Study On The Function Of MicroRNA-125b-5p In Bone Metastasis Of Lung Adenocarcinoma

Objective:This article designed in vivo and in vitro experiments to illustrate the role of miR-125b-5p in the proliferation and metastasis of lung adenocarcinoma.Methods:1.qRT-PCR detects the expression level of miR-125b-5p in lung adenocarcinoma cell lines(XWLC-05,SPC-A1,A549,H1299,95-D)and normal human bronchial epithelial cells HBE.Select the human lung adenocarcinoma cell line with the highest miR-125b-5p expression level was constructed as a stable transgenic cell line.2.The miR-125b-5p mimic and miR-125b-5p inhibitor lentiviral plasmids were constructed.After transfection and screening,cell lines with over-expression and silent expression of miR-125b-5p and empty vectors were obtained,named miR-125b-5p mimic,miR-125b-5p inhibitor,Neg control,the transfection efficiency was detected by qRT-PCR.The MTT method was used to observe the changes in cell proliferation activity;the cell invasion assay(Cell invasion assay)was used to detect the changes in the in vitro invasion ability of each group of cells;the cell migration assay(Cell migration assay)was used to detect the changes in the in vitro migration ability of each group of cells;Through the cell clone formation assay(Cell clone formation assay),by counting the number of colonies formed,the proliferation potential of each group of cells was detected;through the apoptosis assay(Apoptosis assay),the morphological characteristics of each group of apoptotic cells were detected;3.A nude mouse model of lung adenocarcinoma bone metastasis was constructed,and the role of miR-125b-5p in lung adenocarcinoma bone metastasis was verified by in vivo experiments.Before the experiment,cardiac injection,tail vein injection,and in situ injection were used to uniformly inoculate miR-125b-5p mimic cells.The three experimental methods were used to evaluate the tumorigenic ability of nude mice.After comprehensive evaluation,follow-up experiments were carried out by in-situ injection.The experiment was divided into two groups:miR-125b-5p mimic group and Neg control group.Cells were inoculated by in situ injection and weighed every 3 days.After 7 weeks,X-rays were taken,and they were sacrificed.Repeated and photographed,dissected tumor in situ and bone metastases,and evaluated the role of miR-125b-5p in lung adenocarcinoma bone metastasis by monitoring body weight,comparing tumor formation in situ,and comparing the number of bone metastases.Results:1.The results of qRT-PCR showed that compared with normal human bronchial epithelial cells,miR-125b-5p was significantly higher expressed in XWLC-05,SPC-Al,A549,H1650,95-D lung adenocarcinoma cells,and in A549 the highest expression in the cell line.2.We successfully constructed a stably transfected A549 cell line with overexpression and silent expression of miR-125b-5p,and tested the cell transfection efficiency by virus titer measurement.The transfection efficiency of the overexpression group,silence group and control group reached more than 90%.qRT-PCR results showed that compared with the Neg control group,the miR-125b-5p expression level in the miR-125b-5p mimic group was significantly increased(P<0.05),and the miR-125b-5p in the miR-125b-5p inhibitor group the expression level of is significantly down-regulated(P<0.05).3.MTT experiment results:Compared with the Neg control group,the miR-125b-5p mimic group grew the fastest,and the miR-125b-5p inhibitor group grew the slowest,and there was a statistical difference(P<0.05).4.The results of cell invasion and migration experiments showed that compared with the Neg control group,the miR-125b-5p mimic group promoted the invasion and migration ability of A549 cells,while the miR-125b-5p inhibitor group inhibited the invasion and migration of A549 cells.Migration ability,the difference was statistically significant(P<0.05).5.The results of apoptosis experiments showed that compared with the Neg control group,the miR-125b-5p mimic group inhibited the apoptosis of A549 cells;while the miR-125b-5p inhibitor group promoted the apoptosis of A549 cells.6.The results of the clone formation experiment showed that the clonal bodies formed by cells in the miR-125b-5p mimic group were significantly higher than those in the miR-125b-5p inhibitor group and the Neg control group,and there was a statistical difference(P<0.05).7.The results of in vivo experiments suggest that compared with the Neg control group,the tumor mass of the tumor in situ in the miR-125b-5p mimic group was significantly increased(P<0.05),and the bone metastases were obvious.After anatomy,it was pathologically verified to be lung adenocarcinoma bone metastatic lesions.Conclusion(s):1.Successfully established the A549 cell line with over-expression and silent expression of miR-125b-5p,providing a cell model for subsequent studies on the function and molecular mechanism of miR-125b-5p.2.Through in vitro functional experiments,it was found that in the Neg control group,the overexpression of miR-125b-5p can promote the proliferation and invasion and migration of A549 cells,and the silent expression of miR-125b-5p promotes the apoptosis of A549 cells.These results suggest that miR-125b-5p may be a potential tumor-promoting factor in lung adenocarcinoma.3.In vivo experiments further verified the role of miR-125b-5p overexpression in bone metastasis of lung adenocarcinoma.Consistent with the results of in vitro experiments,the overexpression of miR-125b-5p can significantly promote tumor formation in situ and the ability of distant bone metastasis.As mentioned in the review,this article is expected to lay the foundation for the subsequent in-depth study of the biological behavior and mechanism of miR-125b-5p gene in lung adenocarcinoma bone metastasis,and provide part of miR-125b-5p targeted therapy for lung adenocarcinoma bone metastasis theoretical basis.