弥漫性大B细胞淋巴瘤患者血清和细胞中miR-21的表达及其临床意义

Clinical significance and expression of microRNA-21 in diffuse large B-cell lymphoma cell lines and serum of patients

目的探讨微小RNA-21（miR-21）在弥漫性大B细胞淋巴瘤（DLBCL）细胞系和患者血清中的表达及其在DLBCL早期诊断、基因分型及预后判断中的意义。方法用实时荧光定量聚合酶链反应（FQ—PCR）从细胞水平检测9种DLBCL细胞系（OCI—Lyl、OCI—Ly3、OCI-Ly4、OCl一Ly7、OCI—Ly8、OCI—LylO、OCI—Ly18、OCI—Ly19和HBL）中miR-21的表达，及临床确诊的62例DLBCL患者（DLBCL组）、50名健康体检者（健康对照组）血清miR-21表达水平；同时，随访62例DLBCL患者无复发生存期（RFS），应用Kaplan—Meier生存曲线分析62例DLBCL患者血清miR-21表达与预后的关系。结果健康对照组B淋巴细胞miR-21的相对表达量为1．04±0．02，9种DLBCL细胞系miR-21相对表达量依次为2．30±0．35、237．97±56．19、5．27±0．83、3．40±0．30、11．22±2．70、133．55±16．78、6．63±0．24、4．91±0．37、81．59±6．64，9种DLBCL细胞系的miR．21表达水平均高于健康对照组（t值分别为7．3、13．7、21．0、6．2、8．8、13．6、6．5、39．5、18．1，P均〈0．01）；且ABC亚型细胞系（OCI—Ly3、OCI—Lrr10、HBL）显著高于GCB亚型细胞系（OCI—Ly1、OCI—Ly4、OCI—Ly7、OCl一Ly8、OCI-Ly18、OCI—Ly19；t=11．18，P〈0．01）。DLBCL组血清中miR-21的表达量为21．38（10．26—45．21），健康对照组为1．87（L05～3．97），DLBCL组血清miR一21的表达显著高于健康对照组（U=168，P=0．000）。DLBCL患者中GCB亚型miR-21的表达为18．30（7．32～33．46），ABC亚型miR-21的表达为28．68（14．92～98．44），ABC亚型miR-21的表达高于GCB亚型（U：336，P=0．043）。此外，GCB型I＋Ⅱ期患者血清miR-21表达为24．75（16．08—50．38），而Ⅲ＋Ⅳ期为11．96（4．10～21．05）；ABC型I＋Ⅱ期患者血清miR-21表达为47．49（25．65～295．41），而Ⅲ＋Ⅳ期为16．66（5．35—44．30），Ⅰ＋Ⅱ期明显高于Ⅲ＋Ⅳ期（GCB型U=62，P=0．013；ABC型U=53，P=0．014）。随访DLBCL患者的RFS，miR-21高表达患者预后明显好于miR-21低表达患者（U=259，P=0．035）。结论DLBCL患者血清miR-21高表达，且与DLBCL临床分级分期、基因型和预后判断密切相关，miR-21有可能成为DLBCL早期诊断、基因分型及预后判断的新分子标志。

Objective To study the expression of microRNA-21 （ miR-21 ） in serum of patient with diffuse large B cell lymphoma （DLBCL） and DLBCL cell lines and validate the significance of miR-21 in early diagnosis, genotyping and prognosis estimates of DLBCL. Methods miR-21 expression were detected by fluorescent quantity polymerase chain reaction （FQ-PCR） in 9 lymphoma cell lines （ OCI-Ly1, OCI-Ly3, OCI-Ly4, OCI-Ly7, OCI-Ly8, OCI-Ly10, OCI-Ly18, OCI-Ly19 and HBL）, the serum from DLBCL patients （ n = 62 ） and health controls （ n = 50 ）. Kaplan-Meier survival analysis was carried out during the relapsefree survival period of DLBCL patients to explore the relationship between the prognosis and microRNA expression level. Results Real time FQ-PCR result indicated that miR-21 expression was higher in DLBCL cell lines than that in normal B cells （BC）. miR-21 expression in normal B cell and 9 DLBCL cell lines separately were 1.04 ± 0. 02,2. 30 ± 0. 35,237.97 ± 56. 19,5.27 ± 0. 83,3.40 ± 0. 30, 11.22 ± 2. 70, 133.55 ± 16. 78,6. 63 ±0. 24,4. 91 ±0. 37 and 81.59 ±6. 64. Compared with BC, the expression of miR-21 were higher in all 9 DLBCL cell lines （ t = 7.3,13.7,21.0,6. 2,8.8,13.6,6. 5,39. 5,18. 1 ; P 〈 0. 01 ）. miR-21 expression segregates with specific molecular subgroups of DLBCL. The expression was higher in the ABC type cell lines （ OCI-Ly3, OCI-Ly10, HBL） than GCB type cell lines （ OCI-Ly1, OCI-Ly4, OCI-Ly7, OCI-Ly8, OCI-LylS, OCI-Lyl9; t = 11.18, P 〈 0. 01 ） . Consistent with the cell line models, miR-21 expression levels were higher in serum from DLBCL patients [ 21.38 （10. 26-45.21 ）] than from controls [ 1.87（ 1.05-3.97）, U = 168, P =0. 000] , and the levels were higher in DLBCL cases with an ABC-type [ 28.68 （ 14. 92-98.44 ） ] than those in GCB-type [ 18.30 （ 7.32-33.46 ）, U = 336, P = 0. 043 ]. MiR-21 expression levels were different in sera from different clinical stage DLBCL patients. The miR-21 level in serum of patients with subgroup ABC and subgroup GCB in stage I and U were 47.49（25.65-295.41 ） and 24. 74（ 16. 08-50. 38） respectively and in stage m and IV were 16. 66 （ 5.35-44. 30） and 11.96 （4. 10- 21.05） respectively. The levels were higher in DLBCL cases with I -II stage than those with III-IV stage （U =62, P = 0. 013 in GCB type; U = 53, P = 0. 014 in ABC type）. Moreover, compare with relapse-free survival in DLBCL patients, high miR-21 expression was associated with well prognosis （ U = 259, P = 0. 035 ）. Conclusions MiR-21 is high expression in DLBCL cell lines and DLBCL patients serum, miR-21 level in sera from DLBCL patients is associated with clinical stage, molecular subgroup and prognosis estimates. MiR-21 may serve as a new biomarker to early detection, genotyping and prognosis estimates of DLBCL.