Kindlin-2‒miR-1258‒HPSE信号轴调控细胞骨架结构促肝细胞癌转移的作用及机制研究

The Kindlin family is an essential regulator required for integrin activation and signal transduction. Kindlin-2, a member of the Kindlin family, has been found to correlate with cancer progression; however, the mechanisms underlying the involvement of Kindlin-2 in cancer invasion and metastasis have not been demonstrated. Our previous in-vivo, ex-vivo and in-vitro studies showed that Kindlin-2 increased the migration of cancer cells and promoted hepatocellular carcinoma invasion and metastasis, and high-throughput sequencing revealed a strong molecular network among Kindlin-2, integrin, miR-1258, HPSE and cytoskeleton, which was involved in the mediation of cancer cells. Based on our previous studies, this project aims to investigate the effect of Kindlin-2 on the expression of integrin subunits, PI3K/AKT signaling molecules and cytoskeleton related proteins, and explore the molecular mechanisms underlying the Kindlin-2/miR-1258/HPSE axis promoting hepatocellular carcinoma invasion and metastasis, and finally, the role of Kindlin-2/miR-1258/HPSE axis in hepatocellular carcinoma invasion and metastasis will be validated in clinical specimens, so as to elucidate the mechanisms underlying the mediation of Kindlin-2/integrin crosstalk in cancer progression. The results of this project will provide theoretical basis for developing novel target drugs and provide new insights into the precision diagnosis, treatment and follow-up of hepatocellular carcinoma.

Kindlin家族是整合素（integrin）活化与信号转导必要的调节因子，其成员Kindlin-2与肿瘤恶性进展有关，但具体作用机制尚未完全阐明。课题组前期通过临床标本、细胞和动物实验证实：Kindlin-2可增强癌细胞运动迁移能力，促进肝癌侵袭转移；高通量测序及预实验结果提示：Kindlin-2与integrin、miR-1258、HPSE和细胞骨架之间存在紧密的分子网络关系参与对癌细胞的调控。本项目是在此基础上，进一步检测Kindlin-2对integrin各亚型、PI3K/AKT信号分子及骨架相关蛋白的表达调控，深入阐明Kindlin-2‒miR-1258‒HPSE信号轴促肝癌侵袭转移的作用及具体机制，并最终在临床标本中进行验证。从全新角度揭示Kindlin-2协同integrin对肿瘤恶性进展过程的调控，为新型靶向药物开发奠定理论基础，也为肝癌精准诊断、治疗和随访提供新思路。

背景 肝细癌 (HCC) 中Kindlin-2表达上调与肝细胞癌 (HCC) 转移和预后不良相关，本项目研究Kindlin-2在HCC中发挥作用的分子机制。.方法 通过microRNA测序探索Kindlin-2下游信号通路。应用亚硫酸盐测序、荧光素酶报告实验、qRT-PCR和回复实验等验证了Kindlin-2-miR-1258-TCF4信号轴，通过启动子活性分析和染色质免疫沉淀等证实TCF4与Kindlin-2启动子区结合。.结果 miRNA 测序发现miR-1258可能是Kindlin-2下游的效应分子。Kindlin-2敲降后miR-1258表达增加，Kindlin-2过表达后miR-1258表达减少，进而转录因子7 类似物2（TCF7L2 或 TCF4）被证实为miR-1258的靶基因，HCC中Kindlin-2可通过表观遗传抑制miR-1258进而上调TCF4的表达。此外，TCF4可与Kindlin-2的启动子区结合并增强其转录。因此，Kindlin-2-miR-1258-TCF4形成了一个正反馈调节环路。细胞功能实验和动物实验证实miR-1258和TCF4在体HCC细胞体外迁移和体内转移中起到关键作用。在HCC组织中，Kindlin-2的表达与miR-1258的表达呈负相关，与TCF4的表达呈正相关，同时，miR-1258的表达与TCF4的表达呈负相关。.结论 本项目阐明了Kindlin-2的一条全新的不依赖于intergrin的信号通路，Kindlin-2-miR-1258-TCF4环路可调控HCC侵袭和转移，Kindlin-2亦有望成为HCC的治疗靶点。