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王彦霞, 张春强, 韩菲. HOXA1基因反义寡核苷酸对食管癌细胞生长的影响[J]. koko体育app 学报（医学版）, 2020, 51(1): 24-29. DOI:
引用本文: 王彦霞, 张春强, 韩菲. HOXA1基因反义寡核苷酸对食管癌细胞生长的影响[J]. koko体育app 学报（医学版）, 2020, 51(1): 24-29. DOI:
WANG Yan-xia, ZHANG Chun-qiang, HAN Fei. Effects of HOXA1 Gene Antisense Oligonucleotides on Growth of Esophageal Cancer Cells[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(1): 24-29. DOI:
Citation: WANG Yan-xia, ZHANG Chun-qiang, HAN Fei. Effects of HOXA♑1 Gene Antisense Oligonucleotides on Growth of Esophageal Cancer Cells[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(1): 24-29. DOI:

HOXA1基因反义寡核苷酸对食管癌细胞生长的影响

Effects of HOXA1 Gene Antisense Oligonucleotides on Growth of Esophageal Cancer Cells

  • 摘要:
      目的  探讨同源异形盒A1(HOXA1)基因反义寡核苷酸（ASODN）对食管癌细胞增殖、凋亡、侵袭和迁移的影响及机制。
      方法  采用蛋白免疫印迹（Western blot）检测正常食管上皮细胞Het-1A和人食管鳞癌TE-1、EC9706和Eca109细胞HOXA1蛋白的表达，筛选高表达的食管鳞癌细胞进行后续实验；设计合成HOXA1 ASODN链、正义寡核苷酸(SODN)链及无义寡核苷酸（N-ODN）链；将筛选出的高表达的食管鳞癌细胞分为HOXA1 ASODN组（5、10、15 μmol/L的HOXA1 ASODN转染Eca109细胞）、对照组（细胞常规培养，不进行细胞转染）、SODN组（细胞转染15 μmol/L的SODN）和N-ODN组（细胞转染15 μmol/L的N-ODN）。采用噻唑蓝(MTT)法、流式细胞术、Transwell小室分别检测细胞活力、凋亡率及侵袭迁移能力；Western blot检测HOXA1、磷酸化的丝氨酸苏氨酸激酶（p-AKT）、细胞增殖核抗原(PCNA)、基质金属蛋白酶2(MMP-2)、B细胞淋巴瘤-2蛋白（B-cell lymphoma 2，Bcl-2）和Bcl-2相关X蛋白(Bax)蛋白表达。
      结果  与正常食管上皮细胞Het-1A比较，人食管鳞癌TE-1、EC9706和Eca109细胞HOXA1蛋白表达均升高，差异有统计学意义（P<0.05）。HOXA1蛋白在Eca109细胞表达最高，因此选用Eca109细胞进行后续实验。转染HOXA1 ASODN的Eca109细胞HOXA1蛋白表达降低（P<0.05）。随着Eca109细胞转染HOXA1 ASODN浓度及时间增加，Eca109细胞活力降低，与对照、SODN、N-ODN组比较差异有统计学意义（P<0.05）。15 μmol/L的HOXA1 ASODN转染Eca109细胞后，与对照组比较，细胞侵袭和迁移能力降低，凋亡率升高，p-AKT、PCNA和MMP-2蛋白表达降低，Bax蛋白表达升高（P<0.05）。
      结论  HOXA1基因反义寡核苷酸可抑制食管癌细胞增殖、侵袭和迁移能力，并诱导凋亡，机制与抑制磷脂酰肌醇3-激酶（PI3K）/AKT信号通路可能有关。
     
    Abstract:
      Objective  To investigate the effect of antisense oligodeoxynucleotides (ASODN) of Homeobox A1 gene (HOXA1) on proliferation, apoptosis, invasion and migration of esophageal carcinoma cells.
      Methods  The expression of HOXA1 protein in normal esophageal epithelial cells Het-1A and esophageal cancer TE-1, EC9706 and Eca109 cells was detected by Western blot. Screening of highly expressed of HOXA1 protein esophageal squamous cell carcinoma cells for follow-up experiments. HOXA1 antisense oligonucleotide (ASODN) chains, sense oligodeoxynucleotides (SODN) chain, and nonsense oligodeoxy nucleotides (N-ODN) chain were designed. The screened esophageal squamous cell carcinoma cells with high expression were divided into HOXA1 ASODN group (5, 10, 15 μmol/L HOXA1 ASODN transfected Eca109 cells), control group (conventional culture medium, no cell transfection), SODN group (cells transfected with 15 μmol/L of SODN) and N-ODN group (cells transfected with 15 μmol/L N-ODN). Cell viability, apoptosis rate and invasion and migration ability were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, flow cytometry, transwell chamber respectively; The expression of HOXA1, phosphorylation serine/threonine kinase (p-AKT), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and B-cell lymphoma2 (Bcl-2) associated X protein (Bax) protein was detected by Western blot.
      Results  Compared with normal esophageal epithelial cells Het-1A, the expression of HOXA1 protein in human esophageal squamous cell carcinoma cells TE-1, EC9706 and Eca109 was significantly higher (P<0.05). The expression of HOXA1 protein was the highest in Eca109 cells, therefore, Eca109 cells were selected for follow-up experiments. The expression of HOXA1 protein in Eca109 cells transfected with HOXA1 ASODN was significantly decreased (P<0.05). After transfection of Eca109 cells with HOXA1 ASODN, the viability of Eca109 cells decreased with the increase of concentration and time, the difference was significant compared with the control, SODN and N-ODN groups (P<0.05). 15 μmol/L HOXA1 ASODN significantly inhibited cell viability. After 15 μmol/L HOXA1 ASODN was transfected into Eca109 cells, the invasion and migration abilities of cells were significantly decreased, the apoptosis rate was increased, the expressions of p-AKT, PCNA and MMP-2 were significantly decreased, and the expression of Bax was significantly increased (P<0.05).
      Conclusion  Antisense oligodeoxynucleotides of HOXA1 gene can inhibit the proliferation, invasion and migration of esophageal cancer cells, and induce apoptosis. The mechanism is related to the inhibition of PI3K/AKT signaling pathway.
     
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