长链非编码RNA-GK3P在食管癌中的表达及其对癌细胞生物学功能的影响
Expression of long non-coding RNA-GK3P in esophageal cancer and its effect on biological function of cancer cells
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摘要:
背景 甘油激酶3假基因（
GK3P ）是位于人类染色体4q32.3区域的长链非编码RNA（lncRNA），其亲本基因甘油激酶基因与肿瘤发展有关。然而，lncRNA-GK3P 在食管癌中的表达与作用机制尚不清楚。目的 分析lncRNA-
GK3P 在食管癌组织、癌细胞中的表达情况，并探讨其对食管癌EC109细胞增殖、周期、凋亡的影响及机制。方法 应用实时定量逆转录聚合酶链反应（qRT-PCR）技术检测lncRNA-
GK3P 在食管癌细胞（EC109）与永生化正常食管上皮细胞（Het-1A）以及41例食管癌组织与癌旁组织中的表达情况；采用siRNA敲低技术、质粒转染技术分别构建lncRNA-GK3P 敲低和过表达的EC109细胞株，并将其分为4组：si-GK3P组（siRNAGK3P 转染）、si-NC组（siRNAGK3P 的空载体对照）、plasmid-GK3P组（GK3P 过表达质粒转染）、plasmid-NC组（GK3P 空载质粒对照）；CCK-8法检测细胞活性、EdU法检测细胞的增殖；流式细胞术检测细胞凋亡、细胞周期，Western blotting测定细胞周期和凋亡相关蛋白Cyclin E1、Cyclin B1、Bcl2和Bax的表达变化。结果 与癌旁组织相比，lncRNA-
GK3P 在食管癌组织中表达水平为前者的（2.91±0.61）倍（t =19.38，P < 0.05）；且相对于食管上皮细胞Het-1A，lncRNA-GK3P 在食管癌EC109细胞中表达水平为前者的（3.15±0.27）倍（t =13.06，P < 0.05）。lncRNA-GK3P 敲低后，细胞增殖受到抑制si-GK3P组vs si-NC组，（27.21±1.11）% vs（37.95±0.93）%，P < 0.05；细胞G0/G1期比例增加（P < 0.05），S期比例增加（P < 0.05），G2/M期比例减少（P < 0.05）；细胞凋亡增加si-GK3P组vs si-NC组，（10.19±0.91）% vs（7.46±0.18）%，P < 0.05；Cyclin E1、Cyclin B1、Bcl2表达下调，Bax表达上调。lncRNA-GK3P 过表达后，细胞增殖增加plasmid-GK3P组vs plasmid-NC组，（37.00±1.71）% vs（29.63±1.45）%，P < 0.05；细胞G0/G1期比例减少（P < 0.05），S期比例增加（P < 0.05），G2/M期比例减少（P < 0.05）；细胞凋亡下降plasmid-GK3P组vs plasmid-NC组，（5.27±0.07）% vs（9.71±0.19）%，P < 0.05；Cyclin E1、Cyclin B1、Bcl2表达上调，Bax表达下调。结论 lncRNA-
GK3P 在食管癌中高表达，通过调控Cyclin E1、Cyclin B1、Bcl2及Bax的表达，在食管癌EC109细胞中发挥促进细胞增殖、抑制凋亡的作用。Abstract:Background Glycerol kinase 3 pseudogene (
GK3P ) is a long non-coding RNA (lncRNA) located in the 4q32.3 region of human chromosome. Its parent gene glycerol kinase is related to tumor development. However, the expression and mechanism of lncRNA-GK3P in esophageal cancer are still unclear.Objective This experiment aims to investigate the expression of lncRNA-
GK3P in esophageal cancer tissues and cancer cells, and its effects on the proliferation, cycle, and apoptosis of esophageal cancer EC109 cells.Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology was used to detect expression of lncRNA-
GK3P in esophageal cancer cells (EC109), immortalized esophageal epithelial cells (Het-1A), and 41 esophageal cancer tissues and paired adjacent tissues. siRNA technology and plasmid transfection were used to construct lncRNA-GK3P knockdown and lncRNA-GK3P overexpressed EC109 cell lines, respectively. They were divided into four groups: si-GK3P group (siRNAGK3P transfection), si-NC group (siRNAGK3P empty vector control), plasmid-GK3P group (GK3P overexpression plasmid transfection), and plasmid-NC group (GK3P empty plasmid control). CCK8 method was used to detect cell viability, EdU method for the proliferation of EC109 cells, flow cytometry for apoptosis and cycle of EC109 cells, and Western blotting for expression of proteins related to cell cycle and apoptosis (Cyclin E1, Cyclin B1, Bcl2, and Bax).Results Compared with adjacent tissues, the expression of lncRNA-
GK3P in esophageal cancer tissues was 2.91±0.61 times higher (t =19.38,P < 0.05); and compared with esophageal epithelial Het-1A cells, the expression of lncRNA-GK3P in EC109 cells was 3.15±0.27 times higher (t =13.06,P < 0.05). After the knockdown of lncRNA-GK3P , the cell proliferation was inhibitedsi-GK3P group vs si-NC group, (27.21±1.11)% vs (37.95±0.93)%,P < 0.05; the proportion of cells in G0/G1 phase increased (P < 0.05), the proportion of cells in S phase increased (P < 0.05), and the proportion of cells in G2/M phase decreased (P < 0.05); the overall apoptosis rate increasedsi-GK3P group vs si-NC group, (10.19±0.91)% vs (7.46±0.18)%,P < 0.05; moreover, the expressions of Cyclin E1, Cyclin B1, and Bcl2 were down-regulated, while the expression of Bax was up-regulated. After the overexpression of lncRNA-GK3P , the cell proliferation was promotedplasmidGK3P group vs plasmid-NC group, (37.00±1.71)% vs (29.63±1.45)%,P < 0.05; the proportion of G0/G1 phase cells decreased (P < 0.05), the proportion of S phase cells increased (P < 0.05), and the proportion of G2/M phse cells decreased (P < 0.05); the overall apoptosis rate reducedplasmid-GK3P group vs plasmid-NC group, (5.27±0.07)% vs (9.71±0.19)%,P < 0.05; furthermore, the expressions of Cyclin E1, Cyclin B1, and Bcl2 were up-regulated, and the expression of Bax was down-regulated.Conclusion LncRNA-
GK3P is highly expressed in esophageal cancer, and it promotes proliferation and inhibits apoptosis in EC109 cells by regulating the expressions of Cyclin E1, Cyclin B1, Bcl2, and Bax.
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