miR-1246靶向ATG4D调控自噬对宫颈癌发生发展的影响及机制研究

A number of studies have shown that microRNA(miRNA) regulates autophagy is closely ralated to the tumor progression. However, this mechanism is not clear. In our pilot study, we found that the cervical squamous cancer cell SiHa’proliferation and invasion ability were also enhanced after transfecting miR-1246 mimics in vitro experiments. Biological information displays that ATG4D is a target genes related with autophagy of miR-1246 , and it promotes the autophagy process with ATG3,ATG7.Through the analysis of the data of biological specimens,ATG4D was found lower expression in cervical cancer. Accordingly, we suspect that miR-1246 decrease autophagy activity by down regulated ATG4D expression, which promotes the progression of cervical cancer.To prove this,we plan to continue the study, including: identifying the target sites of miR-1246 on the ATG4D by dual-luciferase reporter system; through down or up regulating miR-1246/ATG4D expression, studing the invasiveness, proliferation and autophagy functions of SiHa cell via experiments in vivo and vitro, and verifying the regulation for downstream gene ATG3and ATG7;detecting the expression of miR-1246 and ATG4D in clinical specimens, expounds its relationship with the progression and prognosis of disease. Our subject is to explore the mechanism of miR-1246/ATG4D -ATG3-ATG7 autophagy pathways influence on occurrence and development of cervical squamous carcinoma, which could provide the new basis for the target of tumor therapy.

多项研究表明miRNA介导自噬参与肿瘤的发展，但作用机制还不清楚。我们前期研究发现：miR-1246能促进宫颈鳞癌SiHa细胞的生长和侵袭；生物信息显示ATG4D是miR-1246的一个自噬相关的靶基因，与ATG3、ATG7共同促进自噬的发生。生物标本库显示ATG4D在宫颈癌中呈低表达。我们假设：miR-1246通过下调ATG4D的表达，抑制自噬水平，从而促进宫颈癌的生长和转移。为了阐明假设，用双荧光素酶法明确miR-1246在ATG4D上的作用靶点；通过上调/下调ATG4D、miR-1246，研究SiHa细胞在体外和体内生长转移能力及自噬水平的变化，并验证其对下游基因ATG3、ATG7的调控；检测临床标本中miR-1246、ATG4D的表达，阐明其与疾病发展和预后的关系,探讨miR-1246/ATG4D-ATG3-ATG7自噬通路对宫颈癌发生发展的作用机制，为肿瘤的靶向治疗提供新依据。

多项研究表明miRNA介导自噬参与肿瘤的发生发展，但相关的作用机制尚不清楚。我们前期研究发现，下调miR-1246表达的宫颈癌SiHa细胞增殖、侵袭能力均增强。生物软件预测多个自噬相关蛋白（ATG4D、GAS1等）为miR-1246的靶基因，且生物数据库显示这些蛋白在宫颈癌组织中为低表达（通过筛选，明确GAS1作为靶基因）。本课题目的在于探讨miR-1246/GAS1自噬通路对宫颈鳞癌发生发展的影响及作用机制，为肿瘤的靶向治疗提供新依据。.主要研究有：.（1）证实miR-1246促进宫颈癌Siha细胞的自噬作用：检测miR-1246上调/下调后，宫颈癌Siha细胞的自噬关键蛋白的变化，LC3-II/LC3–I与miR-1246的表达正相关，而P62与miR-1246的表达负相关。.（2）明确GAS1为miR-1246的自噬相关的靶基因：检测上调/下调miR-1246后，细胞中GAS1蛋白的表达变化，并用双荧光素酶法确定GAS1作为miR-1246靶基因的具体靶点。.（3）证实GAS1抑制宫颈癌Siha细胞的自噬作用以及对细胞功能的抑制作用：GAS1过表达后，发现Siha细胞的LC3-II/LC3–I与GAS1的表达负相关，而P62与GAS1的表达正相关。CCK8、划痕愈合、transwell迁移及侵袭、裸鼠移植瘤实验表明， GAS1抑制了Siha细胞的增殖、迁移和侵袭能力。.（4）发现GAS1在宫颈鳞癌组织中为低表达，并与临床高危因素密切相关，且GAS1的低表达预示患者不良预后。 .（5）通过人全转录组芯片对GAS1下游通路进行初步筛选，利用显著性差异结合显著富集信号通路分析，选择MAPK signaling pathway作为GAS1下游通路的验证。Western blot检测发现，GAS1过表达组的ERK、JNK、P38表达降低，但p-ERK、p-JNK、p-P38的表达明显增高。