RNA sequencing of Rnaseh2c knockdown Mvt1 mammary cancer cells

To better understand the etiology of metastatic disease, we investigated the impact of genetic polymorphism on metastatic progression. Using RNAseq and exome data analysis, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. Mechanistic studies demonstrated that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity and it functions through engagement of the T cell-mediated adaptive immune response. Mvt1 mouse mammary carcinoma cells with an shRNA-mediated knockdown of Rnaseh2c were generated along with Mvt1 cells expressing a scramble control shRNA. Cell lines or primary mammary tumors generated from syngeneic orthotopic implantations of the control and knockdown cells were anlayzed. mRNA seq samples included RNA from 3 tumors, 1 from each of 3 experiments, for both scramble and knockdown (sh4). Total RNA seq samples included RNA from 3 biological replicates of cultured control or knockdown Mvt1 cells. Exome seq samples included DNA from 4 pulmonary metastases from 3 mice each scramble and knockdown.

为深入阐明转移性疾病的致病机制，本研究探究了遗传多态性对肿瘤转移进程的影响。通过RNA测序（RNAseq）与外显子组测序（exome seq）分析，我们鉴定出异源三聚体核糖核酸酶H2（RNase H2）核糖核酸内切酶复合物的支架蛋白Rnaseh2c，其为新型转移易感因子。机制研究显示，Rnaseh2c在转移性疾病中的功能不依赖RNase H2的酶促活性，而是通过激活T细胞介导的适应性免疫应答发挥作用。本研究构建了经短发夹RNA（shRNA）介导Rnaseh2c敲低的Mvt1小鼠乳腺癌细胞系，同时构建了携带乱序对照短发夹RNA的Mvt1对照细胞系。对由对照与敲低细胞经同基因原位移植所获得的细胞系及原代乳腺肿瘤样本开展了分析。mRNA测序样本包含乱序对照组与Rnaseh2c敲低（sh4）组的肿瘤组织RNA：每组各3例，分别来自3次独立实验的单份样本。总RNA测序样本则涵盖培养状态下的对照或敲低Mvt1细胞的3份生物学重复样本的总RNA。外显子组测序样本包含两组的肺转移灶DNA：每组均取自3只小鼠的4个肺转移灶，即乱序对照组与敲低组各有来自3只小鼠的4份肺转移灶DNA样本。