过表达miR-141-3p通过靶向下调PTEN并激活PI3K/Akt信号通路促进卵巢癌A2780细胞的恶性生物学行为
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摘要
目的:探讨mi R-141-3p通过靶向PTEN并调控PI3K/Akt通路对卵巢癌细胞增殖、侵袭和凋亡的影响。方法:收集2014年4月至2017年10月河南省人民医院妇产科收治的资料完整的28例卵巢癌患者肿瘤组织和相应的癌旁组织,采用q PCR检测卵巢癌组织和细胞系中miR-141-3p的表达水平,双荧光素酶报告基因实验验证miR-141-3p和PTEN的靶向关系;过表达或敲降miR-141及PTEN基因后,采用CCK-8、Transwell和Annexin V-FITC/PI双染流式术检测卵巢癌A2780细胞增殖、侵袭和凋亡水平,WB实验进一步检测mi R-141-3p对PTEN-PI3K/Akt信号通路的调控作用。结果:miR-141-3p在卵巢癌组织和细胞系中高表达(P<0.05或P<0.01)。双荧光素酶报告基因证实miR-141-3p靶向作用于PTEN并下调其表达水平(P<0.01)。与对照组相比,敲降miR-141-3p后A2780细胞的增殖受到显著抑制(48 h时,0.36±0.04 vs 0.82±0.06,P<0.05)、侵袭能力明显降低[穿膜细胞数(45.14±7.88)vs(215.32±16.04)个,P<0.01]、细胞凋亡率显著升高[(9.29±0.65)%vs(1.85±0.26)%,P<0.01]。过表达PTEN显著抑制了A2780细胞中p-Akt的表达(均P<0.01)、抑制细胞增殖和侵袭能力(均P<0.01)而明显促进细胞凋亡(均P<0.01),在过表达PTEN的同时过表达mi R-141-3p或添加IGF-1后可逆转上述的变化。结论:miR-141-3p能够促进A2780细胞增殖、侵袭和诱导凋亡,其机制可能与靶向调控PTEN并激活PI3K/Akt通路有关。
Objective: To explore the effect of miR-141-3 p on the proliferation, invasion and apoptosis of ovarian cancer cells via targeting PTEN and regulating PI3 K/Akt pathway. Methods: Collecting twenty-eight cases pairs of ovarian cancerovarian cancer patients with tumor tissues and adjacent tissues were collected from patients, who from April 2014 to October 2017 were treated in the Department of Obstetrics and Gynecology. qPCR was applied to detect the expression of miR-141-3 p in ovarian cancer tissues and cell lines.The relationship between miR-141-3 p and PTEN was verified by dual-luciferase reporter gene assay. After over-expression or knockdown of miR-141 and PTEN genes, the cell viability, invasion and apoptosis of ovarian cancer A2780 cells were examined by CCK-8 assay, Transwell assay and Annexin V-FITC/PI double staining flow cytometry assay, respectively. Furthermore, the effect of miR-141-3 p on PTEN-PI3 K/Akt signaling pathway was measured by WB. Results: miR-141-3 p is was highly expressed in ovarian cancer tissues and cell lines(P<0.05 or P<0.01). The dual luciferase reporter gene assay confirmed that miR-141-3 p targets PTEN was a target of miR-141-3 p and downregulates its expression level was down-regulated(P<0.01). Compared with the control group, after knockdown of miR-141-3 p, the proliferation of A2780 cells was significantly inhibited after knockdown of miR-141-3 p(at 48 h, 0.36±0.04 vs 0.82±0.06, P<0.05), and the invasive ability of A2780 cells was significantly reduced(number of transmembrane cells: 215.32 ± 16.04 vs45.14±7.88, P<0.01), while the apoptotic rate was significantly increased([1.85±0.26]% vs [9.29±0.65]%, P<0.01). Over-expression of PTEN significantly inhibited the expression of p-Akt and cell proliferation and invasion in A2780 cells(all P<0.01), inhibited cell proliferation and invasion(all P<0.01) and significantly promoted apoptosis(all P<0.01). However, simultaneous over-expression of miR-141-3 p or addition of IGF-1 wile over-expressing PTEN can offset the above effects. Conclusion: miR-141-3 p facilitates the proliferation, invasion and decreases apoptosis of A2780 cells. The mechanism may be related to targeted regulation of PTEN and activation of PI3 K/Akt pathway.
Objective: To explore the effect of miR-141-3 p on the proliferation, invasion and apoptosis of ovarian cancer cells via targeting PTEN and regulating PI3 K/Akt pathway. Methods: Collecting twenty-eight cases pairs of ovarian cancerovarian cancer patients with tumor tissues and adjacent tissues were collected from patients, who from April 2014 to October 2017 were treated in the Department of Obstetrics and Gynecology. qPCR was applied to detect the expression of miR-141-3 p in ovarian cancer tissues and cell lines.The relationship between miR-141-3 p and PTEN was verified by dual-luciferase reporter gene assay. After over-expression or knockdown of miR-141 and PTEN genes, the cell viability, invasion and apoptosis of ovarian cancer A2780 cells were examined by CCK-8 assay, Transwell assay and Annexin V-FITC/PI double staining flow cytometry assay, respectively. Furthermore, the effect of miR-141-3 p on PTEN-PI3 K/Akt signaling pathway was measured by WB. Results: miR-141-3 p is was highly expressed in ovarian cancer tissues and cell lines(P<0.05 or P<0.01). The dual luciferase reporter gene assay confirmed that miR-141-3 p targets PTEN was a target of miR-141-3 p and downregulates its expression level was down-regulated(P<0.01). Compared with the control group, after knockdown of miR-141-3 p, the proliferation of A2780 cells was significantly inhibited after knockdown of miR-141-3 p(at 48 h, 0.36±0.04 vs 0.82±0.06, P<0.05), and the invasive ability of A2780 cells was significantly reduced(number of transmembrane cells: 215.32 ± 16.04 vs45.14±7.88, P<0.01), while the apoptotic rate was significantly increased([1.85±0.26]% vs [9.29±0.65]%, P<0.01). Over-expression of PTEN significantly inhibited the expression of p-Akt and cell proliferation and invasion in A2780 cells(all P<0.01), inhibited cell proliferation and invasion(all P<0.01) and significantly promoted apoptosis(all P<0.01). However, simultaneous over-expression of miR-141-3 p or addition of IGF-1 wile over-expressing PTEN can offset the above effects. Conclusion: miR-141-3 p facilitates the proliferation, invasion and decreases apoptosis of A2780 cells. The mechanism may be related to targeted regulation of PTEN and activation of PI3 K/Akt pathway.
引文
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[17]ISHIBASHI O,AKAGI I,OGAWA Y,et al.MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2,a negative regulator of the PI3K/AKT pathway[J].Biochem Biophys Res Commun,2018,501(2):507-513.DOI:10.1016/j.bbrc.2018.05.025.
[18]SUN D,WANG X,SUI G Q,et al.Downregulation of miR-374b-5p promotes chemotherapeutic resistance in pancreatic cancer by upregulating multiple anti-apoptotic proteins[J/OL].Int J Oncol,2018,[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873836/.DOI:10.3892/ijo.2018.4315.
[19]CHEN P,GUO X F,ZHOU H D,et al.SPLUNC1 regulates cell progression and apoptosis through the miR-141-PTEN/p27 pathway,but is hindered by LMP1[J/OL].PLoS One,2013,8(3):e56929[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589440/.DOI:10.1371/journal.pone.0056929.
[20]LIU Y,ZHAO R,WANG H,et al.MiR-141 is involved in BRD7-mediated cell proliferation and tumor formation through suppression of the PTEN/AKT pathway in nasopharyngeal carcinoma[J/OL].Cell Death Dis,2016,7:e2156[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823963/.DOI:10.1038/cddis.2016.64.
[21]MENG Q C,SHI S,LIANG C,et al.Abrogation of glutathione peroxidase-1 drives EMT and chemoresistance in pancreatic cancer by activating ROS-mediated Akt/GSK3β/Snail signaling[J].Oncogene,2018,37(44):5843-5857.DOI:10.1038/s41388-018-0392-z.
[22]WANG S,LEI Y Q,CAI Z L,et al.Girdin regulates the proliferation and apoptosis of pancreatic cancer cells via the PI3K/Akt signalling pathway[J/OL].Oncol Rep,2018,40(2):599-608[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072288/.DOI:10.3892/or.2018.6469.
[2]眭蕊,米雪,苗劲蔚.miR-214、miR-503在上皮性卵巢癌组织及血清外泌小体中表达的研究[J].癌症进展,2017,15(2):128-131.DOI:10.11877/j.issn.1672-1535.2017.15.02.08.
[3]HUANG S,WA Q D,PAN J C,et al.Downregulation of miR-141-3p promotes bone metastasis via activating NF-κB signaling in prostate cancer[J/OL].J Exp Clin Cancer Res,2017,36(1):173[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716366/.DOI:10.1186/s13046-017-0645-7.
[4]ZHANG G X,ZHANG W,LI B J,et al.MicroRNA-200c and microRNA-141 are regulated by a FOXP3-KAT2B axis and associated with tumor metastasis in breast cancer[J/OL].Breast Cancer Res,2017,19(1):73[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480201/.DOI:10.1186/s13058-017-0858-x.
[5]CHEN X,WANG X G,RUAN A M,et al.miR-141 is a key regulator of renal cell carcinoma proliferation and metastasis by controlling EphA2 expression[J].Clin Cancer Res,2014,20(10):2617-2630.DOI:10.1158/1078-0432.CCR-13-3224.
[6]LI J H,ZHANG Z,DU M Z,et al.microRNA-141-3p fosters the growth,invasion,and tumorigenesis of cervical cancer cells by targeting FOXA2[J].Arch Biochem Biophys,2018,657(11):23-30.DOI:10.1016/j.abb.2018.09.008.
[7]LONG Z H,BAI Z G,SONG J N,et al.mir-141 inhibits proliferation and migration of colorectal cancer SW480 cells[J].Anticancer Res,2017,37(8):4345-4352.DOI:10.21873/anticanres.11828.
[8]SHA M,LIN M,WANG J,et al.Long non-coding RNA MIAT promotes gastric cancer growth and metastasis through regulation of miR-141/DDX5 pathway[J/OL].J Exp Clin Cancer Res,2018,37(1):58[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852965/.DOI:10.1186/s13046-018-0725-3.
[9]LI C,WAN L,LIU Z Y,et al.Long non-coding RNA XIST promotes TGF-β-induced epithelial-mesenchymal transition by regulating miR-367/141-ZEB2 axis in non-small-cell lung cancer[J].Cancer Lett,2018,418(4):185-195.DOI:10.1016/j.canlet.2018.01.036.
[10]POLISENO L,PANDOLFI P P.PTEN ceRNA networks in human cancer[J].Methods,2015,77/78:41-50.DOI:10.1016/j.ymeth.2015.01.013.
[11]ZHOU X Y,XIA Y,SU J,et al.Down-regulation of miR-141 induced by helicobacter pylori promotes the invasion of gastric cancer by targeting STAT4[J].Cell Physiol Biochem,2014,33(4):1003-1012.DOI:10.1159/000358671.
[12]DING L,YU L L,HAN N,et al.miR-141 promotes colon cancer cell proliferation by inhibiting MAP2K4[J/OL].Oncol Lett,2017,13(3):1665-1671[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403415/.DOI:10.3892/ol.2017.5653.
[13]GAO Y C,WU J.MicroRNA-200c and microRNA-141 as potential diagnostic and prognostic biomarkers for ovarian cancer[J].Tumour Biol,2015,36(6):4843-4850.DOI:10.1007/s13277-015-3138-3.
[14]ZHU S H,HE X C,WANG L.Correlation analysis of miR-200b,miR-200c,and miR-141 with liver metastases in colorectal cancer patients[J].Eur Rev Med Pharmacol Sci,2017,21(10):2357-2363.
[15]ZHANG R P,LI F X,WANG W J,et al.The effect of antisense inhibitor of miRNA 106b-25 on the proliferation,invasion,migration,and apoptosis of gastric cancer cell[J].Tumour Biol,2016,37(8):10507-10515.DOI:10.1007/s13277-016-4937-x.
[16]MEI Z F,HE Y C,FENG J J,et al.MicroRNA-141 promotes the proliferation of non-small cell lung cancer cells by regulating expression of PHLPP1 and PHLPP2[J].FEBS Lett,2014,588(17):3055-3061.DOI:10.1016/j.febslet.2014.06.020.
[17]ISHIBASHI O,AKAGI I,OGAWA Y,et al.MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2,a negative regulator of the PI3K/AKT pathway[J].Biochem Biophys Res Commun,2018,501(2):507-513.DOI:10.1016/j.bbrc.2018.05.025.
[18]SUN D,WANG X,SUI G Q,et al.Downregulation of miR-374b-5p promotes chemotherapeutic resistance in pancreatic cancer by upregulating multiple anti-apoptotic proteins[J/OL].Int J Oncol,2018,[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873836/.DOI:10.3892/ijo.2018.4315.
[19]CHEN P,GUO X F,ZHOU H D,et al.SPLUNC1 regulates cell progression and apoptosis through the miR-141-PTEN/p27 pathway,but is hindered by LMP1[J/OL].PLoS One,2013,8(3):e56929[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589440/.DOI:10.1371/journal.pone.0056929.
[20]LIU Y,ZHAO R,WANG H,et al.MiR-141 is involved in BRD7-mediated cell proliferation and tumor formation through suppression of the PTEN/AKT pathway in nasopharyngeal carcinoma[J/OL].Cell Death Dis,2016,7:e2156[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823963/.DOI:10.1038/cddis.2016.64.
[21]MENG Q C,SHI S,LIANG C,et al.Abrogation of glutathione peroxidase-1 drives EMT and chemoresistance in pancreatic cancer by activating ROS-mediated Akt/GSK3β/Snail signaling[J].Oncogene,2018,37(44):5843-5857.DOI:10.1038/s41388-018-0392-z.
[22]WANG S,LEI Y Q,CAI Z L,et al.Girdin regulates the proliferation and apoptosis of pancreatic cancer cells via the PI3K/Akt signalling pathway[J/OL].Oncol Rep,2018,40(2):599-608[2018-11-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072288/.DOI:10.3892/or.2018.6469.