RNA干扰LOXL2基因对头颈部鳞状细胞癌凋亡及PD-L1表达的影响
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摘要
目的:探讨抑制赖氨酰氧化酶样蛋白2(LOXL2)基因表达对头颈部鳞状细胞癌(HNSCC)增殖凋亡及PD-L1表达的影响研究。方法:通过Western blot检测人HNSCC细胞YCU-H891、YCU-N861和KB中LOXL2的蛋白表达。以Lipo-fectamineTM2000为载体,参照其说明将合成的LOXL2 siRNA转染KB细胞,转染48 h后,Western blot检测LOXL2、PD-L1、STAT3、p-STAT3、PCNA和Bax的蛋白表达; CCK8及流式细胞仪分别检测细胞活力及凋亡率。结果:LOXL2在YCU-H891、YCU-N861和KB中表达均升高。转染LOXL2 siRNA的KB细胞LOXL2的蛋白表达明显降低,与空白组和NC组比较差异均具有统计学意义(P<0. 05)。转染LOXL2 siRNA的KB细胞活力明显降低,凋亡率明显升高,PD-L1、p-STAT3和PCNA的蛋白表达均明显降低,Bax的蛋白表达明显升高,与NC组比较,差异均具有统计学意义(P<0. 05)。结论:RNA干扰抑制LOXL2基因可降低HNSCC细胞活力和诱导凋亡,下调PD-L1表达,机制可能与抑制STAT3信号通路有关。
Objective: To investigate the effect of LOXL2 gene expression was inhibited on proliferation,apoptosis and PD-L1 expression in the head and neck squamous cell carcinoma( HNSCC). Methods: LOXL2 protein expression in human HNSCC cells YCU-H891,YCU-N861 and KB were detected by Western blot. LipofectamineTM2000 was used as the carrier,the LOXL2 siRNA was transfected into KB cells according to its instructions. Cells were transfected for 48 h,the expression of LOXL2,PD-L1,STAT3,p-STAT3,PCNA protein were detected by Western blot,and the cell viability and apoptosis rate were detected by CCK8 and flow cytometry. Results: LOXL2 expression was increased in YCU-H891,YCU-N861 and KB cells. The protein expression of LOXL2 in KB cells transfected with LOXL2 siRNA decreased significantly compared with blank group and NC group( P<0. 05). The activity of KB cells transfected with LOXL2 siRNA decreased obviously,the apoptosis rate increased obviously,the protein expression of PD-L1,p-STAT3 and PCNA decreased obviously,and the protein expression of Bax increased obviously,the difference was statistically significant compared with NC group( P<0. 05). Conclusion: Inhibition of LOXL2 gene expression by RNA interference can reduce HNSCC cell viability and induce apoptosis,and down regulate PD-L1 expression. The mechanism may be related to inhibition of STAT3 signaling pathway.
Objective: To investigate the effect of LOXL2 gene expression was inhibited on proliferation,apoptosis and PD-L1 expression in the head and neck squamous cell carcinoma( HNSCC). Methods: LOXL2 protein expression in human HNSCC cells YCU-H891,YCU-N861 and KB were detected by Western blot. LipofectamineTM2000 was used as the carrier,the LOXL2 siRNA was transfected into KB cells according to its instructions. Cells were transfected for 48 h,the expression of LOXL2,PD-L1,STAT3,p-STAT3,PCNA protein were detected by Western blot,and the cell viability and apoptosis rate were detected by CCK8 and flow cytometry. Results: LOXL2 expression was increased in YCU-H891,YCU-N861 and KB cells. The protein expression of LOXL2 in KB cells transfected with LOXL2 siRNA decreased significantly compared with blank group and NC group( P<0. 05). The activity of KB cells transfected with LOXL2 siRNA decreased obviously,the apoptosis rate increased obviously,the protein expression of PD-L1,p-STAT3 and PCNA decreased obviously,and the protein expression of Bax increased obviously,the difference was statistically significant compared with NC group( P<0. 05). Conclusion: Inhibition of LOXL2 gene expression by RNA interference can reduce HNSCC cell viability and induce apoptosis,and down regulate PD-L1 expression. The mechanism may be related to inhibition of STAT3 signaling pathway.
引文
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[2]Wang Z,Chen J,Zhang W,et al.Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma[J].Oncotarget,2016,7(5):6048-6062.
[3]Cheng Y,Wang Y,Li J,et al.A novel read-through transcript JM-JD7-PLA2G4B regulates head and neck squamous cell carcinoma cell proliferation and survival[J].Oncotarget,2016,8(2):1972-1982.
[4]Hiroaki K,Masakazu Y,Haruhito K,et al.Lysyl oxidase-like 2(LOXL2)from stromal fibroblasts stimulates the progression of gastric cancer[J].Cancer Lett,2014,354(2):438-446.
[5]Fukumoto I,Kikkawa N,Matsushita R,et al.Tumor-suppressive microRNAs(miR-26a/b,miR-29a/b/c and miR-218)concertedly suppressed metastasis-promoting LOXL2 in head and neck squamous cell carcinoma[J].J Hum Genet,2015,61(2):109-118.
[6]马宝镇,高全立.抗PD-1及PD-L1在肿瘤治疗中的进展[J].中国免疫学杂志,2017,33(5):796-800.Ma BZ,Gao QL.The progress of anti PD-1 and PD-L1 in the treatment of tumor[J].Chin J Immunol,2017,33(5):796-800.
[7]Nguyen LTT,Song YW,Cho SK.Baicalein inhibits epithelial to mesenchymal transition via downregulation of Cyr61 and LOXL-2in MDA-MB231 breast cancer cells[J].Mol Cells,2016,39(12):909-914.
[8]Ahn SG,Dong SM,Oshima A,et al.LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients[J].Breast Cancer Res Treat,2013,141(1):89-99.
[9]Rost T,Pyritz V,Rathcke IO,et al.Reduction of LOX-and LOXL2-mRNA expression in head and neck squamous cell carcinomas[J].Anticancer Res,2003,23(2B):1565-1573.
[10]Martin A,Salvador F,Moreno-Bueno G,et al.Lysyl oxidase-like 2represses Notch1 expression in the skin to promote squamous cell carcinoma progression[J].EMBO J,2015,34(8):1090-1109.
[11]叶丽平,马静方,仇会会.RNA干扰c AMP反应元件结合蛋白对碱性成纤维细胞生长因子诱导的人卵巢癌细胞Bcl-2表达的影响[J].中国老年学杂志,2017,37(2):280-282.Ye LP,Ma JF,Qiu HH.Effect of c AMP response element binding protein by RNA interference on on the expression of Bcl-2 in human ovarian cancer cell line induced by basic fibroblast growth factor[J].Chin J Gerontol,2017,37(2):280-282.
[12]Guan HP,Sun JZ,Feng XL,et al.Effects of RNA interferencemediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells[J].Mol Med Rep,2016,13(2):1821-1826.
[13]Bergeat D,Fautrel A,Turlin B,et al.Impact of stroma LOXL2overexpression on the prognosis of intrahepatic cholangiocarc-inoma[J].J Surg Res,2016,203(2):441-450.
[14]Chen X,Tan M,Xie Z,et al.Inhibiting ROS-STAT3-dependent autophagy enhanced capsaicin-induced apoptosis in human hepatocellular carcinoma cells[J].Free Radic Res,2016,50(7):1-31.
[15]Jia L,Song Q,Zhou C,et al.Dihydroartemisinin as a putative STAT3 inhibitor,suppresses the growth of head and neck squamous cell carcinoma by targeting Jak2/STAT3 signaling[J].PLo S One,2016,11(1):e0147157.
[16]张红霞,吴广胜.β-arrestin1激活STAT3信号通路影响白血病细胞K562体外迁移侵袭能力[J].中国免疫学杂志,2016,32(12):1773-1776.Zhang HX,Wu GS.β-arrestin1 promotes leukemia cell K562 migration and invasion by activating STAT3 signaling pathway[J].Chin J Immunol,2016,32(12):1773-1776.
[17]Feng J,Yan PF,Zhao HY,et al.SIRT6 suppresses glioma cell growth via induction of apoptosis,inhibition of oxidative stress and suppression of JAK2/STAT3 signaling pathway activation[J].Oncol Rep,2016,35(3):1395-1402.
[18]Zeng J,Dong DD,Li FH.Expression of IL-6 in colorectal cancer and correlation with PCNA expression[J].Zhonghua Bing Li Xue Za Zhi,2017,46(2):112-113.
[19]Ye Z,Hao R,Cai Y,et al.Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer[J].Tumour Biol,2016,37(4):4509-4515.
[20]Ruffner RS,Ramsey A,O'Malley BW,et al.Abstract 5474:PD-L1 regulates cisplatin chemoresistance in head and neck squamous cell carcinoma[J].Cancer Res,2015,75(15 Supplement):5474-5474.
[21]Jiang Z,Pan Z,Ren X.Progress of PD-1/PD-L1 inhibitors in nonsmall cell lung cancer[J].Zhongguo Fei Ai Za Zhi,2017,20(2):138-142.
[22]Zhang X,Zeng Y,Qu Q,et al.PD-L1 induced by IFN-γfrom tumor-associated macrophages via the JAK/STAT3 and PI3K/AKT signaling pathways promoted progression of lung cancer[J].Int J Clin Oncol,2017,22(6):1-8.