The Research Of NUCKS1 Gene Expression In Lung Adenocarcinoma

Background and AimLung cancer is a leading cause of cancer-related death worldwide,and is the most commonly diagnosed cancer.Like other cancers,it is a complex and highly heterogeneous disease involving multiple signaling pathways.The basic mechanisms underlying lung cancer initiation and progression remain largely unknown.Molecular biological research,through immunohistochemistry(IHC),PCR,cell culture and other proprietary technologies,to reveal the metastasis-related genes and markers of lung cancer has important theoretical and clinical significance for early prediction and target-based precise treatment.The aim of the study is to investigate NUCKS1 expression in human lung adenocarcinoma(LA)tissues and evaluated the corresponding clinicopathologic features and survival analysis.Material and Methods1.Bioinformatic analysisUsing bioinformatic methods,we analyzed colorectal cancer(CRC)gene expression signatures(GSE 27854)in the Gene Expression database(http://www.ncbi.nlm.nih.gov/geo/and Gene Expression Omnibus,GEO)to obtain the recurrent metastasis-related genes in gene-expression profiles.We use TCGA analysis website(www.cbioportal.org)to predict NUCKS1 gene status in LA.We also used the bioinformatics survival prediction website(kmplot.com)to predict the correlation between NUCKS1 expression and overall survival(OS)in LA.2.IHC methods to evaluate the expression of NUCKS1 in paraffin-embedded LA tissuesWe collected 70 cases of human LA in paraffin-embedded samples from Qingdao Central Hospital,China,between 2014 and 2015.Patients were followed up for 5 years from the end of surgery.The overall survival(OS)of the patients was defined as the time from surgery to death or the last follow-up.All slides were reviewed and one block from each tumor was examined.The slides were evaluated by two experienced pathologists.Phosphate-buffered saline(PBS)-treated slides served as a negative control.For each sample,we observed the entire slide to distinguish adenocarcinoma from normal alveoli.Staining intensity was scored as follows:0=negative,1=weak,2=moderate,and 3=strong.The positive ratio per tumor area was defined as(0=0%,1=1%–10%,2=11%–20%,3=21%–30%,4=31%–40%,5=41%–50%,6=51%–60%,7=61%–70%,8=71%–80%,9=81%–90%,10=91%–100%).The positive cell score multiplied by the intensity score was considered to be the final score,which ranged from 0 to 30.As described by our team before~6,we used receiver operator characteristic(ROC)curves to demonstrate the cut-off point for NUCKS1low expression and high expression to distinguish 5-year OS,where specificity plus sensitivity obtained the maximal value.3.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of NUCKS1 in fresh frozen samples of LATwenty-five fresh LA samples and paracancerous tissues from Qingdao Central Hospital(2018-2020)were collected during the operation.A small part was used for liquid nitrogen quick-frozen tissue retention,while the other part was used for rapid freezing diagnosis and paraffin specimen diagnosis after formaldehyde fixation,and the diagnosis was determined by pathologists.Total RNA was extracted and the relative expression level of NUCKS1 mRNA was detected by qRT-PCR.4.Statistical analysisWe used SPSS software(Version 20.0)and Graph Pad Prism 6(Graph Pad Software,Inc.,San Diego,CA,USA)for statistical analysis.A two-tailed Student's t-test was used to evaluate the final score between two different groups,and a two-tailed Chi-squared test and Fisher's exact test was used to compare the correlation between clinicopathologic parameters and NUCKS1 expression.The distributions of OS and disease-free survival(DFS)were estimated with the Kaplan–Meier method and compared using the log-rank test.The univariant analyses were used in the 5-year OS of LA patients.Results1.Predictive capability of the NUCKS1 gene using cancer genomicsBy GEO database analysis,Nuclear casein kinase and cyclin-dependent kinase substrate1(NUCKS1)mRNA expression in the recurrent metastasis group was shown to be significantly higher than in the non-recurrent metastasis group.In our TCGA cancer investigation bases on tissue classification,LA was found to be one of the most common type of cancer showing alterations in the NUCKS1 gene—including amplification,mutation,and deep deletion.According to bioinformatics survival prediction,the OS rate was predicted to be significantly lower in the NUCKS1 high-expression group compared with the low-expression group.2.Quantitative analysis of NUCKS1 expression with IHC confirmed that high expression of NUCKS1 in LA was associated with poor prognosis,and was an important prognostic factor for OS in LA patientsIHC results showed that NUCKS1 expression was markedly elevated in lung adenocarcinoma components(LACs),compared to normal lung alveoli(NLA)in human LA samples.NUCKS1expression was much higher in the progression group and the dead group.Expression of NUCKS1 was also observed in NLA,although to a much lower degree than in LACs,associated with poor prognosis.There were no significant correlation between clinicopathologic features and NUCKS1 expression in LACs.In the 70 cases of LA,high NUCKS1 expression was not significantly correlated with either patient sex,age,TNM,or whether or not there were interstitial lymphocytes present.Based on the histologic classification of LA in the 70 samples,high NUCKS1 expression was correlated with a low incidence of with solid,micropapillary,or spread through air space(STAS)components,but there was no statistical difference.NUCKS1 expression in LACs was proven to be an important prognostic factor for OS in LA patients by Univariate analysis.3.qRT-PCR further confirmed that high expression of NUCKS1 gene was associated with lymph node metastasis and high histological gradeConclusionCollectively,the aforementioned findings indicated that NUCKS1 was overexpressed in LACs relative to NLA;and that its expression was significantly correlated with patient5-year DFS and OS.NUCKS1 expression in LACs was proven to be an independent prognostic factor for OS in LA patients,and can be used as a biomarker for LA.It may be a therapeutic target and prognostic factor for LA.The mechanisms underlying NUCKS1action in LA still remain to be clarified,however,and further studies are therefore needed to elucidate the process by which NUCKS1 leads to oncogenesis.