| Objective :Globally,lung cancer has been the most common and serious type of cancer,seriously threatening human health,among which non-small cell lung cancer(NSCLC)accounts for about 85% of lung cancer pathological types.Lung adenocarcinoma(LUAD)is an important subtype of NSCLC and is a common subtype causing death in lung cancer patients.Micro RNAs(miRNA)are a class of short-stranded non-coding RNAs that are generally dysregulated in tumors by their expression,thus exerting a proor anti-cancer effect.At the same time,micro RNA /miRNA also h affects the expression of various signaling pathways and cellular processes,and has an important role in regulating tumor development.miR-486-3p,a newly discovered miRNA in recent years,is lowly expressed in lung adenocarcinoma,and its biological function in lung adenocarcinoma is still unknown.Pre-experimentation,bioinformatics analysis revealed that miR-486-3p is down-regulated in lung adenocarcinoma and has sequence complementarity with DVL1 and Wnt5 b in the Wnt/β-catenin signaling pathway,which may be the target gene regulated by miR-486-3p,therefore,it can be hypothesized that miR-486-3p has an important role in the development of lung adenocarcinoma and the abnormal activation of Wnt/β-catenin signaling pathway and its function and molecular mechanism in the development of lung adenocarcinoma can be explored to provide a new theoretical basis for early diagnosis and effective treatment of lung adenocarcinoma.Methods:1.The expression of miR-486-3p in lung adenocarcinoma was firstly predicted by bioinformatics star Base website,and then the sequence complementary relationship between miR-486-3p and DVL1 and Wnt5 b in Wnt/β-catenin signaling pathway was obtained by Target Scan analysis.2.Real Time Quantitative PCR(RT-q PCR)was used to detect the expression of miR-486-3p in lung adenocarcinoma A549 and H358 cell lines and human normal bronchial epithelial Beas-2b cells.3.in vitro transfection of cellular lung adenocarcinoma A549 and H358 cell lines using lentiviral miR-486-3p-mimic/mimic-NC and validation of RNA transfection efficiency using RT-q PCR.4.transfection of miR-486-3p-mimic/mimic-NC by lentiviral grouping of lung adenocarcinoma A549 and H358 cell lines,followed by Western Blot assay to detect changes in the expression of DVL1,Wnt5 b protein in Wnt/β-catenin signaling pathway with miR-486-3p complementary sequence in each group of transfected cells.5.lentiviral group transfection of miR-486-3p-mimic/mimic-NC by lentiviral group transfection of lung adenocarcinoma A549 and H358 cell lines,followed by CCK8 proliferation assay and cell cloning assay to detect the effect of miR-486-3p on the proliferation ability of A549 and H358 cell lines.6.detecting the effect of miR-486-3p on the migration ability of A549 and H358 cell lines by lentiviral grouping transfection of miR-486-3p-mimics/mimic-NC in lung adenocarcinoma A549 and H358 cell lines,using scratch assay.7.detecting the effect of miR-486-3p on the invasive ability of A549 and H358 cell lines by lentiviral grouping transfection of miR-486-3p-mimics/mimic-NC in lung adenocarcinoma A549 and H358 cell lines,using Transwell cell invasion assay.8.By lentiviral group transfection of miR-486-3p-mimics/mimic-NC in lung adenocarcinoma A549 and H358 cell lines,the effect of miR-486-3p on the apoptosis level of A549 and H358 cell lines was detected by flow cytometry,followed by western blot assay to detect the change of expression of anti-apoptotic protein Bcl2 in each group.Results:1.bioinformatics analysis showed that miR-486-3p expression was down-regulated in lung adenocarcinoma.miR-486-3p targeting relationship prediction showed that miR-486-3p had complementary relationship with DVL1 and Wnt5 b in Wnt/β-catenin signaling pathway.2.RT-q PCR results showed that miR-486-3p expression was lower in lung adenocarcinoma A549 and H358 cell lines than in human normal bronchial epithelial Beas-2b cells.The miR-486-3p-mimic group of lentivirally transfected lung adenocarcinoma A549 and H358 cell lines was significantly up-regulated(P < 0.05)compared with the miR-486-3p-mimic-NC group.3.The results of Western Blot experiments showed that the expression of DVL1 and Wnt5 b in lung adenocarcinoma A549 and H358 cell lines was significantly downregulated in the miR-486-3p-mimic group compared with the miR-486-3p-mimicNC group,and the expression of anti-apoptotic protein Bcl2 in A549 and H358 cells was also significantly downregulated(P < 0.05).4.The results of cell proliferation assay showed that the proliferation ability of lung adenocarcinoma A549 and H358 cell lines was significantly decreased(P < 0.05)when miR-486-3p-mimic group was compared with miR-486-3p-mimic-NC group,indicating that upregulation of miR-486-3p expression level significantly inhibited the proliferation ability of lung adenocarcinoma A549 and H358 cells.5.The results of cell migration assay showed that the migration ability of lung adenocarcinoma A549 and H358 cell lines was significantly downregulated in the miR-486-3p-mimic group compared with the miR-486-3p-mimic-NC group(P < 0.05),which indicated that upregulation of miR-486-3p expression level significantly inhibited the migration of lung adenocarcinoma A549 and H358 cells ability.6.The results of cell invasion assay showed that the invasion ability of lung adenocarcinoma A549 and H358 cell lines was significantly downregulated(P < 0.05)when comparing the miR-486-3p-mimic group with the miR-486-3p-mimic-NC group,which indicated that upregulation of miR-486-3p expression level could significantly inhibit the invasion of lung adenocarcinoma A549 and H358 cell lines ability.7.The results of apoptosis assay by flow cytometry showed that the apoptosis rate of lung adenocarcinoma A549 and H358 cell lines was significantly higher in the miR-486-3p-mimic group compared with the miR-486-3p-mimic-NC group(P < 0.05),indicating that upregulation of miR-486-3p expression level could significantly promote the apoptosis level of lung adenocarcinoma A549 and H358 cell lines.Conclusion:The experimental results showed that upregulation of miR-486-3p expression level could significantly inhibit the biological behaviors of proliferation,migration and invasion of lung adenocarcinoma A549 and H358 cell lines,and could promote apoptosis of lung adenocarcinoma A549 and H358 cell lines,and significantly downregulate the expression of DVL1 and Wnt5 b,the key targets in Wnt/β-catenin signaling pathway,indicating that miR 486-3p could target the expression of DVL1 and Wnt5b to regulate the activation of Wnt/β-catenin signaling pathway and thus inhibit the evolution of lung adenocarcinoma,indicating the role of miR-486-3p as an oncogene in the development of lung adenocarcinoma,and therefore miR-486-3p is expected to be an effective biological marker for the early diagnosis of lung adenocarcinoma and a potential therapeutic target. |
