MiRNA-31 Regulates Lung Adenocarcinoma Cancer Stem Cells Via MET-PI3K-Akt Signaling Pathway

Lung cancer remains to be one of the leading cancers worldwide,80% of which is non-small cell lung cancer (NSCLC). NSCLC includes adenocarcinoma, squamous cell carcinoma and large cell lung cancer with adenocarcinoma being the major subtype. Almost all patients succumb to drug-resistant recurrences within 2-3 years. Another major cause of death from NSCLC is metastases. Recent reports show that recurrence and metastasis may largely be attributed to drug resistance of cancer stem cells (CSCs).CSCs are a group of cells that are capable of self-renewal, differentiation and development of drug resistance. Our previous studies showed that CD 133+cells isolated from A549 cells (human lung adenocarcinoma cell line) are able to increase the size of the tumor spheres and their metastasis potential. These CSCs are determined to be the origin of tumors as they both expand the CSCs pool and give rise to all the cell types in a tumor by symmetric or asymmetric division. Therefore, development of new agents which suppress CSCs’ growth is needed to facilitate clinical treatment. MicroRNAs have been considered as the potential regulators targeting CSCs due to their fine adjustment function as well as their specific expression in individual type of cancer cells.MicroRNAs (miRNAs) are endogenous-23nt non-coding RNAs that regulate mRNA activities via translational inhibition and mRNA degradation. Half of miRNA-encoding genes are mutated during tumorigenesis, indicating its molecular-leveled correlation to tumor development. Recent miRNA expression profiles of normal tissues and tumor cells revealed that miRNA signatures are a good indicator of disease status and clinical outcome. Therefore, miRNAs functioning in mRNA level are of our interest in order to provide a novel therapy for NSCLC patients.In our study, miR-31 was found significantly different expression in lung tumor-adjacent tissues, lung adenocarcinoma cells and lung adenocarcinoma cancer stem-like cells. The differential expression of miR-31 in these three samples may have a role in the regulation of lung adenocarcinoma, especially in lung adenocarcinoma stem cells. A series of assays were performed to unclose the effects and mechanisms of miR-31 in lung adenocarcinoma cells and stem-like cells.Part I Regulatory effect of abnormal expression of miR-31 on lung adenocarcinoma cancer stem cells1. Total RNA was extracted from lung tissues, A549 cells and A549CD133+cells, and all the miRNAs’expressions were detected by the Agilent human miRNA V3 micro array chip. There miRNAs (miR-31, miR-194 and 451) were selected to examnied the expression by real time PCR to verify the results of micro array chip. The results showed that the miR-31 was the highest expression in A549CD133+cells and higher expression in A549 cells but were hardly detected in normal lung tissue;2. MiR-31 over-expression lentiviral vector and miR-31 sponge lentiviral vector were successfully constructed. Generation of lung adenocarcinoma CSC-like cells (MiR-31-knockdown control cells, MiR-31-knockdown cells, MiR-31-overexpression control cells and MiR-31-overexpression cells) was obtained, expressing different levels of miR-31. Real time PCR method was used to determine the contents of miR-31 in these cells. Results showed that the expression of miR-31 in MiR-31-knockdown cells was reduced by 80% compared with the control cells, and miR-31 was double in MiR-31-overexpression cells compared with MiR-31-overexpression control cells.3. The growth activities of MiR-31-knockdown control cells, MiR-31-knockdown cells, MiR-31-overexpression control cells and MiR-31-overexpression cells were detwermined in vitro. The results showed that overexpression of miR-31 could significantly inhibit the proliferation of lung adenocarcinoma CSCs.4. Cell cycles were analyzed by flow cytometry instrument. Experimental results showed that the the proportion of MiR-31-knockdown cells from Gl/GO phase into downstream phases (S phase and G2/M phase) were significantly increased by comparation with MiR-31-knockdown control cells; and the ratio of MiR-31-overexpression cells in Gl/GO phase was significantly higher than that of MiR-31-overexpression control cells. Those results revealed that miR-31 was able to block A549CD133+cells in the Gl/GO phase and delay cell cycle process.5. Flow cytometry was used to analyze the apoptosis and necrosis ratios of Annexin V/7-AAD double stained cells. The results showed that the apoptosis and necrosis proportions of MiR-31-knockdown cells were significantly decreased compared with the control group, while the proportion of apoptosis and necrosis of MiR-31-overexpression cells increased significantly.6. In Vivo assays were used to evaluate the inhibitory effect of miR-31 on the occurrence, growth and metastasis of lung adenocarcinoma CSCs. The data of tumor occurrence showed that inhibition of miR-31 induced lung adenocarcinoma CSCs to form tumors. The results of tumor volume-time curve and the weight of tumors in teriminal implicated that tumors grew faster in miR-31 knockdown lung adenocarcinoma CSCs; MiR-31 long-term analogue (MiR-31 Agomir) and NC were used to kill tumors and the results showed that MiR-31 Agomir had significant inhibitory effect on lung adenocarcinoma;7. The tumors derived from experimental mice were used for histological analysis including hematoxylin-eosin staining and PCNA staining. The results verified that MiR-31 Agomir could effectivly kill lung adenocarcinoma cells.Part Ⅱ Studies on the mechanism of miR-31 in regulating lung CSCs1. We uncovered the oncogene targets of miR-31 associated with lung adenocarcinoma based on starBase V2.0, which summarizing four databanks (PicTar, TargetScan, RNA22 and PITA) and vertif the prediction by real time PCR screening. The results showed that the expressions of MET, Ret, PIK3C2A and GRB10 were regulated by miR-31. Inhibition of miR-31 resulted in increase of mRNA and protein levels of these genes.2. Dual luciferase report gene vectors of wild type and mutant type were used to determine the direct bonding of miR-31 and target genes. Relative fluorescence values of cells transfected wih wild-type reporter gene vector were significantly decreased when incubated with miR-31 mimics. However, there was no significantly difference between miR-31 mimics and negative control in mutant-type reporter vectors transfecting cells. These results revealed that MET, Ret, GRB10 and PIK3C2A were direct target genes of miR-31.3. MET small hairpin lentiviral vectors and control vectors were constructed. Generation of lung adenocarcinoma CSCs (A549CD133+-sh-MET cells and A549CD133+-sh-NC cells) were chosen. Real time PCR and western blot methods were used to determine the content of MET in these cells. The results showed that sh-MET could reduce the intracellular MET mRNA level to 60%, and the translational expression of MET was reduced by 40%.4. Growth activities of A549CD133+-sh-MET cells and A549CD133+-sh-NC cells were detected in vitro. The results showed that downregulation of MET significantly inhibited the proliferation of lung adenocarcinoma CSCs. Knockdown of MET significantly reduced the proportion of cells from G1/GO phase into the S/G2/M phase.5. Animal experiments were used to evaluate the effect of MET on the tumorigenicity, growth and metastasis of lung adenocarcinoma CSCs. The results showed that the growth rate of A549CD133+cells was significantly inhibited in MET-knockdown lung adenocarcinoma tumor stem cells.6. A series of cell cycle and apoptosis related proteins were determined by western blot. Results showed that both overexpression of miR-31 and inhibition of MET reduced the expression of PI3K, Akt, p-Akt, CDK2 and Bax and up-regulated the Bcl-2 in cancer cells and tumor tissues. The results suggested that miR-31 bound to MET and then led to PI3K-Akt signal pathway on A549CD133+cells.In summary, this study revealed that miR-31 was aberrantly expressing in A549CD133+cells. In our report, using A549CD133+cells, we examined the inhibitory effects and the underlying mechanisms of microRNA-31 (miR-31) on the growth of lung adenocarcinoma CSCs. These results suggested that miR-31 might inhibit the growth of lung adenocarcinoma CSCs via down regulation of the MET-PI3K-Akt signaling pathway. Here we suggest a miRNA-based strategy to tackle human lung adenocarcinoma effectively by targeting CSCs.