting USP10 decreased the proliferative and metastatic capability of NSCLC cells.5.The Oncomine and TCGA analysis suggested The TCGA database showed that upregulated FGF18 was closely linked to poor prognosis of NSCLC patients.IHC suggested that the FGF18 were dramatically upregulated in NSCLC tissues than that in paired normal tissue.The FGF18 levels were positively linked to tumor size,lymph node metastases and distant dissemination of NSCLC patients.Survival analysis demonstrated that the NSCLC patients with low FGF18 exhibited prolonged postoperative survival time.And the COX analysis suggested that FGF18 was also the independent risk factor of NSCLC patients.Furthermore,we found that HDAC7/FGF18 co-expression was significantly correlated with subset of NSCLC patients with poorest prognosis.Whereas the low HDAC7/low FGF18 subgroup suffered the longest survival time.In both high or low HDAC7 subgroups,high FGF18 level portended poor prognosis.Conclusions:Based on clinical NSCLC tissues,NSCLC cell lines and nude bearing mice models,we revealed a novel USP10-HDAC7-β-catenin-FGF18 pathway that was involved in NSCLC proliferation and metastasis.Our findings may offer new insights and theoretical support for better understanding NSCLC regulatory mechanisms and future antitumor drugs development. |
PDF Full Text Request