Nonmuscle Myosin II-C and its Isoform

本实验室先前的一份报告显示，新发现的非肌肉肌球蛋白重链II-C(NMHC II-C)包含一个由24个核苷酸组成的替代外显子，编码8个氨基酸。该外显子被剪接到NMHC II-C的环I中，存在于各种组织和细胞系中。与未插入的异构体相比，插入该外显子会导致肌动蛋白激活的镁ATPase活性和重链肌球蛋白II-C的体外运动性增加。RT-PCR分析表明，编码插入异构体的mRNA在许多人上皮性肿瘤细胞系中高表达，如肝细胞系HepG2、前列腺细胞系PC-3、胰腺癌细胞系PANC-1、乳腺癌细胞系MCF-7和肺细胞系A-549。有趣的是，当我们量化所有三种非肌肉肌球蛋白亚型(NMHC II-A、II-B和II-C)在肿瘤细胞系和正常细胞系中的表达水平时，插入的II-C在肿瘤细胞系中的表达比正常细胞系高(超过8倍)。相反，通过扫描免疫印迹检测，肿瘤细胞系中NMHC II-A和II-B的数量分别减少了3倍和4倍以上。对人类乳腺、肾脏和肺肿瘤的分析证实，与正常相关组织相比，这三种肿瘤中NMHC II-C的表达都有所增加。用siRNA使A549肺癌细胞系中插入的异构体减少90%，在156 h时抑制生长达80%，这种抑制几乎完全被外源表达的NMHC II-C所恢复，而非插入的II-C只能部分恢复。这些研究表明，NMHC II-C亚型可能在某些上皮性肿瘤细胞系中起作用。

A previous report from this laboratory revealed that the newly recognized nonmuscle myosin heavy chain II-C (NMHC II-C) contains an alternative exon composed of 24 nucleotides encoding 8 amino acids. This exon is spliced into loop I of NMHC II-C and is present in a wide variety of tissues and cell lines. Insertion of this exon leads to an increase in the actin-activated MgATPase activity and in vitro motility of heavy meromyosin II-C compared to the noninserted isoform. RT-PCR analysis reveals that the mRNA encoding the inserted isoform is highly expressed in many human epithelial tumor cell lines such as liver, HepG2; prostate, PC-3; pancreas, PANC-1; breast, MCF-7, and lung, A-549 cells. Interestingly, when we quantitated the expression level of all three nonmuscle myosin isoforms (NMHC II-A, II-B and II-C) in tumor cell lines vs normal cell lines, the expression of inserted II-C was elevated (greater than 8-fold) in the tumor cell lines compared to the normal cell lines. In contrast, NMHC II-A and II-B were decreased (greater than 3- and greater than 4-fold, respectively) in the tumor cell lines as determined by scanning immunoblots. Analysis of breast, kidney and lung tumors from humans confirmed the increase in NMHC II-C expression in all three tumors compared to their normal associated tissue. Using siRNA to reduce the inserted isoform by 90% in the A549 lung epithelial cancer cell line leads to 80% growth inhibition at 156 h. This inhibition of growth was almost completely rescued by exogenous expression of the inserted NMHC II-C, but only partially rescued by noninserted II-C. These studies suggest a putative role for the NMHC II-C isoform in certain epithelial tumor cell lines.