MiR-133b Targets NCAPH To Inhibit Non-small Cell Lung Cancer Progression

[Objectives]Lung cancer is one of the most common malignant tumors in the world.It has a high morbidity and mortality.According to different histological types,lung cancer is majorly divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Among them,non-small cell lung cancer is further divided into three histological subtypes of squamous cell carcinoma,adenocarcinoma and large cell lung cancer.Patients with early lung cancer can alleviate tumor progression through surgical resection,chemotherapy,etc.However,some patients will still inevitably experience tumor recurrence and drug resistance,and eventually die.During cancer progression,the pathogenesis of lung cancer is complicated.Although there are some signaling pathways and drug targets that have been used for diagnosis and treatment,the clinical efficacy is still limiting.On the one hand,compared with radiotherapy and chemotherapy,surgery is the best way.However,due to the characteristics of tumor cells including the rapid proliferation rate and metastatic abilities,lung cancer patients still have a high mortality rate.In addition,radiotherapy,chemotherapy and drug-target therapy also have limiting therapeutic efficacies due to drug resistance and clinical side effects.Therefore,identifying new biomarkers or potential drug targets for lung cancer and clarifying their underlying molecular mechanisms will help to improve the early diagnosis and clinical treatment in the future.Condensin proteins are multi-protein complexes,which play important roles in the assembly and separation of chromosomes in mitotic and amitotic cells and even in tumors.Most eukaryotic cells have two different condensin complexes,named as condensin I and condensin II,respectively.In the early stage,we applied the integrated omics analysis method(CVAA)to identify novel tumor-related molecules.Among them,one of the three non-SMC subunits of condensin I,namely NCAPH,was found to be highly expressed in different types of solid tumors(including non-small cell lung cancer:NSCLC).Furthermore,we performed the immunohistochemistry assay using tumor tissue array(TMA),was and found that NCAPH is highly expressed in cancerous tissues and cell lines comparing with reciprocal controls,and its high expression negatively correlates with clinical outcome,suggesting that NCAPH plays an important role in non-small cell lung cancer.This thesis work mainly focusing on characterizing the molecular mechanism of NCAPH in NSCLC,thus providing a potential new thearapeutic target in the future.[Methods](1)The expression and clinical significance of NCAPH in non-small cell lung cancer:using GEPIA,UALCAN,KMplot bioinformatic datasets and methods to examine NCAPH expression and its correlaton with clinical outcome.Western blotting was used to detect the expression of NCAPH protein in 17 pairs of NSCLC cancerous tissues and adjacent non-cancerous tissues,also in NSCLC cancerous cell lines compairing with normal lung bronchial epithelial cell line:BEAS-2B.(2)In vitro functional validation:a.Establishing NCAPH stablely knockdown cell lines to verify cell growth/colony formation/BrdU incorporation abilities.b.Using scratch and transwell assays to verify the effect of NCAPH knockdown on cell migration.c.Using flow cytometry and western blotting methods to detect the effect of NCAPH knockdown on the cell cycle.d.Using flow cytometry and western blotting methods to detect the effect of NCAPH knockdown on cellualr apoptosis.e.Using the tumor-sphere formation,self-renewal capacity,qRT-PCR and western blotting assays to examine the effect of NCAPH lung cancer stem cells.CD133 positive cell population was also examined by FACS.(3)Characterizing the underlying mechanism of NCAPH in NSCLC:a.Screening microRNAs which are differentially expressed in NSCLC cancerous tissues using omics analysis,and three miRNAs including miRNA-133b,miRNA-140-3p,miRNA-338-3p were identified,and their correlations with clinical outcome were further validated using TCGA and KM plot datasets.We examined the cell proliferation abilities after overexpressing miRNA-133b,miRNA-140-3p,and miRNA-338-3p in NSCLC cancerous cell lines.Western blotting was used to indentify the specific miRNA(s)targeting NCAPH.b.miRNA-133b specificially targets NCAPH,which was validated by both bioinformatic analysis,dual luciferase assay and western blot.c.Examine the functional roles of miR-13 3b in NSCLC cancerous cell lines both in vitro and in vivo,including cell proliferation,cell migration,cell cycle,cancer stem cell maintenance and exnograft tumor formation assays.(4)Rescue experiment:The reduced cell proliferation,cell migration,cell cycle,cancer stem cell maintenance abilities after miR-13 3b overexpression can be rescued by NCAPH overexpression,supporting the specific role of miR-133b/NCAPH axis.(5)Non-SMC Condensin I Complex Subunit H is abnormally overexpressed in various cancer types.A pro-survival member of the Bcl2 family,Mcl-1,is also frequently overexpressed in multiple cancers and associates with worse clinical outcomes.The association of NCAPH and Mcl-1 proteins with the clinical and pathological features of non-small cell lung cancer(NSCLC)is examined.[Results](1)NCAPH is highly expressed in non-small cell lung cancer cancerous tissues and cell lines,comparing with adjacent non-cancerous tissues and control BEAS-2B cell line,respectively.(2)NCAPH knockdown inhibits tumor cell growth and colony formation abilities.NCAPH knockdown can inhibit the colony formation and BrdU incorporation abilities(cell proliferation assays).Knockdown of NCAPH inhibits the migration ability in tumor cells,promotes cell cycle arrest in G0/G1 phase,and increases celluar apoptosis.In addition,DDP(cisplatin)-induced celluar apoptosis is increased after NCAPH knockdown.NCAPH knockdown inhibits tumor spheres formation and cancer stem cell self-renewal ability,througth inhibiting Wnt/beta-catenin signaling pathway activity and related cancer stem cell marker gene expressions,including CD133,Oct4,Sox2 and et al.(3)miR-133b specifically inhibits NCAPH expression.miR-133b,miRNA-140-3p,miRNA-338-3p were decreased in NSCLC cancerous tissues comparing with paired adjacent non-canceorus tissues.In addition,miR-133b,miRNA-140-3p,miRNA-338-3p were found to potentially inhibit NCAPH expression using Targetscan bioinformatic analysis.miRNA-133b was finally identified to specifically inhibit NCAPH expression in NSCLC,overexpression of which inhibit tumor cell proliferation,migration,cell cycle and xenograft tumor formation.(4)The positive percentage of NCAPH in the non-cancerous lung tissues was found to be higher than that in NSCLC.However,the positive percentage of Mcl-1 in the non-cancerous lung tissues was lower than in NSCLC.Moreover,NCAPH high expression patients had a higher overall survival rate than low expression patients,whereas the Mcl-1 high expression group had a lower survival rate.Pairwise association in 260 cases of NSCLC revealed that overexpression of the NCAPH protein was negatively associated with Mcl-1 expression and vice versa.The results of multivariate Cox proportional hazard regression analysis also indicated that NCAPH and Mcl-1 showed potential as distinct prognostic factors in NSCLC.[Conclusions]1.NCAPH is upregulated in non-small cell lung cancer.2.The expression of NCAPH is negatively correlated with clinical stage/lymph node metastasis/survival time.3 NCAPH knockdown inhibits the cell proliferation and migration abilities of NSCLC tumor cells,leading to cell cycle arrest and increased cellular apoptosis with or without cisplatin treatment.Moreover,the cancer stem cell maintenance ability was also reduced after NCAPH knockdown.4.miRNA-133b binds to the 3'-UTR region of NCAPH to inhibit NCAPH expression.5.Overexpression of miRNA-133b inhibits cell proliferation,migration and cancer stem cell self-renewal ailities leading to cycle arrest,promotes cellluar apoptosis.6.The celluar effect induced by miR-133b overexpression can be rescued by NCAPH overexpression,which is dependent on Wnt/beta-catenin signaling pathway.7.NCAPH and Mcl-1 are highly expressed and negatively correlated in NSCLC,and will be the potential to become tumor diagnostic markers.In summary,the miRNA-133b/NCAPH signal axis plays an important role in non-small cell lung cancer.