表观遗传学诱导的长链非编码RNA1在肝细胞癌中的表达及功能

Expression and function of long non-coding RNA epigenetically-induced 1 in hepatocellular carcinoma

目的观察表观遗传学诱导的长链非编码RNA1(lncRNA EPIC1)在肝细胞肝癌(HCC)的表达及其对肝癌细胞株增殖、侵袭、凋亡的影响。方法通过实时定量反转录聚合酶链反应(RT-qPCR)技术检测EPIC1在7例HCC组织和其癌旁组织和五株肝癌细胞株(HepG2、SK-HEP-1、HuH-7-80、SMMC-7721、SNU-387)和人正常肝细胞株(HL-7702)的表达量。使用携带有EPIC1目的基因的重组慢病毒颗粒[LV-小干扰RNA(siRNA)]转染Hep G2、HuH-7-80作为实验组,转染阴性对照慢病毒颗粒(LV-NC)的Hep G2、HuH-7-80作为对照组。转染后细胞计数试剂盒(CCK-8)增殖实验检测Hep G2、HuH-7-80的增殖能力;Transwell侵袭实验检测HepG2、HuH-7-80的侵袭能力;流式细胞术检测Hep G2、HuH-7-80的凋亡率。结果EPIC1在HCC组织的表达量(8.77±0.36)显著高于其癌旁组织(2.39±0.45),差异有统计学意义(P<0.01)。EPIC1在5株肝癌细胞株(HepG2、SK-HEP-1、HuH-7-80、SMMC-7721、SNU-387)的表达量(7.24±0.81、3.11±0.35、5.17±1.12、4.38±0.48、2.69±0.65)均较人正常肝细胞株HL-7702的表达量(1.29±0.13)明显增高,其中,在HepG2、HuH-7-80中表达量最高,约为HL-7702的(5.61±0.12)、(4.01±0.16)倍,差异有统计学意义(P<0.01)。低表达EPIC1组的HepG2在24、48、72 h细胞增殖能力(LV-siRNA1:0.43±0.02、0.72±0.02、1.31±0.01,P<0.05;LV-siRNA2:0.42±0.02、0.76±0.04、1.03±0.02,P<0.05)比对照组的Hep G2(0.59±0.02、0.98±0.02、1.63±0.02)显著下降;低表达EPIC1组的HuH-7-80在24、48、72 h细胞增殖能力(LV-siRNA1:0.59±0.01、0.73±0.02、1.31±0.05,P<0.05;LV-siRNA2:0.43±0.04、0.74±0.04、1.26±0.04,P<0.05)比对照组的HuH-7-80(0.64±0.02、0.82±0.02、1.78±0.02)显著下降。低表达EPIC1组的HepG2、HuH-7-80穿过基底膜的细胞数量[(43.35±6.16)、(55.40±8.18)个]比对照组相对细胞数量[(80.67±5.14)、(74.73±6.13)个]明显减少,差异有统计学意义(P<0.05)。低表达EPIC1组HepG2、HuH-7-80细胞凋亡率[(23.20±0.37)%、(18.60±0.46)%]较对照组细胞凋亡率[(2.60±0.96)%、(3.60±0.33)%]明显增高,差异有统计学意义(P<0.01)。结论lncRNA EPIC1在HCC组织和细胞中高表达,低表达EPIC1会降低肝癌细胞的增殖和侵袭能力,促进肝癌细胞凋亡。

Objective To observe the expression of long non-coding RNA (lncRNA EPIC1) in hepatocellular carcinoma (HCC) and its effects on the proliferation, invasion and apoptosis of hepatoma cell lines. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was adopted to detect the expression levels of EPIC1 in 7 cases of HCC tissues and their paracancerous tissues, 5 liver cancer cell lines (HepG2, SK-HEP-1, HuH-7-80, SMMC-7721, SNU-387) and normal human liver cell line (HL-7702). Hep G2 and HuH-7-80 cells were transfected with recombinant lentiviral particles carrying the EPIC1 genes [LV-small interfering RNA (siRNA)] as the experimental group, and Hep G2 and HuH-7-80 cells transfected with negative control lentiviral particles (LV-NC) were used as the control group. The proliferative capacity of HepG2 and HuH-7-80 cells was measured by cell counting kit-8 (CCK-8) proliferation assay after transfection. The invasion capacity of HepG2 and HuH-7-80 cells was examined by Transwell invasion assay. The apoptosis rate of HepG2 and HuH-7-80 cells was detected by flow cytometry. Results The expression level of EPIC1 in HCC tissues (8.77±0.36) was significantly higher than that in their adjacent tissues (2.39±0.45), and the difference was statistically significant (P<0.01). The expression levels of EPIC1 in HepG2, SK-HEP-1, HuH-7-80, SMMC-7721 and SNU-387 cells (7.24±0.81, 3.11±0.35, 5.17±1.12, 4.38±0.48 and 2.69±0.65) was significantly [about (5.61±0.12) and (4.01±0.16) times] higher than that of HL-7702 cells (1.29±0.13), among which HepG2 and HuH-7-80 cells had the highest expression level, and the difference was statistically significant (P<0.01). The proliferative capacity of HepG2 cells in the EPIC1 group with low expression at 24, 48, 72 h (LV-siRNA1: 0.43±0.02, 0.72±0.02, 1.31±0.01, P<0.05;LV-siRNA2: 0.42±0.02, 0.76±0.04, 1.03±0.02, P<0.01) was significantly lower than HepG2 cells (0.59±0.02, 0.98±0.02, 1.63±0.02) in the control group. The proliferative capacity of HuH-7-80 cells with low expression in the EPIC1 group at 24, 48, 72 h (LV-siRNA1: 0.59±0.01, 0.73±0.02 and 1.31±0.05, P<0.05;LV-siRNA2: 0.43±0.04, 0.74±0.04 and 1.26±0.04, P<0.01) was significantly lower than that of the HuH-7-80 cells (0.64±0.02, 0.82±0.02 and 1.78±0.02) in the control group. The number of cells across the basement membrane in the EPIC1 group with low expression in HepG2 and HuH-7-80 cells (43.35±6.16, 55.40±8.18) was significantly less than that in the control group (80.67±5.14 and 74.73±6.13)(P<0.05). The apoptosis rate of HepG2 and HuH-7-80 cells with low expression in EPIC1 group was significantly higher than that in the control group [(2.60±0.96)% and (3.60±0.33)%], and the difference was statistically significant (P<0.01). Conclusion lncRNA EPIC1 is highly expressed in HCC tissues and cells. EPIC1 with low expression can reduce the capacity of proliferation and invasion of hepatoma cells and promote the apoptosis of hepatoma cells.