摘要目的 探讨亚砷酸钠(NaAsO2)所致恶性转化人肝细胞L-02分泌的外泌体微小RNA(miR)-191对正常人肝细胞L-02增殖的影响.方法 分别使用2μmol/L NaAsO2恶性转化L-02细胞的培养液、提取的外泌体,处理正常的野生型L-02细胞(受体L-02细胞);利用转染试剂LipofectamineTM 2000将anti-miR-191和对照anti-miR-NC分别转染至恶性转化L-02(T-b02)细胞,同时设置未处理组.采用实时荧光定量PCR检测miR-191表达;CCK-8法检测细胞增殖.结果 与同代对照L-02细胞培养液(CM)处理组相比,NaAsO2恶性转化细胞培养液(T-CM)处理组显著促进受体L-02细胞增殖[(207±24)％比(105±21)％,t=5.462,P＜ 0.01]和miR-191表达[(206±25)％比(105±20)％,t=4.116,P＜0.05].来源于CM的不同浓度外泌体未引起受体L-02细胞增殖和miR-191表达明显变化(F=2.213、2.213,P均＞0.05);来源于T-CM的不同浓度外泌体引起受体L-02细胞增殖和miR-191表达明显增高,且存在一定剂量-效应关系伊=10.910、4]53,P＜ 0.01或＜0.05).20、50 mg/L T-CM来源的外泌体处理的受体L-02细胞相对增殖率高于同浓度CM来源的外泌体处理组[(160±32)％比(102±8)％,(203±7)％比(111±5)％,t=2.999、18.750,P＜0.05或＜0.01],10、20、50 mg/L T-CM来源的外泌体处理的受体L-02细胞miR-191表达高于同浓度CM来源的外泌体处理组[(166±13)％比(113±9)％,(211±55)％比(102±8)％,(206±31)％比(105±6)％,t=5.611、3.357、5.509,P＜0.05或＜0.01].anti-miR-191处理组T-L-02细胞及其分泌的外泌体miR-191表达[(39±10)％、(30±19)％]显著低于未处理组和anti-miR-NC处理组[(100±0)％、(106±17)％,(100±0)％、(104±17)％,P均＜0.01].未处理组外泌体促进受体L02细胞相对增殖率升高[(395±31)％比(100±0)％,t=16.290,P＜ 0.01],且miR-191表达显著增高[(208±47)％比(100±0)％,t=4.015,P＜0.05];而anti-miR-191处理组外泌体处理受体L-02细胞的相对增殖率和miR-191表达[(157±19)％、(103±44)％]显著低于未处理组和anti-miR-NC处理组[(395±31)％、(411±55)％,(208±47)％、(197±37)％,P＜ 0.01或＜0.05].结论 NaAsO2恶性转化L-02细胞分泌的外泌体miR-191促进正常人肝L-02细胞增殖.

abstractsObjective To investigate the role of exosomal microRNA(miR)-191 derived from NaAsO2-transformed cells in proliferation of human liver L-02 cells.Methods The normal wild-type L-02 cells (recipient L-02 cells)were treated with media or exosome derived from 2 μmol/L NaAsO2-transformed L-02 cells.Anti-miR-191 and anti-miR-NC were transfected into NaAsO2-transformed L-02 (T-L-02) cells by lipofectamineTM 2000,respectively,while untreated group was set as control.The expression of miR-191 was detected by qRT-PCR.Cell proliferation was evaluated by CCK-8 assay.Results The proliferation [(207 ± 24)％ vs (105 ± 21)％,t =5.462,P ＜ 0.01] and the expression of miR-191 [(206 ± 25)％ vs (105 ± 20)％,t =4.116,P ＜ 0.05] of recipient L-02 cells were significantly increased in the transformed L-02 cells media (T-CM) treated group compared with in the normal L-02 cells media (CM) group.Several concentrations of exosomes derived from CM did not change the proliferation and miR-191 expression of recipient L-02 cells (F =2.213,2.213,all P ＞ 0.05).Several concentrations of exosomes derived from T-CM increased the proliferation and miR-191 expression of recipient L-02 cells in a dose-response manner (F =10.910,4.553,P ＜ 0.01 or ＜ 0.05).The proliferation [(160 ± 32)％ vs (102 ± 8)％,(203 ± 7)％ vs (111 ± 5)％,t =2.999,18.750,P ＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with 20 or 50 mg/L exosomes derived from T-CM was higher than that treated with the same concentration of exosomes derived from CM.The expression of miR-191 [(166 ± 13)％ vs (113 ±9)％,(211 ± 55)％ vs (102 ± 8)％,(206 ± 31)％ vs (105 ± 6)％,t =5.611,3.357,5.509,P ＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with 10,20 or 50 mg/L exosomes derived from T-CM was higher than that treated with the same concentration of exosomes derived from CM.The miR-191 levels of T-L-02 cells [(39 ± 10)％ vs (100 ± 0)％ or (106 ±17)％,all P ＜ 0.01] or exosomes [(30 ± 19)％ vs (100 ± 0)％ or (104 ± 17)％,all P ＜ 0.01] in the anti-miR-191 treated group were significantly lower than that in the untreated group or anti-miR-NC treated group.The exosomes derived from untreated group promoted the proliferation [(395 ± 31)％ vs (100 ± 0)％,t =16.290,P ＜ 0.01] and miR-191 expression [(208 ± 47)％ vs (100 ± 0)％,t =4.015,P ＜ 0.05] of recipient L-02 cells.The proliferation [(157 ± 19)％ vs (395 ± 31)％ or (411 ± 55)％,P ＜ 0.05] and miR-191 expression of [(103 ± 44)％ vs (208 ± 47)％ or (197 ± 37)％,P＜ 0.05 or ＜ 0.01] of recipient L-02 cells treated with exosomes derived from anti-miR-191 treated group were lower than those treated with exosomes derived from untreated group or anti-miR-NC treated group.Conclusion The exosomal miR-191 secreted by NaAsO2-transformed L-02 cells promotes proliferation of normal human L-02 cells.