Study On The Promotion Of METTL7B On Proliferation And Migration Of Lung Adenocarcinoma Cells And Its Significance As A New Molecular Marker For Clinical Diagnosis

Background:Lung cancer is a cardinal cause of cancer-related mortality in both men and women throughout the world.Amongst all cancers,lung cancer has the highest mortality rate in most countries,with industrialized zones such as Europe and North America having the highest rates.In China,the registered lung cancer mortality rate increased by 464.84 % in the past three decades.The American Cancer Society predicts that 228,820 lung and bronchus cancers will be diagnosed in the United States(US)in 2020,with 135,720 deaths.The new classification of lung tumors was published by the World Health Organization(WHO)in 2015.The primary lung cancer types are small-cell lung carcinoma(SCLC)and non-small-cell lung carcinoma(NSCLC).Around 80% of lung cancer cases are non-small cell lung cancer comprise of adenosquamous carcinoma,adenocarcinoma,squamous cell carcinoma,large cell neuroendocrine carcinoma,and large cell carcinoma.The most frequent histological subtype of lung carcinomas is lung adenocarcinoma,consisting of40% of all lung cancers.Even though the survival rate is improving in 60% of lung adenocarcinoma patients with targetable gene alterations,the mortality is still high because of a delayed identification time,cancer progress to advanced stages with extensive metastasis,and poor drug response.The overall 5-year survival rate for lung adenocarcinoma is less than 20%,owing to untimely diagnosis.Despite many improvements and accomplishments in the basic research concerning lung adenocarcinoma,the underlying molecular mechanisms have not been sufficiently elaborated.The absence of specific and sensitive biomarkers that can provide significance in its early diagnosis still is undiscovered.So,there is a desperate need to identify early specific and sensitive biomarkers or drug targets of lung adenocarcinoma for developing improved diagnosis and clinical management.Methyltransferase-like 7B(METTL7B)belongs to the methyltransferase-like proteins(METTL)family.METTL family consists of more than twenty-seven members,but their roles have not yet been explored in detail.METTL7 A is a closely associated protein with METTL7 B sharing 59% sequence identification and more broadly studied than METTL7 B.It is confirmed to play a vital role in hepatocellular carcinoma(HCC),lung adenocarcinoma,thyroid cancer,and breast cancer.Recently METTL7 B has gained much more attention.Initially,METTL7 B was recognized as a Golgi-interrelated methyltransferase with unidentified purposes.To our information,METTL7 B was linked with diseases such as non-alcoholic steatohepatitis lipid metabolism,severe preeclampsia,and infections.Increasing evidence defined the function of METTL7 B in the progression of malignant tumors,including breast tumor,primary thyroid cancer(PTC),and non-small cell lung cancer(NSCLC).METTL7 B was upregulated in primary thyroid cancer and facilitated thyroid cancer cell migration and invasion through favoring tumor growth factor beta-1(TGF-β1)induced EMT(epithelial-mesenchymal transition).In breast carcinoma,Rho BTB(BTB stands for broad-complex,tramtrack,bric à brac)knockdown led to the fragmentation of the Golgi complex because of lowered METTL7 B expression,which intensified the invasion of breast cancer cells.In lung cancer,a recent study described that METTL7 B was overexpressed in non-small cell lung cancer tissues and increased tumorigenesis by regulating cell cycle progression.However,the clinical expressional significance and diagnostic value of METTL7 B as a potential biomarker for lung adenocarcinoma required further confirmation,and the biological role of METTL7 B in lung adenocarcinoma need further exploration.Objective:1.To explore the biological functions of METTL7 B in lung adenocarcinoma and provide a new therapeutic target for lung adenocarcinoma.2.To clarify the expression and clinical significance of METTL7 B in clinical samples of lung adenocarcinoma and provide evidence for METTL7 B as a potential early diagnostic marker and prognostic predictor of lung adenocarcinoma.Methods:1.The effects of METTL7 B on the proliferation and migration of lung adenocarcinoma cells To investigate the role of METTL7B(methyltransferase-like 7B)in lung adenocarcinoma cells,a series of assays were performed.We performed q PCR and western blot to assess the METTL7 B expression at m RNA and protein level in cell lines.Six human lung adenocarcinoma cell lines(H1299,H1975,H827,A549,SPCA1,PC9)and the normal human lung epithelial cell(BEAS2B),which served as the control group,were included in the experiment.The METTL7 B was knockdown by utilizing lentivirus-mediated two different short hairpin RNAs(sh RNA1 and sh RNA2)in H827 and A549 cells to confirm further whether METTL7 B gene silencing could inhibit cell proliferation.Western blotting and CCK-8 were conducted respectively to evaluate the efficacy of knockdown and assess cell proliferation.To evaluate the impact of METTL7 B on lung adenocarcinoma cell migration and invasion,we performed wound healing assay and invasion assay,respectively.2.The expression of METTL7 B in clinical lung adenocarcinoma tissues and its significance METTL7B’s expression was validated in independent clinical cohort samples of lung adenocarcinoma tissues microarray(TMA)via immunohistochemistry(IHC).Tissue microarray(TMA)holding lung adenocarcinoma tumor samples(n=96)and matched nearby non-tumor tissues(n=77)were used for IHC staining to define METTL7B’s expression in clinical lung adenocarcinoma patients.The clinicopathologic characteristics and prognostic data of tissue microarray(TMA)patients were obtained to explore the relationship between METTL7 B and lung adenocarcinoma patients’ outcomes.3.The expression of METTL7 B in clinical lung adenocarcinoma serum and its significance ELISA was performed by collecting serum samples from the model and validation cohort.First,we created a model/training group by obtaining serum specimens from the dependent cohort,which had the normal healthy control group(n=35)and the lung adenocarcinoma group(n=30).Then,to further confirm our findings,we established a validation group by obtaining serum specimens from another clinical cohort with 100 normal healthy individuals and 90 lung adenocarcinoma patients.The receiver operating characteristic(ROC)curve was used to evaluate the performance of METTL7 B as a serum biomarker for the diagnosis of lung adenocarcinoma,compared with the classical serum biomarkers for lung cancer(CEA,CYFRA21-1,SCC).The sensitivity and specificity of METTL7 B and the optimal cut-off levels for predicting lung adenocarcinoma were also determined by using ROC analysis.Results:1.METTL7 B promoted the proliferation and migration of lung adenocarcinoma cells The quantitative polymerase chain reaction(q PCR)and western blot assays’ results were in accordance,revealing a significantly higher METTL7 B level in all lung adenocarcinoma cell lines compared with the normal human lung epithelial cell.A549 and H827 cell lines presented high endogenous METTL7 B expression in this group and were therefore selected for further study.In short,overexpression of METTL7 B was frequently found in lung adenocarcinoma.Both sh RNAs efficiently knocked down METTL7 B resulting in reduced expression in the lung adenocarcinoma cell lines,as shown in western blot.With METTL7B’s silencing,cell proliferation was notably decreased as related to the control.These findings explain that METTL7 B overexpression aid cell proliferation in lung adenocarcinoma.Our results showed that METTL7 B silencing led to intense impairment in cell migration ability of A549 and H827 relative to the control.In vitro invasion assays exposed that METTL7 B depletion resulted in conspicuous suppression of lung adenocarcinoma cell invasion capability.2.METTL7 B was overexpressed in clinical lung adenocarcinoma tissues and associated with the poor prognosis.Immunohistochemistry results revealed that METTL7 B staining was prominently dominant in the cytoplasm of lung adenocarcinoma tissue,and METTL7B’s expression was significantly higher in lung adenocarcinoma tissues than in the matched non-tumor tissues.Stratification analysis demonstrated that higher METTL7 B expression in lung adenocarcinoma was significantly correlated with sex,TNM stage,and Grade.Kaplan–Meier survival analysis showed that lung adenocarcinoma patients with higher expression of METTL7 B had a significantly reduced survival rate.In the univariate analysis of the factors associated with lung adenocarcinoma patients’ overall survival,high METTL7 B expression was significantly correlated with poor OS(overall survival)and an independent risk factor for poor prognosis.Therefore,the results suggest that METTL7 B overexpression was associated with the poor prognosis of lung adenocarcinoma patients.3.METTL7 B was overexpressed in clinical lung adenocarcinoma serum and possessed potential diagnostic significance.ELISA results determined that the average METTL7 B level of the lung adenocarcinoma patients was significantly higher than the healthy group(control group)in both model and validation groups,indicating the diagnostic significance of serum METTL7 B levels in lung adenocarcinoma.ROC results showed that the screening efficacy of METTL7 B was better than that of CEA,CYFRA21-1,and SCC in the model and validation group.Using the cutoff value of 86.48(ng/ml),the predictive diagnosis model’s sensitivity and specificity in the validation dataset were 76.67% and 76.00%,respectively.Conclusion :1.METTL7 B promote the proliferation and migration of lung adenocarcinoma cells.These findings provide a new target for the treatment of lung adenocarcinoma.2.METTL7 B is significantly overexpressed in lung adenocarcinoma tissue samples and its high expression is an independent risk factor for poor prognosis.3.METTL7 B can be applied as a molecular marker for serological diagnosis of lung adenocarcinoma,providing a new direction for non-invasive early diagnosis of lung adenocarcinoma.