The Mechanism Of MiR-199b-5p Targeting DDR1 In Lung Adenocarcinoma

Objective:The lung adenocarcinoma H1299 cell line was selected as the research object.According to the literature,it was found that miR-199b-5p was under-expressed in lung adenocarcinoma H1299 cells.MiR-199b-5p was overexpressed in H1299 cell lines by transfection of lentiviral vectors.To observe the effects of miR-199b-5p on the biological behaviors of lung adenocarcinoma H1299 cells such as proliferation,colony formation,migration and invasion;secondly,to preliminary explore the interaction between miR-199b-5p and DDR1;finally,to form tumors in nude mice The effects of miR-199b-5p on lung adenocarcinoma in vivo were explored.The aim is to provide new biomarkers and potential targets for the clinical diagnosis and treatment of lung adenocarcinoma.Methods:1.Using miR-199b-5p as the target to synthesize OE vectors and transfect lung adenocarcinoma H1299 cells,and set up the NC group as a negative control group.2.Transfection efficiency was detected by qRT-PCR and fluorescence to screen stable transfected cell lines.3.Cell scratch experiments to study the effect of miR-199b-5p overexpression on the migration ability of lung adenocarcinoma H1299 cells.4.Transwell experiments studied the effect of miR-199b-5p overexpression on the migration and invasion ability of lung adenocarcinoma H1299 cells.5.CCK8 experiments to study the effect of miR-199b-5p overexpression on the proliferation ability of lung adenocarcinoma H1299 cells.6.Plate clone formation experiments explored the effect of miR-199b-5p over-expression on clone formation ability of lung adenocarcinoma H1299 cells.7.The luciferase reporter gene experiment was used to detect and verify if DDR1 is the direct target gene of miR-199b-5p.8.The effect of miR-199b-5p overexpression on DDR1 protein expression in lung adenocarcinoma H1299 cells was detected by Western blot experiments.9.To investigate the effect of miR-199b-5p overexpression on the subcutaneous tumorigenicity of lung adenocarcinoma H1299 cells in nude mice through tumor formation experiments in nude mice.Results:1.Detection of miR-199b-5p-OE by qRT-PCR showed that miR-199b-5p expression level in H1299 lung adenocarcinoma cell lines was significantly increased(P<0.05).2.CCK8 test showed that miR-199b-5p-OE significantly inhibited cell proliferation after transfection into H1299 cells(P<0.05).3.Clone formation test showed that miR-199b-5p-OE was significantly inhibited in cell clone formation after transfection into H1299 cells(P<0.05).4.Cell scratches and Transwell experiments showed that miR-199b-5p-OE transfected H1299 cells significantly reduced the migration speed and invasion ability(P<0.05).5.The luciferase reporter gene experiment showed that the luciferase activity of DDR1 3'UTR-luc-WT in the overexpression group was significantly reduced,and verification of qRT-PCR showed that the expression level of DDR1 mRNA in the overexpression cell line was significantly reduced(P<0.05).6.Western Blotting results showed that the expression of DDR1 protein was reduced after transfection with miR-199b-5p-OE(P<0.05).7.The results of nude mice tumorigenesis experiments show that after transfection with miR-199b-5p-OE,the ability of nude mice to subcutaneous tumorigenesis is significantly inhibited,and the content of DDR1 is significantly reduced by immunohistochemical detection(P<0.05).Conclusions:1.Overexpression of miR-199b-5p can significantly inhibit the proliferation and invasion of lung adenocarcinoma cell line H1299,reduce its migration speed,and weaken its clone formation ability.2.In the miR-199b-5p overexpression group,luciferase activity of DDR13'UTR-luc-WT was significantly reduced,DDR1 mRNA expression level was significantly reduced,and DDR1 expression content was significantly reduced.3.Overexpression of miR-199b-5p can significantly inhibit the ability of lung adenocarcinoma cell line H1299 to subcutaneously develop in nude mice,and the content of DDR1 was significantly reduced by immunohistochemistry4.MiR-199b-5p may play a role in suppressing cancer in lung adenocarcinoma by targeting DDR1,which is a potential target for clinical diagnosis and treatment of lung adenocarcinoma.