Objective: To detect the effect and regulatory mechanism of human ether à go-go related gene 1 (Herg 1) knockdown on the proliferation and invasion of osteosarcoma (OS). Methods: We constructed a recombinant adenovirus vector (Ad5-Herg1-shRNA) expressing short hair RNA (shRNA) against Herg1 and tested the knockdown efficiency. Then, the effects of Herg 1 knockdown on the proliferation, growth and invasion of osteosarcoma were measured by using cell counting kit-8 (CCK-8), wound healing assay, Transwell assay and xenograft model of nude mice, respectively. Tandem affinity purification, mass spectrometry and dual luciferase reporter assay were used to find out the molecules interacted with Herg1. Western blot was used to detect the expressions of large tumor suppressor gene (LATS1), p-LATS1, Yes-associated protein (YAP) and p-YAP in cells after infection of Ad5-Herg1-shRNA. Results: Compared to Ad5-control-shRNA, Ad5-Herg1-shRNA dramatically inhibited the expression of Herg1 in OS cells. The result of CCK8 array demonstrated that 143B cell vitalities of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (65.47±3.90)% and (79.90±1.52)%, significantly lower than (100.00±6.14)% of Ad5-control-shRNA group. Meanwhile, U2OS cell vitality of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (69.69±1.36)% and (76.72±2.75)%, significantly lower than (100.00±3.01)% of Ad5-control-shRNA group (all P<0.001). The results of wound healing array showed that 143B cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (33.03±2.88)% and (36.47±4.16)%, significantly lower than (97.78±2.28)% of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (68.07±0.90)% and (73.97±1.25)%, significantly lower than (96.50±1.12)% of Ad5-control-shRNA group (all P<0.001). The results of Transwell showed that 143B cell invasion numbers of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 36.50±12.15 and 44.83±7.62, significantly lower than 195.33±19.68 of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 21.83±7.99 and 22.85±7.08, significantly lower than 83.33±12.36 of Ad5-control-shRNA group (all P<0.001). The results of xenograft model of OS showed that the tumor volume and weight of Ad5-Herg1-shRNA group were significantly smaller than of Ad5-control-shRNA group after 14 days and 5 weeks of inoculation, respectively (P<0.001). Moreover, knockdown of Herg1 inhibited the metastasis of OS cells. In mechanism, Herg1 protein interacted with NF2 protein. Knockdown of Herg1 significantly suppressed the expression levels of LATS1 and YAP protein, and promoted the phosphorylation of LATS1 and YAP in OS cells (all P<0.001). Conclusion: Our findings suggest that Herg1 participates in the proliferation and motility of OS cells and may serve as a potential therapeutic target for osteosarcoma patients.
目的： 观察沉默Herg1的表达对骨肉瘤增殖和侵袭的影响，并探讨其可能的调控机制。 方法： 构建重组腺病毒载体Ad5－Herg1－shRNA并检测其转染效能。分别采用细胞计数试剂盒8(CCK－8)法、划痕实验、Transwell实验、肿瘤生长曲线和活体成像检测Ad5－Herg1－shRNA转染后人骨肉瘤细胞株143B、U2OS的增殖、迁移和侵袭能力变化，采用串联亲和纯化/质谱法寻找可能与Herg1相互作用的蛋白并进行鉴定，采用Western blot法检测转染前后骨肉瘤细胞中大肿瘤抑制因子1(LATS1)、Yes激酶相关蛋白(YAP)及其磷酸化后的表达变化。 结果： Ad5－Herg1－shRNA转染能有效抑制骨肉瘤细胞中Herg1的表达。CCK－8法检测结果显示，Ad5－Herg1－shRNA1组和Ad5－Herg1－shRNA2组143B细胞的存活率分别为(65.47±3.90)%和(79.90±1.52)%，U2OS细胞的存活率分别为(69.69±1.36)%和(76.72±2.75)%，均明显低于Ad5－control－shRNA组(均P<0.001)。划痕实验显示，Ad5－Herg1－shRNA1组和Ad5－Herg1－shRNA2组143B细胞的迁移率分别为(33.03±2.88)%和(36.47±4.16)%，U2OS细胞的迁移率分别为(68.07±0.90)%和(73.97±1.25)%，均明显低于Ad5－control－shRNA组(均P<0.001)。Transwell侵袭实验显示，Ad5－Herg1－shRNA1组和Ad5－Herg1－shRNA2组143B细胞的侵袭细胞数分别为(36.50±12.15)个和(44.83±7.62)个，U2OS细胞的侵袭细胞数分别为(21.83±7.99)个和(22.85±7.08)个，均明显低于Ad5－control－shRNA组(均P<0.001)。体内实验结果显示，从第14天起，Ad5－Herg1－shRNA1组裸鼠骨肉瘤体积小于Ad5－control－shRNA组(P<0.001)。接种5周后，Ad5－Herg1－shRNA1组瘤重为(0.36±0.10)g，低于Ad5－control－shRNA组(P<0.001)。裸鼠活体成像显示，Ad5－control－shRNA组明显可见其他部位肿瘤转移且荧光信号值高，而Ad5－Herg1－shRNA1未见其他部位的肿瘤转移。Herg1蛋白与2型神经纤维瘤(NF2)蛋白相互作用，沉默Herg1可明显下调骨肉瘤中LATS1和YAP蛋白的表达，并促进LATS1和YAP的磷酸化。 结论： Herg1可能通过调控Hippo信号通路参与骨肉瘤的增殖和侵袭过程，其有望成为骨肉瘤治疗的潜在靶点。.
Keywords: Herg1; Hippo signaling pathway; Motility; Osteosarcoma; Proliferation.