miR-143-3p靶向LASP1抑制胃癌细胞侵袭和迁移的研究

许鑫鑫; 鲁意迅; 李凯; 梁文全; 高云鹤; 郗洪庆; 王鑫鑫; 陈凛

doi:10.3969/j.issn.2095-5227.2022.08.011

摘要:

背景肿瘤远处转移作为影响胃癌患者预后的一大危险因素，常导致治疗失败。因此，亟需探索针对肿瘤转移的新治疗靶点以改善胃癌患者的预后。

目的探讨通过生物信息学筛选的miRNA能否通过靶向LASP1抑制胃癌细胞的侵袭和迁移。

方法通过生物信息学方法预测靶向于LASP1的miRNAs, 基于TCGA数据库分析miRNAs在胃癌中的表达水平及LASP1与miRNAs的相关性。利用双荧光素酶报告实验验证miR-143-3p与LASP1之间的靶向互作关系。以胃癌MGC-803细胞系为实验对象，分别转染miR-143-3p模拟物、抑制剂及其阴性对照，据此将实验分为miR-143-3p模拟物对照组(miR-143-3p mimic NC)、miR-143-3p模拟物组(miR-143-3p mimic)、miR-143-3p抑制剂对照组(miR-143-3p inhibitor NC)和miR-143-3p抑制剂组(miR-143-3p inhibitor)。应用Transwell侵袭实验和划痕愈合实验测定各组MGC-803细胞侵袭和迁移能力的变化。qRT-PCR检测转染后MGC-803细胞中LASP1的mRNA表达水平，Western blot检测MGC-803细胞中LASP1的蛋白表达水平。

结果生物信息学分析提示miR-143-3p可能靶向于LASP1，二者相关性r=-0.284(P＜0.01)。双荧光素酶报告实验进一步证实LASP1是miR-143-3p的一个直接靶基因(P＜0.01)。Transwell侵袭实验和划痕愈合实验证实miR-143-3p对胃癌MGC-803细胞的侵袭和迁移能力有明显抑制作用。qRT-PCR实验结果显示miR-143-3p mimic组的LASP1的表达水平低于miR-143-3p mimic NC组(P＜0.01)，而miR-143-3p inhibitor组的LASP1的表达水平高于miR-143-3p inhibitor NC组(P＜0.05)。此外，Western blot与qRT-PCR的实验结果一致。

结论 miR-143-3p可通过靶向下调LASP1，抑制胃癌细胞的侵袭和迁移。

Abstract:

Background Distant metastasis is a major risk factor affecting the prognosis of gastric cancer (GC) patients and often leads to treatment failure. Therefore, it is urgent to explore new therapeutic targets for tumor metastasis to improve the prognosis of GC patients.

Objective To explore whether the miRNAs we screened by bioinformatics analysis could inhibit the invasion and migration of GC cells by targeting LASP1.

Methods Based on TCGA database, the expression levels of miRNAs in GC and the correlation between LASP1 and miRNAs were analyzed by bioinformatic methods to predict miRNAs targeting LASP1. The targeted interaction between miR-143-3p and LASP1 was verified by dual-luciferase reporter assay. Using GC cell line MGC-803 as experimental object, miR-143-3p mimic, inhibitor and negative controls were transfected, respectively. Accordingly, the experiments were divided into miR-143-3p mimic negative control (NC) group, miR-143-3p mimic group, miR-143-3p inhibitor NC group and miR-143-3p inhibitor group. Transwell invasion assay and wound healing assay were applied to determine the changes of invasion and migration ability of MGC-803 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the messenger RNA (mRNA) expression level of LASP1 in MGC-803 cells after transfection, and western blot (WB) was used to detect the protein expression level of LASP1 in MGC-803 cells.

Results Bioinformatics analysis suggested that miR-143-3p might target LASP1 and the correlation between miR-143-3p and LASP1 was r=-0.284 (P＜0.01). The dual-luciferase reporter assay further confirmed that LASP1 was a direct target gene of miR-143-3p (P＜0.01). Transwell invasion assay and wound healing assay confirmed that miR-143-3p significantly inhibited the invasion and migration ability of gastric cancer MGC-803 cells. The results of qRT-PCR showed that the expression level of LASP1 in miR-143-3p mimic group was lower than that in miR-143-3p mimic NC group (P＜0.01), while the expression level of LASP1 in miR-143-3p inhibitor group had a higher expression level of LASP1 than that of the miR-143-3p inhibitor NC group (P＜0.05). The results of WB showed that protein changes of LASP1 were consistent with that of qRT-PCR.

Conclusion miR-143-3p could inhibit the invasion and migration of GC cells by targeting and down-regulating LASP1.

miR-143-3p靶向LASP1抑制胃癌细胞侵袭和迁移的研究

miR-143-3p inhibits invasion and migration of gastric cancer by targeting LASP1