Expression Of Hsa_circ₀044013 In Lung Adenocarcinoma And Its Regulation Of Proliferation In Lung Adenocarcinoma Cell

Background: Lung cancer has become a malignant tumor with the highest mortality rate and incidence rate in China,and it seriously affect people’s health and life.Lung adenocarcinoma（LAUD）is the most common pathological type of lung cancer.About 40%-50% of newly diagnosed lung cancers are lung adenocarcinomas.Finding new molecular markers and therapeutic targets through the study of tumor pathogenesis and gene level is an important research direction in the field of lung cancer.Circular RNAs（circRNAs）are a special class of endogenous RNAs exhibiting stable structure and high tissue-and developmental-specific expression.Recent studies have found that aberrant expression of circular RNAs plays important roles in carcinogenesis and tumor progression.However,their value in the diagnosis of lung adenocarcinoma remains unknown.Objective: The purpose of this study is to determine the expression of hsa_circ₀044013 in lung adenocarcinoma and analyze its correlation with clinicopathological data.And investigating the expression level of hsa_circ₀044013 in the plasma of lung adenocarcinoma patients and testing its possibility as a diagnostic marker for lung adenocarcinoma.Then exploring the effect of hsa_circ₀044013 on the proliferation,cell cycle and apoptosis of lung adenocarcinoma cell A549 through cell experiments..Methods: 1.Expression levels of hsa_circ₀044013 in 50 paired lung adenocarcinoma tissues and adjacent non-tumor tissues were measured by real-time quantitative reverse transcription polymerase chain reaction（qRT-PCR）.And the association between the expression level of hsa_circ₀044013 and the clinicopathological features of patients with lung adenocarcinoma was further analyzed.2.We also detected expression levels of hsa_circ₀044013 in the plasma samples from 70 lung adenocarcinoma patients and 70 healthy individuals.Differences in expression levels of hsa_circ₀044013 were analyzed using the paired t-test.A receiver operating characteristic（ROC）curve was generated to evaluate the diagnostic value.3.Then we used small interfering RNA（siRNA）of hsa_circ₀044013 to transfect into lung adenocarcinoma cell A549 by Lipo3000,and we determined the best transfection efficiency and the most suitable concentration of siRNA.The proliferation of lung adenocarcinoma cells after transfection was detected by CCK-8 and EdU,and the changes in cell cycle and apoptosis after transfection were examined by flow cytometry.Results: 1.The expression levels of hsa_circ₀044013 in paraffin-embedded tissue specimens of 50 pairs of lung adenocarcinoma tissues and matched paracancer tissues were detected by qRT-PCR.The results showed that the expression level of hsa_circ₀044013 in lung adenocarcinoma tissue was significantly higher than that in the adjacent tissue.The difference was statistically significant（P<0.001）.Then we analyzed the correlation between the expression level of hsa_circ₀044013 and clinicopathologic data.The results suggested that the expression level of hsa_circ₀044013 was correlated with the maximum tumor size（P=0.0216）and TNM stage（P=0.0011）of lung adenocarcinoma.There was no statistically significant difference in gender,the age of patients,smoking history,lymph node metastasis,and tumor differentiation.2.The expression levels of hsa_circ₀044013 in plasma of 70 patients with lung adenocarcinoma and 70 healthy volunteers were detected by qRT-PCR.The results showed that the expression level of hsa_circ₀044013 in plasma of patients with lung adenocarcinoma was higher than that in healthy human plasma,and the difference was statistically significant（P<0.05）.Then we used ROC curve to test the specificity and sensitivity of hsa_circ₀044013 in the diagnosis of lung adenocarcinoma.The results suggested that the sensitivity and specificity of hsa_circ₀044013 as a diagnostic biomarker for lung adenocarcinoma is 61.43% and 91.43%（95% CI: 0.6644-0.8352）,and the AUC is 0.75.3.Transfection of lung adenocarcinoma cells with We designed different siRNAs（si-1,si-2）and different siRNA concentrations to transfect lung adenocarcinoma cells.Using qRT-PCR 24 h after transfection,the results suggest that the siRNA could knockdown hsa_circ₀013958 and si-1 was more effective which was selected for the subsequent experiments,and the transfection efficiency is highest when the siRNA concentration is 50 nM,which can reach 85%.Cellular function experiments were performed 24 hours after transfection.The results of CCK-8 showed that there was a significant difference in the absorbance at 450 nm between the low expression group of hsa_circ₀044013 and the control group（P<0.01）,and the result suggested that the downregulation of hsa_circ₀044013 reduced the proliferation of lung adenocarcinoma cell line A549.The EdU experiments showed that after the down-regulation of hsa_circ₀044013,the proportion of EdU-positive cells was decreased compared with the control group（P<0.05）,and the result suggested that the down-regulation of hsa_circ₀044013 decreased the proliferation of A549 cells.The cell cycle detected by flow cytometry showed that when the expression of hsa_circ₀044013 was down-regulated,there was no significant change in the proportion of cells in S phase,G1/G0 phase,and G2/M phase（P>0.05）.The apoptosis detected by flow cytometry showed that there was no significant change in the percentage of apoptosis in the siRNA transfection group compared to the control group.The results showed that after down-regulating hsa_circ₀044013,the cell cycle and apoptosis has no significant change.Conclusions: 1.Hsa_circ₀044013 was upregulated in lung adenocarcinoma tissues,and its expression levels were significantly correlated with tumor diameter and TNM stage.2.Hsa_circ₀044013 was upregulated in plasma samples from lung adenocarcinoma patients and may act as a novel biomarker of lung adenocarcinoma.3.The down-regulation of hsa_circ₀044013 reduced the cell proliferation of A549,but had no affection to the cell cycle and apoptosis.Hsa_circ₀044013 may affect the development of lung adenocarcinoma by cell proliferation.