Parathyroid hormone type 1 receptor regulates osteosarcoma K7M2 Cell growth by interacting with angiotensinogen

Abstract This study aimed to determine the interactions between parathyroid hormone type 1 receptor (PTHR1) and angiotensinogen (AGT) and the effects of these agents on osteosarcoma (OS). We constructed a stably transfected mouse OS K7M2 cell line (shPTHR1‐ K7M2) using shRNA and knocked down AGT in these cells using siRNA‐AGT. The transfection efficiency and expression of AGT, chemokine C‐C motif receptor 3 (CCR3), and chemokine (C‐C motif) ligand 9 (CCL9) were determined using real‐time quantitative PCR. Cell viability and colony formation were assessed using Cell Counting Kit‐8 and crystal violet staining, respectively. Cell apoptosis and cycle phases were assessed by flow cytometry, and cell migration and invasion were evaluated using Transwell assays. Interference with PTHR1 upregulated the expression of AGT and CCR3, and downregulated that of CCL9, which was further downregulated by AGT knockdown. Cell viability, migration, invasion and colony formation were significantly decreased, while cell apoptosis was significantly increased in shPTHR1‐K7M2, compared with those in K7M2 cells (P < .05 for all). However, AGT knockdown further inhibited cell viability after 72 h of culture but promoted cell migration and invasion. PTHR1 interference decreased and increased the numbers of cells in the G0/G1 and G2/M phases, respectively, compared with those in K7M2 cells. Angiotensinogen knockdown increased the number of cells in the G0/G1 phase compared with that in the shPTHR1‐K7M2 cells. Therefore, PTHR1 affects cell viability, apoptosis, migration, invasion and colony formation, possibly by regulating AGT/CCL9 in OS cells.

particularly in metastatic tissues and samples from patients with recurrent OS samples. [4][5][6] Studies have indicated that PTHR1, can be activated by its ligands, including parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), and can then exert tumour-promoting effects. 7,8 Downregulating PTHR1 expression using shRNA inhibits OS cell proliferation and invasion, and reduces RANK ligand expression, thus suppressing tumour growth. 9 Additionally, Li et al 10 identified 1,163 differentially expressed genes (DEG) in tumour tissues from mice with PTHR1 knockdown and in tumour tissues from mice with control knockdown. A protein-protein interaction (PPI) network revealed that angiotensinogen (AGT) was the hub node gene, and it interacted with CC chemokine receptor 3 (CCR3) and chemokine (C-C motif) ligand 9 (CCL9) genes. However, interactions between PTHR1 and AGT remain unclear.
Angiotensinogen is the only precursor of angiotensin peptides and it is involved in the production of angiotensin II (AngII), which is the primary mediator of the renin-angiotensin system (RAS). 11 Angiotensin II participates in angiogenesis and regulates cell growth, apoptosis, migration, differentiation, extracellular matrix conformation and inflammation. [12][13][14] Angiotensin II simulation activates the epithelial-mesenchymal transition (EMT) in HepG2 cells. 15 The overexpression of AGT induces inflammation via the JAK/STAT signalling pathway, eventually inhibiting A549 cell proliferation and promoting bronchopulmonary dysplasia. 11 Thus, we speculated that downregulated AGT interacts with PTHR1 to reduce the inflammatory response and improve OS progression.
We investigated the effects of PTHR1 interference in mouse K7M2 OS cells and in a stably transfected shPTHR1-K7M2 cell line based on the K7M2 OS line. We also assessed the effects of AGT knockdown on shPTHR1-K7M2 cells. The findings should help to improve understanding of OS development and provide new targets for treating OS.
Subsequently, K7M2 cells (6 × 10 5 /well) were seeded into 6-well plates and cultured overnight. The medium was replaced with serum-free medium, and the cells were infected with the packaged lentivirus supernatant (50 μL). After culture for 2 h, the medium was replaced with complete medium, which was removed after 48 h of infection, and replaced with medium containing puromycin (2 μg/mL) to select resistant cells. 17 We then selected and expanded shPTHR1-K7M2 cells in culture. Transfection efficiency was assessed by measuring PTHR1 expression using real-time quantitative PCR (RT-qPCR). Table 1 shows the sequences of PTHR1.

| Cell transfection
The siRNA-angiotensinogen (si-AGT) and siRNA-negative control (si-NC) cells (Yanzai Biotechnology (Shanghai) Co., Ltd.) were transfected as described option. 18 The shPTHR1-K7M2 cells were seeded in 6-well plates and cultured overnight. The medium was then replaced with serum-free medium, and the shPTHR1-K7M2 cells were transfected with 20 nmol/L si-AGT or si-NC using Lipofectamine 300 (Thermo Fisher Scientific), essentially as described by the manufacturer. After 6 h of transfection, the serum-free medium was replaced with complete medium and cultured for another 48 h.

| Cell viability and cloning assays
Cell viability was determined using Cell Counting Kit-8 (CCK-8; Beyotime Biotechnology) as described by the manufacturer. Harvested cells (1 × 10 4 ) were seeded in 96-well plates, cultured for 24, 48, 72 and 96 h, then the CCK-8 reagent (10 μL) was added to the wells. Absorbance at 450 nm was measured 2 h later using a microplate reader.
We assessed colony formation by seeding cells into 6-well plates and incubating them under a 5% CO 2 atmosphere for 10 days. The supernatant was discarded when colonies became visible. The colonies were washed twice with PBS, then fixed in 4% paraformaldehyde at room temperature for 10 min. After two PBS washes, the colonies were stained with 0.5% crystal violet for 10 min, and visualized by microscopy.

| Cell apoptosis and cycle assays
Cell apoptosis was determined using Annexin V-FITC apoptosis assay kits (Beyotime Biotechnology) as described by the manufac- Cell cycle phases were also determined by flow cytometry. Cells were resuspended in PBS (200 μL), then chilled 70% ethyl alcohol (4 mL) was added. The cells were fixed at 4°C overnight, centrifuged at 1000 × g for 5 min, then washed with PBS and centrifuged. The sediment was incubated at 37°C for 30 min in PBS containing RNase (50 μg/mL), followed by PI (final concentration, 50 μg/mL) in the dark for 30 min. Cell cycles phases were determined using flow cytometry within 24 h.

| Cell migration and invasion assays
Cell migration and invasion of cells were evaluated using Transwell chambers (pore size 8 μm; Guangzhou Jet Bio-Filtration Co., Ltd.).
Transwell chambers were coated with matrix glue to assay cell invasion. Cells were inoculated into the upper inserts of Transwell chambers, and medium with 10% FBS was added into the lower chambers.
The cells were incubated for 48 h, fixed in 4% paraformaldehyde for 20 min, washed with PBS, and then incubated with crystal violet for 20 min. Excess stain was removed, and relative cell numbers were analysed on microscopy images. and CCL9 was calculated using the 2 −ΔΔCt method. 19

| Statistical analysis
All experiments were conducted in triplicate, and data are expressed as mean ± standard deviation (SD). All data were statistically analysed using GraphPad prism 5 (GraphPad Software Inc). Multiple groups were compared using one-way analyses of variance (ANOVA) followed by Tukey tests. Student t-tests was used for comparisons between two groups. Values with P < .05 were considered statistically significant.

| Screening stably transfected shPTHR1-K7M2 cells and effects of PTHR1 interference on AGT, CCR3 and CCL9 expression
Stably transfected cell lines were screened using puromycin, and and shRNA-4 (P < .05, Figure 1A). The expression of PTHR1 was significantly lower after transfection with PTHR1 shRNA-1 compared with the other groups (P < .05, Figure 1A). Therefore, we selected K7M2 cells that were stably transfected with PTHR1 shRNA-1 for subsequent experiments.
Levels of AGT, CCR3 and CCL9 expressed in K7M2 and shPTHR1-K7M2 cells were compared using RT-qPCR. The expression of AGT was significantly upregulated in shPTHR1-K7M2, compared with K7M2 cells (P < .05, Figure 1B). The expression of CCR3 was significantly upregulated after PTHR1 interference compared with K7M2 cells (P < .05, Figure 1C). The trend of CCL9 expression was inverse to that of CCR3 expression ( Figure 1D).

| Effects of PTHR1 interference on K7M2 cell viability, apoptosis, and cycle phase
The viability of shPTHR1-K7M2 cells at 24, 48, 72 and 96 h was significantly inhibited (P < .05, Figure 2A) and the apoptosis rate was significantly increased (P < .05, Figure 2B) compared with K7M2 cells.
The distribution of K7M2 and shPTHR1-K7M2 cells in S phase did not significantly differ (P > .05, Figure 2C). After PTHR1 interference, the

F I G U R E 3 Effects of PTHR1 interference on cell migration, invasion and colony formation. K7M2 and shPTHR-K7M2 cell migration (A)
and invasion (B) assessed using Transwells. (C) Colony formation by K7M2 and shPTHR-K7M2 cells. *: P < .05 vs. K7M2 cells numbers of cells in the G0/G1 and G2/M phases were significantly decreased and increased, respectively, compared with K7M2 cells (P < .05, Figure 2C). These results showed that PTHR1 interference inhibited K7M2 cell viability and promoted apoptosis by affecting the cell cycle.

| Effects of PTHR1 interference on the cell migration, invasion and colony formation in K7M2 Cells
Cell migration and invasion assessed using Transwells showed that the numbers of cells were significantly decreased after transfection with shPTHR1 compared with K7M2 cells (P < .05, Figure 3A and B).
These results suggested that the migration and invasion of shPTHR1-K7M2 cells were inhibited compared with K7M2 cells. Furthermore, significantly fewer colonies were generated by shPTHR1-K7M2, than K7M2 cells (P < .05, Figure 3C). This indicated that PTHR1 interference restrained the formation of cell colonies.

| AGT knockdown in the shPTHR1-K7M2 cells and the expression of related genes
We further explored the relationship between AGT and PTHR1 by knocking down the AGT gene in shPTHR1-K7M2 cells. The expression of AGT did not significantly differ between the blank control and si-NC groups (P > .05, Figure 4A). Transfection with si-AGT-1/2/3 significantly decreased AGT mRNA expression compared with the si-NC group (P < .05, Figure 4A). These results confirmed that AGT was appropriately knocked down in shPTHR1-K7M2 cells.

| Effects of AGT knockdown on shPTHR1-K7M2 cell viability, apoptosis and cycle phase
The viability of shPTHR1-K7M2 cells with and without AGT knockdown did not significantly differ after incubation for 24 and 48 h (P > .05, Figure 5A), but became significantly suppressed in shPTHR1-K7M2 cells with knockdown after incubation for 72 h and 96 h (P < .05, Figure 5A). Apoptosis rates did not significantly differ between shPTHR1-K7M2 cells with and without AGT knockdown (P > .05, Figure 5B). The number of cells in the G0/G1 phase was significantly increased in shPTHR1-K7M2 cells with AGT knockdown.
(P < .05, Figure 5C), but the distribution of the S and G2/M phases did not significantly differ between shPTHR1-K7M2 cells with and without AGT knockdown (P > .05, Figure 5C).

| Effects of AGT knockdown on the shPTHR1-K7M2 cell migration, invasion and colony formation
The Transwell results showed that compared with the shPTHR1-K7M2 cells, the number of shPTHR1-K7M2 cells with AGT knockdown was significantly increased (P < .05, Figure 6A,B). These results without AGT knockdown (P > .05, Figure 6C).

| D ISCUSS I ON
OS is the most prevalent primary malignancy of the skeletal system.
It is characterized by aggressive clinical development and high metastatic potential, and is the leading cause of death in adolescents. 20 Previous studies have indicated that PTHR1 interacts with AGT, and that PTHR1 expression is associated with the progression of OS. 6,10, 21 We created K7M2 stable transfection cell lines with PTHR1 knockdown using shRNA, then knocked down AGT in these lines using siRNA-AGT. Viability, migration, invasion and colony formation were all significantly decreased while cell apoptosis was evidently mice. 28 Therefore, we speculated that PTHR1 regulates cell growth and induces cell apoptosis by interacting with AGT.
The interactive genes of AGT (CCR3 and CCL9) were further determined by RT-qPCR. The hybrid G-protein-coupled receptor CCR3 interacts with various inflammatory chemokines, including high-affinity agonist eotaxin-1 (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26) and CCL5. 29 The processes of many pathological states such as Crohn's disease, 30  immunological processes process. 35 High levels of CCL9 are induced in Gr-1 + CD11B+ immature myeloid cells and in tumour-bearing mice before lung metastasis, and knockdown of CCL9 in bone marrow cells reduces the survival and metastasis of tumour cells. 36 We showed here that CCR3 expression was upregulated, and CCL9 expression was downregulated in cells with PTHR1 interference, and that AGT knockdown did not significantly affect CCR3 expression, but further downregulated CCL9 expression. These results indicated that PTHR1/AGT knockdown affects OS cell growth by regulating CCL9 expression.
This study had several limitations. The relationship between PTHR1 and CCL9 requires verification by a rescue experiment, and AGT, CCR3 and CCL9 protein expression should be assessed by western blotting. Our findings also require confirmation in animal models in vivo. Further studies of preclinical or clinical models are required. In conclusion, PTHR1 interference might inhibit cell viability, migration, invasion and colony formation and promote cell apoptosis by regulating AGT/CCL9 in OS cells, thus improving OS.
Our findings will help to enhance knowledge of OS pathogenesis and provide a theoretical basis for the treatment of OS using PTHR1 and AGT as therapeutic targets.

ACK N OWLED G M ENTS
We are grateful for the generous support by Liaoning Cancer Hospital & Institute (Shenyang) and Dalian Municipal Center Hospital Affiliated of Dalian Medical University (Dalian).

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in the current study are available from the corresponding authors upon reasonable request.