miR-132靶向作用SOX4/E-cadherin信号通路逆转PC-9细胞株吉非替尼耐药性的实验研究
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摘要
目的 探讨miR-132靶向SOX4/E-cadherin信号通路对PC-9吉非替尼耐药细胞株增殖、迁移和凋亡的影响。方法 采用吉非替尼长期浓度梯度递增法体外建立PC-9/GR耐药细胞株,MTT法验证其耐药性。将miR-132模拟物(mimics)或空质粒转染至PC-9/GR细胞作为miR-132组和NC组,另设未处理PC-9/GR作为空白对照组。采用MTT法、Annexin V/PI双染法、划痕实验检测吉非替尼对各组细胞增殖、凋亡和迁移的影响。采用实时荧光定量PCR(QPCR)检测miR-132和SOX4的表达水平。采用双荧光素酶报告实验验证SOX4与miR-132的靶向关系。将miR-132组PC-9/GR细胞分别转染过表达SOX4质粒和空质粒为miR-132+SOX4组和miR-132-NC组,采用Western blotting检测E-cadherin和Vimentin蛋白的表达水平。结果 体外成功建立PC-9/GR耐药细胞株,不同浓度吉非替尼对PC-9/GR细胞增殖抑制率低于PC-9细胞。PC-9和PC-9/GR的IC_(50)分别为157.7 nmol/L和917.9 nmol/L。PC-9/GR的耐药指数(RI)为5.82。PC-9/GR细胞miR-132表达水平明显低于PC-9细胞(P<0.05),转染miR-132 mimics后,miR-132组miR-132表达水平明显高于PC-9/GR组和NC组(P<0.05)。不同浓度吉非替尼对miR-132组的增殖抑制率高于PC-9/GR组和NC组(P<0.05)。miR-132组IC_(50)为357.6 nmol/L。357.6 nmol/L吉非替尼作用于miR-132组24 h后,细胞早期凋亡率和细胞愈合率分别为(23.14±2.23)%、(13.44±1.81)%,与PC-9/GR组和NC组比较,差异具有统计学意义(P<0.05)。双荧光素酶报告实验验证SOX4可能是miR-132的下游靶基因。miR-132组PC-9/GR细胞SOX4 mRNA表达水平低于PC-9/GR组和NC组(P<0.05)。miR-132+SOX4组SOX4和Vimentin蛋白表达水平高于miR-132组和miR-132-NC组(P<0.05);而E-cadherin则相反(P<0.05)。结论 上调miR-132可抑制SOX4水平进而诱导EMT发生,从而逆转PC-9/GR细胞株对吉非替尼的耐药。
Objective To investigate the effects of miR-132 targeting SOX4/E-cadherin signaling pathway on the proliferation, migration and apoptosis of PC-9 gefitinib-resistant cell line. Methods PC-9/GR drug-resistant cell lines were established in vitro by long-term concentration gradient increasing method of gefitinib, and their drug resistance was verified by MTT method. miR-132 mimics or blank plasmids were transfected into PC-9/GR cells as miR-132 group and NC group, and PC-9/GR cells without treatment as blank control group. MTT assay, Annexin V/PI double staining and scratch test were used to detect the effects of gefitinib on cell proliferation, apoptosis and migration. Real-time fluorescence quantitative polymerase chain reaction(QPCR) was used to detect the expression levels of miR-132 and SOX4. The targeting relationship between SOX4 and miR-132 was validated by double luciferase reporter assay. PC-9/GR cells in the miR-132 group were transfected with SOX4 plasmids and empty plasmids as miR-132+SOX4 group and the miR-132-NC group respectively. Western blotting was used to detect the expression of E-cadherin and Vimentin proteins. Results PC-9/GR resistant cell lines were successfully established in vitro. The proliferation inhibition rate of gefitinib at different concentrations on PC-9/GR cell was lower than that of PC-9 cell. IC_(50) of PC-9 and PC-9/GR were 157.7 nmol/L and 1017.9 nmol/L, respectively. The resistance index(RI) of PC-9/GR was 5.82. The expression level of miR-132 in PC-9/GR cells was significantly lower than that in PC-9 cells(P<0.05). After transfection of miR-132 mimimics, the expression level of miR-132 in miR-132 group was significantly higher than that in PC-9/GR group and NC group(P<0.05). The inhibitory rate of gefitinib at different concentrations on the proliferation of miR-132 cells was higher than that of PC-9/GR and NC cells(P<0.05). The IC_(50) of miR-132 group was 357.6 nmol/L. After treatment with 357.6 nmol/L gefitinib for 24 hours, the early apoptotic rate and cell healing rate were(23.14±2.23)% and(13.44±1.81)%, respectively. There was a significant difference between PC-9/GR group and NC group(P < 0.05). Double luciferase reporter assay confirmed that SOX4 may be a downstream target gene of miR-132. The SOX4 expression level of PC-9/GR cells in the miR-132 group was lower than that of PC-9/GR and NC groups(P<0.05). The expression levels of SOX4 and Vimentin in the group of miR-132+SOX4 were higher than those in the group of miR-132 and the group of miR-132-NC(P<0.05), while that in the group of E-cadherin was the opposite(P<0.05). Conclusion Upregulation of miR-132 can inhibit SOX4 levels and induce EMT, thus reversing gefitinib resistance in PC-9/GR cell lines.
Objective To investigate the effects of miR-132 targeting SOX4/E-cadherin signaling pathway on the proliferation, migration and apoptosis of PC-9 gefitinib-resistant cell line. Methods PC-9/GR drug-resistant cell lines were established in vitro by long-term concentration gradient increasing method of gefitinib, and their drug resistance was verified by MTT method. miR-132 mimics or blank plasmids were transfected into PC-9/GR cells as miR-132 group and NC group, and PC-9/GR cells without treatment as blank control group. MTT assay, Annexin V/PI double staining and scratch test were used to detect the effects of gefitinib on cell proliferation, apoptosis and migration. Real-time fluorescence quantitative polymerase chain reaction(QPCR) was used to detect the expression levels of miR-132 and SOX4. The targeting relationship between SOX4 and miR-132 was validated by double luciferase reporter assay. PC-9/GR cells in the miR-132 group were transfected with SOX4 plasmids and empty plasmids as miR-132+SOX4 group and the miR-132-NC group respectively. Western blotting was used to detect the expression of E-cadherin and Vimentin proteins. Results PC-9/GR resistant cell lines were successfully established in vitro. The proliferation inhibition rate of gefitinib at different concentrations on PC-9/GR cell was lower than that of PC-9 cell. IC_(50) of PC-9 and PC-9/GR were 157.7 nmol/L and 1017.9 nmol/L, respectively. The resistance index(RI) of PC-9/GR was 5.82. The expression level of miR-132 in PC-9/GR cells was significantly lower than that in PC-9 cells(P<0.05). After transfection of miR-132 mimimics, the expression level of miR-132 in miR-132 group was significantly higher than that in PC-9/GR group and NC group(P<0.05). The inhibitory rate of gefitinib at different concentrations on the proliferation of miR-132 cells was higher than that of PC-9/GR and NC cells(P<0.05). The IC_(50) of miR-132 group was 357.6 nmol/L. After treatment with 357.6 nmol/L gefitinib for 24 hours, the early apoptotic rate and cell healing rate were(23.14±2.23)% and(13.44±1.81)%, respectively. There was a significant difference between PC-9/GR group and NC group(P < 0.05). Double luciferase reporter assay confirmed that SOX4 may be a downstream target gene of miR-132. The SOX4 expression level of PC-9/GR cells in the miR-132 group was lower than that of PC-9/GR and NC groups(P<0.05). The expression levels of SOX4 and Vimentin in the group of miR-132+SOX4 were higher than those in the group of miR-132 and the group of miR-132-NC(P<0.05), while that in the group of E-cadherin was the opposite(P<0.05). Conclusion Upregulation of miR-132 can inhibit SOX4 levels and induce EMT, thus reversing gefitinib resistance in PC-9/GR cell lines.
引文
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[16] 胡翘廷,周娟,程东海,等.肿瘤干细胞在NSCLC细胞EMT介导的EGFR-TKI获得性耐药中的作用[J].实用医学杂志,2016,36(8):1223-1225.
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[2] Poh ME,Liam CK,Rajadurai P,et al.Epithelial-to-mesenchymal transition (EMT) causing acquired resistance to afatinib in a patient with epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma[J].J Thorac Dis,2018,10(7):560-563.
[3] Lu J,Zhan Y,Feng J,et al.MicroRNAs associated with therapy of non-small cell lung cancer[J].Int J Biol Sci,2018,14(4):390-397.
[4] Arunkumar G,Deva Magendhra Rao AK,Manikandan M,et al.Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma[J].Oncol Lett,2018,15(1):649-657.
[5] Li Y,VandenBoom TG 2nd,Kong D,et al.Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells[J].Cancer Res,2009,69(16):6704-6712.
[6] Ding J,Zhao Z,Song J,et al.MiR-223 promotes the doxorubicin resistance of colorectal cancer cells via regulating epithelial-mesenchymal transition by targeting FBXW7[J].Acta Biochim Biophys Sin (Shanghai),2018,50(6):597-604.
[7] 邹绮明,张玲.miRNA在乙肝相关性肝癌发生发展中的作用研究进展[J].山东医药,2018,58(9):89-91.
[8] Zheng YB,Luo HP,Shi Q,et al.miR-132 inhibits colorectal cancer invasion and metastasis via directly targeting ZEB2[J].World J Gastroenterol,2014,20(21):6515-6522.
[9] Liu Y,Li Y,Liu J,et al.MicroRNA-132 inhibits cell growth and metastasis in osteosarcoma cell lines possibly by targeting Sox4[J].Int J Oncol,2015,47(5):1672-1684.
[10] Masters GA,Temin S,Azzoli CG,et al.Systemic therapy for stage IV non-small-cell lung cancer:American Society of Clinical Oncology Clinical Practice Guideline Update[J].J Clin Oncol,2015,33(30):3488-3515.
[11] Westover D,Zugazagoitia J,Cho BC,et al.Mechanisms of acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors[J].Ann Oncol,2018,29(Suppl 1):10-19.
[12] Li L,Gu X,Yue J,et al.Acquisition of EGFR TKI resistance and EMT phenotype is linked with activation of IGF1R/NF-κB pathway in EGFR-mutant NSCLC[J].Oncotarget,2017,8(54):92240-92253.
[13] 刘海斌,华莹,金洲祥.微小RNA-132转染对体内外肝癌生长和凋亡的作用[J].中国医学科学院学报,2015,37(1):30-36.
[14] Yao ZH,Yao XL,Zhang Y,et al.miR-132 down-regulates Methyl CpG binding protein 2 (MeCP2) during cognitive dysfunction following chronic cerebral hypoperfusion[J].Curr Neurovasc Res,2017,14(4):385-396.
[15] Louren?o AR,Coffer PJ.SOX4:Joining the master regulators of epithelial-to-mesenchymal transition?[J].Trends Cancer,2017,3(8):571-582.
[16] 胡翘廷,周娟,程东海,等.肿瘤干细胞在NSCLC细胞EMT介导的EGFR-TKI获得性耐药中的作用[J].实用医学杂志,2016,36(8):1223-1225.
[17] Goossens S,Vandamme N,Van Vlierberghe P,et al.EMT transcription factors in cancer development re-evaluated:Beyond EMT and MET[J].Biochim Biophys Acta,2017,1868(2):584-591.