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Abstract：
Metastasis is one of the key factors of treatment failure in late-stage colorectal cancer (CRC). Metastatic CRC frequently develops resistance to chemotherapeutic agents. This study aimed to identify the novel regulators from \"hidden\" proteins encoded by long noncoding RNAs (lncRNAs) involved in tumor metastasis and chemoresistance. Methods: CRISPR/Cas9 library functional screening was employed to identify the critical suppressor of cancer metastasis in highly invasive CRC models. Western blotting, immunofluorescence staining, invasion, migration, wound healing, WST-1, colony formation, gain- and loss-of-function experiments, in vivo experimental metastasis models, multiplex immunohistochemical staining, immunohistochemistry, qRT-PCR, and RT-PCR were used to assess the functional and clinical significance of FOXP3, PRDM16-DT, HNRNPA2B1, and L-CHEK2. RNA-sequencing, co-immunoprecipitation, qRT-PCR, RT-PCR, RNA affinity purification, RNA immunoprecipitation, MeRIP-quantitative PCR, fluorescence in situ hybridization, chromatin immunoprecipitation and luciferase reporter assay were performed to gain mechanistic insights into the role of PRDM16-DT in cancer metastasis and chemoresistance. An oxaliplatin-resistant CRC cell line was established by in vivo selection. WST-1, colony formation, invasion, migration, Biacore technology, gain- and loss-of-function experiments and an in vivo experimental metastasis model were used to determine the function and mechanism of cimicifugoside H-1 in CRC. Results: The novel protein PRDM16-DT, encoded by LINC00982, was identified as a cancer metastasis and chemoresistance suppressor. The down-regulated level of PRDM16-DT was positively associated with malignant phenotypes and poor prognosis of CRC patients. Transcriptionally regulated by FOXP3, PRDM16-DT directly interacted with HNRNPA2B1 and competitively decreased HNRNPA2B1 binding to exon 9 of CHEK2, resulting in the formation of long CHEK2 (L-CHEK2), subsequently promoting E-cadherin secretion. PRDM16-DT-induced E-cadherin secretion inhibited fibroblast activation, which in turn suppressed CRC metastasis by decreasing MMP9 secretion. Cimicifugoside H-1, a natural compound, can bind to LEU89, HIS91, and LEU92 of FOXP3 and significantly upregulated PRDM16-DT expression to repress CRC metastasis and reverse oxaliplatin resistance. Conclusions: lncRNA LINC00982 can express a new protein PRDM16-DT to function as a novel regulator in cancer metastasis and drug resistance of CRC. Cimicifugoside H-1 can act on the upstream of the PRDM16-DT signaling pathway to alleviate cancer chemoresistance.
摘要：
转移是晚期结直肠癌（CRC）治疗失败的关键因素之一。转移性CRC经常对化学治疗剂产生抗性。这项研究旨在从涉及肿瘤转移和化学抗性的长链非编码RNA（lncRNAs）编码的“隐藏”蛋白中鉴定新的调节因子。方法：采用CRISPR/Cas9文库功能筛查来鉴定高侵袭性CRC模型中癌症转移的关键抑制剂。西方印迹,免疫荧光染色，入侵,迁移,伤口愈合，WST-1，集落形成，功能增益和丧失实验，体内实验转移模型，多重免疫组织化学染色，免疫组织化学，qRT-PCR,和RT-PCR用于评估FOXP3，PRDM16-DT，HNRNPA2B1和L-CHEK2。RNA测序，免疫共沉淀,qRT-PCR,RT-PCR,RNA亲和纯化，RNA免疫沉淀，MeRIP-定量PCR,荧光原位杂交,进行染色质免疫沉淀和荧光素酶报告基因测定以获得对PRDM16-DT在癌症转移和化学耐药性中的作用的机制见解。通过体内选择建立了耐奥沙利铂的CRC细胞系。WST-1，集落形成，入侵,迁移,Biacore技术,使用功能增益和功能丧失实验以及体内实验转移模型来确定升麻苷H-1在CRC中的功能和机制。结果:新蛋白PRDM16-DT,由LINC00982编码，被鉴定为癌症转移和化学耐药抑制因子。PRDM16-DT水平下调与CRC患者的恶性表型和不良预后呈正相关。在FOXP3的转录调控下，PRDM16-DT直接与HNRNPA2B1相互作用，并竞争性降低HNRNPA2B1与CHEK2外显子9的结合，从而形成长CHEK2（L-CHEK2），随后促进E-cadherin分泌。PRDM16-DT诱导的E-cadherin分泌抑制成纤维细胞活化，进而通过减少MMP9分泌抑制CRC转移。菊苣苷H-1，一种天然化合物，可以结合FOXP3的LEU89，HIS91和LEU92，并显着上调PRDM16-DT表达以抑制CRC转移并逆转奥沙利铂耐药。结论：lncRNALINC00982可表达一种新的蛋白PRDM16-DT，在CRC的肿瘤转移和耐药中起新的调控作用。升麻苷H-1可作用于PRDM16-DT信号通路的上游,减轻肿瘤化疗耐药。
