长链非编码RNA AC003973.4对乳腺癌细胞增殖、迁移和侵袭的影响研究
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摘要
目的检测长链非编码RNA AC003973.4在人乳腺癌组织和乳腺癌细胞株中的表达,探讨其对乳腺癌细胞增殖、迁移和侵袭的影响及作用机制。方法采用实时荧光定量PCR(qRT-PCR)法检测20例乳腺癌组织及配对的癌旁组织,以及检测乳腺癌细胞株(BT-549、MDA-MB-231、MCF-7、T47D)和正常乳腺上皮细胞株MCF-10A中AC003973.4的表达。取AC003973.4表达水平最低的乳腺癌细胞株转染过表达质粒以上调AC003973.4的表达,分别采用MTS法、Transwell迁移和侵袭实验检测AC003973.4对乳腺癌细胞增殖、迁移和侵袭能力的影响;生物信息学法预测AC003973.4互补结合的mi RNA及下游靶基因,qRT-PCR法检测mi RNA和下游靶基因m RNA的表达,Western blot检测相关蛋白的表达。结果乳腺癌组织AC003973.4表达水平明显低于癌旁组织(P<0.01)。乳腺癌细胞株AC003973.4表达水平明显低于人正常乳腺上皮细胞(均P<0.05),MCF-7细胞中AC003973.4的表达水平最低(P<0.01)。上调MCF-7细胞AC003973.4的表达后,细胞增殖、迁移和侵袭能力均明显减弱(均P<0.05)。AC003973.4可互补结合mi R-224-5p,mi R-224-5p可互补结合PTEN。上调MCF-7细胞AC003973.4的表达后,mi R-224-5p的表达均下调(P<0.01),PTENm RNA和蛋白的表达均上调(均P<0.01),细胞增殖相关蛋白CDK4、Cyclin D1与细胞迁移相关蛋白Zeb-1、N-cadherin表达均下调(均P<0.01)。结论乳腺癌组织和细胞株中AC003973.4表达水平降低,上调AC003973.4表达可减弱乳腺癌MCF-7细胞的增殖、迁移和侵袭能力,其作用机制可能与调节mi R-224-5p与PTEN基因的表达有关。
Objective To investigate the effects of long non-coding RNA(LncRNA) AC003973.4 on proliferation, migration and invasion of breast cancer cells. Methods Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect the expression level of LncRNA AC003973.4 in 20 samples of breast cancer tissue and adjacent tissues, and in breast cancer cell lines BT-549, MDA-MB-231, MCF-7,T47 D, and normal mammary epithelial MCF-10 A cells. The over-expressed plasmid was transfected into the breast cancer cell line with low expression level of AC003973.4 and the expression of AC003973.4 was detected. MTS assay, Transwell migration assay and Transwell invasion assay were performed to detect the proliferation, migration and invasion of breast cancer cells, respectively. The complementary microRNA(mi RNA) of AC003973.4 and downstream genes of mi RNA were predicted with bioinformatics. The expression of mi RNA and downstream gene m RNA was detected by qRT-PCR, and the related protein expression was examined by Western blot. Results The expression of AC003973.4 in the breast cancer tissues was significantly lower than that in the adjacent tissues(P<0.01). The expression levels of AC003973.4 in the breast cancer cell lines were significantly lower than that in the normal mammary epithelial cell(P<0.05). The expression of AC003973.4 was lowest in MCF-7 cells(P <0.01). After transfection with AC003973.4 in MCF-7 cells, the cell proliferation, migration and invasion was significantly inhibited(P <0.05). AC003973.4 was complementarily bound with mi R-224-5 p, and the latter was complementarily bound with PTEN. In AC003973.4 up-regulated MCF-7 cells the expression of mi R-224-5 p was decreased(P<0.01), the m RNA and protein expressions of PTEN were increased(P<0.01), and the protein expressions of CDK4, Cyclin D1, Zeb-1, and N-cadherin were decreased. Conclusion The expression of AC003973.4 is down-regulated in breast cancer tissues and breast cancer cell lines. Up-regulation of AC003973.4 expression inhibits the proliferation, migration and invasion of breast cancer cells, which may be related to regulating the mi R-224-5 p and PTEN gene expression.
Objective To investigate the effects of long non-coding RNA(LncRNA) AC003973.4 on proliferation, migration and invasion of breast cancer cells. Methods Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect the expression level of LncRNA AC003973.4 in 20 samples of breast cancer tissue and adjacent tissues, and in breast cancer cell lines BT-549, MDA-MB-231, MCF-7,T47 D, and normal mammary epithelial MCF-10 A cells. The over-expressed plasmid was transfected into the breast cancer cell line with low expression level of AC003973.4 and the expression of AC003973.4 was detected. MTS assay, Transwell migration assay and Transwell invasion assay were performed to detect the proliferation, migration and invasion of breast cancer cells, respectively. The complementary microRNA(mi RNA) of AC003973.4 and downstream genes of mi RNA were predicted with bioinformatics. The expression of mi RNA and downstream gene m RNA was detected by qRT-PCR, and the related protein expression was examined by Western blot. Results The expression of AC003973.4 in the breast cancer tissues was significantly lower than that in the adjacent tissues(P<0.01). The expression levels of AC003973.4 in the breast cancer cell lines were significantly lower than that in the normal mammary epithelial cell(P<0.05). The expression of AC003973.4 was lowest in MCF-7 cells(P <0.01). After transfection with AC003973.4 in MCF-7 cells, the cell proliferation, migration and invasion was significantly inhibited(P <0.05). AC003973.4 was complementarily bound with mi R-224-5 p, and the latter was complementarily bound with PTEN. In AC003973.4 up-regulated MCF-7 cells the expression of mi R-224-5 p was decreased(P<0.01), the m RNA and protein expressions of PTEN were increased(P<0.01), and the protein expressions of CDK4, Cyclin D1, Zeb-1, and N-cadherin were decreased. Conclusion The expression of AC003973.4 is down-regulated in breast cancer tissues and breast cancer cell lines. Up-regulation of AC003973.4 expression inhibits the proliferation, migration and invasion of breast cancer cells, which may be related to regulating the mi R-224-5 p and PTEN gene expression.
引文
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[2]Li WX,Sha RL,Bao JQ,et al.Expression of long non-coding RNAlinc-ITGB1 in breast cancer and its influence on prognosis and survival[J].Eur Rev Med Pharmacol Sci,2017,21(15):3397-3401.
[3]Li Z,Hou P,Fan D,et al.The degradation of EZH2 mediated by lncRNAANCR attenuated the invasion and metastasis of breast cancer[J].Cell Death Differ,2017,24(1):59-71.
[4]王婧,孙妍,廖昆玲,等.长链非编码RNA在结肠癌组织与癌旁组织中的表达研究[J].中国病理生理杂志,2018,34(1):75-80.
[5]Wang Y,Zhou J,Wang Z,et al.Upregulation of SOX2 activated LncR-NA PVT1 expression promotes breast cancer cell growth and invasion[J].Biochem Biophys Res Commun,2017,493(1):429-436.
[6]覃伟峰,仉红刚.lncRNA在心血管疾病中作用的研究进展[J].中国病理生理杂志,2016,32(8):1471-1477.
[7]Betts JA,Moradi MM,Al-Ejeh F,et al.Long Noncoding RNAs CUPID1and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage[J].Am J Hum Genet,2017,101(2):255-266.
[8]裴振,霍小蕾,田向阳,等.长链非编码RNA MIAT在非小细胞肺癌中的表达及功能研究[J].中国病理生理杂志,2018,34(4):592-598.
[9]Li RK,Gao J,Guo LH,et al.PTENP1 acts as a ce RNA to regulate PTENby sponging miR-19b and explores the biological role of PTENP1 in breast cancer[J].Cancer Gene Ther,2017,24(7):309-315.
[10]Kong Q,Qiu M.Long noncoding RNA SNHG15 promotes human breast cancer proliferation,migration and invasion by sponging miR-211-3p[J].Biochem Biophys Res Commun,2018,495(2):1594-1600.
[11]Zhang W,Shi S,Jiang J,et al.LncRNA MEG3 inhibits cell epithelial-mesenchymal transition by sponging miR-421 targeting E-cadherin in breast cancer[J].Biomed Pharmacother,2017,91:312-319.
[12]Cai C,Huo Q,Wang X,et al.SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5[J].Biochem Biophys Res Commun,2017,485(2):272-278.
[13]Fang Y,Wang J,Wu F,et al.Long non-coding RNA HOXA-AS2 promotes proliferation and invasion of breast cancer by acting as a miR-520c-3p sponge[J].Oncotarget,2017,8(28):46090-46103.
[14]Zhu G,Zhou L,Liu H,et al.MicroRNA-224 Promotes Pancreatic Cancer Cell Proliferation and Migration by Targeting the TXNIP-Mediated HIF1alpha Pathway[J].Cell Physiol Biochem,2018,48(4):1735-1746.
[15]Li N,Miao Y,Shan Y,et al.MiR-106b and miR-93 regulate cell progression by suppression of PTEN via PI3K/Akt pathway in breast cancer[J].Cell Death Dis,2017,8(5):e2796.
[16]Li S,Zhang J,Zhao Y,et al.miR-224 enhances invasion and metastasis by targeting HOXD10 in non-small cell lung cancer cells[J].Oncol Lett,2018,15(5):7069-7075.
[17]Huang Y,Li Y,Wang FF,et al.Over-Expressed miR-224 Promotes the Progression of Cervical Cancer via Targeting RASSF8[J].PLoS One,2016,11(9):e162378.
[18]Peng F,Tang H,Liu P,et al.Isoliquiritigenin modulates miR-374a/PTEN/Akt axis to suppress breast cancer tumorigenesis and metastasis[J].Sci Rep,2017,7(1):9022.
[19]Chu S,Liu G,Xia P,et al.miR-93 and PTEN:Key regulators of doxorubicin-resistance and EMT in breast cancer[J].Oncol Rep,2017,38(4):2401-2407.
[20]Dai X,Fang M,Li S,et al.miR-21 is involved in transforming growth factor beta1-induced chemoresistance and invasion by targeting PTENin breast cancer[J].Oncol Lett,2017,14(6):6929-6936.