[Downregulation of Herg1 suppresses osteosarcoma proliferation and invasion by targeting Hippo signaling pathway].

ABSTRACT

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.