Abstract：
KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we investigated the loss of KANK1 function by performing CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and the less the wild-type KANK1 dosage, the greater the number of cells with abnormal numbers of centrosomes and rates of cell-doubling and apoptosis, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. The rescue of KANK1-knockdown cells with a KANK1-expressing plasmid restored the rates of cells exhibiting centrosomal amplification to the control level. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2, and BMP3, which have been reported to show haploinsufficiency when they function. When EGR1 protein expression was reduced by siRNA technology, the number of cells exhibiting centrosomal amplification increased, along with the reduction of KANK1 protein expression, suggesting their functional relationship. Thus, KANK1 haploinsufficiency may contribute to centrosome aberrations through the network of haploinsufficiency-related genes.
摘要：
基于肾细胞癌的频繁缺失和肿瘤细胞增殖的抑制活性，发现KANK1是一种抑癌基因。以前,我们报道了KANK1敲低诱导中心体扩增，导致细胞分裂异常,通过过度激活RhoA小GTP酶。这里,我们通过进行基于CRISPR/Cas9的基因组编辑以敲除基因,研究了KANK1功能的缺失.经过几轮基因组编辑，然而,没有完全丧失KANK1的细胞系，野生型KANK1剂量越少，具有异常中心体数量的细胞数量以及细胞倍增和凋亡的速率越多，提示KANK1单倍体功能不全参与中心体畸变。用表达KANK1的质粒拯救KANK1敲低细胞将显示中心体扩增的细胞的速率恢复到对照水平。对具有减少剂量的功能性KANK1的细胞的RNA测序分析揭示了其他细胞增殖相关基因的潜在参与。如EGR1，MDGA2和BMP3，据报道它们在起作用时显示单倍体不足。当通过siRNA技术降低EGR1蛋白表达时，显示中心体扩增的细胞数量增加，随着KANK1蛋白表达的减少，暗示他们的功能关系。因此，KANK1单倍体不足可能通过单倍体不足相关基因网络导致中心体畸变。
