该研究旨在探索MALAT1与miR-188-5p竞争性结合以上调PSMD10以促进胆管癌细胞迁移和侵袭并抑制凋亡的调节网络。qRT-PCR和荧光原位杂交(FISH)检测MALAT1和miR-188-5p在胆管癌组织和HIBEC中的表达和阳性信号,HCCC-9810,RBE,和QBC939细胞。蛋白质印迹,qRT-PCR,免疫组化检测胆管癌组织和细胞系中PSMD10的表达。采用双荧光素酶报告基因检测验证miR-188-5p靶向MALAT1和PSMD10。qRT-PCR,向下拉,和蛋白质印迹用于检测MALAT1-miR-188-5p-PSMD10轴的调节。Transwell,伤口愈合试验,和Tunel细胞凋亡分别检测MALAT1-miR-188-5p-PSMD10轴对细胞侵袭的调控能力,迁移,和凋亡。Westernblot检测MALAT1对Bax的调控机制,Bcl-2和caspase-3蛋白。建立胆管癌裸鼠皮下移植瘤模型,研究MALAT1对皮下肿瘤生长的影响。采用免疫组织化学方法检测各组中Ki67抗体和SMD10抗体的阳性指标。MALAT1和PSMD10在胆管癌组织和细胞系中高表达,而miR-188-5p低表达。MALAT1可以竞争性结合miR-188-5p,miR-188-5p可以负调控PSMD10。MALAT1,In-miR-188-5p,PSMD10可以促进细胞的侵袭和迁移,抑制细胞凋亡,而siMALAT1,miR-188-5p,和siPSMD10产生相反的结果。MALAT1-miR-188-5p-PSMD10轴可促进RBE细胞侵袭和迁移,抑制细胞凋亡,而siMALAT1-In-miR-188-5p-siPSMD10轴显示相反的结果。另一方面,证实上调/下调MALAT1能抑制/促进Bax和caspase-3蛋白的表达,促进/抑制Bcl-2蛋白的表达。MALAT1可以促进皮下肿瘤生长并增强PSMD10的细胞增殖和阳性信号,而miR-188-5p则相反。MALAT1竞争性结合miR-188-5p,上调PSMD10的mRNA翻译和蛋白表达,从而促进胆管癌细胞的侵袭和迁移,抑制其凋亡。然而,干扰MALAT1-miR-188-5p-PSMD10轴可抑制胆管癌的发生发展。
The study is designed to explore the regulatory network that MALAT1 competitively binds with miR-188-5p to up-regulate PSMD10 to facilitate cholangiocarcinoma cell migration and invasion and suppress apoptosis. qRT-PCR and fluorescence in situ hybridization (FISH) were used to examine the expression and positive signal of MALAT1 and miR-188-5p in cholangiocarcinoma tissues and HIBEC, HCCC-9810,
RBE, and QBC939 cells. Western blot, qRT-PCR, and immunohistochemistry were selected to detect PSMD10 expression in cholangiocarcinoma tissues and cell lines. Dual luciferase reporter gene assay was adopted to verify that miR-188-5p targeted MALAT1 and PSMD10. qRT-PCR, pull down, and western blot were used to examine the regulation of MALAT1-miR-188-5p-PSMD10 axis. Transwell, wound healing assay, and Tunel cell apoptosis were adopted to respectively detect the regulatory abilities of MALAT1-miR-188-5p-PSMD10 axis on cell invasion, migration, and apoptosis. Western blot was used to detect the regulation mechanism of MALAT1 on Bax, Bcl-2, and caspase-3 proteins. Nude mice subcutaneous xenograft model of cholangiocarcinoma was established to examine the impacts of MALAT1 on subcutaneous tumor growth. Immunohistochemistry was adopted to examine the positive indicator of Ki67 antibodies and SMD10 antibodies in each group. MALAT1 and PSMD10 were highly expressed in cholangiocarcinoma tissues and cell lines, while miR-188-5p was lowly expressed. MALAT1 could competitively bind to miR-188-5p, and miR-188-5p could negatively regulate PSMD10. MALAT1, In-miR-188-5p, and PSMD10 could facilitate cell invasion and migration and inhibit apoptosis, while siMALAT1, miR-188-5p, and siPSMD10 produced an opposite result. MALAT1-miR-188-5p-PSMD10 axis could promote
RBE cell invasion and migration and inhibit apoptosis, whereas siMALAT1-In-miR-188-5p-siPSMD10 axis showed an opposite result. On the other hand, it was verified that up-regulation/down-regulation of MALAT1 can inhibit/promote Bax and caspase-3 proteins and promote/inhibit the expression of Bcl-2 protein. MALAT1 could facilitate subcutaneous tumor growth and enhance cell proliferation and positive signal of PSMD10, while miR-188-5p worked in an opposite direction. MALAT1 competitively binds to miR-188-5p to up-regulate mRNA translation and protein expression of PSMD10, thereby facilitating cholangiocarcinoma cell invasion and migration and inhibiting its apoptosis. However, interfering MALAT1-miR-188-5p-PSMD10 axis could inhibit the occurrence and development of cholangiocarcinoma.