| Objective:Small cell lung cancer is a poorly differentiated cancer with highly aggressive and metastatic characteristics,nearly two-thirds of patients have extensive metastatic lesions at the time of diagnosis,and the prognosis is poor.Invasion and metastasis is the main cause of death in patients with small cell lung cancer.However,the mechanism of the invasion and metastasis of small cell lung cancer is not fully confirmed.Therefore,in-depth study of the invasion and metastasis mechanism of small cell lung cancer will lay a preliminary foundation for finding new therapeutic targets in small cell lung cancer.This study aims to explore the correlation between miRNA-34a-5p and the invasion and metastasis ability of small cell lung cancer,and then explore the potential mechanism.Methods:1、High-throughput gene expression database(GEO)was adopted to screen the miRNA associated with the survival of small cell lung cancer.In addition,five pairs of small cell lung cancer patients with significant difference in survival were selected to screen for the differentially expressed miRNA.2、Real-time quantitative PCR was used to detect the expression level of miRNA-34a-5p in small cell lung cancer cells,and miRNA-34a-5p mimics and miRNA-34a-5p inhibitor were transfected into small cell lung cancer cell line NCI-H1688 respectively,and the control group was set up.In addition,the expression level of miRNA-34a-5p in each group was detected by real-time quantitative PCR,Transwell invasion assay was used to detect the invasion and migration ability of transfected cells.3、Bioinformatic softwares were used to predict the target gene of miRNA-34a-5p,and Western blot assay was used to detect the protein expression level of Flotillin-2after transfection in each group.Results:1、The miRNA results related to the prognosis of patients with small cell lung cancer were screened by GEO database.The 5-year survival rate was 18% in patients with low miRNA-34a-5p expression and 70% in patients with high miRNA-34a-5p expression.The expression level of miRNA-34a-5p was correlated with the prognosis of patients with small cell lung cancer(P<0.05).2、Clinical samples were screened for the expression of miRNA.The high expression level of miRNA-34a-5p is related to the prognosis of small cell lung cancer patients with a long survival time.3、Mi RNA-34a-5P mimics and miRNA-34a-5P inhibitor were transfected into small cell lung cancer cell line NCI-H1688,and control group was set up.Forty-eight hours after transfection,PCR results showed that the expression level of miRNA-34a-5p in the mimics group was significantly higher than that of the control group(P<0.05),while the expression level of miRNA-34a-5p in the inhibitor group was significantly lower than that of the control group(P <0.05).The results showed that the transfection successfully regulated the expression level of miRNA-34a-5p in the human small cell lung cancer cell line NCI-H1688.According to Transwell invasion assay,compared with the number of transmembrane cells in the control group(90.66+10.02),the number of transmembrane cells in the mimics group(56.66+09.50)was significantly reduced(P<0.05),and the number of transmembrane cells in the inhibitor group(135.0+10.00)was significantly increased(P< 0.05).As a result,miRNA-34a-5p could inhibit the invasion of small cell lung cancer cells.Overexpression of miRNA-34a-5p could significantly inhibit the invasive ability of NCI-H1688 small cell lung cancer cells.In addition,down-regulating the expression of miRNA-34a-5p,and the invasive ability of small cell lung cancer cells was enhanced.These results indicate that miRNA-34a-5p could inhibit the invasive ability of small cell lung cancer cells.4、Bioinformatics software analysis results showed that the m RNA of flotillin-2contains a putative region that is able to pair to the seed sequence of miRNA-34a-5p.Further Western blot results showed that the protein expression level of flotillin-2 in miRNA-34a-5p mimics group was significantly lower than that in the control group(P<0.05),while the protein expression level of flotillin-2 in miRNA-34a-5p inhibitor group was significantly higher than that in the control group(P<0.05),suggesting that miRNA-34a-5p could down-regulate the protein expression level of flotillin-2.These results indicate that miRNA-34a-5p could down-regulate the expression of Flotillin-2.Conclusions:1、Small cell lung cancer patients with higher expression of miRNA-34a-5p have a better prognosis than those with lower expression of miRNA-34a-5p.2、The miRNA-34a-5p could inhibit the invasion ability of small cell lung cancer NCI-H1688 cells.3、The miRNA-34a-5p could down-regulate the protein expression of Flotillin-2. |
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