ROCK2 is upregulated in OS tissue and cells.
To clarify the role of ROCK2 in the development of OS, ROCK2 expression was detected in OS and corresponding adjacent tissues. Following RT-qPCR and western blotting, the mRNA and protein levels of ROCK2 were revealed in tumour tissue (Fig. 1A and B). IHC was subsequently performed, the results of which revealed an increased expression of ROCK2 in cancer tissue. As presented in Fig. 1C, ROCK2 was overexpressed in 72.09% (31/43) of all OS tissues examined, while weak positive staining was observed in corresponding adjacent non-tumour tissues. The expression of ROCK2 in OS cell lines (MG-63, 143B U2-OS, and Saos-2) and in the normal cell line, hfoBI-19, was determined. The results revealed an increased expression of ROCK2 in OS cells (Fig. 1D and E). These data indicated that ROCK2 is overexpressed in OS tissue and cells.
ROCK2 promotes the growth of OS in vitro and in vivo
To further explore the functional role of ROCK2 in OS, ROCK2 shRNA was used to knockdown the expression of ROCK2 in U2OS and MG-63 OS cells (Fig. 2A and B; Fig. S1). As presented in Fig. 2C-D and Fig. S4 A-B, the results of the CCK8 and EdU assays revealed that ROCK2 downregulation significantly inhibited the growth of OS cells in vitro. Additionally, a colony formation assay was performed to detect OS cell clones in shROCK2- and shNC-treated cells (Fig. 2E). The results of flow cytometry demonstrated a significant increase in apoptosis following ROCK2 downregulation (Fig. 2F and Fig. S4 C). Additionally, the expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2-assocaited protein X was analysed via western blotting (Fig. S2). The expression of ROCK2 following ROCK2 overexpression was subsequently evaluated (Fig. 3A and B). As Presented in Fig. 3C-D and Fig. S5 A-B, OS cell proliferation was significantly increased in P-ROCK2 cells. Consistent with the aforementioned results, the colony formation assay revealed that ROCK2 overexpression increased OS cell growth (Fig. 3E). Furthermore, the results of flow cytometry indicated a significant decrease in apoptosis following ROCK2 upregulation (Fig. 3F and Fig. S5 C).
To confirm the role of ROCK2 in tumour formation in vivo, OS cells (U2-OS and 143B) were over- and under-expressed with ROCK2. The effect of ROCK2 on tumour formation in nude mice was subsequently established. After 5 weeks of growth, ROCK2-knockeddown U2-OS cells (U2-OS/shROCK2) exhibited reduced tumour growth in nude mice (Fig. 2H; Fig. S3). Consistent with this result, the average tumour weights of mice bearing U2-OS/shROCK2 cells were significantly decreased (Fig. 2I and J), while ROCK2-overexpressing 143B cells (143B/p-ROCK2) resulted in significantly increased tumour growth compared with respective control cells (Fig. 3H). Tumours in mice bearing 143B/p-ROCK2 cells were markedly increased in size compared with those of their respective controls (Fig. 3I and J). In summary, these data indicated that ROCK2 regulated the growth of OS in vivo and in vitro.
ROCK2 Affects The Level Of Glycolysis In OS Cells
Given that aerobic glycolysis is indicative of changes to cellular metabolism, this shift is ubiquitous in tumour cells. Therefore, the current study investigated whether ROCK2 affected the metabolism OS cells. As presented in Fig. 4A-D, glucose-6 phosphate levels, glucose consumption, lactate production and ATP levels were all significantly reduced in OS cells where ROCK2 expression was downregulated. In contrast, ROCK2 overexpression produced the opposite affects in Saos-2 cells (Fig. 4E-H).
To further confirm the role of ROCK2 in the glycolytic hydrolysis of OS, OS cell glycolysis was determined via ECAR. As presented in Fig. 4I, ROCK2 knockdown significantly decreased the glycolytic rate and capacity of U2-OS cells, whereas ROCK2 overexpression increased ECAR in Saos-2 cells (Fig. 4K). The current study assessed OCR, an indicator of mitochondrial respiration. The results revealed that U2-OS/shROCK2 cells exhibited an increased OCR (Fig. 4J), whereas ROCK2 overexpression produced the opposite effect in Saos-2 cells (Fig. 4L). In summary, the results indicated that ROCK2 may promote aerobic glycolysis and inhibit mitochondrial respiration in OS cells.
ROCK2 Positively Regulates HKII Expression
Hexokinase, a key enzyme in glycolysis, has been revealed to serve a crucial role in the regulation of glycolysis. Among the enzymes that participate in glycolysis, HKII is of particularly importance. Previous studies have confirmed that HKII is upregulated in liver , colorectal  and lung cancer . Since HKII is closely associated with the proliferation of tumour cells and in order to confirm the expression of HKII in OS, the current study detected the expression of HKII in OS tissues via RT-qPCR and western blotting (Fig. 5A-C). The results of IHC confirmed the increased expression of HKII in OS (Fig. 5D). Scatter plots revealed that ROCK2 and HKII mRNA and protein levels were positively correlated with OS tissues (Fig. 5E and F). The expression of HKII in OS cells was further confirmed via RT-qPCR and western blotting (Fig. 5G-H).
To verify whether ROCK2 regulates HKII expression, ROCK2 was up- and down-regulated in OS cells, the results of which demonstrated that ROCK2 knockdown significantly reduced the mRNA and protein expression of HKII in U2-OS and MG-63 cells. In contrast, ROCK2 overexpression significantly increased HKII protein and mRNA levels in Saos-2 and 143B cells (Fig. 5I and J). Taken together, these results indicated that ROCK2 positively regulates the expression of HKII in OS cells.
ROCK2 regulates glycolysis and cell proliferation depending on the expression of HKII
As aforementioned, HKII was determined to be a downstream gene of glycolysis. The current study therefore upregulated the expression of HKII in U2-OS cells that were previously treated to knockdown ROCK2. Successful HKII overexpression was confirmed via immunoblotting (Fig. 6A). The results of CCK8 and EdU assays revealed that HKII overexpression restored the proliferation of shROCK2-treated cells (Fig. 6B-C and Fig. S6 A). The results also demonstrated that HKII overexpression restored glucose-6 phosphate levels, glucose consumption, lactate production and ATP levels in shROCK2-treated cells (Fig. 6D-G).
The expression of HKII was silenced in p-ROCK2 OS cells. Western blot analysis confirmed that HKII expression was increased in P-ROCK2 cells and that its expression was inhibited following HKII silencing (Fig. 6H). Similarly, OS cell proliferation was also inhibited (Fig. 6I-J and S6 B). Further studies confirmed that silencing HKII in P-ROCK2 inhibited glucose-6 phosphate levels, glucose consumption, lactate production and ATP levels (Fig. 6K-N). In conclusion, ROCK2 regulated glucose metabolism and proliferation in OS cells depending on the expression of HKII.
ROCK2 regulates HKII expression by activating the phosphorylated PI3K/AKT signalling pathway
To further investigate the mechanism by which ROCK2 regulates HKII expression, it was determined whether ROCK2 and HKII could be intrinsically bound. However, the results of Co-IP revealed no intrinsic link between them (Fig. 7A). Additionally, it has been reported that HKII is affected by PI3K/AKT signalling pathways  and that AKT phosphorylates HKII at Thr-473 . Previous studies have also confirmed that ROCK2 acts as a protein kinase that affects the phosphorylation of substrate proteins [16, 17]. Therefore, the current study hypothesized that ROCK2 may promote HKII expression by activating the PI3K/AKT signalling pathway. To verify this hypothesis, ROCK2 expression was reduced and immunoblotting revealed that total AKT and PI3K levels did not change. However, levels of phosphorylated AKT and PI3K decreased significantly (Fig. 7B). Furthermore, AKT and PI3K phosphorylation was significantly increased in cells in which ROCK2 was upregulated (Fig. 7C). To further verify whether ROCK2 regulates the expression of HKII by activating phosphorylated PI3K/AKT and whether it affects OS growth, ROCK2 was overexpressed and OS cells were treated with the PI3K inhibitor LY294002 (10 µM). Western blot analysis confirmed that HKII expression was inhibited (Fig. 7D). The results of the CCK8 and EdU assay also indicated that OS growth was inhibited (Fig. 7E and F; Fig. S4). The results indicated that ROCK2 activated AKT and PI3K via phosphorylation, which induced HKII expression and further promoted the growth of OS cells.