Nonmuscle Myosin II-C and its Isoform

本实验室先前的报告显示，新识别的非肌肉肌球蛋白重链II-C（NMHC II-C）包含一个由24个核苷酸编码8个氨基酸组成的选择性外显子。该外显子剪接到NMHC II-C的环I中，并且存在于多种组织和细胞系中。插入这个外显子导致肌动蛋白激活的MgATP酶活性的增加，并在体外运动的重裂肌球蛋白II-C相比，noninserted亚型。RT-PCR分析显示，编码插入的同种型的mRNA在许多人上皮肿瘤细胞系中高度表达，如肝HepG 2、前列腺PC-3、胰腺PANC-1、乳腺MCF-7和肺A-549细胞。有趣的是，当我们定量肿瘤细胞系与正常细胞系中所有三种非肌肉肌球蛋白亚型（NMHC II-A，II-B和II-C）的表达水平时，与正常细胞系相比，肿瘤细胞系中插入的II-C的表达升高（大于8倍）。相比之下，NMHC II-A和II-B在肿瘤细胞系中减少（分别大于3倍和大于4倍），如通过扫描免疫印迹所确定的。对来自人类的乳腺、肾脏和肺肿瘤的分析证实，与其正常相关组织相比，所有三种肿瘤中NMHC II-C表达增加。使用siRNA将A549肺上皮癌细胞系中插入的同种型减少90%，导致在156 h时80%的生长抑制。这种生长抑制几乎完全由插入的NMHC II-C的外源性表达拯救，但只有部分由非插入的II-C拯救。这些研究表明NMHC II-C亚型在某些上皮肿瘤细胞系中的假定作用。

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.