Study On The Function Of MiR-516a-3p In Lung Adenocarcinoma Cells By Targeting PTPRD

[Background and Objective]:Lung cancer is one of the malignant tumors severely endangering human health.The morbidity and mortality of lung cancer remains top among various malignant tumors.The pathological types of lung cancer include non-small cell lung cancer(NSCLC)and small cell lung cancer.Lung adenocarcinoma is one of the main pathological types in NSCLC,with low overall survival(OS)rate but increasing incidence in recent years.Although the research of lung cancer has made great progress,the pathogenesis of lung cancer has not been fully elucidated because of its biological characteristics and high heterogeneity of invasion.MicroRNAs(miRNAs),a class of endogenous non-coding small RNAs with approximately 23 nt in length,can inhibit the transcription process to decrease the expression of its target genes by binding to mRNA of the target gene.At present,multiple studies have confirmed that miRNAs play important roles in biological processes.Recent studies have shown that miR-516a-3p plays an important role in a variety of tumors.However,the mechanism of miR-516a-3p in lung adenocarcinoma is rarely reported.In recent years,Receptor-type tyrosine-protein phosphatase 8(PTPRD)has been widely concerned by researchers.It has been found that PTPRD is down-regulated in a variety of tumors and plays a role of tumor suppressor gene.In previous studies,we found that mir-516a-3p was up-regulated in lung adenocarcinoma,while PTPRD was down-regulated in lung adenocarcinoma.Using bioinformatics prediction,we found that miR-516a-3p and PTPRD mRNA 3 'untranslated region had complementary base pairing sequence.We speculated that there might be some biological relationship between them,so as to affect the process of tumor developmentIn this study,the major objectives are as follows:To detect the expression of miR-516a-3p and PTPRD in lung adenocarcinoma tissues and cells,and to analyze the correlation of the expression levels of them with the clinicopathological characteristics.To investigate the effects of miR-516a-3p on the proliferation,invasion,migration and apoptosis in lung adenocarcinoma cells and explore the relevant mechanisms.To verify that whether PTPRD was a direct target gene of miR-516a-3p and explore the mechanism of mir-516a-3p targeting PTPRD.In vivo assays will be conducted to observe the effect of miR-516a-3p on tumorigenicity in nude mice.Taken together,we intend to elucidate the role and mechanism of miR-516a-3p in lung adenocarcinoma by targeting PTPRD.Section ?:The expression of miR-516a-3p and PTPRD in lung adenocarcinoma and their correlation with clinicopathological characteristics[Objective]:The expression of miR-516a-3p and PTPRD in lung adenocarcinoma tissues and cells was determined by qPCR.Meanwhile,clinical samples were collected to analyze the correlation of the expression of miR-516a-3p and PTPRD with clinicopathological characteristics.[Methods]:(1)qPCR was used to determine the expression of miR-516a-3p and PTPRD in lung adenocarcinoma.Chi-square test was used to analyze the correlation of the expression of miR-516a-3p and PTPRD with clinicopathological features.Spearman correlation analysis was used to explore the correlation between miR-516a-3p and PTPRD expression.(2)The starBase database was adopted to further analyze the expression of PTPRD in lung adenocarcinoma compared with normal lung tissue.Online Kaplan Meier plotter database was adopted to analyze the relationship between the expression of PTPRD and the OS as well as PFS of patients with lung adenocarcinoma using.[Results]:(1)Compared with matched normal lung tissue,miR-516a-3p was highly expressed in lung adenocarcinoma tissues and PTPRD was low in lung adenocarcinoma tissue.The expression of miR-516a-3p and PTPRD was negatively correlated.(2)Compared with normal bronchial epithelial cells,miR-516a-3p was highly expressed in lung adenocarcinoma cells and PTPRD was low in lung adenocarcinoma cells.(3)To analyze the correlation between the expression of miR-516a-3p and clinicopathological characteristics,we found that miR-516a-3p was closely associated with TNM clinical staging,that is,the earlier the staging,the higher the expression level of miR-516a-3p.(4)To analyze the correlation between the expression of PTPRD and clinicopathological characteristics,we found that PTPRD was closely correlated with TNM clinical staging,that is,the earlier the staging,the lower the expression level of PTPRD.(5)The starBase database revealed the low expression of PTPRD in lung adenocarcinoma compared with normal lung tissue.KM plotter analysis further showed prolonged OS and PFS of patients with high PTPRD expression(HR=0.7,P=2.2×10-8)and longer(HR=0.79,P=0.013).[Conclusion]:Compared with normal lung tissues and bronchial epithelial cells,miR-516a-3p was highly expressed in lung adenocarcinoma tissues and cells,while PTPRD was lowly expressed in lung adenocarcinoma tissues and cells.The expression of miR-516a-3p and PTPRD was negatively correlated in tumor tissue.The expression level of miR-516a-3p and PTPRD was not associated with sex,age,smoking status,tumor size and lymph node metastasis of patients with lung adenocarcinoma,but was correlated with TNM stage.Moreover,the higher expression of miR-516a-3p indicated the more advanced TNM stage of patients,while the lower expression of PTPRD suggested the more advanced TNM stage of patients.Section ?:Effect of miR-516a-3p on biological behavior of lung adenocarcinoma cell lines H1299 and SPC-A1 and preliminary study of its mechanism[Objective]:In this section,we aimed to investigate the effects of up-and down-regulation of miR-516a-3p on the proliferation,migration,invasion and apoptosis of lung adenocarcinoma cell lines H1299 and SPC-A1.We also explored the mechanism of miR-516a-3p regulating the proliferation,invasion,metastasis and apoptosis of lung adenocarcinoma cells.[Methods]:(1)Inhibitor and mimics were used to for knockdown and overexpression of miR-516a-3p.The transfection efficiency was determined by qPCR and fluorescence microscope,followed by assessment of cell proliferation,migration,invasion and apoptosis.(2)The expression of p-STAT3,STAT3,MMP-9,cleaved-caspase-3,Bcl-2 and Bax was detected by Western blot.[Results](1)Compared with the negative control group,transfection of inhibitor and mimics led to knockdown and overexpression of miR-516a-3p in SPC-A1 and H1299.(2)Knockdown of miR-516a-3p could inhibit the proliferation of lung adenocarcinoma cells H1299 and SPC-Al.Overexpression of miR-516a-3p could promote the proliferation of lung adenocarcinoma cells H1299 and SPC-Al.(3)The migration and invasion of lung adenocarcinoma cells H1299 and SPC-A1 can be inhibited by miR-516a-3p knockdown.Overexpression of miR-516a-3p can promote the migration and invasion of lung adenocarcinoma cells H1299 and SPC-A1.(4)Knockdown of miR-516a-3p could promote the apoptosis of lung adenocarcinoma cells H1299 and SPC-A1.Overexpression of miR-516a-3p can inhibit the apoptosis of lung adenocarcinoma cells H1299 and SPC-A1.(5)Down-regulation of miR-516a-3p can inhibit the expression of p-STAT3 in lung adenocarcinoma cells,while up-regulation of miR-516a-3p can promote the expression of p-STAT3 in lung adenocarcinoma cells.However,either up-regulation or down-regulation of miR-516a-3p has no significant effect on the expression of STAT3 in lung adenocarcinoma cells.(6)Down-regulation of miR-516a-3p can promote the expression of Bax and cleaved Caspase-3,and inhibit the expression of Bcl-2.However,up regulation of miR-516a-3p had no significant effect on the expression of Bax,cleaved Caspase 3 and Bcl-2.(7)Down-regulation of miR-516a-3p can inhibit the expression of MMP-9 in lung adenocarcinoma cells,while up-regulation of miR-516a-3p can promote the expression of MMP-9 in lung adenocarcinoma cells.[Conclusion]:Down-regulation of miR-516a-3p can inhibit the proliferation,migration and invasion,but promote the apoptosis of lung adenocarcinoma cells H1299 and SPC-A1.Up regulation of miR-516a-3p can promote the proliferation,migration and invasion,but inhibit the apoptosis of lung adenocarcinoma cells H1299 and SPC-A1.Together,it is suggested that miR-516a-3p may play a role in promoting lung adenocarcinoma.The mechanism of miR-516a-3p promoting the proliferation and invasion of lung adenocarcinoma cells might be mediated by regulating MMP-9.And the mechanism of miR-516a-3p inhibiting apoptosis of lung adenocarcinoma can be mediated by regulating the expression of p-STAT3,MMP-9,cleaved-Caspase 3,Bcl-2 and Bax.Section ?:Preliminary study on the mechanism of miR-516a-3p targeting PTPRD in lung adenocarcinoma[Objective]:In this section,we aimed to predict the target gene of miR-516a-3p in lung adenocarcinoma,followed by further validation.[Methods]:(1)Bioinformatics was used to predict the target gene of miR-516a-3p,which revealed that PTPRD was a candidate target gene.(2)After up-and down-regulation of miR-516a-3p in lung adenocarcinoma cells H1299 and SPC-A1,the mRNA expression of PTPRD was detected by qPCR,and the protein expression of PTPRD was detected by Western blotting.(3)Luciferase reporter assay was used to further validate whether PTPRD was a direct target gene of miR-516a-3p.(4)The low expression PTPRD cell line was constructed by small interfering RNA(siRNA)technology,followed by qPCR and WB to determine the transfection efficiency,(5)Based on the down-regulation of miR-516a-3p,down-regulation of PTPRD expression was conducted to observe whether it could reverse the effect of down-regulation of miR-516a-3p alone on the expression of PTPRD in lung adenocarcinoma cells(6)Based on the down-regulation of miR-516a-3p,down-regulation of PTPRD expression was performed to investigate whether it could reverse the effect of down-regulation of miR-516a-3p alone on proliferation,migration,invasion and apoptosis of lung adenocarcinoma cells.(7)Based on the down-regulation of miR-516a-3p,down-regulation of PTPRD expression was carried out to assess whether it can rescue the effect of down-regulation of miR-516a-3p alone on the expression of p-STAT3,MMP-9,cleaved-Caspase 3,Bcl-2 and Bax protein in lung adenocarcinoma cells.[Results]:(1)Bioinformatics analysis showed that PTPRD was a potential target gene of miR-516a-3p.(2)After knocking down miR-516a-3p in lung adenocarcinoma cell line H1299 and SPC-A1,qPCR and Western blotting showed that the expression of PTPRD was up-regulated.After overexpression of miR-516a-3p in lung adenocarcinoma cell line H1299 and SPC-A1,qPCR and Western blotting showed that the expression of PTPRD was down-regulated.(3)Luciferase reporter assay confirmed that PTPRD was a direct target gene of miR-516a-3p.(4)Based on the down-regulation of miR-516a-3p expression,down regulating the expression of PTPRD can partially recover the effect of miR-516a-3p down-regulation on the expression of PTPRD in lung adenocarcinoma cells(5)Based on the down-regulation of miR-516a-3p expression,down regulating the expression of PTPRD can partially restore the promoting effect of miR-516a-3p down-regulation on the migration and invasion of lung adenocarcinoma cells.(6)Based on the down-regulation of miR-516a-3p expression,down regulating the expression of PTPRD can partially restore the effect of miR-516a-3p down-regulation on promoting apoptosis of lung adenocarcinoma cells.(7)Based on the down-regulation of miR-516a-3p expression,down-regulation of PTPRD expression can rescue the effect of miR-516a-3p down-regulation alone on the expression of p-STAT3,MMP-9,cleaved-Caspase 3,Bcl-2 and Bax protein in lung adenocarcinoma cells.[Conclusion]:PTPRD is a direct target gene of miR-516a-3p.miR-516a-3p may directly target PTPRD to regulate the expression of p-STAT3,MMP-9,cleaved-Caspase 3,Bcl-2 and Bax in lung adenocarcinoma.Section ?:The effect of miR-516a-3p on the growth of lung adenocarcinoma in nude mice[Objective]:The effect of up-regulation and down-regulation of miR-516a-3p on the tumorigenesis and growth of lung adenocarcinoma cells in nude mice was investigated in vivo.[Methods]:(1)The cells were transfected with miR-516a-3p antigomir,miR-516a-3p agomir,NC and siPTPRD.(2)Tumor size in nude mice was observed and measured every three days.After 4 weeks,the tumors were sacrificed,weighed and photographed.(3)The expression of PTPRD in tumors of mice was detected by qPCR and immunohistochemistry.[Results]:(1)The tumor volume of miR-516a-3p antigomir group were significantly smaller than those of NC group.The tumor volume of miR-516a-3p agomir group were significantly larger than those of NC group.(2)The tumor weight of miR-516a-3p antigomir group were significantly lower than those of NC group.The tumor weight of miR-516a-3p agomir group were significantly higher than those of NC group.(3)The tumor weight of miR-516a-3p antigomir group was significantly smaller than that of NC group.(4)qPCR and immunohistochemistry showed that the expression of PTPRD in miR-516a-3p antigomir group was significantly higher than that in NC group,while the expression of PTPRD in miR-516a-3p agomir group was significantly lower than that in NC group.[Conclusion]:Up-regulation of miR-516a-3p can promote the tumorigenicity of lung adenocarcinoma cells in nude mice and inhibit the expression of PTPRD,while down-regulation of miR-516a-3p can inhibit the tumorigenicity of lung adenocarcinoma cells in nude mice and promote the expression of PTPRD.