miR-140-5p调控Nrf2影响结肠癌细胞5-FU耐药性
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摘要
目的观察miR-140-5p对结肠癌耐药细胞株SW480/5-FU及其亲本细胞株SW480 5-FU敏感性的影响及其与核因子NF-E2相关因子2(NF-E2-related factor 2,Nrf2)的关系,探讨miR-140-5p调控结肠癌细胞5-FU耐药的可能机制。方法用结肠癌细胞株SW480构建结肠癌耐药细胞株SW480/5-FU,qRT-PCR法检测两株细胞中miR-140-5p的表达,CCK-8法和细胞集落形成实验评估miR-140-5p对两株细胞5-FU敏感性的影响;采用qRT-PCR法、Western blot法双荧光素酶报告基因验证miR-140-5p与Nrf2的关系,CCK-8法、细胞集落形成实验探讨miR-140-5p调控结肠癌细胞5-FU敏感性与Nrf2的关系。结果成功构建结肠癌耐药细胞株SW480/5-FU,其miR-140-5p表达水平显著低于亲本细胞株SW480(P<0.05);CCK-8法、细胞集落形成实验结果显示,miR-140-5p可增强SW480/5-FU细胞对5-FU的敏感性,而降低SW480对5-FU的耐药性;qRT-PCR法、Westren blot法、双荧光素酶报告基因实验共同证实miR-140-5p可结合并抑制Nrf2的表达;CCK-8、细胞集落形成实验表明,Nrf2过表达能逆转miR-140-5p对结肠癌耐药细胞SW480/5-FU的5-FU敏感性调控结果。结论 miR-140-5p可能通过结合并抑制Nrf2表达,增加结肠癌耐药细胞株SW480/5-FU对5-FU的敏感性。
Objective To evaluate the effect of miR-140-5 p on the 5-FU sensitivity of SW480 cells and 5-FU-resistant SW480 cells,and to explore whether the effects of miR-140-5 p involve Nrf2. Methods We constructed the 5-FU-resistant cell line SW480/5-FU from SW480 colon cancer cells. Expression of miR-140-5 p was detected using qRT-PCR,and sensitivity of the two cell lines to 5-FU was measured using the CCK-8 and cell colony formation assays. Possible relationships between miR-140-5 p and Nrf2 expression were explored using qRT-PCR,Western blotting and a dual-luciferase reporter gene assay. The involvement of Nrf2 in regulation of 5-FUresistance by miR-140-5 p was tested in CCK-8 and cell colony formation assays. Results SW480/5-FU cells showed strong 5-FU resistance,which qRT-PCR linked to much lower miR-140-5 p expression. CCK-8 and cell colony formation assays showed that miR-140-5 p expression correlated with significantly higher 5-FU sensitivity in SW480/5-FU cells. The combination of qRT-PCR,Western blotting and dual-luciferase reporter gene assays suggested that miR-140-5 p binds directly to Nrf2 and inhibits it. Overexpression of Nrf2 in SW480/5-FU cells rescued 5-FU sensitivity,based on CCK-8 and cell colony formation assays. Conclusion It appears that miR-140-5 p expression is associated with higher 5-FU sensitivity in SW480 cells,and that miR-140-5 p sensitizes cells by binding and inhibiting Nrf2.
Objective To evaluate the effect of miR-140-5 p on the 5-FU sensitivity of SW480 cells and 5-FU-resistant SW480 cells,and to explore whether the effects of miR-140-5 p involve Nrf2. Methods We constructed the 5-FU-resistant cell line SW480/5-FU from SW480 colon cancer cells. Expression of miR-140-5 p was detected using qRT-PCR,and sensitivity of the two cell lines to 5-FU was measured using the CCK-8 and cell colony formation assays. Possible relationships between miR-140-5 p and Nrf2 expression were explored using qRT-PCR,Western blotting and a dual-luciferase reporter gene assay. The involvement of Nrf2 in regulation of 5-FUresistance by miR-140-5 p was tested in CCK-8 and cell colony formation assays. Results SW480/5-FU cells showed strong 5-FU resistance,which qRT-PCR linked to much lower miR-140-5 p expression. CCK-8 and cell colony formation assays showed that miR-140-5 p expression correlated with significantly higher 5-FU sensitivity in SW480/5-FU cells. The combination of qRT-PCR,Western blotting and dual-luciferase reporter gene assays suggested that miR-140-5 p binds directly to Nrf2 and inhibits it. Overexpression of Nrf2 in SW480/5-FU cells rescued 5-FU sensitivity,based on CCK-8 and cell colony formation assays. Conclusion It appears that miR-140-5 p expression is associated with higher 5-FU sensitivity in SW480 cells,and that miR-140-5 p sensitizes cells by binding and inhibiting Nrf2.
引文
[1]Li ZH,Rana TM.Therapeutic targeting of micro RNAs:current status and future challenges[J].Nat Rev Drug Discov,2014,13(8):622-638.
[2]Rupaimoole R,Slack FJ.Micro RNA therapeutics:towards a new era for the management of cancer and other diseases[J].Nat Rev Drug Discov,2017,16(3):203-222.
[3]van Beijnum JR,Giovannetti E,Poel D,et al.mi RNAs:micro-managers of anticancer combination therapies[J].Angiogenesis,2017.
[4]谢学成,邱海,覃宇周.micro RNAs与结直肠癌[J].中国癌症防治杂志,2014,6(3):310-314.
[5]Jeddi F,Soozangar N,Sadeghi MR,et al.Contradictory roles of Nrf2/Keap1 signaling pathway in cancer prevention/promotion and chemoresistance[J].DNA Repair(Amst),2017,54:13-21.
[6]Penning TM.Aldo-Keto reductase regulation by the Nrf2 system:implications for stress response,chemotherapy drug resistance,and carcinogenesis[J].Chem Res Toxicol,2017,30(1):162-176.
[7]Ryoo IG,Lee SH,Kwak MK.Redox modulating NRF2:a potential mediator of cancer stem cell resistance[J].Oxid Med Cell Longev,2016:2428153.
[8]Samatiwat P,Prawan A,Senggunprai L,et al.Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents[J].Tumour Biol,2016,37(8):11495-11507.
[9]Flamini Valentina,Jiang Wen G,Cui Yuxin.Therapeutic Role of Mi R-140-5p for the treatment of non-small cell Lung cancer[J].Anticancer Res,2017,37(8):4319-4327.
[10]Zhao Ke,Chen Bao-Jun,Chen Zhi-Guo.Erb B4 as a potential molecular target in the treatment of esophageal squamous cell cancers[J].Scientific World Journal,2014,124105.
[11]胡万乐,何占红,蒋芳,等.5-Fu诱导人大肠癌SW480细胞株耐药与自噬活性的变化[J].中国中西医结合外科杂志,2013,19(3):279-282.
[12]Mosakhani N,Lahti L,Borze I,et al.Micro RNA profiling predicts survival in anti-EGFR treated chemorefractory metastatic colorectal cancer patients with wild-type KRAS and BRAF[J].Cancer Genet,2012,205(11):545-551.
[13]Zhai H,Fesler A,Ba Y,et al.Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-mi R-140-5p mediated suppression of Smad2 and autophagy[J].Oncotarge,2015,6(23):19735-19746.
[14]Zhang W,Zou C,Pan L,et al.Micro RNA-140-5p inhibits the progression of colorectal cancer by targeting VEGFA[J].Cell Physiol Biochem,2015,37(3):1123-1133.
[15]De Nicola GM,Karreth FA,Humpton TJ,et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis[J].Nature,2011,475(7354):106-109.
[2]Rupaimoole R,Slack FJ.Micro RNA therapeutics:towards a new era for the management of cancer and other diseases[J].Nat Rev Drug Discov,2017,16(3):203-222.
[3]van Beijnum JR,Giovannetti E,Poel D,et al.mi RNAs:micro-managers of anticancer combination therapies[J].Angiogenesis,2017.
[4]谢学成,邱海,覃宇周.micro RNAs与结直肠癌[J].中国癌症防治杂志,2014,6(3):310-314.
[5]Jeddi F,Soozangar N,Sadeghi MR,et al.Contradictory roles of Nrf2/Keap1 signaling pathway in cancer prevention/promotion and chemoresistance[J].DNA Repair(Amst),2017,54:13-21.
[6]Penning TM.Aldo-Keto reductase regulation by the Nrf2 system:implications for stress response,chemotherapy drug resistance,and carcinogenesis[J].Chem Res Toxicol,2017,30(1):162-176.
[7]Ryoo IG,Lee SH,Kwak MK.Redox modulating NRF2:a potential mediator of cancer stem cell resistance[J].Oxid Med Cell Longev,2016:2428153.
[8]Samatiwat P,Prawan A,Senggunprai L,et al.Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents[J].Tumour Biol,2016,37(8):11495-11507.
[9]Flamini Valentina,Jiang Wen G,Cui Yuxin.Therapeutic Role of Mi R-140-5p for the treatment of non-small cell Lung cancer[J].Anticancer Res,2017,37(8):4319-4327.
[10]Zhao Ke,Chen Bao-Jun,Chen Zhi-Guo.Erb B4 as a potential molecular target in the treatment of esophageal squamous cell cancers[J].Scientific World Journal,2014,124105.
[11]胡万乐,何占红,蒋芳,等.5-Fu诱导人大肠癌SW480细胞株耐药与自噬活性的变化[J].中国中西医结合外科杂志,2013,19(3):279-282.
[12]Mosakhani N,Lahti L,Borze I,et al.Micro RNA profiling predicts survival in anti-EGFR treated chemorefractory metastatic colorectal cancer patients with wild-type KRAS and BRAF[J].Cancer Genet,2012,205(11):545-551.
[13]Zhai H,Fesler A,Ba Y,et al.Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-mi R-140-5p mediated suppression of Smad2 and autophagy[J].Oncotarge,2015,6(23):19735-19746.
[14]Zhang W,Zou C,Pan L,et al.Micro RNA-140-5p inhibits the progression of colorectal cancer by targeting VEGFA[J].Cell Physiol Biochem,2015,37(3):1123-1133.
[15]De Nicola GM,Karreth FA,Humpton TJ,et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis[J].Nature,2011,475(7354):106-109.