| Lung cancer is one of the most common cancers in the world.It is a leading cause of death in men and women among all cancers per year.The principal risk factors include smoking,infection,air pollution,and genetic factors.There are two main types of lung cancer:non-small cell lung cancer and small cell lung cancer.Non-small cell lung cancer accounts for about 80%of all lung cancers,including adenocarcinoma,squamous cell carcinoma,and large cell carcinoma.Lung adenocarcinoma accounts for about half of all lung cancer cases.In recent decades,great efforts have been made in the treatment of lung adenocarcinoma,especially in the development and application of targeted drugs and immunotherapeutics.However,the overall survival rate of lung adenocarcinoma is still poor.Exploring the molecular mechanisms underlying lung adenocarcinoma progression is helpful for identifying novel potential molecular therapeutic targets.MicroRNAs(miRNAs)are small non-coding RNAs containing 22-25 nucleotides that cause mRNA degradation or translational inhibition by directly interacting with the 3’-untranslated regions(3’-UTRs)of their target genes.Therefore,miRNAs can participate in the regulation of various biological processes,including cell proliferation,differentiation,apoptosis,cell cycle,and migration,by regulating the expression of a large number of genes.In addition,by regulating the expression of oncogenes or tumor suppressor genes,many miRNAs play an important role in the development and progression of malignant tumors including lung adenocarcinoma.Therefore,exploring the expression and function of miRNAs in lung adenocarcinoma can help identify novel biomarkers and therapeutic targets.Recently,miR-148a has been reported to be disregulatedly expressed in various malignant tumors,and exhibits tumor suppressing or promoting effects.For example,miR-148a is significantly down-regulated in gastric cancer tissues and cell lines,functioning as a tumor suppressor in the development of gastric cancer by inactivating STAT3 and Akt via targeting CCK-BR.The miR-148 levels in serum of non-small cell lung cancer were significantly lower than those in serum of benign lung disease and healthy controls.Multiple studies have shown that miR-148a plays an inhibitory role in non-small cell lung cancer.However,the regulatory mechanism of miR-148a in the growth of lung adenocarcinoma remains unclear.This study was conducted to investigate the clinical significance of miR-148a expression in lung adenocarcinoma and to explore its regulatory role in the growth of lung adenocarcinoma in vitro.Methods1.From June 2011 to October 2013,lung adenocarcinoma tissues and adjacent non-tumor tissues were collected from 53 patients with lung adenocarcinoma.RNA was extracted and the expression levels of miR-148a were evaluated by RT-PCR.2.The 53 patients with lung adenocarcinoma were divided into miR-148a high expression group and miR-148a low expression group.Then,we studied the association between the miR-148a expression and age,sex,tumor size,smoking history,tumor differentiation,clinical TNM stage,metastasis and survival time.3.Lung adenocarcinoma cell lines(H23,H1975,H2228 and H2085)and normal bronchial epithelial cell line BEAS-2B were subcultured.RNA was extracted and the expression levels of miR-148a were evaluated by RT-PCR.4.miR-148a mimics were transfected into H23 and H1975 lung adenocarcinoma cells.After transfection for 48 hours,RT-PCR was used to detect the expression of miR-148a.CCK-8 assay was used to detect cell proliferation.Colony formation assay was used to determine the cloning formation ability.Flow cytometry was used to examine the cell cycle distribution.5.Bioinformatics analysis and dual luciferase reporter gene assays were used to verify the target relationship between miR-148a and E2F3.6.The regulation of miR-148a on E2F3 in lung adenocarcinoma cells was studied.7.RT-PCR was used to detect the expression levels of E2F3 in 53 pairs of lung adenocarcinoma tissues and adjacent non-tumor tissues,as well as in lung adenocarcinoma cell lines(H23,H1975,H2228 and H2085)and normal bronchial epithelial cell line BEAS-2B.We then investigated the correlation between E2F3 and miR-148a expression in 53 lung adenocarcinoma tissues.8.H23 and H1975 lung adenocarcinoma cells were transfected with miR-148a mimics+E2F3 expression plasmid(or blank vector).After 48 hours of transfection,RT-PCR and Western Blot were used to detect the expression levels of E2F3.The cell proliferation was detected by CCK-8 assay.Colony formation assay was used to determine the cloning formation ability.Flow cytometry was used to examine the cell cycle distribution.Results1.The lung adenocarcinoma tissues,and adjacent non-tumor tissues were obtained from 53 adenocarcinoma patients by surgery.The expression levels of miR-148a in tissue samples were detected by RT-PCR.The results showed that the expression levels of miR-148a in lung adenocarcinoma tissues were significantly lower than those in adjacent tissues.2.Four lung adenocarcinoma cell lines(H23,H1975,H2228 and H2085)and one normal bronchial epithelial cell line(BEAS-2B)were selected,and the expression level of miR-148a in each cell line of RT-PCR was utilized.The results showed that the expression level of miR-148a in lung adenocarcinoma cells was significantly lower than that in normal bronchial epithelial cell lines.3.Chi-square test was used to study the association between miR-148a expression and clinicopathological features in patients with lung adenocarcinoma.The results showed that low miR-148a expression was significantly associated with high TNM stage and lymph node metastasis.4.Patients with lung adenocarcinoma with low expression of miR-148a have shorter survival time than patients with lung adenocarcinoma with high expression of miR-148a.5.The miR-148a mimics were transfected into lung adenocarcinoma cell lines H23 and H1975 cells.The control group was transfected with the miR-control mimic.After 48 hours of transfection,RT-qPCR detection was performed.The results showed that the expression levels of miR-148a were significantly up-regulated in the miR-148a group compared with the control group.6.Compared with the control group,the proliferationof lung adenocarcinoma cells transfected with miR-148a mimics was significantly decreased,the ability of colony formation was weakened,and the cell cycle was significantly blocked in G1 phase.7.Bioinformatics and dual luciferase reporter gene assay results indicate that there is a target relationship between miR-148a and E2F3.8.The miR-148a mimic and E2F3 expression plasmid were co-transfected into lung adenocarcinoma cell lines H23 and H1975 cells.Cells in the control group were co-transfected with miR-148a mimics and blank vector.Compared with the control group,the E2F3 mRNA and protein levelswere significantly increasedin the experimental group.9.The lung adenocarcinoma tissues,and adjacent non-tumor tissues were obtained from 53 adenocarcinoma patients by surgery.The expression levels of E2F3 in tissue samples were detected by RT-PCR.The results showed that the expression levels of E2F3 in lung adenocarcinoma were significantly higher than those in adjacent tissues.10.The miR-148a mimics and E2F3 expression plasmid were co-transfected into lung adenocarcinoma cell lines H23 and H1975 cells.The control group was co-transfected with the miR-148a mimics and blank vector.Compared with the control group,cell proliferation and colony formation were significantly up-regulated,and cell cycle progression was acceleratedin the experimental group.Conclusion1.The expression levels of miR-148a in lung adenocarcinoma are significantly decreased,and it is closely related to the malignant degree and poor prognosis of lung adenocarcinoma.2.Up-regulation of miR-148a can significantly inhibit the proliferation and colony formation capacity of lung adenocarcinoma cell lines,which may be due to cell cycle arrest at G1 phase.3.E2F3 is a target gene of miR-148a.In lung adenocarcinoma cell lines,miR-148a can negatively regulate the expression of E2F3.4.In lung adenocarcinoma,the expression level of miR-148a and E2F3 was significantly inversely correlated,suggesting that the increase of E2F3 expression may be caused by the decrease of miR-148a expression.5.E2F3 is likely to be a downstream target gene of miR-148a,involved in the regulation of miR-148a on the proliferation of lung adenocarcinoma cells. |
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