Supplementary Material for: Long non-coding RNA brain cytoplasmic RNA 1 induces cisplatin-resistance of cervical cancer cells by sponging microRNA-330-5p and upregulating high-mobility group box 3

Objectives: To find out the function of long non-coding RNA (lncRNA) brain cytoplasmic RNA 1 (BCYRN1) in cisplatin (DDP)-resistance of cervical cancer (CC) cells. Design and Materials, Setting, Methods: BCYRN1 expression in CC and DDP-resistant cells was evaluated, with the association of BCYRN1 and prognosis analyzed. Then, DDP-resistant cells with BCYRN1 knockdown were established and the DDP-resistance of these cells was assessed. BCYRN1 subcellular localization was detected and confirmed. Besides, binding relation of BCYRN1 and microRNA (miR)- 330-5p and between miR-330-5p and high-mobility group box 3 (HMGB3) were examined and verified. Moreover, role of miR-330-5p and HMGB3 in the mechanism of BCYRN1 modulating DDP-resistance of CC cells was detected. In addition, xenograft transplantation was conducted to confirm the effect of BCYRN1 in CC cell DDP-resistance. Results: BCYRN1 was overexpressed in CC, which resulted in poor prognosis and DDP-resistance. BCYRN1 knockdown in DDP-resistant cells downregulated DDP-resistance. Mechanically, BCYRN1 sponged miR-330-5p to strengthen HMGB3 mRNA level. Besides, miR-330-5p underexpression or HMGB3 overexpression reversed the function of BCYRN1 knockdown in inhibiting DDP-resistance of CC cells. Eventually, BCYRN1 knockdown reduced DDP-resistance of CC cells in vivo. Limitations: There are still some deficiencies in the research; for example, whether there are other miRs working as the downstream genes of BCYRN1 in the ceRNA interaction is not fully clarified; nor the other downstream mechanism of miR-330-5p. Besides, the experimental findings and application into practice need extensive validation. Conclusions: BCYRN1 knockdown could disrupt DDP-resistance of CC cells through upregulating miR-330-5p to suppress HMGB3 mRNA level.

研究目的：探究长链非编码RNA（long non-coding RNA, lncRNA）脑胞质RNA 1（brain cytoplasmic RNA 1, BCYRN1）在宫颈癌细胞（cervical cancer, CC）顺铂（cisplatin, DDP）耐药中的作用。研究设计、材料、实验环境与方法：首先检测BCYRN1在宫颈癌及顺铂耐药细胞中的表达水平，并分析其与患者预后的相关性；随后构建BCYRN1敲低的顺铂耐药细胞株，评估该细胞株的顺铂耐药性；检测并验证BCYRN1的亚细胞定位；此外，验证BCYRN1与微小RNA（microRNA, miR）-330-5p的靶向结合关系，以及miR-330-5p与高迁移率族蛋白B3（high-mobility group box 3, HMGB3）的靶向结合关系；同时探究miR-330-5p与HMGB3在BCYRN1调控宫颈癌细胞顺铂耐药的分子机制中所发挥的作用；此外，通过异种移植瘤实验验证BCYRN1对宫颈癌细胞顺铂耐药的体内调控效果。研究结果：BCYRN1在宫颈癌组织及细胞中呈高表达，且与患者不良预后及顺铂耐药性密切相关；敲低BCYRN1可显著降低顺铂耐药细胞的顺铂耐药性。分子机制层面，BCYRN1作为miRNA分子海绵吸附miR-330-5p，从而上调HMGB3的mRNA表达水平；此外，miR-330-5p低表达或HMGB3过表达，可逆转BCYRN1敲低对宫颈癌细胞顺铂耐药的抑制作用。最终体内实验证实，敲低BCYRN1可降低宫颈癌细胞的顺铂耐药性。研究局限性：本研究仍存在若干不足，例如未完全阐明BCYRN1在内源竞争RNA（competing endogenous RNA, ceRNA）互作中是否存在其他下游miRNA靶点，亦未明确miR-330-5p的其他下游调控机制；此外，本研究的实验发现及临床转化应用仍需开展大规模验证。研究结论：敲低BCYRN1可通过上调miR-330-5p的表达水平、抑制HMGB3的mRNA表达，从而破坏宫颈癌细胞的顺铂耐药性。