| Background and Objectives:Lung cancer is one of the deadliest malignant tumors, with both the highest incidence and mortality rate occurring in China, which serious threat the safety and quality of people’s life. It is one of the major public health problems in our country. The key to reducing the incidence and mortality of lung cancer is to explore the pathogenesis of lung cancer and to find effective biomarkers for lung cancer screening and early diagnosis. In this study,high-throughput screening technique of miRNA and IncRNA microarray was used to screen the candidate biomarkers of lung cancer in combination with TCGA database and bioinformatics analysis. After further expanding the population size, the real-time quantitative PCR technique was used to detect and analyze the effect of miRNA and lncRNA on the risk of lung cancer and its diagnostic value. Finally, we selected miR-30a-3p, miR-30a-5p, and BRE-AS1 to further explore its biological function and possible regulatory mechanism in lung cancer cells. The purpose of this paper is to provide a theoretical basis for further understanding the pathogenesis of lung cancer and screening for the diagnosis and treatment of lung cancer.Methods:1. miRNA expression microarray was used to screen differentially expressed miRNA in lung cancer tissues and adjacent tissues. Construction of miRNA-mRNA expression network in combination with TCGA database and bioinformatics analysis, and a further selection of lung cancer-associated miRNA.RT-qPCR detected the expression of miRNA. Logistic regression analysis was used to analyze the relationship between the abnormal expression of miRNA and the risk of lung cancer. The area under receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of candidate miRNA in lung cancer. Independent sample t-test analyzed the relationship between the level of candidate miRNA expression and the clinical features of lung cancer.2. lncRNA expression microarray was used to screen differentially expressed miRNAs in lung cancer tissues and adjacent tissues. Construction of lncRNA-mRNA expression network in combination with TCGA database and bioinformatics analysis, and a further selection of lung cancer-associated IncRNA. The expression of IncRNA was detected by RT-qPCR in lung cancer population. Logistic regression analysis was used to analyze the relationship between the abnormal expression of lncRNA and the risk of lung cancer. The area under receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of candidate lncRNA in lung cancer. Independent sample t-test analyzed the relationship between the level of candidate lncRNA expression and the clinical features of lung cancer.3. The exogenous miR-30a-3p mimic and miR-30a-5p mimic were transfected into A549 cell. The expression of miR-30a-3p and miR-30a-5p in A549 cells was detected by RT-qPCR technique to determine the transfection efficiency. By this, the effects of miR-30a-3p and miR-30a-5p on the biological function of lung cancer cells and the possible regulatory mechanism were analyzed. MTT assay detected the cell growth and proliferation. Flow cytometry detected cell cycle. Annexin-V FITC& PI assay detected apoptosis. Western Blot was used to detect the expression of the key protein in the PI3K-AKT-mTOR pathway.4. The lentiviral lncRNA BRE-AS1 overexpression vector was infected into the A549 cell. The expression of BRE-AS1 in A549 cells was detected by RT-qPCR technique to determine the transfection efficiency. By this, the effects of BRE-AS1 on the biological function of lung cancer cells and the possible regulatory mechanism were analyzed. The cell proliferation was detected by MTT assay. Cell cycle was detected by flow cytometry. Apoptosis was detected by Annexin-V APC & 7-ADD double staining. Cell scratches were used to detect cell migration ability. Western Blot was used to detect the expression of the key protein in the PI3K-AKT-mTOR pathway.Results1 Identification of microRNA associated with lung cancer1.1 Analysis of microRNA differential expression profiles in lung cancermiRNA/mRNA microarray analysis identified a total of 116 miRNAs and 502 mRNA that distinguish lung cancer tissues from adjacent non-tumor tissues. TCGA database analysis identified a total of 154 miRNAs and 5322 mRNA that distinguish lung cancer tissues from adjacent non-tumor tissues. In combination with TCGA analysis, we identified 29 miRNAs and 417 mRNA related to lung cancer. Based on the above results, the miRNA-mRNA network was constructed via Cytoscape 3.0. The miRNA pathway analysis was indicative of that the regulation of the miRNA target genes mainly involved in signaling pathways related to Environmental Information Processing and Human Diseases, such as the Hippo, TGF-beta, Wnt, ErbB, MAPK signaling pathway and non-small cell lung cancer.1.2 Study on the relationship between the expression level of candidate microRNA and the risk and clinical characteristics of lung cancerThe expression of 6 candidate miRNAs was detected by RT-qPCR technique in 91 cases. The results showed that miR-205-5p was significantly up-regulated,and miR-27a-5p,miR-30a-3p,miR-30a-5p, miR-30c-2-3p and miR-30d-5p were significantly down-regulated (p<0.001), which was consistent with the results obtained from the microarray and TCGA database. Logistic regression analysis showed that the expression level of miR-205-5p was positively correlated with the risk of lung cancer, and increase of the expression level of miR-205-5p could increase the risk of lung cancer(OR = 1.287,p <0.01). The expression levels of miR-27a-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p and miR-30d-5p were negatively correlated with the risk of lung cancer and increased its expression level could decrease the risk of lung cancer (OR values were 0.846, 0.700, 0.585, 0.745,0.479, p <0.01). The results of the ROC curve showed that individual diagnoses AUC of miR-205-5p,miR-27a-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p and miR-30d-5p were found to be 0.728,0.637,0.758, 0.772, 0.734, 0.776 (p <0.001), respectively. Combined diagnosis of AUC was 0.898(p<0.001), combined diagnostic sensitivity and specificity were 87.7% and 84.9%, suggesting that these miRNAs have a certain diagnostic effect of lung cancer, combined diagnosis better. The independent sample t-test results show that the abnormal expression levels of miR-205-5p, miR-30a-3p, miR-30c-2-3p and miR-30d-5p were correlated with pathologic type of lung cancer (p <0.05).The difference in expression was significantly higher in lung squamous cell carcinoma than in lung adenocarcinoma. The abnormal expression level of miR-30a-3p and miR-30c-2-3p was related to the sex of lung cancer patients (p <0.05), and the difference was significant in males. MiR-30a-5p, miR-30c-2-3p abnormal expression levels were associated with age in lung cancer patients (p <0.05), and their differences were significantly higher in patients over 50 years of age than in patients younger than 50 years. It is suggested that the expression of miRNAs in different lung cancer pathogens, age and sex groups is different, that is, the expression of miRNAs in specific population may be more meaningful for the diagnosis, treatment and prognosis of lung cancer.2 Identification of lncRNA associated with lung cancer2.1 Analysis of IncRNA differential expression profiles in lung cancerA total of 385 lung cancer-related differentially expressed lncRNA were screened by lncRNA expression profiles microarray, of which 124 were up-regulated and 261 were down-regulated. A total of 337 lung cancer-related differentially expressed lncRNA were screened from the TCGA database,of which 191 were up-regulated and 146 were down-regulated. In combination with TCGA analysis,we identified 11 lncRNA related to lung cancer. There are three lncRNA expressing down-regulation,namely MCTS2P, SCARNA12 and LOC728554; and eight IncRNA expressing up-regulation, namely LHFPL3-AS2, SFTA1P,LINC00472, GVINP1,BRE-AS1,MIR22HG,LINC00312 and MEIS3P1,respectively. Based on 11 candidate lncRNA and 417 mRNA, the lncRNA-mRNA network was constructed via Cytoscape 3.0.2.2 Study on the relationship between the expression level of candidate lncRNA and the risk and clinical characteristics of lung cancerThe expression of two candidate IncRNAs was detected in 91 cases of lung cancer by RT-qPCR.The results showed that the expressions of LINC00312 and BRE-AS1 were down-regulated (p<0.05),which was consistent with the results of microarray and TCGA. Logistic regression analysis showed that the expression level of LINC00312 and BRE-AS 1 was negatively correlated with the risk of lung cancer. Increasing the expression level of LINC00312 and BRE-AS 1 could reduce the risk of lung cancer (OR = 0.739 and 0.362,p <0.001). ROC curve analysis showed that individual diagnoses AUC of LINC00312 and BRE-AS I were 0.784 and 0.916 (p <0.05), respectively. The combined diagnosis of AUC was 0.922 (p <0.05). The diagnostic sensitivity and specificity of the two were 87.8% and 84.6%, respectively, suggesting that combined diagnosis can improve the diagnostic efficiency of lung cancer. The abnormal expression level of LINC00312 was correlated with gender and age of patients with lung cancer (p <0.05). The difference was statistically significant in patients with male patients greater than 50 years old. The abnormal expression level of BRE-AS 1 was related to the sex,age and pathological type of lung cancer patients (p <0.05). The difference was significantly higher in patients with lung squamous cell carcinoma than that of 50 years old. The expression of lncRNA in different lung cancer pathogens, age and sex groups is different, that is, the expression of lncRNA in specific population may be more meaningful for the diagnosis, treatment and prognosis of lung cancer.3 Study on biological function and mechanism of miR-30a-3p and miR-30a-5p in lung cancer3.1 MiR-30a-3p and miR-30a-5p bioinformatics analysisBioinformatics analysis of miR-30a-3p and miR-30a-5p using DIANA-mirPath 3.0 revealed that the two were closely related to the development of multiple tumors such as non-small cell lung cancer and were also involved in PI3K-Akt signaling pathway, Fox signaling community, p53 signaling pathway and other tumor-related pathway regulation. In addition, through the action sites of miR-30a-3p and miR-30a-5p potential target genes in Small cell lung cancer and non-small cell lung cancer pathways. Further analysis showed that miR-30a-3p and miR-30a-5p could affect the biological processes of cell proliferation and apoptosis through the regulation of PI3K-Akt pathway-related proteins.3.2 Study on the biological function of miR-30a-3p and miR-30a-5p in A549 cellsMiR-30a-3p and miR-30a-5p cell background values of RT-qPCR test results show that,compared with 16HBE cells, miR-30a-3p and miR-30a-5p were down-regulated in A549 cells.Compared with the negative control group, the expression levels of miR-30a-3p and miR-30a-5p were significantly increased in mimic transfection group (p<0.05). MTT assay showed that the cell proliferation ability of miR-30a-3p and miR-30a-5p in mimic transfection group was significantly lower than that of negative control group at 24h, 36h and 48h (p <0.01), suggesting that overexpression of miR-30a-3p and miR-30a-5p in A549 cells could inhibit cell growth. The cell cycle results showed that the proportion of S phase in miR-30a-3p mimic transfection group was significantly higher than that in the negative control group (p<0.05), suggesting that the overexpression of miR-30a-3p in A549 cells could be induced cell cycle were arrested in S phase.The apoptotic rate of miR-30a-3p and miR-30a-5p in mimic transfection group was significantly higher than that of the negative control group (p<0.05), suggesting that the overexpression of miR-30a-3p and miR-30a-5p could induce A549 cells apoptosis were increased.3.3 Study on the regulation of key protein of miR-30a-3p and miR-30a-5p in PI3K-AKT-mTOR signaling pathwayThe expression of key protein in PI3K-AKT-mTOR signaling pathway was detected by Western Blot. The results showed that compared with the negative control group, the expression levels of AKT3, mTOR and S6K in miR-30a-3p mimic and miR-30a-5p mimic transfection groups were significantly decreased. It is suggested that miR-30a-3p and miR-30a-5p may influence the development of lung cancer through PI3K-AKT3-mTOR pathway.4 Study on biological function and mechanism of lncRNABRE-AS1 in lung cancer4.1 LncRNABRE-AS1 overexpression lentiviral vector constructionThe sequencing results of the positive clones were compared with the target sequences, and the sequencing results were consistent with the target sequences. The BRE-AS1 overexpression lentiviral vector was constructed successfully.4.2 Study on the biological function of IncRNABRE-AS1 in A549 cellsLentivirus BRE-AS 1 overexpressing vector was transfected into A549 cells, and its expression level was detected by RT-qPCR. Results showed that the expression level of BRE-AS 1 in LV-BRE-AS1 group was 59.756 times (p<0.05) in negative control group, and the transfection efficiency of lentivirus was better. MTT results showed that the proliferation of LV-BRE-AS1 cells were significantly decreased compared with the negative control group (p<0.05), suggesting that overexpression of BRE-AS 1 could inhibit the proliferation of A549 cells. Cell cycle results showed that the ratio of S phase in LV-BRE-AS1 group was decreased and the ratio of G2/M phase increased compared with the negative control group (p<0.01), suggesting that the overexpression of BRE-AS1 could be induced A549 cells were arrested in G2/M phase. Apoptotic results showed that the ratio of late and total apoptosis in LV-BRE-AS1 group was significantly higher than that in negative control group (p <0.05), suggesting that overexpression of BRE-AS1 could induce A549 cells apoptosis were increased. The results of cell scratches showed that the migration ability of A549 cells in LV-BRE-AS1 group was slightly lower than that in negative control group, but not statistically significant (p>0.05).4.2 Study on the regulation of key protein of lncRNA BRE-AS1 in PI3K-AKT-mTOR signaling pathwayThe expression of key protein in PI3K-AKT-mTOR signaling pathway was detected by Western Blot. The results showed that compared with the negative control group, the expression levels of PI3K,AKT3, mTOR, S6K and BCL-XL in LV-BRE-AS1 group were significantly decreased. Suggesting that BRE-AS 1 may be involved in the development of lung cancer by influencing the expression of key proteins in PI3K-AKT3-mTOR signaling pathway.Conclusions:1. In this study, 29 differentially expressed miRNAs were screened in lung cancer tissues by miRNA microarray, TCGA database, and miRNA-mRNA expression network analysis. Results of RT-qPCR showed that expression of miR-205-5p was up-regulated in lung cancer tissues and positively correlated with the risk of lung cancer. Moreover, expression of miR-27a-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p, and miR-30d-5p were down-regulated in lung cancer tissues and negatively correlated with the risk of lung cancer. Suggesting that miRNA co-analysis can improve the diagnostic efficacy of lung cancer and have a high application value. It may be more practical to further develop lung cancer-related miRNAs in specific population of different sex, age and pathological types.2. In this study, 11 differentially expressed IncRNAs were screened in lung cancer tissues by IncRNA microarray, TCGA database and IncRNA-mRNA expression analysis, which including MCTS2P,SCARNA12, LOC728554, LHFPL3-AS2, SFTA1P,LINC00472, GVINP1,BRE-AS1,MIR22HG,LINC00312, MEIS3P1. Results of RT-qPCR showed that expression of LINC00312 and BRE-AS1 was down-regulated in lung cancer tissues, and it was negatively correlated with the risk of lung cancer. Suggesting that IncRNA co-analysis can improve the diagnostic efficacy of lung cancer and have a high application value. It may be more practical to further develop lung cancer-related IncRNAs in specific population of different sex,age and pathological types.3. Overexpression of miR-30a-3p / 5p can inhibit the proliferation of A549 cells, cause cell S phase arrest and induce apoptosis increased. Besides, both can participate in the regulation of lung cancer by influencing the expression of key proteins in the PI3K-AKT-mTOR signaling pathway.4. This study first studied the biological function and mechanism of BRE-AS1 in lung cancer cells.Overexpression of BRE-AS1 could inhibit the proliferation of A549 cells, change the cell G2 / M cycle distribution, and induce the increase of apoptosis. BRE-AS1 could be involved in the development of lung cancer by influencing the expression of key proteins in the PI3K-AKT-mTOR signaling pathway. |
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