| Objectives Analyze and identify the direct target micro RNA(mi RNA)of brain cytoplasmic RNA1(BCYRN1)in SCLC cells;analyze the effects of BCYRN1/mi RNA on the proliferation,migration and invasion of small cell lung cancer(SCLC)cells.Methods The serums of patients with SCLC(20 cases with involuntary process transfer,10 cases with defensive process transfer)who are pathologically diagnosed at the Tangshan People’s Hospital affiliated to North China University of Technology,as well as the serums of healthy people(10 cases),were collected from November 1,2019 to December 30,2020.The BCYRN1 levels in serums were analyzed by Quantitative Realtime PCR.2 The expression of BCYRN1 in lung cancer cell lines(NCI-H460,NCI-H1299,NCI-SBC-2)and bronchial epithelioid cell line(16-HBE)were detected by RT-q PCR.3The knockdown effect on endogenous BCYRN1 in SBC-2 cells was confirmed by RTq PCR.4 The mi RNAs differentially expressed between SBC-2 cells transfected with psh RBCYRN1 and the p Silencer-NC were analyzed using mi RNA microarray.The targeting relationship between BCYRN1 and mi RNA was confirmed with dual-luciferase reporter assay.5 The effects of BCYRN1 knockdown on proliferation,migration and invasion abilities were analyzed with colony formation assay and transwell experiment with or without matrigel.6 The protein levels of proliferation marker Ki-67 and key proteins of epithelial-mesenchymal transition(EMT)(E-cadherin,Vimentin,Snail,Slug)were detected by Western blot.Results BCYRN1 levels in the serum of SCLC patients with and without distant metastasis were all significantly higher than that of healthy subjects(P<0.001;P=0.003).There was no significant difference of serum BCYRN1 levels between patients without distant metastatic and patients with distant metastatic(P=0.089).2 There was a significant difference of BCYRN1 expression in the four cell lines(F=1096.81,P<0.001).The expression of BCYRN1 in lung cancer cell lines was significantly higher than that in 16-HBE cells(NCI-H1299 vs 16-HBE: P=0.009;NCI-H460 vs 16-HBE: P=0.001;SBC-2 vs16-HBE: P<0.001).3 The psh R-BCYRN1 plasmid was constructed.Electrophoresis after restriction endonuclease digestion showed that the size of the inserted fragment was consistent with the known BCYRN1 sequence.The BCYRN1 level in the psh R-BCYRN1 group was significantly lower than that in the p Silencer-NC group(t=12.87,P<0.001).4Five mi RNAs up-regulated morethan 3 folds in the psh R-BCYRN1 group were screened:hsa-mi R-378 d,hsa-let-7c-5p,hsa-mi R-421,hsa-mi R-378 c and hsa-mi R-22-3p.Based on bioinformatics analysis,hsa-mi R-378 c was selected for further study.The mi R-378 c expression in SBC-2 cells was significantly lower than that of 16-HBE cells(t=5.242,P=0.006).The mi R-378 c level in psh R-BCYRN1 group was significantly higher than that in p Silencer-NC group(t=6.81,P=0.0024).The luciferase activity of 293 T cells in pmi RGLO-BCYRN1-WT+mi R-378 c mimics group was significantly lower than that in pmi RGLO-BCYRN1-WT group+mimics-NC(P<0.001).5 There were differences in the clone formation,migration and invasion abilities of SBC-2 cells among p Silencer-NC group,psh R-BCYRN1 group and psh R-BCYRN1+anti-sense oligo A targeting mi R-378c(ASO-mi R-378c)group(F=14.611 P=0.005;F=28.83,P=0.0015;F=30.403,P=0.0013).The clone formation,migration and invasion abilities of psh R-BCYRN1 group were significantly lower than those in the p Silencer-NC group(P=0.0004;P<0.001;P=0.0015).The clone formation,migration and invasion abilities of psh R-BCYRN1+ASO-mi R-378 c group were markedly higher than those in sh R-BCYRN1 group(P=0.0014;P=0.009;P=0.003),and those abilites also were significantly higher than those of p Silencer-NC group(P=0.0098;P=0.009;P=0.0018).6 There were significant differences of protein levels of E-cadherin,Vimentin,Snail and Slug among p Silencer-NC,psh R-BCYRN1 and psh R-BCYRN1+ASO-mi R-378 c groups(F=36.788,P<0.001;F=31.015,P=0.001;F=34.662,P=0.0012;F=55.181,P<0.001).The E-cadherin protein level in psh R-BCYRN1 group was significantly higher than that in p Silencer-NC group(P=0.0015)and psh RBCYRN1+ASO-mi R-378 c group(P=0.0013),while there was no significant difference between the p Silencer-NC group and the psh R-BCYRN1+ASO-mi R-378 c group(P=0.628).The expressions of Vimentin,Snail and Slug in psh R-BCYRN1 group were markedly lower than those in p Silencer-NC group(P=0.0073;P=0.008;P=0.0017),and the expressions of Vimentin,Snail and slug in psh R-BCYRN1+ASO-mi R-378 c group lower than those in psh R-BCYRN1 group(P=0.004;P=0.0013;P=0.021).There were no differences of Vimentin and Snail expressions between psh R-BCYRN1+ASO-mi R-378 c group and p Silencer-NC group(P=0.053;P=0.059).Compared with the p Silencer-NC group,the expression of Slug in psh R-BCYRN1+ASO-mi R-378 c group was significantly decreased(P=0.002).Conclusions 1.The level of BCYRN1 in serum of SCLC patients and SBC-2 cell line is higher than that of normal human serum and human bronchial epithelioid cells.2.BCYRN1 in SCLC cells directly binds to mi R-378 c to make mi R-378 c low expression.3.Knockdown of BCYRN1 can inhibit Ki-67 and EMT by up-regulating mi R-378 c,thereby inhibiting the proliferation,migration and invasion of SCLC cells.Figure 8;Table 1;Reference 105... |
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