The Clinical Significance And Effect Of RNA Editing In Lung Adenocarcinoma

Objective:1. To investigate RNA editing of gene’s coding region in the early stage of lung adenocarcinoma.2. To investigate the expression of adenosine deaminase acting on RNA 1(ADAR1) 、 adenosine deaminase acting on RNA 2(ADAR2) and Ornithine Decarboxylase(ODC), observe their correlation with clinicopathological characteristics and prognosis in lung adenocarcinoma.3. To explore the effect of RNA editing of AZIN1 on proliferation and invasiveness of A549.Methods:1. RNA editing of gene’s coding region in the early stage of lung adenocarcinoma was detected by microfluidics-based multiplex PCR and RNA sequencing.Real-time quantitative polymerase chain reaction(RT-q PCR) was applied to detect the expression of ADAR1 m RNA、ADAR2 m RNA and ODC m RNA.2. The expression of ADAR1 protein, ADAR2 protein and ODC protein in lung adenocarcinoma tissues and adjacent normal lung tissues was detected by immunohistochemistry. Correlation of the expression of ADAR1 and ODC with clinicopathological characteristics and overall survival of lung adenocarcinoma patients was analyzed.3. Establish stably expressed wild-type(A549-wt/AZIN1) or edited-type(A549-edt/AZIN1) AZIN1 A549. Proliferation, and invasive capability of A549-wt/AZIN1 and A549-edt/AZIN1 were assessed by XTT proliferation assay and in vitro invasion assay. Expression of ODC was detected by Western Blot.Results:Results:1. RNA editing level was elevated in lung adenocarcinoma compared to normal lung. The top editing genes, which RNA editing resided in coding region, were COG3, SRP9, COPA, CACNA1 D, RICTOR, ZNF669, METTL6, FLNA, AZIN1,C20orf30, PODXL, FLNB, PDZD7.2. The expression of ADAR1 m RNA and ODC m RNA were significantly higher in lung adenocarcinoma tissues than in adjacent normal lung tissues(P(27)0.05). The expression of ADAR2 m RNA was not significantly different between lung adenocarcinoma tissues and adjacent normal lung tissues(P(29)0.05).3. The expression of ADAR1 protein was mainly in nucleus. High ADAR1 protein expression was detected in 60.4% of lung adenocarcinoma tissues and 20.8% of adjacent normal lung tissues. The expression of ADAR1 protein was significantly correlated with TNM stage(P(27)0.05) and lymph node metastasis(P(27)0.05), but not gender, age, smoking history, pleural invasion, tumor diameter and tumor differentiation(P(29)0.05).4. The expression of ADAR2 protein was mainly in nucleus. High ADAR2 protein expression was detected in 10.4% of lung adenocarcinoma tissues and 8.3% of adjacent normal lung tissues. The expression of ADAR2 protein was not significantly different between lung adenocarcinoma tissues and adjacent normal lung tissues(P(29)0.05).5. The expression of ODC protein was mainly in cytoplasm. High ODC protein expression was detected in 56.3% of lung adenocarcinoma tissues and 12.5% of adjacent normal lung tissues. The expression of ODC protein was significantly correlated with TNM stage(P(27)0.05), but not lymph node metastasis, gender, age,smoking history, pleural invasion, tumor diameter and tumor differentiation(P(29)0.05).6. There was a positive correlation between the protein expression of ADAR1 and ODC in lung adenocarcinoma tissues(r=0.832, P(27) 0.01). The median overall survival time was longer in the low ADAR1 expression group than in the high expression group(P(27) 0.05) and low ODC expression group than in the high expression group(P(27) 0.05) by Kaplan-Meier survival analysis. The cox multivariate analysis showed that ADAR1 expression and TNM stage were independent prognostic factors(P(27)0.05).7. The XTT viability and proliferation experiments showed that cell proliferation of A549-edt/AZIN1 and A549-wt/AZIN1 increased compared with control at day three and six(P(27) 0.05). Also cell proliferation of A549-edt/AZIN1 increased compared with A549-wt/AZIN1 at day six(P(27)0.05).8. The transwell invasion assay showed that the number of invasive cells were signi?cantly increased in A549-edt/AZIN1(477±68) compared with A549-wt/AZIN1(327±42)(P(27)0.05).9. Western blot showed that ODC protein expression of A549-edt/AZIN1 was higher than A549-wt/AZIN1(P(27)0.05).Conclusions:1. RNA editing level was elevated in lung adenocarcinoma compared to normal lung. The top editing genes, which RNA editing resided in coding region, were COG3, SRP9, COPA, CACNA1 D, RICTOR, ZNF669, METTL6, FLNA, AZIN1,C20orf30, PODXL, FLNB, PDZD7.2. The expression of ADAR1 m RNA and ODC m RNA were significantly higher in lung adenocarcinoma tissues than in adjacent normal lung tissues(P(27)0.05). The expression of ADAR2 m RNA was not significantly different between lung adenocarcinoma tissues and adjacent normal lung tissues(P(29) 0.05). The expression of ADAR1 protein was mainly in nucleus. The expression of ADAR1 protein was significantly correlated with TNM stage(P(27)0.05) and lymph node metastasis(P(27) 0.05). The expression of ADAR2 protein was not significantly different between lung adenocarcinoma tissues and adjacent normal lung tissues(P(29) 0.05). The expression of ODC protein was mainly in cytoplasm. The expression of ODC protein was significantly correlated with TNM stage(P(27)0.05).The median overall survival time was longer in the low ADAR1 expression group than in the high expression group(P(27)0.05) and low ODC expression group than in the high expression group(P(27)0.05) by Kaplan-Meier survival analysis.The cox multivariate analysis showed that ADAR1 expression and TNM stage were independent prognostic factors(P(27)0.05).3. The proliferation and invasiveness capability of A549-edt/AZIN1 was increased compared with A549-wt/AZIN1. ODC protein expression of A549-edt/AZIN1 was higher than A549-wt/AZIN1(P(27)0.05).