血清外泌体介导lncRNA HOTTIP通过miR‑138‑5p/TJP1轴调控胃癌细胞顺铂耐药的研究

韩明阳; 白军伟; 聂洁伟; 李媛媛; 张超

doi:10.3760/cma.j.cn115610-20231123-00214

血清外泌体介导lncRNA HOTTIP通过miR‑138‑5p/TJP1轴调控胃癌细胞顺铂耐药的研究

Serum exosome‑mediated lncRNA HOTTIP regulates cisplatin resistance in gastric cancer cells through the miR‑138‑5p/TJP1 axis

摘要

摘要:
目的探讨胃癌患者血清外泌体对胃癌细胞AGS顺铂耐药、克隆形成、迁移、侵袭和凋亡的影响,并探讨其分子机制。
方法分离胃癌患者血清外泌体,qRT‑PCR检测血清外泌体中lncRNA HOTTIP的表达。体外培养正常胃上皮细胞株GES1、胃癌细胞株AGS、人胚肾细胞株293T。AGS细胞与外泌体（Exo）孵育,以PBS处理作为对照,随后转染si‑NC或si‑HOTTIP‑3,设为Exo组、PBS组、si⁃NC+Exo组、si‑HOTTIP‑3+Exo组。AGS细胞分别转染si‑NC、si‑HOTTIP‑1、si‑HOTTIP‑2、si‑HOTTIP‑3、oe⁃HOTTIP、vector、oe‑HOTTIP+miR‑138‑5p mimic、oe‑HOTTIP+mimic NC、miR⁃138‑5p inhibitor、inhibitor NC、miR‑138‑5p inhibitor+si‑TJP1及miR‑138‑5p inhibitor+si‑NC,设为si‑NC组、si‑HOTTIP‑1组、si⁃HOTTIP‑2组、si‑HOTTIP‑3组、oe‑HOTTIP组、vector组、oe‑HOTTIP+miR‑138‑5p mimic组、oe‑HOTTIP+mimic NC组、miR‑138‑5p inhibitor组、inhibitor NC组、miR‑138‑5p inhibitor+si‑TJP1组及miR‑138‑5p inhibitor+si‑NC组。293T细胞转染mimic NC+HOTTIP wt、miR‑138‑5p mimic+HOTTIP wt、mimic NC+HOTTIP mut、miR‑138‑5p mimic+HOTTIP mut、mimic NC+TJP1 3'UTR wt、miR⁃138⁃5p mimic+TJP1 3'UTR wt、mimic NC+TJP1 3'UTR mut、miR‑138‑5p mimic+TJP1 3'UTR mut分别设为mimic NC+HOTTIP wt组、miR‑138‑5p mimic+HOTTIP wt组、mimic NC+HOTTIP mut组、miR‑138‑5p mimic+HOTTIP mut组、mimic NC+TJP1 3'UTR wt组、miR‑138‑5p mimic+TJP1 3'UTR wt组、mimic NC+TJP1 3'UTR mut组、miR‑138‑5p mimic+TJP1 3'UTR mut组。CCK8实验检测细胞的顺铂敏感性,平板克隆检测细胞克隆形成,Transwell实验检测细胞迁移和侵袭,流式细胞仪检测细胞凋亡,Western blot实验检测外泌体标志蛋白CD63和CD81、TJP1及耐药相关蛋白P‑gp、MCL‑1的表达,双荧光素酶实验验证lncRNA HOTTIP、miR‑138‑5p和TJP1之间的靶向关系。观察指标：（1）lncRNA HOTTIP的表达情况。（2）血清外泌体介导lncRNA HOTTIP对胃癌细胞顺铂的耐药情况。（3）过表达lncRNA HOTTIP通过miR⁃138⁃5p对胃癌细胞顺铂耐药的调控情况。（4） miR‑138‑5p靶向TJP1 3′非编码区检测情况。（5）抑制miR⁃138‑5p通过TJP1对胃癌细胞顺铂耐药的调控情况。正态分布的计量资料以x±s表示,组间比较采用t检验,多组间比较采用单因素方差分析,进一步两两比较采用Tukey′s检验。计数资料以绝对数表示,组间比较采用χ²检验。相关性分析采用Pearson检验。
结果（1）lncRNA HOTTIP的表达情况。lncRNA HOTTIP在胃癌患者血清外泌体中的表达高于在健康志愿者血清外泌体中的表达,差异有统计学意义（P<0.05）。透射电子显微镜结果显示：血清外泌体呈圆形或卵圆形；Western blot检测结果显示：血清外泌体中存在标志性蛋白CD63和CD81的表达。（2）血清外泌体介导lncRNA HOTTIP对胃癌细胞顺铂的耐药情况。与PBS组比较,Exo组半抑制浓度（IC50）、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均升高,细胞凋亡率降低,差异均有统计学意义（P<0.05）。与si‑NC+Exo组比较,si‑HOTTIP‑3+Exo组IC50、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均降低,细胞凋亡率升高,差异均有统计学意义（P<0.05）。（3）过表达lncRNA HOTTIP通过miR‑138‑5p对胃癌细胞顺铂耐药的调控情况。与vector组比较,oe‑HOTTIP组IC50、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均升高,细胞凋亡率降低,差异均有统计学意义（P<0.05）。与oe‑HOTTIP+mimic NC组比较,oe‑HOTTIP+miR‑138‑5p mimic组IC50、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均降低,细胞凋亡率升高,差异均有统计学意义（P<0.05）。（4）miR‑138‑5p靶向TJP1 3′非编码区检测情况。双荧光素酶实验显示：mimic NC+TJP1 3′UTR wt组、miR‑138‑5p mimic+TJP1 3′UTR wt组的荧光素酶活性分别为1.00±0.09、0.21±0.03,差异有统计学意义（t=15.02,P<0.05）。（5）抑制miR‑138‑5p通过TJP1对胃癌细胞顺铂耐药的调控情况。与inhibitor NC组比较,miR‑138‑5p inhibitor组IC50、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均升高,细胞凋亡率降低,差异均有统计学意义（P<0.05）。与miR‑138‑5p inhibitor+si‑NC组比较,miR‑138‑5p inhibitor+si‑TJP1组IC50、细胞克隆形成数、侵袭细胞数、迁移细胞数、P‑gp蛋白表达和MCL‑1蛋白表达均降低,细胞凋亡率升高,差异均有统计学意义（P<0.05）。
结论血清外泌体介导lncRNA HOTTIP通过调控胃癌细胞中miR‑138‑5p/TJP1的表达,促进胃癌细胞的顺铂耐药性、克隆形成、迁移和侵袭,抑制细胞凋亡。

Abstract:
Objective To investigate the effects of serum exosomes of patients with gastric cancer on cisplatin resistance, clonal formation, migration, invasion and apoptosis of the AGS gastric cancer cells, and the corresponding molecular mechanisms.
Methods The experimental study was conducted. The exosomes of patients with gastric cancer was separated from their serum, and the expression of lncRNA HOTTIP was analyzed using the quantitative real time polymerase chain reaction (qRT‑PCR). Normal gastric epithelial cell line GES1, gastric cancer cell line AGS and human embryonic kidney cell 293T were cultured in vitro. AGS cells were incubated with exosomes (Exo),with phos-phate buffered saline (PBS) treatment as control, and transfected with si-NC or si-HOTTIP-3, named as Exo group, PBS group, si-NC+Exo group, and si-HOTTIP-3+Exo group. The AGS cells were trans-fected with si-NC, si-HOTTIP-1, si-HOTTIP-2, si-HOTTIP-3, oe-HOTTIP, vector, oe-HOTTIP+miR-138-5p mimic, oe-HOTTIP+mimic NC, miR-138-5p inhibitor, inhibitor NC, miR-138-5p inhibitor+si-TJP1 and miR-138-5p inhibitor+si-NC. They were recorded as si-NC group, si-HOTTIP-1 group, si-HOTTIP-2 group, si-HOTTIP-3 group, oe-HOTTIP group, vector group, oe-HOTTIP+miR-138-5p mimic group, oe-HOTTIP+mimic NC group, miR-138-5p inhibitor group, inhibitor NC group, miR-138-5p inhibitor+si-TJP1 group and miR-138-5p inhibitor+si-NC group. The 293T cells transfected with mimic NC+HOTTIP wt, miR-138-5p mimic+HOTTIP wt, mimic NC+HOTTIP mut, miR-138-5p mimic+HOTTIP mut, mimic NC+TJP1 3'UTR wt, miR-138-5p mimic+TJP1 3'UTR wt, mimic NC+TJP1 3'UTR mut, miR-138-5p mimic+TJP1 3'UTR mut were recorded as the mimic NC+HOTTIP wt group, miR-138-5p mimic+HOTTIP wt group, mimic NC+HOTTIP mut group, miR-138-5p mimic+HOTTIP mut group, mimic NC+TJP1 3'UTR wt group, miR-138-5p mimic+TJP1 3'UTR wt group, mimic NC+TJP1 3'UTR mut group, miR-138-5p mimic+TJP1 3'UTR mut group. The cell counting kit‑8 (CCK8) was used to analyze the cisplatin sensitivity of gastric cancer cells. The colony formation experiment was used to analyze the colony formation of gastric cancer cells. The Transwell experiment was used to analyzed cell migration and invasion of gastric cancer cells. The flow cytometry experiment was used to analyze cell apoptosis of gastric cancer cells. The Western bolt assay was used to analyze the expression of exosome marker proteins, including the CD63 and CD81, and the protein of TJP1, the drug‑resistance related proteins, including the P‑gp and MCL‑1. The dual‑luciferase assay was used to analyze the targeted relationships among lncRNA HOTTIP, miR‑138‑5p and TJP1. Observation indicators: (1) expression of lncRNA HOTTIP; (2) resistance of gastric cancer cells to cisplatin regulated by exosome-mediated lncRNA HOTTIP; (3) regulation of cisplatin resistance in gastric cancer cells mediated by miR‑138‑5p through lncRNA HOTTIP overexpression; (4) targeting of TJP1 gene 3′‑untranslated region (UTR) by miR‑138‑5p; (5) regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR‑138‑5p inhibition. Measurement data with normal distribution were represented as Mean±SD, and comparison between groups was conducted using the t test. The one‑way ANOVA was used for comparison for multiple groups and the Tukey′s test was used for further pairwise compari-son. Count data were described as absolute numbers, and the chi‑square test was used for comparison. Correlation analysis was conducted using the Pearson′s test.
Results (1) Expression of lncRNA HOTTIP. The expression of lncRNA HOTTIP in the serum exosome of patients with gastric cancer was higher than that in healthy volunteers, showing a significant difference (P<0.05). Results of transmi-ssion electron microscopy examination showed that the serum exosomes were circular or oval in shape. Results of Western bolt assay showed the expression of marker proteins of CD63 and CD81 in serum exosomes. (2) Resistance of gastric cancer cells to cisplatin regulated by exosome‑mediated lncRNA HOTTIP. Compared with the PBS group, the biochemical half maximal inhibitory concentra-tion (IC50), the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the Exo group increased, while the cell apoptosis rate decreased, showing significant differences between them (P<0.05). Compared with the si-NC+Exo group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the si-HOTTIP-3+Exo group decreased, while the cell apoptosis rate increased, showing significant differences between them (P<0.05). (3) Regulation of cisplatin resistance in gastric cancer cells mediated by miR‑138‑5p through lncRNA HOTTIP overexpression. Compared with the vector group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the oe-HOTTIP group increased, while the cell apoptosis rate decreased, showing significant differences between them (P<0.05). Compared with the oe-HOTTIP+mimic NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the oe-HOTTIP+miR-138-5p mimic group increased, while the cell apoptosis rate decreased, showing significant differences between them (P<0.05). (4) Targeting of TJP1 gene 3′‑UTR by miR‑138‑5p. Results of dual‑luciferase assay showed that the luciferase activity in 293T cells treatment with mimics of control+vectors of wild type of TJP1 gene 3′‑UTR and 293T cells treatment with mimics of miR-138-5p+vectors of wild type of TJP1 gene 3′‑UTR was 1.00±0.09 and 0.21±0.03, respectively, showing a significant difference between them (t=15.02, P<0.05). (5) Regulation of cisplatin resistance in gastric cancer cells by TJP1 through miR‑138‑5p inhibition. Compared with the inhibitor group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the miR-138-5p inhibitor group increased, while the cell apoptosis rate decreased, showing significant differences between them (P<0.05). Compared with the miR-138-5p inhibitor+si-NC group, the IC50, the number of clone formation, the number of invasive cell, the number of migratory cell, expression of P‑gp protein, expression of MCL‑1 protein in the miR-138-5p inhibitor+si-TJP1 group decreased, while the cell apoptosis rate increased, showing significant differences between them (P<0.05).
Conclusion Serum exosomes-mediated lncRNA HOTTIP can promote cisplatin resistance, clonal formation, migration, invasion and apoptosis of gastric cancer cells and inhibit cell apoptosis of gastric cancer cells through regulating the expression of miR‑138‑5p/TJP1.

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