Expression Of HEG1 And Its Significance In Diagnosis And Prognosis Of Lung Adenocarcinoma

Background: Lung cancer is one of the most lethal tumors,and lung adenocarcinoma is the most common type of lung cancer.The early diagnosis rate of lung cancer is relatively low,and most of patients are found in the middle or advanced stage.Therefore,early diagnosis and accurate prediction of lung cancer are helpful to timely adjust treatment and improve clinical prognosis.Thus,it is of great clinical significance to actively explore and search for appropriate molecular markers for early diagnosis and prognosis.Heart development protein with EGF-like domains 1,or protein HEG homolog 1(HEG1)was the homologue of HEG(Heart of glass)in human and mice,discovered by Mably in 2003 when he was studying the mechanism of concentric growth of zebrafish heart.HEG1 has been shown to play important roles in angiogenesis,vascular integrity and embryo development.Recent studies have revealed that HEG1 plays an important role in tumor invasion and metastasis,and serves as a specific diagnostic marker for malignant mesothelioma.However,the effect and mechanism of HEG1 in lung adenocarcinoma remains unclear.Objective: To explore the expression of HEG1 in lung adenocarcinoma,and investigate its association with clinicopathological features,diagnosis and prognosis of lung adenocarcinoma,so as to provide theoretical basis for clarifying the role of HEG1 in the occurrence and development of lung adenocarcinoma and experimental basis for early diagnosis and timely treatment of lung adenocarcinoma.Methods: A total of 32 pairs of specimens of primary lung adenocarcinoma and adjacent tissues,12 specimens of benign lung disease were collected from the Department of Thoracic Surgery of Qingdao Municipal Hospital from June 2019 to June2020.Another 59 lung adenocarcinoma blood samples and 28 healthy controls blood samples were also collected.All patients had no history of radiation and chemotherapy or other anti-tumor treatment before surgical operation.Lung adenocarcinoma cell lines A549,H1299 and normal alveolar epithelial cell lines BEAS-2B were cultured.The mRNA expression of HEG1 in tissues and cells were detected by real-time quantitative reverse transcription polymerase chain reaction(QRT-PCR).The protein expression of HEG1 in tissues were detected by Western blot and immunofluorescence.The level of HEG1 in plasma was detected by enzyme-linked immunosorbent assay(ELISA).The expression of HEG1 in lung adenocarcinoma and its influence on prognosis were explored by analyzing public bioinformatics databases.SPSS 24.0 and Graph Pad Prism8.0 were used for statistical analysis.Results:1.Bioinformatic analysis showed that HEG1 was downregulated in lung adenocarcinoma tissues.The results from analyzing the public bioinformatic dataset showed that the m RNA and protein expression level of HEG1 in lung adenocarcinoma tissues were significantly lower than that in normal tissues(P=1.62E-12、7.61E-51).However,there was no significant difference in the expression of HEG1 in different tumor stages.2.HEG1 was downregulated in lung adenocarcinoma tissues and cell lines.Verification was performed on the collected specimens.The expression of HEG1 m RNA in lung adenocarcinoma tissue was significantly lower than that in benign lung disease tissues by QRT-PCR(P<0.001).Western blot and immunofluorescence analyses showed that the expressionof HEG1 protein in lung adenocarcinoma tissues was also significantly lower than that in benign lung disease tissues(P=0.044).The results from ELISA revealed that compared with healthy controls,HEG1 was downregulated in lung adenocarcinoma patients(P=0.007).In addition,the expression of HEG1 m RNA was markedly lower in lung adenocarcinoma cell lines A549 and H1299 than that in the normal alveolar epithelial cell line BEAS-2B(P<0.001,P=0.002).These results suggested that HEG1 was lowly expressed in lung adenocarcinoma tissues and cell lines.3.HEG1 was downregulated in lung adenocarcinoma tissues than that in adjacent tissues.The expression of HEG1 m RNA in lung adenocarcinoma tissues was significantly lower than that in adjacent tissues by QRT-PCR(P<0.001).Western blot and immunofluorescence analyses showed that the expression of HEG1 protein in lung adenocarcinoma tissues was significantly lower than that in adjacent tissues(P=0.007).These results suggested that HEG1 was downregulated in lung adenocarcinoma tissues compared to adjacent tissues.4.HEG1 may act as a potential serum biomarker for lung adenocarcinoma.To explore the diagnostic significance of HEG1 in lung adenocarcinoma,we detected the relationship between HEG1 and CEA.Pearson correlation analysis indicated a negative correlation between CEA and HEG1expression(r =-0.636,P<0.001).Receiver operating characteristic curve(ROC)analysis showed that the area under the ROC curve(AUC)of HEG1 in the diagnosis of lung adenocarcinoma was 0.776(95%CI: 0.646-0.886,P<0.001),the cut-off value was0.464,and the sensitivity and specificity were 77.6% and 66.7%,respectively.These data suggested that HEG1 could be used as a diagnostic biomarker for lung adenocarcinoma.5.HEG1 can be used as a prognostic biomarker for lung adenocarcinoma.To evaluate the role of HEG1 in the prognosis of lung adenocarcinoma,the patients were divided into Ⅰ-Ⅱstage and Ⅲ-Ⅳstage according to the clinicopathological stage.The m RNA expression of HEG1 in Ⅲ-Ⅳ stage was significantly lower than that in Ⅰ-Ⅱstage(P=0.010).ROC curve analysis showed that the AUC of HEG1 in distinguishing tumor stage was 0.903(0.776-1.000),the cut off value was 0.257,and the sensitivity and specificity were 92.3% and 83.3% respectively.According to the mean m RNA expression level of HEG1,lung adenocarcinoma patients were divided into high expression group and low expression group.we found that the expression of HEG1 was correlated with tumor stage(P=0.025),but was not correlated with age,gender,lymph node metastasis or distant metastasis.The rate of high expression of HEG1 in Ⅰ-Ⅱgroup(50.0%)was significantly higher than that in Ⅲ-Ⅳ group(0%).It suggested that the prognosis of patients with low HEG1 expression were relatively poor.Then,survival analysis from Kaplan-Meier Plotter demonstrated that patients with high HEG1 expression had longer OS(HR=0.51,95%CI: 0.40-0.65,P=2.6e-08).These results demonstrated that HEG1 closely correlated with favorable survival and could be used as a prognostic biomarker for lung adenocarcinoma.Conclusion:HEG1 is down-regulated in lung adenocarcinoma tissues and lung adenocarcinoma cell lines,which may have an inhibitory effect on the occurrence and development of lung adenocarcinoma.HEG1 may serve as a new diagnostic and prognostic biomarker for lung adenocarcinoma.