长链非编码RNA UCA1通过miR-23b-3p/ZNF281轴促进结肠癌细胞对5-氟尿嘧啶的耐药性。

lncRNA UCA1 Contributes to 5-Fluorouracil Resistance of Colorectal Cancer Cells Through miR-23b-3p/ZNF281 Axis.

作者信息

Xian Zhenyu, Hu Bang, Wang Ting, Zeng Junyi, Cai Jinlin, Zou Qi, Zhu Peixuan

机构信息

Graceland Medical Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, People's Republic of China.

Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University (Gastrointestinal and Anal Hospital), Guangzhou, Guangdong, People's Republic of China.

出版信息

Onco Targets Ther. 2020 Jul 31;13:7571-7583. doi: 10.2147/OTT.S258727. eCollection 2020.

PURPOSE

The chemoresistance of 5-fluorouracil (5-FU) limited the application of chemotherapy in colorectal cancer (CRC) treatment. Herein, we aimed to uncover the potential mechanism behind the 5-FU resistance of CRC cells.

METHODS

The abundance of long noncoding RNA urothelial carcinoma associated 1 (lncRNA UCA1), microRNA-23b-3p (miR-23b-3p) and zinc finger protein 281 (ZNF281) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. Western blot was conducted to examine autophagy-related proteins, apoptosis-associated proteins and ZNF281 in CRC tissues and cells. Cell counting kit-8 (CCK8) assay was performed to detect the viability and inhibitory concentration 50% (IC50) value of 5-FU of CRC cells. The apoptosis of CRC cells was measured by flow cytometry. The binding sites between miR-23b-3p and UCA1 or ZNF281 were predicted by miRcode and Starbase software, respectively, and the combination was confirmed by dual-luciferase reporter assay and RIP assay. Murine xenograft model was established to verify the role of UCA1 on the 5-FU resistance of CRC in vivo.

RESULTS

The 5-FU resistance of CRC was positively related to the level of UCA1 and autophagy. UCA1 accelerated the 5-FU resistance of CRC cells through facilitating autophagy and suppressing apoptosis. MiR-23b-3p was a target of UCA1 in 293T and CRC cells. The knockdown of miR-23b-3p reversed the inhibitory effects of UCA1 interference on the 5-FU resistance and autophagy and the promoting impact on the apoptosis of CRC cells. ZNF281 could bind to miR-23b-3p in 293T cells. MiR-23b-3p elevated the 5-FU sensitivity through down-regulating ZNF281 in CRC cells. UCA1 interference enhanced the 5-FU sensitivity of CRC through miR-23b-3p/ZNF281 axis in vivo.

CONCLUSION

UCA1 mediated 5-FU resistance of CRC cells through facilitating autophagy and inhibiting apoptosis via miR-23b-3p/ZNF281 axis in vivo and in vitro.

目的

5-氟尿嘧啶（5-FU）的化疗耐药性限制了其在结直肠癌（CRC）治疗中的应用。在此，我们旨在揭示CRC细胞对5-FU耐药的潜在机制。

方法

通过定量实时聚合酶链反应（qRT-PCR）检测CRC组织和细胞中长链非编码RNA尿路上皮癌相关1（lncRNA UCA1）、微小RNA-23b-3p（miR-23b-3p）和锌指蛋白281（ZNF281）的丰度。采用蛋白质免疫印迹法检测CRC组织和细胞中自噬相关蛋白、凋亡相关蛋白和ZNF281。使用细胞计数试剂盒-8（CCK8）检测CRC细胞的活力和5-FU的半数抑制浓度（IC50）值。通过流式细胞术检测CRC细胞的凋亡情况。分别使用miRcode和Starbase软件预测miR-23b-3p与UCA1或ZNF281之间的结合位点，并通过双荧光素酶报告基因检测和RNA免疫沉淀（RIP）检测进行验证。建立小鼠异种移植模型以验证UCA1在体内对CRC细胞5-FU耐药性的作用。

结果

CRC对5-FU的耐药性与UCA1水平和自噬呈正相关。UCA1通过促进自噬和抑制凋亡加速CRC细胞对5-FU的耐药性。在293T细胞和CRC细胞中，miR-23b-3p是UCA1的靶标。敲低miR-23b-3p可逆转UCA1干扰对5-FU耐药性和自噬的抑制作用以及对CRC细胞凋亡的促进作用。在293T细胞中，ZNF281可与miR-23b-3p结合。在CRC细胞中，miR-23b-3p通过下调ZNF281提高5-FU敏感性。在体内，UCA1干扰通过miR-23b-3p/ZNF281轴增强CRC对5-FU的敏感性。