Functional Study Of MicroRNA-199b-5p In Bone Metastasis Of Lung Adenocarcinoma

Objective(s):The downstream targets of miR-199b-5p were identified by bioinformatic database prediction combined with dual luciferase assay.Four plasmids,miR-199b-5p mimic empty-loaded plasmid(miR-199b-5p-mimic-NC),miR-199b-5p mimic plasmid(miR-199b-5p-mimic),miR-199b-5p inhibitor empty-loaded plasmid(miR-199b-5p-inhibitor-NC)and miR-199b-5p inhibitor plasmid(miR-199b-5p-inhibitor),were imported into A549 lung adenocarcinoma cells to construct transient cell lines,based on which cell function experiments were conducted to explore the effect of miR-199b-5p on the proliferation,invasion,migration and apoptosis of A549 lung adenocarcinoma cell.Western blot was performed to explore the possible mechanism of miR-199b-5p affecting the function of A549 lung adenocarcinoma cells,and transmission electron microscopy was used to observe the alteration of submicroscopic structure autophagosomes and autophago-lysosomes in A549 lung adenocarcinoma cells to verify the above mentioned possible mechanism.Clinically,immunohistochemistry was used to demonstrate whether miR-199b-5p affected lung adenocarcinoma bone metastasis through down-regulation of autophagy.In combination with the functional effects of miR-199b-5p on A549 lung adenocarcinoma cells and the corresponding mechanisms,we investigated and elucidated whether miR-199b-5p could be a molecular marker for the early diagnosis of lung adenocarcinoma bone metastasis in the clinical setting,providing an important theoretical basis for the clinical study of lung adenocarcinoma bone metastasis.Methods:1.It was predicted that AMPK was a downstream target gene of miR-199b-5p by bioinformatic databases and it was verified that the targeted regulatory relationship between miR-199b-5p and AMPK existed by dual luciferase assay.2.Four plasmids,miR-199b-5p-mimic-NC,miR-199b-5p-mimic,miR-199b-5pinhibitor-NC,and miR-199b-5p-inhibitor,were introduced into A549 lung adenocarcinoma cells to construct transient cell lines.QRT-PCR was used to verify that miR-199b-5p-mimic up-regulated miR-199b-5p expression in A549 lung adenocarcinoma cells relative to miR-199b-5p-mimic-NC to verify the transfection efficiency of miR-199b-5p-mimic;Western blot was used to verify that miR-199b-5pinhibitor up-regulated the protein expression levels of downstream target gene AMPK of miR-199b-5p in A549 lung adenocarcinoma cells compared with miR-199b-5pinhibitor-NC to verify the transfection efficiency of miR-199b-5p-inhibitor;Meanwhile,the possible mechanism of miR-199b-5p affecting the function of A549 lung adenocarcinoma cells was explored by Western blot.3.The effect of miR-199b-5p on the proliferation ability of A549 lung adenocarcinoma cells was detected by CCK-8 kit and plate cell clone formation assay;the effect of miR-199b-5p on the invasion ability of A549 lung adenocarcinoma cells was detected by cell invasion assay.The effect of miR-199b-5p on the migration ability of A549 lung adenocarcinoma cells was detected by cell scratch assay;the effect of miR-199b-5p on the apoptosis ability of A549 lung adenocarcinoma cells was detected by Annexin V-FITC Apoptosis Detection Kit.4.The quantity alteration of submicroscopic structure in A549 lung adenocarcinoma cells was observed by transmission electron microscopy to verify the above possible mechanism.5.Specimens of primary lung adenocarcinoma foci and their normal lung tissues from lung adenocarcinoma patients without bone metastasis and bone metastatic cancer tissues from lung adenocarcinoma patients with bone metastasis were collected from the clinic for immunohistochemical experiments to explore the possible mechanism of miR-199b-5p affecting bone metastasis of lung adenocarcinoma.Results:1.The prediction results from bioinformatic databases combined with dual luciferase assay results showed that miR-199b-5p targeted AMPK(PRKAA)and AMPK was a downstream target of miR-199b-5p.2.QRT-PCR and subsequent Western Blot assays showed that miR-199b-5p-mimic up-regulated miR-199b-5p expression in A549 lung adenocarcinoma cells relative to miR-199b-5p-mimic-NC(the transfection efficiency of miR-199b-5p-mimic was effective),and miR-199b-5p-inhibitor up-regulated the protein expression levels of downstream target gene AMPK of miR-199b-5p in A549 lung adenocarcinoma cells compared with miR-199b-5p-inhibitor-NC(the transfection efficiency of miR-199b-5p-inhibitor was effective);Meanwhile,miR-199b-5p maybe affect the function of A549 lung adenocarcinoma cells by down-regulating autophagy.3.The results of CCK-8 cell proliferation assay and flat panel cell clone formation assay showed that miR-199b-5p promoted the proliferation of A549 lung adenocarcinoma cells.4.The results of cell invasion assay showed that miR-199b-5p promoted the invasion of A549 lung adenocarcinoma cells.5.The results of cell scratching assay showed that miR-199b-5p promoted the migration of A549 lung adenocarcinoma cells.6.The results of apoptosis assay showed that miR-199b-5p inhibited the apoptosis of A549 lung adenocarcinoma cells.7.It was observed that miR-199b-5p inhibited the generation of autophagosomes and autophago-lysosomes in the cytoplasm of A549 lung adenocarcinoma cells by transmission electron microscopy.8.It was revealed that miR-199b-5p may promote bone metastasis of lung adenocarcinoma through downregulating the expression of autophagy-related genes AMPK by immunohistochemical experiments.Conclusion(s):MiR-199b-5p promotes the proliferation,invasion,and migration of lung adenocarcinoma cells and inhibits the apoptosis of lung adenocarcinoma cells.The alteration of this function may be related to the event that miR-199b-5p down-regulates autophagy by targeting AMPK.Mi R-199b-5p may promote lung adenocarcinoma bone metastasis by targeting AMPK to down-regulate autophagy.Mi R-199b-5p is expected to be a molecular marker for early diagnosis of lung adenocarcinoma bone metastasis in clinical practice.