LncRNA AC087645调控SPHK1促进胰腺癌细胞增殖的分子机制

It is well-known that SPHK1 promotes tumor progression. LncRNA is involved in tumor progression; however, whether it participates in SPHK1 regulation is unclear. Under the support of previous NSFC, we found that miR-506 inhibited pancreatic adenocarcinoma cell proliferation via down-regulating SPHK1 (Article has been accepted). Bioinformatics analysis showed that lncRNA AC087645, a SPHK1 nearby genes, has several complementary sequences with miR-506 seed region and AC087645 and miR-506 expression was negatively correlated. Studies have shown that lncRNA could bind to miRNA by complementary base pairing and reduce the amount of free miRNA. Therefore, it is reasonable for us to speculate: AC087645 could bind to miR-506 by complementary base pairing and reduce the amount of free miR-506, up-regulate SPHK1 and finally promote the proliferation of pancreatic adenocarcinoma. We are planning to do the following experiments: to identify AC087645 and miR-506 seed binding region sequence by luciferase reporter gene, Pull-down and fluorescence in situ hybridization; to detect proliferation of pancreatic adenocarcinoma in cell lines and animal models and examine SPHK1 expression and its downstream molecule using lentiviral vector; to verify reversely. This study is expected to clarify the molecular mechanisms regarding AC087645 promoting proliferation of pancreatic adenocarcinoma, and provide new ideas for diagnosis and treatment of pancreatic adenocarcinoma.

已知SPHK1促进肿瘤进展。最近发现lncRNA参与肿瘤发生发展，而其是否参与SPHK1调控尚不清楚。前一项国家基金资助下发现miR-506下调SPHK1抑制胰腺癌增殖（文章已接收）。生物信息学分析发现：SPHK1基因附近的lncRNA AC087645与miR-506的种子区存在数个互补序列，且AC087645与miR-506表达呈明显负相关。研究表明lncRNA可经碱基互补配对吸附miRNA从而降低其游离量发挥效应。故推测：AC087645通过碱基互补配对吸附miR-506从而降低其游离量，上调SPHK1促进胰腺癌增殖。拟采用报告基因、Pull-down和荧光原位杂交证明并鉴定AC087645与miR-506种子区结合序列；采用慢病毒载体在细胞株及动物模型中检测胰腺癌增殖、SPHK1及下游分子表达并行反向验证。本研究有望阐明AC087645促进胰腺癌增殖的分子机制，为其防治提供新思路。

本课题顺利按时完成了相关的科研计划任务。进一步扩大样本检测了胰腺癌中lncRNA AC087645、miR-506、SPHK1的表达情况，分析其与患者分期分级、生存时间等临床资料的相关性。通过构建lncRNA慢病毒荧光表达载体，体外细胞增殖实验、克隆及流式细胞周期检测证明lncRNA AC087645促进胰腺癌细胞增殖；荧光素酶报告实验等证实lncRNA AC087645与miR-506种子区结合，降低miR-506游离量，上调SPHK1；裸鼠荷瘤模型中检测lncRNA促进胰腺癌生长及miR-506、SPHK1等下游分子表达，综合阐明lncRNA AC087645调控SPHK1促进胰腺癌增殖的机制。同时我们研究发现胰腺癌中DCLK1通过调控miRNA和EMT促进胰腺癌进展。通过相关实验成功培养博士研究生1名，硕士研究生1名，发表SCI论著2篇，申请重庆市科技新星培育工程项目1项（编号：KJXX2017022），申请国家发明专利 1 项（公开号：CN105497919A）。