Effects And Related Mechanisms Of MiR-22regulated Radiosensitivity Of Small Cell Lung Cancer

Recently,the morbidity and mortality of lung cancer have risen rapidly,which have been seriously threaten human life.Small cell lung cancer(SCLC),accounting for about 20% of all lung cancer,is a very aggressive malignancy characterized by high cellular proliferation and early metastatic spread.In fact,although SCLC is a chemosensitive and radiosensitive disease,the initial responsiveness to chemotherapy is usually followed by development of resistance and the prognosis remains poor.With the rapid development of stereotactic radiosurgery,radiotherapy equipment and technology,radiotherapy provides a very effective treatment for patients with lung cancer.Unfortunately,tumor cells often produces radiation tolerance and resistance in the later stages of radiation therapy,which would decrease the efficacy of radiation therapy.miRNAs are a kind of endogenous non-encoding small molecule RNA.miRNA play important roles in the formation,cell proliferation,cell apoptosis,drug resistance and radiation resistance of tumor,which markedly influences the diagnosis and treatment of tumors.mi R-22 is a tumor suppressor and enhanced expression of miRNA-22 significantly inhibit the proliferation and invasion of tumor cells,which is as a tumor suppressor.This study is aimed to explore effects and related molecular mechanisms of miR-22 regulated radiosensitivity in small cell lung cancer,providing new ideas for improving the radiotherapy effect of SCLC.The main contents are as follows.1.RT-qPCR was used to detect the mRNA expression level of miR-22 in human normal lung epithelial cell line BEAS-2B and SCLC cell line NCI-H446.The results showed that the expression of miR-22 in SCLC cell line NCI-H446 was significantly decreased.2.NCI-H446 cells with miR-22 mimics and mi R-22 inhibitors by transient transfection were established,respectively.They could achieve the effect of miR-22 overexpression and knockdown.At the same time,NCI-H446 cells with their control nc and inhibitors NC were also established.Additionally,the miR-22 overexpression recombinant plasmid was constructed with the vector of pLKO.1 and transfected intoNCI-H446 with lentivirus transfection method.The positive cell lines were detected by RT-qPCR and showed that miR-22 were higher expression than control,suggesting that they were successfully constructed.3.NCI-H446 cells with miR-22 overexpression and knockdown were irradiated with 0Gy,2Gy,4Gy ?-ray,respectively.MTS assay,colony forming assay and Ki-67 antibody assay were performed to detected effect of miR-22 on the proliferation of NCI-H446.The results showed that miR-22 overexpression significantly inhibited NCI-H446 cell proliferation,and miR-22 knockdown significantly promoted NCI-H446 cell proliferation,and they were dose dependent.APC Annexin V/PI(propidium iodide,PI)dual-staining,PI staining and flow cytometric analysis were used to detect the influence of miR-22 on NCI-H446 cell apoptosis and cell cycle.The results showed that miR-22 overexpression significantly promote cell apoptosis and miR-22 had little effect on NCI-H446 cell cycle.Wound healing test was used to detect the effect of miR-22 on NCI-H446 cell migration.It showed that miR-22 overexpression could significantly inhibit the migration ability of NCI-H446 cell.4.Two bioinformatics databases Target Scan and Pictar were employed to screen the potential targeting genes of miR-22 and the overlapping genes were identified.According to the results of NCBI and literatures,WRNIP1 was selected for further study,which was associated with DNA damage repair.RT-qPCR,Western blot analysis and dual-luciferase reporter assay were used to validate the regulatory effect of miR-22 on WRNIP1 expression.The results showed that WRNIP1 was a direct target of miR-22.Alteration of miR-22 expression had a negative regulatory effect on WRNIP1 mRNA level and protein level.5.The miR-22 overexpression stable cell and its vector control were used for high-throughput RNA sequencing.Five differentially expressed genes,which were related with the proliferation,migration and apoptosis of tumor cells(KLK8?PC?SCUBE1?STC1 and GPM6A)were selected for RT-qPCR.The results showed that KLK8 down-regulated mRNA and other four genes up-regulated m RNAs in mi R-22 overexpression stable cell,which was in accodance with the theoretical results.It was suspected that KLK8,PC and SCUBE1 related to the inhibition of proliferation andmigration and STC1 and GPM6 A related to the promotion of cell apoptosis by miR-22 in small cell lung cancer.The related molecular mechanisms of miR-22 regulated radiosensitivity of small cell lung cancer were preliminarily explained.