hsa_circ_0013058促进食管鳞状细胞癌侵袭和迁移的机制研究

谈艳; 陶菲儿; 王成海

doi:10.7619/jcmp.20233957

hsa_circ_0013058促进食管鳞状细胞癌侵袭和迁移的机制研究

Mechanism of hsa_circ_0013058 in promoting invasion and migration of esophageal squamous cell carcinoma

摘要

摘要:
目的探讨食管鳞状细胞癌(ESCC)中hsa_circ_0013058的作用及其促进ESCC细胞侵袭和迁移的机制。
方法收集75例患者的ESCC组织及癌旁正常组织。采用实时荧光定量聚合酶链反应(qRT-PCR)和RNA原位杂交技术(RISH)检测hsa_circ_0013058表达情况，并分析其与ESCC患者临床病理资料的关系。采用划痕实验和Transwell实验检测ESCC细胞侵袭和迁移能力。采用生物信息学方法预测hsa_circ_0013058的靶基因。采用Rescue实验检测hsa_circ_0013058对下游基因的调控及对ESCC细胞侵袭、迁移的影响。
结果 qRT-PCR结果显示, ESCC组织中hsa_circ_0013058表达量高于癌旁正常组织，差异有统计学意义(t=5.078, P < 0.05)。ESCC细胞株KYSE70中hsa_circ_0013058表达量高于人正常食管上皮细胞Het-1A, 差异有统计学意义(P < 0.05)。在RNA水平上, hsa_circ_0013058与miR-548p呈负相关(r=-0.254 2, P < 0.05)。ESCC组织中, hsa_circ_0013058 RNA阳性表达率为72.00%, 高于癌旁正常组织的17.33%, 差异有统计学意义(χ²=6.862, P < 0.05)。hsa_circ_0013058阳性表达与肿瘤浸润深度、分化程度和淋巴结转移均密切相关(P < 0.05)。划痕实验结果表明，过表达hsa_circ_0013058增强KYSE70细胞的迁移能力(P < 0.05); 敲低hsa_circ_0013058表达减弱KYSE70细胞的迁移能力(P < 0.05)。过表达hsa_circ_0013058后, ESCC细胞侵袭和迁移细胞数目增加，敲低hsa_circ_0013058表达后，侵袭和迁移细胞数目下降(P < 0.05)。qRT-PCR实验证实, hsa_circ_0013058可调控微小RNA-548p(miR-548p)表达。双荧光素酶报告基因实验表明, miR-548p与 LPAR3 为特异性结合, LPAR3 是miR-548p的直接靶基因。Rescue实验结果表明, hsa_circ_0013058通过miR-548p/LPAR3信号轴调控ESCC细胞的侵袭、转移。
结论 ESCC中, hsa_circ_0013058抑制miR-548p表达，进而激活LPAR3信号通路，促进ESCC细胞的侵袭、迁移。

Abstract:
Objective To investigate the role of hsa_circ_0013058 in esophageal squamous cell carcinoma (ESCC) and the mechanism in promoting the invasion and migration of ESCC cells.
Methods ESCC tissues and adjacent normal tissues of 75 patients were collected. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and RNA in situ hybridization (RISH) were used to detect the expression of hsa_circ_0013058, and the relationship between hsa_circ_0013058 and clinicopathological data of ESCC patients was analyzed. The invasion and migration ability of ESCC cells were detected by scratch test and Transwell test. The target gene of hsa_circ_0013058 was predicted by bioinformatics. The regulation of hsa_circ_0013058 on downstream genes and its effect on invasion and migration of ESCC cells were detected by Rescue experiment.
Results The results of qRT-PCR showed that the expression level of hsa_circ_0013058 in the ESCC tissues was significantly higher than that in the adjacent normal tissues (t=5.078, P < 0.05). The expression level of hsa_circ_0013058 in KYSE70 was significantly higher than that in human normal esophageal epithelial cells HTT-1A (P < 0.05). At the RNA level, hsa_circ_0013058 was negatively correlated with miR-548p (r=-0.254 2, P < 0.05). The positive expression rate of hsa_circ_0013058 RNA in the ESCC tissues was 72.00%, which was significantly higher than 17.33% in the adjacent normal tissues (χ²=6.862, P < 0.05). The positive expression of hsa_circ_0013058 was closely related to the depth of tumor invasion, differentiation degree and lymph node metastasis (P < 0.05). Scratch test results showed that overexpression of hsa_circ_0013058 enhanced the migration ability of KYSE70 cells (P < 0.05); knockdown of hsa_circ_0013058 expression decreased the migration ability of KYSE70 cells (P < 0.05). After overexpression of hsa_circ_0013058, the number of invasion and migration cells of ESCC increased, and after knockdown of hsa_circ_0013058 expression, the number of invasion and migration cells decreased (P < 0.05). The qRT-PCR confirmed that hsa_circ_0013058 could regulate the expression of microRNA-548p (miR-548p). Dual luciferase reporter gene assay showed that miR-548p was specifically bound to LPAR3, which was the direct target gene of miR-548p. Rescue experiment results indicated that hsa_circ_0013058 regulated the invasion and migration of ESCC cells through the miR-548p/LPAR3 signaling axis.
Conclusion In ESCC, hsa_circ_0013058 inhibits the expression of miR-548p, thereby activating the LPAR3 signaling pathway and promoting the invasion and migration of ESCC cells.

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