长链非编码RNA-PKD2-2-3/miR-328/GPAM竞争性内源RNA网络诱导胆管癌增殖、侵袭及5-氟尿嘧啶化疗耐药
Lnc-PKD2-2-3/miR-328/GPAM ceRNA Network Induces Cholangiocarcinoma Proliferation, Invasion and 5-FU Chemoresistance.
作者信息
Zhang Lei, Ma Donglai, Li Fujun, Qiu Gongcai, Sun Dongsheng, Zeng Zhaolin
机构信息
Department of General Surgery, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China.
出版信息
Front Oncol. 2022 Jul 29;12:871281. doi: 10.3389/fonc.2022.871281. eCollection 2022.
PURPOSE
Our previous study observed that long non-coding RNA PKD2-2-3 (lnc-PKD2-2-3) is related to advanced tumor features and worse prognosis in cholangiocarcinoma (CCA). Then, this study aimed to further explore the linkage between lnc-PKD2-2-3, miR-328, and GPAM, as well as their effects on regulating CCA viability, mobility, and chemosensitivity.
METHODS
Lnc-PKD2-2-3, miR-328, and GPAM expression in 30 pairs of CCA tumor and adjacent tissues, as well as in CCA cell lines, were determined. Two CCA cell lines (HuCCT1 and TFK1) were transfected by lnc-PKD2-2-3 overexpression plasmid, lnc-PKD2-2-3 siRNA, miR-328 inhibitor, and GPAM siRNA alone or in combination, followed by cell proliferation, apoptosis, invasion, and 5-FU chemosensitivity detection. Besides, xenograft mice were established for validation.
RESULTS
Lnc-PKD2-2-3 and GPAM were higher, whereas miR-328 was lower in CCA tissues versus adjacent tissues and also in CCA cell lines versus control cells; meanwhile, they were correlated with each other (all 0.05). Lnc-PKD2-2-3 knockdown decreased CCA cell proliferation, invasion, and increased apoptosis (all 0.05), but lnc-PKD2-2-3 overexpression exhibited the opposite and weaker effect. MiR-328 knockdown induced CCA cell proliferation and invasion and also attenuated the effect of lnc-PKD2-2-3-knockdown in these functions (all 0.05). Subsequently, GPAM knockdown reduced CCA cell proliferation and invasion and also weakened the effect of miR-328-knockdown in these functions (all 0.05). Additionally, lnc-PKD2-2-3 positively regulated GPAM while negatively regulating miR-328. MiR-328 negatively modified GPAM in CCA cells. Luciferase gene reporter assays verified that lnc-PKD2-2-3 directly bound miR-328 and miR-328 directly bound GPAM. Finally, the lnc-PKD2-2-3/miR-328/GPAM network also regulated the 5-FU chemosensitivity of CCA cells. experiments further revealed that lnc-PKD2-2-3 overexpression promoted tumor volume and weight but repressed tumor apoptosis in xenograft mice; meanwhile, it increased GPAM expression but decreased miR-328 expression (all 0.05). Conversely, lnc-PKD2-2-3 knockdown exhibited the opposite effects (all 0.05).
CONCLUSION
Lnc-PKD2-2-3/miR-328/GPAM ceRNA network promotes CCA proliferation, invasion, and 5-FU chemoresistance.
目的
我们之前的研究观察到长链非编码RNA PKD2-2-3（lnc-PKD2-2-3）与胆管癌（CCA）的肿瘤进展特征及较差预后相关。因此，本研究旨在进一步探究lnc-PKD2-2-3、miR-328和甘油磷酸酰基转移酶（GPAM）之间的联系，以及它们对CCA细胞活力、迁移能力和化疗敏感性的调控作用。
方法
检测30对CCA肿瘤组织及癌旁组织以及CCA细胞系中lnc-PKD2-2-3、miR-328和GPAM的表达。分别单独或联合转染lnc-PKD2-2-3过表达质粒、lnc-PKD2-2-3小干扰RNA（siRNA）、miR-328抑制剂和GPAM siRNA至两种CCA细胞系（HuCCT1和TFK1），随后检测细胞增殖、凋亡、侵袭及5-氟尿嘧啶（5-FU）化疗敏感性。此外，建立异种移植小鼠模型进行验证。
结果
与癌旁组织相比，CCA组织中lnc-PKD2-2-3和GPAM表达较高，而miR-328表达较低；与对照细胞相比，CCA细胞系中lnc-PKD2-2-3和GPAM表达较高，而miR-328表达较低；同时，它们之间相互关联（均P<0.05）。lnc-PKD2-2-3敲低可降低CCA细胞增殖、侵袭能力并增加细胞凋亡（均P<0.05），但lnc-PKD2-2-3过表达表现出相反且较弱的作用。miR-328敲低可诱导CCA细胞增殖和侵袭，并减弱lnc-PKD2-2-3敲低对这些功能的影响（均P<0.05）。随后，GPAM敲低可降低CCA细胞增殖和侵袭，并减弱miR-328敲低对这些功能的影响（均P<0.05）。此外，lnc-PKD2-2-3正向调控GPAM，同时负向调控miR-328。miR-328在CCA细胞中负向调节GPAM。荧光素酶基因报告实验证实lnc-PKD2-2-3直接结合miR-328，miR-328直接结合GPAM。最后，lnc-PKD2-2-3/miR-328/GPAM网络也调节CCA细胞对5-FU的化疗敏感性。体内实验进一步显示，lnc-PKD2-2-3过表达可促进异种移植小鼠肿瘤体积和重量增加，但抑制肿瘤细胞凋亡；同时，增加GPAM表达，但降低miR-328表达（均P<0.05）。相反，lnc-PKD2-2-
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