■这项研究对结直肠癌(CRC)中CXCL1依赖性调节机制进行了系统描述。
■应用生物信息学方法获得靶mRNACXCL1和相应的上游miRNA。进行qRT-PCR和Western印迹以测量CRC组织和细胞中CXCL1和miR-302e的水平。实验包括CCK-8,伤口愈合试验,Transwell侵袭试验,和流式细胞术评估细胞生物学行为。为了验证CXCL1和miR-302e之间的靶向关系,进行了双荧光素酶报告基因测定。JAK-STAT信号通路的抑制剂AG490用于鉴定CXCL1/JAK-STAT在CRC进展中的作用机制。和肿瘤异种移植实验进行进一步验证。
■CXCL1在CRC组织和细胞中高表达,而miR-302e表达较差。沉默CXCL1或过表达miR-302e可导致细胞增殖抑制,迁移,侵袭但促进CRC细胞凋亡。此外,CXCL1被鉴定为miR-302e的直接靶标,CXCL1可以逆转miR-302e对细胞增殖的影响,迁移,入侵,和凋亡。此外,CXCL1通过激活JAK-STAT信号通路对CRC细胞生物学行为起作用。
■CXCL1可能受miR-302e调控而使JAK-STAT信号通路失活,进而影响细胞增殖,迁移,入侵,以及CRC的凋亡。我们的结果为CRC治疗提供了潜在的治疗靶点。
OBJECTIVE: This study made a systemic description for the CXCL1-dependent regulatory mechanism in colorectal cancer (CRC).
METHODS: Bioinformatics methods were applied to obtain target mRNA CXCL1 and corresponding upstream miRNA. qRT-PCR and Western blot were performed to measure the levels of CXCL1 and miR-302e in CRC tissue and cells. Experiments including CCK-8, wound healing assay, Transwell invasion assay, and flow cytometry were conducted to assess cell biological behaviors. Dual-luciferase reporter assay was carried out for verification of the targeting relationship between CXCL1 and miR-302e. The inhibitor AG490 of JAK-STAT signaling pathway was used to identify the functional mechanism of CXCL1/JAK-STAT underlying progression of CRC, and tumor xenograft experiments were performed for further validation.
RESULTS: CXCL1 was highly expressed in CRC tissue and cells, while miR-302e was poorly expressed. Silencing CXCL1 or overexpressing miR-302e could lead to inhibition of cell proliferation, migration, invasion but promotion of cell apoptosis of CRC. Besides, CXCL1 was identified as a direct target of miR-302e, and CXCL1 could reverse the effect of miR-302e on cell proliferation, migration, invasion, and apoptosis. Furthermore, CXCL1 functioned on CRC cell biological behaviors via activation of JAK-STAT signaling pathway.
CONCLUSIONS: CXCL1 could be regulated by miR-302e to inactivate JAK-STAT signaling pathway, in turn affecting cell proliferation, migration, invasion, and apoptosis of CRC. Our result provides a potential therapeutic target for CRC treatment.