Tian Ke, Sun Dongxia, Chen Min, Yang Yifei, Wang Fang, Guo Taotao, Shi Zhimin
Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan 056000, Hebei Province, People's Republic of China.
Department of Pathology, Handan Maternal and Child Health Hospital, Handan 056000, Hebei Province, People's Republic of China.
Onco Targets Ther. 2020 Feb 12;13:1277-1287. doi: 10.2147/OTT.S225661. eCollection 2020.
Although X-inactive specific transcript (XIST) is known to play a critical role in the pathogenesis of melanoma, the mechanisms through which this remains unclear.
RNAseq, immunohistochemistry, and qRT-PCR were used to identify the levels of XIST, miR-139-5p, and Rho-Associated Coiled-Coil Containing Protein Kinase-1 (ROCK1) in melanoma tissues and cells. A subcellular fractionation assay was used to determine the location of XIST. CCK-8 and colony formation assays were used to evaluate cellular proliferation. Cell migration and wound healing assays were used to detect the effects on cell migration. RNA pull-down was used to confirm the interaction between XIST and miR-139-5p. Besides, the xenograft tumor experiment was performed to further verify the roles of XIST in melanoma.
In this study, an increased level of XIST was revealed in melanoma tissues and cells, which was associated with higher TNM stage and positive lymph node metastasis. XIST was found to function as a "molecular sponge" of miR-139-5p to facilitate cellular functions. Moreover, these consequences could be partially reversed by inhibition of miR-139-5p. MiR-139-5p was found to target ROCK1 directly, leading to suppression of ROCK1 expression; this effect could be partially reversed by inhibiting XIST expression. Furthermore, the deletion of ROCK1 induced anti-oncogenic effects similar to those seen with knockout of XIST. Upregulation of miR-139-5p and knockdown of XIST could inhibit cell functions in melanoma.
Our findings suggested that the lncRNA XIST facilitates cellular functions in melanoma via the miR-139-5p/ROCK1 pathway.
虽然已知X染色体失活特异性转录本（XIST）在黑色素瘤发病机制中起关键作用，但其具体机制尚不清楚。
采用RNA测序、免疫组织化学和定量逆转录聚合酶链反应（qRT-PCR）来检测黑色素瘤组织和细胞中XIST、miR-139-5p和含Rho相关卷曲螺旋蛋白激酶-1（ROCK1）的水平。采用亚细胞分级分离试验来确定XIST的定位。采用细胞计数试剂盒-8（CCK-8）和集落形成试验来评估细胞增殖。采用细胞迁移和伤口愈合试验来检测对细胞迁移的影响。采用RNA下拉试验来证实XIST与miR-139-5p之间的相互作用。此外，进行异种移植瘤实验以进一步验证XIST在黑色素瘤中的作用。
在本研究中，黑色素瘤组织和细胞中XIST水平升高，这与更高的TNM分期和阳性淋巴结转移相关。发现XIST作为miR-139-5p的“分子海绵”来促进细胞功能。此外，抑制miR-139-5p可部分逆转这些结果。发现miR-139-5p直接靶向ROCK1，导致ROCK1表达受抑制；抑制XIST表达可部分逆转这种作用。此外，敲除ROCK1诱导的抗肿瘤作用类似于敲除XIST所见的作用。上调miR-139-5p和敲低XIST可抑制黑色素瘤细胞的功能。
我们的研究结果表明，长链非编码RNA XIST通过miR-139-5p/ROCK1途径促进黑色素瘤细胞的功能。
