Expression And Clinical Significance Of Circular RNA TMX4 (hsa＿circ＿0001130) In Lung Adenocarcinoma

Objective:Lung cancer is still one of the main causes of mortality from cancer-related diseases worldwide according to the new cancer statistics.Although the diagnosis and treatments are improving,the survival of patients is still very low.circ RNAs are the popular noncoding RNAs that can be detected in kinds of cells and tissues.They are a stable and conserved,and also exhibit tissue/developmental-stage-specificity,which have been reported as potential diagnostic markers in different cancers including lung cancer.They regulate tumorigenesis and development in vivo and has certain correlation with the clinical characteristics of lung cancer.It is expected to be a new diagnostic marker and therapeutic target for lung cancer.circTMX4 was overexpressed in lung adenocarcinoma(LAC)tissues.We studied the effects on lung cancer cell proliferation and invasion,and the differential expression between lung adenocarcinoma tissues and normal tissue.We wish to provide a basis for the diagnosis of lung adenocarcinoma.Methods:Microarray technique was used to evaluate the circ RNA profiles of three paired LAC samples,59 abnormally expressed circ RNAs were detected in patients with LAC,including 39 up-regulated and 20 down-regulated circ RNAs.circTMX4(hsa＿circ＿0001130)with a fold change of 2.94 was selected from it.Firstly quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the expression level of circTMX4 in LAC tissues and adjacent tissues specimens.A paired t-test was used to analyze the differences in circTMX4 expression between LAC tissues and adjacent tissues.Chi-square test was used to reveal the correlation between the expression level of circTMX4 and clinicopathological features in LAC patients,and the receiver operating characteristic curve was used to evaluate the diagnostic value of circTMX4 in LAC patients.Then qRT-PCR was used to detect the circTMX4 expression in plasma pre-operation of 30 stage I-III LAC patients and the plasma of healthy volunteers.A paired t-test was used to analyze the differences in circTMX4 expression between LAC plasma and normal plasma.We used ROC curve to see the efficiency of circTMX4 in the plasma,which to be a potential biomarker of LAC.Finally,we detected the expression level of circTMX4 in LAC cell lines,and further sh RNA interfered with the expression of the circular RNA to detect its effects on lung cancer cells proliferation and migration.Result:1.The expression levels of circTMX4 in paraffin-embedded tissue specimens of 60 pairs of LAC tissues and matched paracancer tissues were detected by qRT-PCR.The results showed that the expression level of circTMX4 in LAC tissue was significantly higher than that in the adjacent tissue(P<0.001).Then we analyzed the relationship between the expression level of circTMX4 in LAC tissue and clinicopathologic feature.The results suggested that the expression level of circTMX4 was correlated with the tumor size(P = 0.0207),lymph node metastasis(P = 0.0413)and TNM stage(P=0.0435)of LAC.There was no statistically significant difference in gender,age and smoking history.We used ROC curve to test the efficiency of circTMX4 distinguishing LAC tissue from normal one.The results suggested that the sensitivity and specificity are 70% and 66.7%(P<0.01,95%CI0.56-0.76),and the AUC is 0.66.2.The expression levels of circTMX4 in plasma of 30 patients with LAC and 30 healthy volunteers were detected by qRT-PCR.The results showed that the expression level of circTMX4 in plasma of patients with LAC was higher than that in healthy human(P<0.001).The expression level of circTMX4 in pre-operation plasma was correlated with the lymph node metastasis(P = 0.0227)and TNM stage(P =0.0147)of LAC.There was no statistically significant difference in gender,age,smoking history and tumor size.We used ROC curve to test the diagnosis efficiency as a biomarker of circTMX4 in LAC.The results suggested that the sensitivity and specificity are 76.7%and 73.3%(P<0.01,95%CI 0.69-0.92),and the AUC is 0.81.3.The expression of circTMX4 in LAC cells is higher than that in HBE.Transfection of LAC cells with designed sh RNA,qRT-PCR results suggested that the si RNA could knockdown expression of circTMX4.CCK-8 、Ed U and colony formation assay suggested downregulation of circTMX4 decreased the proliferation of LAC cells.Transwell assay showed the migration of LAC cells was inhibited.Conclusion:1.circTMX4 was upregulated in LAC tissues,and the expression level was significantly correlated with tumor diameter,lymph node metastasis and TNM stage.circTMX4 has low efficiency in distinguishing LAC tissue from normal one.2.circTMX4 was upregulated in plasma samples from LAC patients and may act as a novel biomarker of LAC.3.The down-regulation of circTMX4 inhibited the cell proliferation and migration,which may affect the development of LAC as a cancer-promoting factor.