Downregulation of CD44 Inhibits Proliferation, Invasion and Migration of Osteosarcoma Cells by Regulating the Expression of Cathepsin S


 BackgroundOsteosarcoma (OS) is a malignant bone tumour of mesenchymal origin. These tumours are characterised by rich vascularisation, therefore promoting rapid proliferation and facilitating metastasis. CD44 has been reported to be involved in OS, but its role and molecular mechanisms in the pathogenesis of the disease are not fully determined. MethodsIn this study, we investigated the antitumor effect of CD44 on the development of OS and further explored the molecular mechanisms. The expression of CD44, cathepsin S and MMP-9 was detected by Western blot (WB) and reverse transcription-polymerase chain reaction (RT-qPCR) in different cell lines (MG63, U2OS OS and hFOB 1.19). To elucidate the role of CD44 in OS, MG63 and U2OS cells were treated with small interference RNA (siRNA) to knock down CD44, and the knockdown efficiency was validated with GFP and RT-qPCR. Furthermore, cell proliferation was assayed using Cell Counting Kit‑8 (CCK-8) and colony formation assays, and cell migration and invasion were assayed by transwell and wound-healing assays. ResultsWe found that CD44 expression in the MG63 and U2OS OS cell lines was markedly increased compared to that of the human osteoblast hFOB 1.19 cell line. Knockdown of CD44 inhibited proliferation, migration, and invasion of MG63 and U2OS cells, possibly by regulating the expression of cathepsin S in OS. Conclusion﻿Taken together, our data reinforced the evidence that CD44 knockdown inhibited cell proliferation, migration, and invasion of OS cells accompanied by altered expression of cathepsin S. These findings offer new clues for OS development and progression, suggesting CD44 as a potential therapeutic target for OS.


Introduction
Osteosarcoma (OS) is the most common primary bone tumour, mainly occurring in children and adolescents, and the third most frequent in adults, following chondrosarcoma and chordoma. The overall incidence of OS is 3.4 per million cases per year worldwide [1], and the principal cause of death in patients suffering from OS is pulmonary metastasis [2]. Osteosarcoma is a primary bone cancer characterised by cancer cells that produce calci ed osteoid extracellular matrix and inducing lung metastases with a high frequency. Despite recent advances in treating osteosarcoma with a combination of chemotherapy and surgery, the 5-year survival rate remains low, and the prognosis for patients is poor [3]. The cellular and molecular mechanisms underlying the progression of osteosarcoma, including the rate of cancer cell proliferation, the formation of metastatic lesions and the development of drug resistance, remain unclear [4].
Cluster of differentiation 44 (CD44) is a complex transmembrane adhesion glycoprotein considered an essential bridge molecule as it links the extracellular matrix and intracellular skeletal proteins and participates in intracellular signal transduction, affecting cell deformation or movement through cytoskeletal changes [5]. Numerous studies have reported that CD44 not only participates in normal cellular functions but also plays pivotal roles in pathological processes [6]. For example, CD44-RhoA-YAP signalling mediates mechanics-induced broblast activation, and targeting this pathway could ameliorate crystalline silica-induced silicosis and provide a potential therapeutic strategy to mitigate brosis [7]. It is noteworthy that CD44 expression was found upregulated in different tumours [8][9][10], promoting cancer cell invasion and migration [11,12]. However, the role and molecular mechanisms of CD44 in the development and progression of OS remain uncertain.
The present study analysed the CD44 expression pattern in OS cell lines using reverse transcription-quantitative PCR (RT-qPCR) and Western blot (WB). Furthermore, loss-of-function experiments were performed to investigate the biological roles of CD44 in OS. The results revealed that CD44 expression was upregulated in OS cell lines. In addition, in vitro assays revealed that CD44 downregulation inhibited cell proliferation, migration, and invasion, probably by regulating cathepsin S. These ndings suggest that CD44 functions as an oncogene and future research may contribute to the development of new tools for the diagnosis and treatment of OS.

Cell culture
The human OS MG63 and U2OS cell lines were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and cultured in modi ed Eagle's medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 ˚C with 5% CO 2 .
The normal human osteoblastic cell line hFOB 1.19 was purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and maintained in D-MEM/F-12 (Gibco) supplemented with 10% FBS (Gibco) and 0.3 mg/mL Geneticin (G418; Gibco) at 37 ˚C with 5% CO 2 .

Small interference RNA transfection
Small interference RNA (siRNA) for transfection were purchased from Ribobio (Guangzhou, China).
RT-qPCR analysis MG63 and U2OS cells were treated with si-CD44 or si-NC for 24 h, and total RNA was extracted from the OS cell lines and the normal human osteoblastic cell line hFOB 1.19 using Trizol (Supersmart, China).
Next, 2 μg of RNA was used to synthesise the complementary DNA (cDNA) by reverse transcriptase (ABclonal, China). The resulting complementary cDNA was used for PCR analysis. The relative levels of genes were detected by RT-qPCR using SYBR Premix Ex Taq™ (ABclonal, China). The PCR cycling conditions were 95 ˚C for 5 min, followed by denaturation for 10 sec at 95 ˚C and extension for 20 sec at 60 ˚C for 40 cycles. GAPDH was used as an internal loading control. All reactions were performed in triplicates. Fold changes were calculated using the 2-ΔΔCq method. The primers were as follows: CD44 Colony formation assay MG63 and U2OS cells were treated with CD44 siRNA or negative control for 24 h. Cells were then resuspended and seeded in 6-well plates at a density of 500 cells per well and cultured for 15 days.
Subsequently, cells were xed with pre-cooled methanol for 30 min at RT and stained with 0.1% crystal violet for 20 min at RT, washed twice with PBS and twice with double distilled water. The colonies were counted and analysed under a light microscope.

Wound-healing assay
To evaluate the role of CD44 in OS cell migration, MG63 and U2OS cells were transfected with si-CD44 or si-NC for 24 h. Cells were resuspended and seeded in 6-well plates at a density of 1×10 6 cells per well, and 2 ml of culture medium supplemented with 10% FBS was added. Cells were grown to 90% con uence, and then a uniform and consistent wound was scraped on the bottom of the 6-well plate with a 200 μL plastic pipette tip (time set as 0 h). PBS was used to remove oating cells. Subsequently, cells were incubated in fresh complete medium (1% FBS) for 0, 24 and 48 h and the number of migrated cells were observed and counted under a light microscope.

Transwell assay
Migration and invasion abilities of MG-63 and U2OS cells were measured using a transwell assay. The Matrigel was incubated at 37˚C for 5 h before testing. OS cells were transfected with si-CD44 or si-NC for 24 h. 1x10 5 transfected cells were resuspended in serum-free medium and seeded in the upper chamber with or without Matrigel (BD Biosciences) for the invasion and migration assays, respectively. Subsequently, medium containing 20% FBS was added to the lower chambers. Following a 24 h incubation, the cells from the upper compartments were scraped off with cotton swabs, while the cells that migrated to or invaded the lower surface of the membrane were xed with pre-cooled methanol at RT for 20 min and stained with 0.1% crystal violet at RT for 20 min. The stained cells were counted in ve random elds off view under a light microscope at x200 magni cation, and all experiments were repeated three times.

Statistical analysis
The results are presented as the mean ± SD. Statistical analyses were performed using SPSS 23.0 (IBM Corp, USA) and GraphPad Prism 9.0 (La Jolla, CA, USA) software. ANOVA test was applied to compare differences among multiple groups. P < 0.05 was considered to indicate a statistically signi cant difference. 2. CD44 knockdown in MG63 and U2OS cells in vitro. MG63 and U2OS cells were transfected with si-CD44 or si-NC for 24 h, and the transfection e ciency was detected using uorescence microscopy ( Fig. 2A). CD44 mRNA and protein expression was quanti ed by RT-PCR and Western blot, respectively, after CD44 knockdown in MG63 and U2OS cells. As shown in Figure 2B and C, the results revealed that the siRNA transfection decreased CD44 expression, but as expected, no signi cant difference was observed between the si-NC and control groups. For subsequent experiments, two (siCD44-1, siCD44-2) of the three CD44 siRNA with high transfection e ciency were selected.

CD44 knockdown inhibited the proliferation of MG63 and U2OS cells. To assess the role of CD44 in
MG63 and U2OS cell proliferation, siRNA was transfected to silence CD44 expression. Subsequently, cell proliferation was assessed using CCK-8 and colony formation assays. As demonstrated by the result of the CCK-8 assay, cell growth was suppressed in CD44-silenced MG63 and U2OS cells compared with the si-NC-transfected cells (Fig. 3A). In addition, the colony formation ability of si-CD44-transfected cells was decreased (Fig. 3B). These results revealed that downregulation of CD44 markedly decreased the proliferation of MG63 and U2OS cells.

CD44 knockdown inhibited the migration and invasion of MG63 and U2OS cells.
To investigate the role of CD44 in the migration and invasion of OS cells, the wound-healing assay were used at 0, 12, or 24 h after transfection. The result of the wound-healing assay showed that the migration distances of cells transfected with si-NC was compared to the migration distances in CD44-silenced cells (Fig. 4A). The result of the transwell migration and invasion assay showed that the number of control si-NC-transfected cells was more than the number of CD44-silenced cells (Fig. 4B). Furthermore, Western blot was applied to evaluate the matrix metalloproteinase MMP-9 protein levels. As shown in Figure 4C, silencing of CD44 decreased MMP-9 expression in MG-63 and U2OS cells compared with the si-NC group. Therefore, the results suggested that the migration and invasion abilities of MG-63 and U2OS cells were suppressed following CD44 knockdown.

CD44 knockdown downregulated the expression of cathepsin S in MG63 and U2OS cells. To further
con rm the underlying mechanism of CD44 in OS, we detected the mRNA and protein expression of cathepsin S by RT-PCR and Western blot after CD44 knockdown in MG63 and U2OS cells. The mRNA and protein levels of cathepsin S in the CD44-silenced OS cells were markedly reduced compared with the control cells (si-NC) at 24 and 48 h after transfection (p < 0.01) (Fig. 5A-5B). These data indicated that CD44 exerted its effects in OS in part by regulating cathepsin S.

Discussion
The current treatment for osteosarcoma is surgical resection and combined neoadjuvant chemotherapy, which has increased the 5-year overall survival rate for osteosarcoma patients from less than 20% to more than 60% [13]. However, survival rates for patients with metastatic or recurrent osteosarcoma have remained virtually unchanged over the past 30 years, with an overall 5-year survival rate as low as 20% [3,14]. The molecular mechanisms underlying the development of OS have not been fully explored. Therefore, it is crucial to elucidate the predictive markers of OS and their potential regulatory mechanisms.
CD44, also known as homing cell adhesion molecule, is a cell surface transmembrane glycoprotein molecule involved in cell-cell and cell-extracellular matrix communication. In humans, CD44 proteins are encoded by a highly conserved gene located on the short arm of chromosome 11 (11p13) [15]. CD44 can affect cell growth, proliferation and motility through changes in the cytoskeleton, as well as being involved in intracellular signal transduction [16]. Therefore, CD44 is a critical bridging molecule between the extracellular matrix and intracellular skeletal proteins.
CD44 expression is elevated in a wide range of malignant tumours [17], such as colon tumours [18], ovarian clear cell carcinoma [19], and glioblastoma [20]. Overexpression of CD44 splice mesenchymal isoform in OS cells induced EMT and invasion, followed by the gain of stem-like characteristics and chemoresistance [21]. In gastric cancer, CD44V6 regulates the transformation of normal mucosal epithelial cells into tumour cells and is associated with gastric cancer differentiation, lymph node metastasis, and pathological staging [22]. CD44 potentiates AKT activation to induce phosphorylation and nuclear translocation of Mdm2, which terminates the p53 genomic surveillance response. This allows DNA-damaged hepatocytes to escape p53-induced death and go on to become HCC progenitors [23]. We con rmed that CD44 is highly expressed in osteosarcoma cell lines compared to hFOB 1.19 human osteoblasts. Hence, we next evaluated the effects of CD44 on the proliferation, invasion, and migration of osteosarcoma cells.
We found that downregulation of CD44 inhibited the proliferation, invasion, and migration ability of osteosarcoma cells MG63 and U2OS, which is consistent with the reported role of CD44 in most tumours [8,10,17], suggesting that CD44 may be a pro-oncogene in osteosarcoma.
To know more about the molecular mechanisms, we next investigated the expression of cathepsin S. Like matrix metalloproteinases, members of the histone family have been associated with metastasis and cancer recurrence [24]. Cathepsin S (CTSS), a member of the histone family of enzymes, is highly expressed in renal clear cell carcinoma [25], hepatocellular carcinoma [26], cervical cancer [27], lung cancer [28] and other tumours and is an essential regulator of tumour growth and invasion. Suppression of cell migration and invasion by modulation of Ca 2+ -dependent downstream effectors after CTSS inhibition [29]. The expression of CTSS was regulated by PI3K/Akt and Ras/Raf/MAPK signalling pathways, is a candidate target for blocking the metastasis of breast and oral cancers [30,31]. However, it is not clear what role cathepsin S plays in osteosarcoma. Previous experiments found that cathepsin S was highly expressed in osteosarcoma cells and that silencing inhibited the invasion and metastasis of osteosarcoma cells.
Previous studies have reported that CD44 can promote tumour stem cell properties in triple-negative breast cancer by regulating the PI3K/AKT pathway [32]. Interaction of hyaluronan and CD44 enhances neutrophil phagocytosis and IL-8 production via the p38 and ERK1/2-MAPK signalling pathways [33]. Activation of the receptor complex CD74/CD44 can lead to activation of the ERK1/2, PI3K-Akt signalling cascade, NFκB and AMP-activated protein kinase (AMPK) pathways [34]. Therefore, we speculate that CD44 may exert its biological effects on osteosarcoma cells through the regulation of cathepsin S. The experimental results showed that cathepsin S expression was downregulated after CD44 silencing compared to the si-NC group. Therefore, we suggest that CD44 may exert regulation of osteosarcoma cell proliferation, invasion, and metastasis through the regulation of cathepsin S. However, the speci c regulatory mechanisms of the two still need to be further explored, and this will be the focus of our subsequent work.

Conclusion
This study shows that inhibition of CD44 attenuates cell proliferation, migration, and invasion, possibly by regulating the expression of cathepsin S in OS cells. These ndings suggest that CD44 may be an oncogenic factor in the progression of OS and may be a promising molecular marker for the diagnosis and treatment of OS.    Expression levels of migration and invasion related proteins (MMP 9) were detected by Western blotting.