| Objectives:Study the expression of miR-370 in lung cancer and the effect of miR-370 on the growth and invasion of lung cancer cells,confirm the regulation of miR-370 on EGFR signaling pathway,and explore the inhibitory effect of miR-370 on lung cancer xenografts and metastatic.Method:1.Use web to predict the binding site of miR-370 on EGFR.The relative quantity of miR-370 and EGFR of four lung cancer cell line,a normal bronchial epithelial cell line and lung cancer tissue,normal lung tissue from patients were detected by real-time PCR and western blot.The relative quantity of miR-370 and EGFR of lung cancer tissue and normal lung tissue were detected by real-time PCR,and the correlation between miR-370 and EGFR expression was analysis.EGFR 3 'UTR wild type and EGFR 3 'UTR mutant fragments were cloned into the pmiRGLO dual-luciferase-expressing vector.XWLC-05 and H157 cells were co-transfected with pmiRG-EGFR-UTR-wt,pmiRG-EGFR-UTR-mu or pmiRG-miR-370-inhibitor-PC,together with miR-370 mimic or miR-NC,the luciferase activities of individual cell samples were analyzed using the Dual Luciferase Reporter Assay System.2.miR-370 mimics and miR-370 inhibitor were synthesized,and H157 and XWLC-05 cells were transfected with miR-370 mimics and miR-370 inhibitor.The effects of miR-370 on proliferation,migration,cloning and metastasis of lung cancer cells were studied by MTT experiment,scratch test,plate cloning and transwell experiment.The changes of EGFR,AKT/PAKT,ERK/PERK,and HIF-1? were detected by western-blot,and the mechanism of miR-370 molecules was discussed.3.XWLC-05 were transfected with miR-370 stabley,and the proliferation,migration,colony formation and metastasis ability of the cells were detected.The expression of AKT/PAKT,ERK/PERK,HIF-1? were detected by western-blot.Mice were implanted subcutaneously or through tail vain with XWLC-05-miR-370 or XWLC-05-miR-NC cells.The growth of formed solid tumors and body weights of individual mice were measured every three days up to 20 days post implantation.At the end of the experiment,all of mice were sacrificed and their tumors were dissected,imaged and weighed.The tumor sections were examined by histology and immunohistochemistry.4.Mice were implanted subcutaneously or through tail vain with XWLC-05 cells.The tumor was injected with miR-370 mimics and its negative control.The growth of formed solid tumors and body weights of individual mice were measured every three days up to 20 days post implantation.At the end of the experiment,all of mice were sacrificed and their tumors were dissected,imaged and weighed.The tumor sections were examined by histology and immunohistochemistry.After subcutaneous transplantation,the tumor was injected with miR-370 mimics and its negative control.Result:1.The websites predict that EGFR has miR-370 binding sites.Results of real time quantitative PCR show that miR-370 is low and EGFR is highly expressed in lung cancer tissue,and the two were negatively correlated.H157 and XWLC-05 were co-transfected miR-370 mimics and wild type EGFR 3'UTR,48 hours after transfection the fluorescence expression decreased(p<0.05),suggesting that miR-370 can be combined with EGFR 3' UTR.2.miR-370 mimics decreased the expression of EGFR protein in H157 and XWLC-05 cells(p<0.05),inhibited the proliferation,migration,invasion and clone formation ability of H157 and XWLC-05 cells(p<0.05);weaken the expression of phosphorylation of AKT,phosphorylation of ERK and the expression of HIF-la,miR-370 inhibitor can enhance the expression of EGFR protein(p<0.05),promoted the proliferation migration,invasion and clone formation of lung cancer cells significantly(p<0.05),and increase the expression of phosphorylation of AKT,ERK and the expression of HIF-1 alpha.3.Establishment of XWLC-05 cell line which miR-370 high expression,confirm the proliferation,migration,invasion and clone formation ability of miR-370-XWLC-05 decreased(P<0.05);western-blot showed the expression of EGFR,activation AKT and ERK,and expression of HIF-1 alpha decreased;the mice were transplantated with cancer cell subcutaneously,the volume and the weight of tumor were observed every three days,but the difference between groups was not significant(P>0.05).Compared with the negative control group,the growth of tumor in experimental group significantly inhibited,the difference was statistically significant(p=0.009);tumor weight of miR-370 group was 0.275±0.048g,significantly lower than the negative control group 0.725±0.103g(p=0.0075);immunohistochemical staining showed:EGFR Ki-67 and HIF-1? protein and microvessel density(MVD)expression of tumor in experiment group was significantly lower than the control group(p<0.05);micrometastasis of miR-370 group(14.75±3.95)were lower than the control group(39.50±5.852)(p=0.0127).4.In the experiment of injection of miR-370 mimics,the weight of two groups of mice was increased,the weight of the experimental mice was higher than control group at 17 days after innoculation(p<0.05);compared with the control group,the tumor of experimental group mice grew inhibited obviously,the difference is significant(p=0.000);The tumor size of experiment mice(240.3±65.51cm3)less than that of the control group(4981±75.14 cm3)at 19th day.The weight of the tumor of the experimental group was(0.40±0.07)g,which was significantly lower than that in the control group(0.65±0.07)g(p=0.04).Immunohistochemical staining showed that the expression of EGFR,HIF-1alpha,Ki-67 and microvessel density in the experimental group were significantly lower than those in the control group(p<0.05).In the metastatic tumor model,the metastatic tumor score of the experimental group 10.50±8.35 was lower than that of the control group(35.75±7.72)(p<0.05).Conclusion:1.miR-370 expression lower in lung cancer tissue and can inhibiting the proliferation,migration,clonogenic and invasive ability of lung cancer cell.2.miR-370 can regulate the expression of EGFR by combining with EGFR3'UTR in lung cancer cells.3.miR-370 downregulated EGFR expression,inhibited activation of AKT and ERK signaling pathway,reduced the expression of HIF-1 alpha.4.miR-370 inhibits the expression of EGFR in the tissues of lung cancer in vivo,reduces the expression of HIF-1 alpha,and inhibits the growth,angiogenesis and metastasis of lung cancer. |
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