miR-140靶向抑制PD-L1表达增强宫颈癌HeLa和Caski细胞对奥沙利铂的敏感性
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摘要
目的:探究miR-140能否通过靶向抑制程序性细胞死亡蛋白-1(programmed death-1, PD-L1)表达增强宫颈癌(cervical cancer, CC)细胞对奥沙利铂的敏感性。方法:采用qPCR检测miR-140在人正常宫颈细胞、CC细胞株及其奥沙利铂耐药细胞株中的表达情况;采用miR-140模拟物转染细胞,CCK-8法检测CC正常细胞株及其奥沙利铂耐药细胞株的增殖情况、克隆形成实验检测细胞的克隆形成率。通过Starbase及TargetScan在线分析软件预测miR-140与PD-L1的靶向结合位点,并通过双荧光素酶报告基因验证miR-140与PD-L1的靶向结合关系。采用Annexin V FITC/PI双染色法和Wb法分别检测过表达miR-140或同时过表达PD-L1,且在使用奥沙利铂处理后CC细胞的凋亡、迁移及凋亡相关蛋白的表达情况。建立裸鼠CC移植瘤模型,检测miR-140加强肿瘤对奥沙利铂处理敏感性的效果。结果:miR-140在奥沙利铂耐药性CC细胞中表达显著下调(P<0.01),过表达miR-140能够明显上调奥沙利铂耐药CC细胞对奥沙利铂的敏感性(P<0.05),抑制CC细胞的增殖及克隆形成(均P<0.01)。miR-140与PD-L1 3'-UTR靶向结合并抑制其表达。过表达miR-140显著促进CC细胞的迁移及凋亡(P<0.01),过表达miR-140的同时过表达PD-L1明显减弱了miR-140对CC细胞迁移的抑制及细胞凋亡的促进作用(均P<0.05)。小鼠异体移植瘤模型验证了miR-140促进肿瘤对奥沙利铂的敏感性。结论:miR-140通过靶向抑制PD-L1表达增加CC细胞对奥沙利铂的敏感性,上调miR-140或抑制PD-L1的表达再与奥沙利铂联合处理有可能作为奥沙利铂耐药CC治疗的新策略。
Objective: To investigate whether miR-140 could increase the sensitivity of cervical cancer(CC) to oxaliplatin by downregulating the expression of programmed death-1(PD-L1). Methods: q PCR was used to analyze miR-140 expression in normal human cervical cells, CC cells and oxaliplatin-resistant CC cells. Cells were transfected with miR-140 mimic, and then, the proliferation of CC cells and oxaliplatin-resistant CC cells was detected by using CCK-8 assay, and the colony formation rate of CC cells was obtained by using colony formation assay. Starbase and TargetScan were used to predict the targeted binding site of miR-140 and PD-L1, and the influence of miR-140 on the expression of PD-L1 was validated by dual luciferase reporter gene assay. Annexin V FITC/PI double staining and Wb assays were used to detect the effect of over-expression of miR-140 or both over-expression of PD-L1 and miR140 on the apoptosis, migration and expression of apoptosis-related proteins in CC cells after treatment with oxaliplatin. Moreover, transplantation tumor of CC cell lines was established in nude mice to assess the effects of miR-140 on enhancing the sensitivity of tumors to oxaliplatin. Results: The expression of miR-140 was significantly decreased in oxaliplatin-resistant CC cells(P<0.01). Over-expression of miR-140 could significantly increase the sensitivity of oxaliplatin-resistant CC cells to oxaliplatin(P<0.05), and inhibit the CC cells proliferation and colony formation(P<0.01). miR-140 showed targeted binding to PD-L1 3'-UTR and inhibited its expression. Over-expression of miR-140 significantly promoted CC cell migration and apoptosis(P<0.01). However, co-transfection of PD-L1 counteracts the effects of miR-140 on cell metastasis and apoptosis(all P<0.05). In addition, xenograft tumor model in mice also verified that miR-140 could promote the sensitivity of tumors to oxaliplatin. Conclusion: miR-140 increases the sensitivity of CC to oxaliplatin through inhibition of PD-L1 expression. Therefore, up-regulation of miR-140 or down-regulation of PD-L1 in combination with oxaliplatin may be a novel strategy for the treatment of Oxaliplatin-resistant CC.
Objective: To investigate whether miR-140 could increase the sensitivity of cervical cancer(CC) to oxaliplatin by downregulating the expression of programmed death-1(PD-L1). Methods: q PCR was used to analyze miR-140 expression in normal human cervical cells, CC cells and oxaliplatin-resistant CC cells. Cells were transfected with miR-140 mimic, and then, the proliferation of CC cells and oxaliplatin-resistant CC cells was detected by using CCK-8 assay, and the colony formation rate of CC cells was obtained by using colony formation assay. Starbase and TargetScan were used to predict the targeted binding site of miR-140 and PD-L1, and the influence of miR-140 on the expression of PD-L1 was validated by dual luciferase reporter gene assay. Annexin V FITC/PI double staining and Wb assays were used to detect the effect of over-expression of miR-140 or both over-expression of PD-L1 and miR140 on the apoptosis, migration and expression of apoptosis-related proteins in CC cells after treatment with oxaliplatin. Moreover, transplantation tumor of CC cell lines was established in nude mice to assess the effects of miR-140 on enhancing the sensitivity of tumors to oxaliplatin. Results: The expression of miR-140 was significantly decreased in oxaliplatin-resistant CC cells(P<0.01). Over-expression of miR-140 could significantly increase the sensitivity of oxaliplatin-resistant CC cells to oxaliplatin(P<0.05), and inhibit the CC cells proliferation and colony formation(P<0.01). miR-140 showed targeted binding to PD-L1 3'-UTR and inhibited its expression. Over-expression of miR-140 significantly promoted CC cell migration and apoptosis(P<0.01). However, co-transfection of PD-L1 counteracts the effects of miR-140 on cell metastasis and apoptosis(all P<0.05). In addition, xenograft tumor model in mice also verified that miR-140 could promote the sensitivity of tumors to oxaliplatin. Conclusion: miR-140 increases the sensitivity of CC to oxaliplatin through inhibition of PD-L1 expression. Therefore, up-regulation of miR-140 or down-regulation of PD-L1 in combination with oxaliplatin may be a novel strategy for the treatment of Oxaliplatin-resistant CC.
引文
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[20]FANG Z,YIN S,SUN R,et al.miR-140-5p suppresses the proliferation,migration and invasion of gastric cancer by regulating YES1[J].Mol Cancer,2017,16(1):139-150.DOI:10.1186/s12943-017-0708-6.
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[22]LI Q,YAO Y,EADES G,et al.Downregulation of mi R-140 promotes cancer stem cell formation in basal-like early stage breast cancer[J].Oncogene,2014,33(20):2589-2600.DOI:10.1038/onc.2013.226.
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[2]MUKAMA T,NDEJJO R,MUSABYIMANA A,et al.Women's knowledge and attitudes towards cervical cancer prevention:a cross sectional study in Eastern Uganda[J].BMC Womens Health,2017,17(1):9-17.DOI:10.1186/s12905-017-0365-3.
[3]ZHAO H,SU W,KANG Q,et al.Natural killer cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p[J/OL].Am J Cancer Res,2018,8(5):824-834.eCollection 2018[2018-08-10].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992516/.
[4]KASHIWAGI E,IZUMI H,YASUNIWA Y,et al.Enhanced expression of nuclear factor I/B in oxaliplatin-resistant human cancer cell lines[J].Cancer Sci,2011,102(2):382-386.DOI:10.1111/j.1349-7006.2010.01784.x.
[5]LEE J,YOON Y,HAN Y,et al.Melatonin promotes apoptosis of oxaliplatin-resistant colorectal cancer cells through inhibition of cellular prion protein[J].Anticancer Res,2018,38(4):1993-2000.DOI:10.21873/anticanres.12437.
[6]LEE J,AHN J,CHOI M,et al.Gemcitabine-oxaliplatin plus prednisolone is active in patients with castration-resistant prostate cancer for whom docetaxel-based chemotherapy failed[J].Br J Cancer,2014,110(10):2472-2478.DOI:10.1038/bjc.2014.204.
[7]WEN Q,CHEN Z,CHEN Z,et al.EphA2 affects the sensitivity of oxaliplatin by inducing EMT in oxaliplatin-resistant gastric cancer cells[J].Oncotarget,2017,8(29):47998-48011.DOI:10.18632/oncotarget.18208.
[8]MONTOPOLI M,RAGAZZI E,FROLDI G,et al.Cell-cycle inhibition and apoptosis induced by curcumin and cisplatin or oxaliplatin in human ovarian carcinoma cells[J].Cell Prolif,2009,42(2):195-206.DOI:10.1111/j.1365-2184.2009.00585.x.
[9]JENG L,KUMAR VELMURUGAN B,CHEN M,et al.Fisetin mediated apoptotic cell death in parental and Oxaliplatin/irinotecan resistant colorectal cancer cells in vitro and in vivo[J].J.Cell Physiol,2018,233(9):7134-7142.DOI:10.1002/jcp.26532.
[10]PUJADE-LAURAINE E,FUJIWARA K,DYCHTER S,et al.Avelumab(anti-PD-L1)in platinum-resistant/refractory ovarian cancer:JAVELIN Ovarian 200 Phase III study design[J].Future Oncol,2018,14(21):2103-2113.DOI:10.2217/fon-2018-0070.
[11]WANGPAICHITR M,KANDEMIR H,LI Y,et al.Relationship of metabolic alterations and PD-L1 expression in cisplatin resistant lung cancer[J].Cell Dev Biol,2017,6(2):1-12.DOI:10.4172/2168-9296.1000183.
[12]FANKHAUSER C,SCHüFFLER P,GILLESSEN S,et al.Comprehensive immunohistochemical analysis of PD-L1 shows scarce expression in castration-resistant prostate cancer[J].Oncotarget,2018,9(12):10284-10293.DOI:10.18632/oncotarget.22888.
[13]JIN Y,CHEN Q,XU K,et al.Involvement of microRNA-141-3p in5-fluorouracil and oxaliplatin chemo-resistance in esophageal cancer cells via regulation of PTEN[J].Mol Cell Biochem,2016,422(1/2):161-170.DOI:10.1007/s11010-016-2816-9.
[14]TAN S,SHI H,BA M,et al.mi R-409-3p sensitizes colon cancer cells to oxaliplatin by inhibiting Beclin-1-mediated autophagy[J].Int J Mol Med,2016,37(4):1030-1038.DOI:10.3892/ijmm.2016.2492.
[15]TANAKA S,HOSOKAWA M,UEDA K,et al.Effects of decitabine on invasion and exosomal expression of miR-200c and miR-141 in oxaliplatin-resistant colorectal cancer cells[J].Biol Pharm Bull,2015,38(9):1272-1279.DOI:10.1248/bpb.b15-00129.
[16]MENG Y,GAO R,MA J,et al.Micro RNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy[J].Sci Rep,2017,7(1):416-429.DOI:10.1038/s41598-017-00405-3.
[17]SONG B,WANG Y,XI Y,et al.Mechanism of chemoresistance mediated by mi R-140 in human osteosarcoma and colon cancer cells[J].Oncogene,2009,28(46):4065-4074.DOI:10.1038/onc.2009.274.
[18]NANDY S,SUBRAMANI R,RAJAMANICKAM V,et al.microR-NA alterations in ALDH positive mammary epithelial cells:a crucial contributing factor towards breast cancer risk reduction in case of early pregnancy[J/OL].BMC Cancer,2014,14:644-657[2018-08-21].https://wwww.ncbi.nlm.nhi.gov/pmc/articles/PMC4167510.DOI:10.1186/1471-2407-14-644.
[19]FLAMINI V,JIANG W,and CUI Y.Therapeutic role of miR-140-5p for the treatment of non-small cell lung cancer[J].Anticancer Res,2017,37(8):4319-4327.DOI:10.21873/anticanres.11825.
[20]FANG Z,YIN S,SUN R,et al.miR-140-5p suppresses the proliferation,migration and invasion of gastric cancer by regulating YES1[J].Mol Cancer,2017,16(1):139-150.DOI:10.1186/s12943-017-0708-6.
[21]ZHAI H,FESLER A,BA Y,et al.Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy[J].Oncotarget,2015,6(23):19735-19746.DOI:10.18632/oncotarget.3771.
[22]LI Q,YAO Y,EADES G,et al.Downregulation of mi R-140 promotes cancer stem cell formation in basal-like early stage breast cancer[J].Oncogene,2014,33(20):2589-2600.DOI:10.1038/onc.2013.226.
[23]WU D,ZHANG J,LU Y,et al.miR-140-5p inhibits the proliferation and enhances the efficacy of doxorubicin to breast cancer stem cells by targeting Wnt1[J/OL].Cancer Gene Ther,2018[Epub ahead of print][2018-10-11].https://www.ncbi.nlm.nih.gov/pubmed/?term=30032164.DOI:10.1038/s41417-018-0035-0.