miRNA-21靶向调控Wnt/β-catenin对非小细胞肺癌细胞增殖与侵袭的影响
|
摘要
目的研究microRNA-21(miR-21)是否通过调控wnt/β-catenin信号通路从而影响人非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞株NCI-H1975细胞体外增殖与侵袭能力。方法预测和构建含有miR-21候选靶基因和β-连环蛋白基因的双荧光素酶报告质粒。将miR-21模拟物通过Lipofactamine 2000脂质体转染法转染NCI-H1975细胞,应用qRTPCR检测细胞中miR-21和β-catenin mRNA表达情况,应用Western blot检测β-catenin蛋白水平,CCK-8法检测miR-21mimic对NCI-H1975细胞增殖能力的影响,Transwell侵袭实验检测miR-21 mimic对NCI-H1975细胞侵袭能力的影响。结果qRT-PCR肺癌组织中的miR-21与邻近正常组织相比表达明显升高;双荧光素酶报告基因结果显示miR-21能和β-catenin的3’UTR端结合且显著促进荧光素酶活性;上调miR-21可使β-catenin的mRNA和蛋白表达水平显著升高,同时上调miR-21可显著增强NCI-H1975细胞的增殖与侵袭能力。结论 miR-21在转录后水平调控β-catenin表达来促进NCI-H1975细胞的增殖与侵袭能力,可能是NSCLC潜在的治疗靶点。
Objective To investigate whether miR-21 affects the proliferation and invasion of non-small-cell lung cancer(NSCLC) cell line NCI-H1975 by regulating Wnt/β-catenin signaling pathway in vitro. Methods The dual luciferase reporter plasmids containing the candidate target gene of miR-21 and the β-catenin gene were predicted and constructed. miR-21 mimics were transfected into NCI-H1975 cells by lipofection with lipofactamine 2000. The expression level of miR-21 and β-catenin mRNA in non-small lung cancer tissues and adjacent normal lung tissues was detected by quantitative real-time PCR(qRT-PCR). The expression level ofβ-catenin protein was detected by Western blot. The effect of miR-21 mimics on the proliferation of NCI-H1975 cells was detected by CCK-8 method and on the invasion ability was detected by transwell invasion assay. Results Compared with adjacent normal lung tissues, miR-21 detected by qRT-PCR was highly expressed in non-small cell lung cancer tissues. Dual luciferase reporter gene assay showed that miR-21 could directly bind to the 3′-untranslated region(3' UTR) of the β-catenin gene and significantly enhance luciferase activity. qRT-PCR and western blot results indicated that the upregulation of miR-21 expression could significantly increase the mRNA and protein expression levels of β-catenin in NCI-H1975 cell line. Moreover, CCK-8 and Transwell invasion assay showed that the upregulation of miR-21 expression significantly promoted the proliferation and invasion of NCI-H1975 cell line. Conclusion miR-21 can promote the proliferation and invasion of NSCLC cell line NCI-H1975 by regulating the expression of β-catenin at the post-transcriptional level, which may be a potential treatment target of NSCLC.
Objective To investigate whether miR-21 affects the proliferation and invasion of non-small-cell lung cancer(NSCLC) cell line NCI-H1975 by regulating Wnt/β-catenin signaling pathway in vitro. Methods The dual luciferase reporter plasmids containing the candidate target gene of miR-21 and the β-catenin gene were predicted and constructed. miR-21 mimics were transfected into NCI-H1975 cells by lipofection with lipofactamine 2000. The expression level of miR-21 and β-catenin mRNA in non-small lung cancer tissues and adjacent normal lung tissues was detected by quantitative real-time PCR(qRT-PCR). The expression level ofβ-catenin protein was detected by Western blot. The effect of miR-21 mimics on the proliferation of NCI-H1975 cells was detected by CCK-8 method and on the invasion ability was detected by transwell invasion assay. Results Compared with adjacent normal lung tissues, miR-21 detected by qRT-PCR was highly expressed in non-small cell lung cancer tissues. Dual luciferase reporter gene assay showed that miR-21 could directly bind to the 3′-untranslated region(3' UTR) of the β-catenin gene and significantly enhance luciferase activity. qRT-PCR and western blot results indicated that the upregulation of miR-21 expression could significantly increase the mRNA and protein expression levels of β-catenin in NCI-H1975 cell line. Moreover, CCK-8 and Transwell invasion assay showed that the upregulation of miR-21 expression significantly promoted the proliferation and invasion of NCI-H1975 cell line. Conclusion miR-21 can promote the proliferation and invasion of NSCLC cell line NCI-H1975 by regulating the expression of β-catenin at the post-transcriptional level, which may be a potential treatment target of NSCLC.
引文
[1]Salander P, Lilliehorn S. To carry on as before:A meta-synthesis of qualitative studies in lung cancer. Lung cancer(Amsterdam, Netherlands), 2016, 99:88-93.
[2]Rothschild SI. Advanced and Metastatic Lung Cancer-What is new in the Diagnosis and Therapy? Praxis, 2015, 104(14):745-750.
[3]Xue J, Yang J, Luo M, et al. MicroRNA-targeted therapeutics for lung cancer treatment. Expert opinion on drug discovery, 2017, 12(2):141-157.
[4]Parikh M, Riess J, Lara PN, et al. New and emerging developments in extensive-stage small cell lung cancer therapeutics. Current opinion in oncology, 2016, 28(2):97-103.
[5]Levasseur N, Clemons M, Hutton B, et al. Bone-targeted therapy use in patients with bone metastases from lung cancer:A systematic review of randomized controlled trials.Cancer treatment reviews, 2016, 50:183-193.
[6]Montani F, Bianchi F. Circulating Cancer Biomarkers:The Macro-revolution of the Micro-RNA. EBioMedicine, 2016,5:4-6.
[7]Fan B, Zhang L, Ma C. Relation of micro-RNA and carcinogenesis. Zhonghua zhong liu za zhi[Chinese journal of oncology], 2012, 34(6):401-404.
[8]Fabris L, Ceder Y, Chinnaiyan AM, et al. The Potential of Micro RNAs as Prostate Cancer Biomarkers. Eur Urol, 2016,70(2):312-322.
[9]Cairo S, Wang Y, De Reynies A, et al. Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(47):20471-20476.
[10] Chruscik A, Lam AK. Clinical pathological impacts of microRNAs in papillary thyroid carcinoma:A crucial review.Experimental and molecular pathology, 2015, 99(3):393-398.
[11] Heegaard N H, Schetter AJ, Welsh J A, et al. Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer. International journal of cancer, 2012, 130(6):1378-1386.
[12] Nagasaka M, Gadgeel SM. Role of chemotherapy and targeted therapy in early-stage non-small cell lung cancer. Expert review of anticancer therapy, 2018, 18(1):63-70.
[13] Levy B, Hu ZI, Cordova KN, et al. Clinical Utility of Liquid Diagnostic Platforms in Non-Small Cell Lung Cancer. The oncologist, 2016, 21(9):1121-1130.
[14] Hong QY, Wu GM, Qian GS, et al. Prevention and management of lung cancer in China. Cancer, 2015, 121 Suppl 17:3080-3088.
[15] Warth A. Diagnosis, prognosis, and prediction of non-small cell lung cancer. Importance of morphology, immunohistochemistry and molecular pathology. Der Pathologe, 2015, 36Suppl 2:194-200.
[16] Kayser G. Non-small cell lung cancer. New biomarkers for diagnostics and therapy. Der Pathologe, 2015, 36 Suppl 2:189-193.
[17] Shah S, Jadhav K, Shah V, et al. miRNA 21:Diagnostic Prognostic and Therapeutic Marker for Oral Cancer. Micro RNA(Shariqah, United Arab Emirates), 2016, 5(3):175-179.
[18] Qu K, Lin T, Pang Q, et al. Extracellular miRNA-21 as a novel biomarker in glioma:Evidence from meta-analysis,clinical validation and experimental investigations. Oncotarget, 2016, 7(23):33994-34010.
[19] Wang G, Wang JJ, Tang HM, et al. Targeting strategies on miRNA-21 and PDCD4 for glioblastoma. Arch Biochem Biophys, 2015, 580:64-74.
[20] Wang P, Guan Q, Zhou D, et al. miR-21 Inhibitors Modulate Biological Functions of Gastric Cancer Cells via PTEN/PI3K/mTOR Pathway. DNA Cell Biol, 2018, 37(1):38-45.
[21] Marts LT, Green DE, Mills ST, et al. MiR-21-Mediated Suppression of Smad7 Induces TGFbeta1 and Can Be Inhibited by Activation of Nrf2 in Alcohol-Treated Lung Fibroblasts.Alcohol Clin Exp Res, 2017, 41(11):1875-1885.
[22] Hua Y, Jin Z, Zhou F, et al. The expression significance of serum MiR-21 in patients with osteosarcoma and its relationship with chemosensitivity. Eur Rev Med Pharmacol Sci, 2017, 21(13):2989-2994.
[23] An F, Liu Y, Hu Y. miR-21 inhibition of LATS1 promotes proliferation and metastasis of renal cancer cells and tumor stem cell phenotype. Oncol Lett, 2017, 14(4):4684-4688.
[24] Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell, 2006, 9(3):189-198.
[25] Jiang LP, He CY, Zhu ZT. Role of microRNA-21 in radiosensitivity in non-small cell lung cancer cells by targeting PDCD4 gene. Oncotarget, 2017, 8(14):23675-23689.
[26] Li B, Ren S, Li X, et al. MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung cancer. Lung cancer(Amsterdam, Netherlands), 2014,83(2):146-153.
[27] Svoronos A A, Engelman D M, Slack F J. OncomiR or Tumor Suppressor? The Duplicity of MicroRNAs in Cancer.Cancer Res, 2016, 76(13):3666-3670.
[28] Luo G, Luo W, Sun X, et al. MicroRNA21 promotes migration and invasion of glioma cells via activation of Sox2 and betacatenin signaling. Mol Med Rep, 2017, 15(1):187-193.
[29] Lin PL, Wu DW, Huang CC, et al. MicroRNA-21 promotes tumour malignancy via increased nuclear translocation of beta-catenin and predicts poor outcome in APC-mutated but not in APC-wild-type colorectal cancer. Carcinogenesis,2014, 35(10):2175-2182.
[30] Kawakita A, Yanamoto S, Yamada S, et al. MicroRNA-21promotes oral cancer invasion via the Wnt/beta-catenin pathway by targeting DKK2. Pathol Oncol Res, 2014, 20(2):253-261.
[2]Rothschild SI. Advanced and Metastatic Lung Cancer-What is new in the Diagnosis and Therapy? Praxis, 2015, 104(14):745-750.
[3]Xue J, Yang J, Luo M, et al. MicroRNA-targeted therapeutics for lung cancer treatment. Expert opinion on drug discovery, 2017, 12(2):141-157.
[4]Parikh M, Riess J, Lara PN, et al. New and emerging developments in extensive-stage small cell lung cancer therapeutics. Current opinion in oncology, 2016, 28(2):97-103.
[5]Levasseur N, Clemons M, Hutton B, et al. Bone-targeted therapy use in patients with bone metastases from lung cancer:A systematic review of randomized controlled trials.Cancer treatment reviews, 2016, 50:183-193.
[6]Montani F, Bianchi F. Circulating Cancer Biomarkers:The Macro-revolution of the Micro-RNA. EBioMedicine, 2016,5:4-6.
[7]Fan B, Zhang L, Ma C. Relation of micro-RNA and carcinogenesis. Zhonghua zhong liu za zhi[Chinese journal of oncology], 2012, 34(6):401-404.
[8]Fabris L, Ceder Y, Chinnaiyan AM, et al. The Potential of Micro RNAs as Prostate Cancer Biomarkers. Eur Urol, 2016,70(2):312-322.
[9]Cairo S, Wang Y, De Reynies A, et al. Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(47):20471-20476.
[10] Chruscik A, Lam AK. Clinical pathological impacts of microRNAs in papillary thyroid carcinoma:A crucial review.Experimental and molecular pathology, 2015, 99(3):393-398.
[11] Heegaard N H, Schetter AJ, Welsh J A, et al. Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer. International journal of cancer, 2012, 130(6):1378-1386.
[12] Nagasaka M, Gadgeel SM. Role of chemotherapy and targeted therapy in early-stage non-small cell lung cancer. Expert review of anticancer therapy, 2018, 18(1):63-70.
[13] Levy B, Hu ZI, Cordova KN, et al. Clinical Utility of Liquid Diagnostic Platforms in Non-Small Cell Lung Cancer. The oncologist, 2016, 21(9):1121-1130.
[14] Hong QY, Wu GM, Qian GS, et al. Prevention and management of lung cancer in China. Cancer, 2015, 121 Suppl 17:3080-3088.
[15] Warth A. Diagnosis, prognosis, and prediction of non-small cell lung cancer. Importance of morphology, immunohistochemistry and molecular pathology. Der Pathologe, 2015, 36Suppl 2:194-200.
[16] Kayser G. Non-small cell lung cancer. New biomarkers for diagnostics and therapy. Der Pathologe, 2015, 36 Suppl 2:189-193.
[17] Shah S, Jadhav K, Shah V, et al. miRNA 21:Diagnostic Prognostic and Therapeutic Marker for Oral Cancer. Micro RNA(Shariqah, United Arab Emirates), 2016, 5(3):175-179.
[18] Qu K, Lin T, Pang Q, et al. Extracellular miRNA-21 as a novel biomarker in glioma:Evidence from meta-analysis,clinical validation and experimental investigations. Oncotarget, 2016, 7(23):33994-34010.
[19] Wang G, Wang JJ, Tang HM, et al. Targeting strategies on miRNA-21 and PDCD4 for glioblastoma. Arch Biochem Biophys, 2015, 580:64-74.
[20] Wang P, Guan Q, Zhou D, et al. miR-21 Inhibitors Modulate Biological Functions of Gastric Cancer Cells via PTEN/PI3K/mTOR Pathway. DNA Cell Biol, 2018, 37(1):38-45.
[21] Marts LT, Green DE, Mills ST, et al. MiR-21-Mediated Suppression of Smad7 Induces TGFbeta1 and Can Be Inhibited by Activation of Nrf2 in Alcohol-Treated Lung Fibroblasts.Alcohol Clin Exp Res, 2017, 41(11):1875-1885.
[22] Hua Y, Jin Z, Zhou F, et al. The expression significance of serum MiR-21 in patients with osteosarcoma and its relationship with chemosensitivity. Eur Rev Med Pharmacol Sci, 2017, 21(13):2989-2994.
[23] An F, Liu Y, Hu Y. miR-21 inhibition of LATS1 promotes proliferation and metastasis of renal cancer cells and tumor stem cell phenotype. Oncol Lett, 2017, 14(4):4684-4688.
[24] Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell, 2006, 9(3):189-198.
[25] Jiang LP, He CY, Zhu ZT. Role of microRNA-21 in radiosensitivity in non-small cell lung cancer cells by targeting PDCD4 gene. Oncotarget, 2017, 8(14):23675-23689.
[26] Li B, Ren S, Li X, et al. MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung cancer. Lung cancer(Amsterdam, Netherlands), 2014,83(2):146-153.
[27] Svoronos A A, Engelman D M, Slack F J. OncomiR or Tumor Suppressor? The Duplicity of MicroRNAs in Cancer.Cancer Res, 2016, 76(13):3666-3670.
[28] Luo G, Luo W, Sun X, et al. MicroRNA21 promotes migration and invasion of glioma cells via activation of Sox2 and betacatenin signaling. Mol Med Rep, 2017, 15(1):187-193.
[29] Lin PL, Wu DW, Huang CC, et al. MicroRNA-21 promotes tumour malignancy via increased nuclear translocation of beta-catenin and predicts poor outcome in APC-mutated but not in APC-wild-type colorectal cancer. Carcinogenesis,2014, 35(10):2175-2182.
[30] Kawakita A, Yanamoto S, Yamada S, et al. MicroRNA-21promotes oral cancer invasion via the Wnt/beta-catenin pathway by targeting DKK2. Pathol Oncol Res, 2014, 20(2):253-261.