Circ-CDYL通过miR-223-3p/PHF19轴调节多发性骨髓瘤对硼替佐米的敏感性  

Circ-CDYL regulates the sensitivity of multiple myeloma to bortezomib via the miR-223-3p/PHF19 axis

作　　者：周燕[1] 唐云龙 刘佳琦 吴蕾蕾 孙乃同 ZHOU Yan;TANG Yunlong;LIU Jiaqi;WU Leilei;SUN Naitong(Department of Hematology,Yancheng Clinical College of Medicine,Nanjing Medical University/Yancheng Third People′s Hospital,Yancheng,Jiangsu 224000,China;Medical School,Nantong University,Nantong,Jiangsu 226000,China)

机构地区：[1]南京医科大学盐城临床医学院/盐城市第三人民医院血液内科,江苏盐城224000 [2]南通大学医学院,江苏南通226000

出　　处：《国际检验医学杂志》2023年第10期1229-1234,共6页International Journal of Laboratory Medicine

基　　金：2019年盐城市医学科技发展计划项目(YK2019097)。

摘　　要：目的 探讨环状RNA Circ-CDYL调节多发性骨髓瘤(MM)对硼替佐米(BTZ)的敏感性,以及对miR-223-3p/PHD锌指蛋白19(PHF19)轴的影响。方法 收集盐城市第三人民医院118例MM患者血清标本,其中60例患者未接受任何化疗(BTZ敏感组),58例患者接受BTZ治疗并产生化疗耐药性(BTZ耐药组),比较两组Circ-CDYL及miR-223-3p、PHF19的表达情况。将MM1.R细胞根据细胞转染情况分为C组、sh-Circ-C组、sh-Circ-CDYL组、sh-Circ-CDYL+anti-miR-223-3p组和sh-Circ-CDYL+pcDNA-PHF19组,对5组细胞进行相应转染。采用荧光定量PCR(RT-qPCR)测定Circ-CDYL、miR-223-3p和PHF19 mRNA水平,CCK-8法测定MM1.R细胞活性。采用克隆形成实验和流式细胞术检测细胞增殖和凋亡情况,采用免疫印迹法(Western blotting)检测凋亡蛋白[增殖细胞核抗原(PCNA)、Ki67、剪切化半胱氨酸天冬氨酸蛋白激酶3(C-caspase 3)]及PHF19蛋白的表达水平,采用双荧光素酶报告基因实验检测Circ-CDYL与miR-223-3p/PHF19的靶向关系。结果 Circ-CDYL和PHF19在BTZ耐药组患者血清标本和MM1.R细胞中均呈高表达(P<0.05),miR-223-3p呈低表达(P<0.05)。下调Circ-CDYL表达下调Circ-CDYL表达可明显降低MM1.R细胞对BTZ的半数抑制浓度(IC_(50)),抑制增殖活力,使凋亡蛋白PCNA、Ki67表达水平均下调,细胞凋亡率、C-caspase 3蛋白表达水平上调,差异均有统计学意义(P<0.05)。抑制miR-223-3p或过表达PHF19可部分逆转敲低Circ-CDYL后的MM1.R细胞对BTZ敏感性(P<0.05)。双荧光素酶报告基因实验显示,miR-223-3p可抑制Circ-CDYL、PHF19的表达,Circ-CDYL、PHF19是miR-223-3p的靶基因。结论 敲低Circ-CDYL可通过miR-223-3p/PHF19轴而提高MM1.R细胞对BTZ的敏感性。Objective To explore the regulation of the sensitivity of multiple myeloma(MM)to bortezomib(BTZ)and the influence of Circ-CDYL on miR-223-3p/PHD finger protein 19(PHF19)axis.Methods Serum samples were collected from 118 MM patients in Yancheng Third People′s Hospital,among which 60 patients did not receive any chemotherapy treatment(BTZ-sensitive group)and 58 patients received BTZ treatment and developed chemotherapy resistance(BTZ-resistance group).The expressions of Circ-CDYL,miR-223-3p and PHF19 in the two groups were compared.The mRNA levels of Circ-CDYL,miR-223-3p and PHF19 were determined by fluorescence quantitative PCR(RT-qPCR),and the activity of MM1.R cells was determined by CCK-8 method.Clone formation assay and flow cytometry were used to detect cell proliferation and apoptosis,while the expression levels of apoptosis-related proteins(PCNA,Ki67,C-caspase 3)and PHF19 protein were detected by Western blotting.Meanwhile,double luciferase reporting experiments was used to detect the targeting relationship between Circ-CDYL and miR-223-3p/PHF19.Results Circ-CDYL and PHF19 were highly expressed in serum specimens of BTZ resistant group and MM1.R cells(P<0.05),while miR-223-3p was low expressed(P<0.05).Down-regulation of Circ-CDYL expression significantly reduced the median inhibition concentration(IC 50)of MM1.R cells to BTZ,inhibited the proliferation activity,down-regulated the expression levels of apoptotic proteins PCNA and Ki67,and up-regulated the apoptosis rate and C-caspase 3 protein expression levels,with statistical significance(P<0.05).Inhibition of miR-223-3p or overexpression of PHF19 could partially reverse the sensitivity of MM1.R cells to BTZ after Circ-CDYL knockdown(P<0.05).Double luciferase reporting experiments showed that miR-223-3p could inhibit the expression of Circ-CDYL and PHF19,and Circ-CDYL and PHF19 were target genes of miR-223-3p.Conclusion Knocking down Circ-CDYL can increase the sensitivity of MM1.R cells to BTZ through miR-223-3p/PHF19 axis.

关 键 词：Circ-CDYL miR-223-3p PHD锌指蛋白19 多发性骨髓瘤 硼替佐米 

分 类 号：R733.3[医药卫生—肿瘤]