miR-19a-3p通过靶向PIK3CB参与调控雌激素受体阳性乳腺癌化疗耐药的相关研究
|
摘要
目的:研究miR-19a-3p在雌激素受体(ER)阳性乳腺癌化疗耐药中的作用及可能的调控机制。方法:用10μmol/L顺铂处理ER阳性乳腺癌耐药细胞株和敏感细胞株,MTT法检测细胞活性;RNA测序分析ER阳性乳腺癌耐药细胞株和敏感细胞株中表达差异的microRNA;实时荧光定量PCR验证miR-19a-3p在ER阳性乳腺癌耐药细胞株和敏感株中的表达差异;合成miR-19a-3p类似物和抑制物,分别转染ER阳性乳腺癌耐药细胞株和敏感株,检测耐药细胞株对顺铂耐药的药物作用浓度是否发生改变;通过Starbase v2.0调取TCGA数据分析预测miR-19a-3p的靶标基因及相互调控关系;实时荧光定量PCR进一步验证miR-19a-3p与靶标基因在ER阳性乳腺癌耐药细胞株和敏感株中的表达。结果:顺铂处理后不影响ER阳性乳腺癌耐药细胞株的细胞活性;小RNA测序发现miR-19a-3p在ER阳性乳腺癌耐药细胞株中低表达,qPCR进一步确认miR-19a-3p在ER阳性乳腺癌耐药细胞株中的表达量相比于敏感株较低;miR-19a-3p过表达促进ER阳性乳腺癌耐药株的顺铂耐药作用浓度下调,而miR-19a-3p表达被抑制后会上调ER乳腺癌敏感细胞株的顺铂敏感作用浓度;通过Starbase v2.0预测表明miR-19a-3p负调控PIK3CB,qPCR及Western印迹证实miR-19a-3p过表达抑制PIK3CB的表达。结论:miR-19a-3p通过抑制PIK3CB的表达,负调控ER阳性乳腺癌化疗耐药,增强ER阳性乳腺癌细胞对顺铂化疗的敏感性。
Objective: To explore the possible mechanism of miR-19a-3p involved in the regulation of chemotherapeutic resistance of estrogen receptor(ER) positive breast cancer. Methods: MTT assay was used to detect cell activity in the treatment of chemoresistance ER positive breast cancer cells and the control with 10 μmol/L cisplatin. RNA sequencing was used to analyze microRNA expression differences between chemoresistance ER positive breast cancer cells and the control. Real-time quantitative PCR was used to verify the expression difference of miR-19a-3p in chemoresistance cells and the control. miR-19a-3p mimic and inhibitor were synthesized and transfected into chemoresistance ER positive breast cancer cells and the control strains respectively, and drug resistance concentration of cisplatin was detected. The target genes of miR-19a-3p and their regulatory relationship were predicted via the analysis of TCGA data obtained through Starbase v2.0. Real-time quantitative PCR was used to further verify the regulatory relationship between miR-19a-3p and the target gene in chemoresistance ER positive breast cancer cells and the control. Results: Cisplatin treatment did not affect the cell activity of ER positive breast cancer chemoresistance cells. RNA sequencing found that miR-19a-3p was low expressed in chemoresistance ER positive breast cancer cells, and qPCR further confirmed that miR-19a-3p expression in chemoresistance ER positive breast cancer cells was very low compared with that in the controls. Overexpression of miR-19a-3p decreased the drug resistance concentration of cisplatin in chemoresistance ER positive breast cancer cells,while inhibition of miR-19a-3p promoted the drug resistance concentration of cisplatin in ER positive breast cancer control cells. Starbase v2.0 predicted that miR-19a-3p negatively regulated PIK3CB, and qPCR verified that overexpression of miR-19a-3p inhibited the expression of PIK3CB. Conclusion: miR-19a-3p is involved in the negative regulation of ER positive breast cancer chemotherapy resistance, promoting ER positive breast cancer chemotherapy sensitivity possibly through the negative regulation of PIK3CB.
Objective: To explore the possible mechanism of miR-19a-3p involved in the regulation of chemotherapeutic resistance of estrogen receptor(ER) positive breast cancer. Methods: MTT assay was used to detect cell activity in the treatment of chemoresistance ER positive breast cancer cells and the control with 10 μmol/L cisplatin. RNA sequencing was used to analyze microRNA expression differences between chemoresistance ER positive breast cancer cells and the control. Real-time quantitative PCR was used to verify the expression difference of miR-19a-3p in chemoresistance cells and the control. miR-19a-3p mimic and inhibitor were synthesized and transfected into chemoresistance ER positive breast cancer cells and the control strains respectively, and drug resistance concentration of cisplatin was detected. The target genes of miR-19a-3p and their regulatory relationship were predicted via the analysis of TCGA data obtained through Starbase v2.0. Real-time quantitative PCR was used to further verify the regulatory relationship between miR-19a-3p and the target gene in chemoresistance ER positive breast cancer cells and the control. Results: Cisplatin treatment did not affect the cell activity of ER positive breast cancer chemoresistance cells. RNA sequencing found that miR-19a-3p was low expressed in chemoresistance ER positive breast cancer cells, and qPCR further confirmed that miR-19a-3p expression in chemoresistance ER positive breast cancer cells was very low compared with that in the controls. Overexpression of miR-19a-3p decreased the drug resistance concentration of cisplatin in chemoresistance ER positive breast cancer cells,while inhibition of miR-19a-3p promoted the drug resistance concentration of cisplatin in ER positive breast cancer control cells. Starbase v2.0 predicted that miR-19a-3p negatively regulated PIK3CB, and qPCR verified that overexpression of miR-19a-3p inhibited the expression of PIK3CB. Conclusion: miR-19a-3p is involved in the negative regulation of ER positive breast cancer chemotherapy resistance, promoting ER positive breast cancer chemotherapy sensitivity possibly through the negative regulation of PIK3CB.
引文
[1]顾玺,张文海.miRNA在乳腺癌化疗耐药中的作用[J].中国肿瘤临床,2014,41(8):538-541.
[2]李文静,赵建华.miRNAs与肿瘤化疗耐药[J].国际检验医学杂志,2011,32(21):2481-2483.
[3]Fendler W,Malachowska B,Meghani K,et al.Evolutionarily conserved serum microRNAs predict radiationinduced fatality in nonhuman primates[J].Sci Transl Med,2017,9(379).doi:10.1126/scitranslmed.aal24088.
[4]Ward A,Balwierz A,Zhang J D,et al.Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer[J].Oncogene,2013,32(9):1173-1182.
[5]Croce C M.Causes and consequences of microRNA dysregulation in cancer[J].Nat Rev Genet,2009,10(10):704-714.
[6]Musgrove E A,Sutherland R L.Biological determinants of endocrine resistance in breast cancer[J].Nat Rev Cancer,2009,9(9):631-643.
[7]Zhu Y,Liu Y,Zhang C,et al.Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1[J].Nat Commun,2018,9(1):1595.
[8]Cancer Genome Atlas Network.Comprehensive molecular portraits of human breast tumours[J].Nature,2012,490(7418):61-70.
[9]Osako T,Horii R,Matsuura M,et al.Common and discriminative clinicopathological features between breast cancers with pathological complete response or progressive disease in response to neoadjuvant chemotherapy[J].J Cancer Res Clin Oncol,2010,136(2):233-241.
[10]Kim J W,Mori S,Nevins J R.Myc-induced miRNAs integrate Myc-mediated cell proliferation and cell fate[J].Cancer Res,2010,70(12):4820-4828.
[11]Chen J,Tian W,Cai H,et al.Down-regulation of microRNA-200c is associated with drug resistance in human breast cancer[J].Med Oncol,2012,29(4):2527-2534.
[12]Liang Z,Li Y,Huang K,et al.Regulation of mi R-19 to breast cancer chemoresistance through targeting PTEN[J].Pharm Res,2011,28(12):3091-3100.
[13]Cochrane D R,Spoelstra N S,Howe E N,et al.microRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents[J].Mol Cancer Ther,2009,8(5):1055-1066.
[2]李文静,赵建华.miRNAs与肿瘤化疗耐药[J].国际检验医学杂志,2011,32(21):2481-2483.
[3]Fendler W,Malachowska B,Meghani K,et al.Evolutionarily conserved serum microRNAs predict radiationinduced fatality in nonhuman primates[J].Sci Transl Med,2017,9(379).doi:10.1126/scitranslmed.aal24088.
[4]Ward A,Balwierz A,Zhang J D,et al.Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer[J].Oncogene,2013,32(9):1173-1182.
[5]Croce C M.Causes and consequences of microRNA dysregulation in cancer[J].Nat Rev Genet,2009,10(10):704-714.
[6]Musgrove E A,Sutherland R L.Biological determinants of endocrine resistance in breast cancer[J].Nat Rev Cancer,2009,9(9):631-643.
[7]Zhu Y,Liu Y,Zhang C,et al.Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1[J].Nat Commun,2018,9(1):1595.
[8]Cancer Genome Atlas Network.Comprehensive molecular portraits of human breast tumours[J].Nature,2012,490(7418):61-70.
[9]Osako T,Horii R,Matsuura M,et al.Common and discriminative clinicopathological features between breast cancers with pathological complete response or progressive disease in response to neoadjuvant chemotherapy[J].J Cancer Res Clin Oncol,2010,136(2):233-241.
[10]Kim J W,Mori S,Nevins J R.Myc-induced miRNAs integrate Myc-mediated cell proliferation and cell fate[J].Cancer Res,2010,70(12):4820-4828.
[11]Chen J,Tian W,Cai H,et al.Down-regulation of microRNA-200c is associated with drug resistance in human breast cancer[J].Med Oncol,2012,29(4):2527-2534.
[12]Liang Z,Li Y,Huang K,et al.Regulation of mi R-19 to breast cancer chemoresistance through targeting PTEN[J].Pharm Res,2011,28(12):3091-3100.
[13]Cochrane D R,Spoelstra N S,Howe E N,et al.microRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents[J].Mol Cancer Ther,2009,8(5):1055-1066.