Exosome-mediated Hic-5 regulates proliferation and apoptosis of osteosarcoma via Wnt/β-catenin signal pathway

Hic-5 is up-regulated in osteosarcoma tissues and cells

RT-qPCR was performed to detect Hic-5 expression in osteosarcoma tissues and cell lines. The results showed miR-496 expression was up-regulated in the OS tissues than that in the normal group (Figure 1A). Compared with hFOB1.19 cells, the expression level of Hic-5 was significantly increased in MG-63, HOS, U2-OS and SAOS-2 cells (Figure 1B). The protein level of Hic-5 was measured by western blot, which was upregulation in four OS cell lines (Figure 1C). In adjacent normal bone tissue biopsies (53.3%, 8/15), only a low level of Hic-5 was present. More frequency (46.7%, 7/15) of higher expressed Hic-5 was present in low grade osteosarcoma tissues. In contrast with low grade osteosarcoma tissues, a decreased staining for Hic-5 was observed in most high grade osteosarcoma patient tissues (53.3%, 8/15) (Figure 1D).

Figure 1. Hic-5 was up-regulation in human osteosarcoma tissues and cell lines. (A) RT-PCR was employed to detect the expression of Hic-5 in tumor and normal tissues. n=15, **P<0.01. (B and C) The mRNA and protein expression of Hic-5 was measured in MG-63, HOS, U2-OS and SAOS-2 cells, hFOB1.19 cells were described as control. n=8, *P<0.05. (D) Hic-5 staining in slides from osteosarcoma (high grade and low grade) and adjacent normal bone tissues.

Knockdown of Hic-5 prevents growth and induces apoptosis of OS

For further explore the role of Hic-5, we created red fluorescence plasmid (RFP)-shRNA for knockdown Hic-5 (RFP-sh-Hic-5), RFP-sh-NC was described as an negative control. RT-PCR and western blot were performed to detect the transfection efficiency (Figure 2A and 2B), and immunofluorescence experiment also verified the RFP-shRNA functional entered the OS cells (Figure 2C). Cell viability was evaluated in MG-63 and HOS cells, as Figure 2D shown, downregulation of Hic-5 inhibited the cell viability of OS cells. We observed that knockdown of Hic-5 prevented the proliferation of OS cells, which was indicated by Edu assay (Figure 2E). Then we investigated the effect of Hic-5 on apoptosis of OS cells. Flow cytometry was employed to detect the apoptosis rate of MG-63 and HOS cells after RFP-shRNA transfection. RFP-sh-Hic-5 dramatically increases the number of apoptosis cell (Figure 2F). Compared with RFP-sh-NC, the caspase3 activity was increasing in RFP-sh-Hic-5 group, which was measured by caspase3 activity kit (Figure 2G). Meanwhile, we detected the expression of apoptosis-relative protein, Bcl2, Bax and Cleaved-caspase3. The results revealed the downregulation of Bcl2 and upregulation of Bax, Cleaved-caspase3 (Figure 2H).

Figure 2. Dysfunction of Hic-5 regulated the proliferation and apoptosis in osteosarcoma. The transfection efficiency was verified by RT-PCR (A, n=6) western blot (B, n=3), and immunofluorescence (C, n=6). *P<0.05. (D) MTT assay was performed to detected cell viability in OS cells. n=10, *P<0.05. (E) The proliferation ability of OS cells was measured by Edu assay. (green, Edu⁺, blue, DAPI). n=6, *P<0.05. (F) The apoptosis rate was detected by flow cytometry. n=5, *P<0.05. (G) The caspase3 activity was assessed in OS cells. n=6, *P<0.05. (H) The apoptosis-associated protein (Bcl2, Bax and Cleaved-caspase3) level was detected by western blot. n=6, *P<0.05. (I) Effects of Hic-5 knockdown on the size of MG-63 xenograft tumors in nude mice. (J) The tumor volume and weights of removed tumor were calculated. n=10, *P<0.05. (K) Representative Ki-67 staining of tumor sections in different group.

Subsequently, we constructed a stable MG-63 cell line with low expression of Hic-5 for in vivo tumor experiment. MG-63 cells were subcutaneously injected in right lower limb of the nude mice. Tumor size was measured every 3 days. After another 15 d of injection, the tumors were removed (Figure 2I). Tumor volume and weight were calculated, the tumors from RFP-sh-Hic-5-MG-63 cells injection mice were smaller and lighter than tumor from RFP-sh-NC-MG-63 cells injection mice (Figure 2J). Further, immunohistochemical analysis of Ki-67 in two group samples performed that knockdown of Hic-5 inhibited the tumor progression (Figure 2K). Taken together, knockdown of Hic-5 would inhibit the proliferation and promote apoptosis of OS.

Extraction, purification and confirmation of exosomes

Exosome-specific membrane structures and contents are widely involved in material exchange and information exchange between osteosarcoma cells, such as regulating the microenvironment of osteosarcoma, regulating the expression of Wnt/β-catenin signal and TGF-β signal pathway, inducing tumor cell immune escape and so on. The exosome body of osteosarcoma can also cooperate with antigen presenting cells to activate the immune response of the body and play an anti-tumor role. We speculated that Hic-5 may also affect the development of OS in the form of exosomes. Then we isolated the exosomes from MG-63 supernatant. Exosomes were identified by transmission electron microscopy (TEM, Figure 3A), biomarkers (CD63, Alix, Tsg101 and GM130, Figure 3B) and particle size (about 30-150 nm, Figure 3C) analysis. Then we extracted exosomes from MG-63 cells transfection with RFP-shRNA. Then the exosomes from sh-Hic-5-MG-63 (sh-Hic-5-exo) and RFP-sh-NC-MG-63 (sh-NC-exo) cells supernatant were identified by TEM (Figure 3D), biomarkers (Figure 3E) and particle size (Figure 3F). RT-PCR assay was performed to detect the expression level of Hic-5 from sh-Hic-5-exo and sh-NC-exo. Hic-5 was decreased in sh-Hic-5-exo (Figure 3G).

Figure 3. Exosomes derived from OS supernatant were identified. (A) The morphology of exosome was observed by TEM. (B) The biomarker (CD63, Alix, Tsg101 and GM130) of exosome were detected by western blot. (C) Exosome particle size (about 30-150 nm) were analyzed. The characteristics of sh-Hic-5-exo and sh-NC-exo were confirmed by TEM (D), biomarker (E) and particle size (F). (G) The mRNA expression of Hic-5 was measured in sh-Hic-5-exo and sh-NC-exo. n=4, *P<0.05.

Exosomal-Hic-5 regulates the proliferation and apoptosis of OS

For further explore the function of exosomal-Hic-5 in OS, we co-cultured sh-Hic-5-exo/sh-NC-exo with MG-63 and HOS cells. Then immunofluorescence assay was performed to detect Hic-5. Hic-5 was decreased in co-cultured with sh-Hic-5-exo cells (Figure 4A), the results revealed exosomal-Hic-5 could enter into OS cells. sh-Hic-5-exo also reduced cell viability in OS cells (Figure 4B), and inhibited proliferation of cells (Figure 4C). Apoptosis level was measured by flow cytometry. sh-Hic-5-exo promotes apoptosis rate of OS cells (Figure 4D), and increased caspase3 activity (Figure 4E). The down-regulated of Bcl2 and up-regulated of Bax, Cleaved-caspase3 once indicated that sh-Hic-5-exo could elevate apoptosis level (Figure 4F).

Figure 4. Exosomal-Hic-5 effected the proliferation and apoptosis of OS. (A) Immunofluorescence assays was performed to detect co-cultured results in OS cells. (B) MTT assay was performed to detected cell viability in co-cultured OS cells. n=10, *P<0.05. (C) The proliferation ability of co-cultured OS cells was measured by Edu assay. (green, Edu⁺, blue, DAPI). n=6, *P<0.05. (D) The apoptosis rate was detected by flow cytometry. n=5, *P<0.05. (E) The caspase3 activity was assessed in co-cultured OS cells. n=6, *P<0.05. (F) The apoptosis-associated protein (Bcl2, Bax and Cleaved-caspase3) level was detected by western blot. n=6, *P<0.05. (G) The size of exosomes co-cultured MG-63 xenograft tumors in nude mice. (H) The tumor volume and weights of removed tumor were calculated. n=15, *P<0.05. (I) Representative Ki-67 staining of tumor sections in different group.

Then the MG-63 cells were collected after co-cultured with sh-Hic-5-exo/sh-NC-exo. The collected cells were subcutaneously injected in right lower limb of the nude mice. Tumor size was measured after another 15 d of injection, the tumors were removed (Figure 4G). Tumor volume and weight were calculated, the tumors from sh-Hic-5-exo-MG-63 cells injection mice were smaller and lighter than tumor from sh-NC-exo-MG-63 cells injection mice (Figure 4H). Further, immunohistochemical analysis of Ki-67 in two group samples performed that sh-Hic-5-exo-MG-63 cells injection mice showed poor ability of proliferation (Figure 4I). Taken together, knockdown of Hic-5 would inhibit the proliferation and promote apoptosis of OS via exosome pathway.

Exosomal-Hic-5 regulates OS through Wnt/β-catenin signal pathway

Wnt pathway is the key pathway for cell development and growth regulation. Wnt/β-catenin pathway is the most classical Wnt signal pathway, and its abnormal activation plays an important role in the formation of malignant tumors. CoIP assay was carried out for detect the relationship between Hic-5 and Wnt/β-catenin signal pathway. We observed that Hic-5 could interact with smad4 in OS cells. Downregulation of sFRP1 and Smad4 could activate Wnt/β-catenin signaling pathway, thereby promoting tumor cell invasion and metastasis. Then we detected the expression of pathway elements (sFRP1, Smad4, Wnt1and β-catenin), upregulation of sFRP1, Smad4 and downregulation of Wnt1, β-catenin was performed in MG-63 cells transfection with RFP-sh-Hic-5 (Figure 5A and 5B). Similar results were achieved in vivo mice injection with RFP-sh-Hic-5-MG-63 cells (Figure 5C). Subsequently, we detected the expression of sFRP1, Smad4, Wnt1and β-catenin in MG-63 cells co-cultured with sh-Hic-5-exo/sh-NC-exo. Increased level of sFRP1, Smad4 and decreased level of Wnt1, β-catenin was performed in MG-63 cells co-cultured with sh-Hic-5-exo (Figure 5D). The mice injection with MG-63 cells co-cultured with sh-Hic-5-exo were showed consistent results (Figure 5E). Further, Knockdown of Hic-5 decreased the TOP luciferase activity which indicated the inhibition of Wnt/β-catenin signal activation (Figure 5F). Therefore, we found that protein expression of active β-catenin, but not total β-catenin, was reduced in RFP-sh-Hic-5-transfected MG-63 cells by immunofluorescence (Figure 5G).

Figure 5. Exosomal-Hic-5 regulated the development of OS via inhibiting Wnt/β-catenin signal pathway. (A) CoIP assay was performed to confirm the relationship between Hic-5 and smad4. (B) The expression of Wnt/β-catenin signal pathway elements (sFRP1, Smad4, Wnt1and β-catenin) were detected in MG-63 cells after RFP-shRNA transfection. n=6, *P<0.05. (C) The expression of sFRP1, Smad4, Wnt1, β-catenin were detected in tumor from treated-MG-63 cell injection mice. n=10, *P<0.05. (D) The expression of Wnt/β-catenin signal pathway elements (sFRP1, Smad4, Wnt1and β-catenin) were detected in MG-63 cells after exosomes co-cultured. n=6, *P<0.05. (E) The expression of sFRP1, Smad4, Wnt1, β-catenin were detected in tumor from exosomes co-cultured-MG-63 cell injection mice. n=10, *P<0.05. (F) Wnt pathway activation by treatment with RFP-sh-Hic5/RFP-sh-NC regulates ß-Catenin signaling mediated TCF/LEF reporter activity. n=3, *P<0.05. (G) Subcellular localization of active β-catenin and total β-catenin in MG-63 cells by immunofluorescence staining.

Clinical samples

Fresh tissue samples and adjacent normal tissue samples were collected from 15 osteosarcoma patients at People’s Hospital of Rizhao (data shown in Table 1). This experiment has got the approval of the Medical Ethics Committee of People’s Hospital of Rizhao and this study is in line with the Declaration of Helsinki.

Cell culture

MG-63, HOS, U2-OS, SAOS-2 and hFOB1.19 cell lines were purchased from the Science Cell Laboratory. Cells were cultured in MEM (GIBCO, USA) supplemented with 10 % fetal bovine serum (Cromwell, USA) and 100 μL/mL penicillin and streptomycin (Sigma-Aldrich, USA) and placed at 37°C with 5% CO2.

Cell transfection

RFP-shRNA was produced by Genomeditech, Co. (Shanghai, China). A scrambled shRNA was used as the negative control (sh-NC). About 5×10⁵ cells per well were seeded in 6 well plates, transfection of siRNA into the cells was performed using lipo2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations. Cells were transfected with 20 nmol/L siRNA for 48 h, and then siRNA transfection in the best condition was performed.

Real time-PCR

Total RNA was isolated from cells and tissues according to a standard protocol. And then, the purity and concentration of RNA was detected and all the samples were converted into cDNA using reverse transcription kit. We used SYBR Green (Thermo Fisher Scientific) system to perform the qRT-PCR.

Western blot

Total protein was collected from cells with RIPA lysis Mix. Briefly, 50 μg protein extraction was loaded via SDS-PAGE and transferred onto nitrocellulose membranes (Life Sciences, Mexico), the membranes were incubated in 5% non-fat milk blocking buffer for 2 h. Then incubated with primary antibodies for 2 h at room temperature, then plated at 4 °C overnight, After incubation with secondary antibodies, the membranes were readied using an Odyssey, and data were analyzed with Odyssey software (LI-COR, USA). The primary antibody brand and usage concentration were as follow. Hic-5 (proteintech, 10565-1-AP, 1:500), Bcl2 (proteintech, 12789-1-AP, 1:500), Bax (proteintech, 60267-1-Ig, 1:500), Cleaved-caspase3 (CST, Asp175 1:1000), CD63 (proteintech, 25682-1-AP, 1:500), Alix (proteintech, 12422-1-AP, 1:500), Tsg101 (proteintech,14497-1-AP, 1:500), GM130 (proteintech,11308-1-AP, 1:500), smad4 (proteintech, 10231-1-AP, 1:500), sFRP1 (proteintech, 26460-1-AP, 1:500), wnt1 (proteintech, 27935-1-AP, 1:500), β-catenin (proteintech, 17565-1-AP, 1:500), and GAPDH (proteintech, 60004-1-Ig, 1:2000) was used as an internal control.

Cell viability assay

MTT assay was used to assess cell viability. MG-63 and HOS cells were cultured in the 96-well plates for 1×10³/well. Overnight, cells were starved for 12 h. transfection reagent was added to the cells. Then cells were treated with 5 mg/mL MTT (20 μL/well) for 4 h. Removing supernatant, and adding DMSO (150 μL/well), the plate was shaking 30 min in room temperature. The absorbance was read at 490 nm using an Infinite 200PRO microplate spectrophotometer (BioTek, USA). The absorbance values were normalized to the control.

In vivo tumor growth assay

Nude mice were purchased from Beijing Charles River. The nude mice were injected with treated-MG-63, MG-63 cells (5 × 10⁶) were subcutaneously injected in right lower limb of the nude mice. Tumor size was measured every 3 days. After another 15 d of injection, the tumor was removed for follow-up study.

EDU assay

EdU incorporation. For EdU labeling, a 1:1,000 dilution of EdU-labeling reagent (Beyotime, China) was added to islet culture medium during the last 18 h of cell culture.

Immunofluorescence staining

Cells were plated in a 24-well cell culture plate. The cells were washed by PBS and fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton-X-100 solution in PBS. Next, we blocked cell using goat serum. Then, the cells were incubated with Hic-5 antibody at 4 °C overnight followed with FITC-conjugated goat anti-mouse antibodies incubation for 1h. After three washes with PBS, we incubated cells by DAPI.

Isolation and identification of exosome

When the cell fusion reached 70%, it was washed three times with 0.01mol/L phosphate buffer (PBS) and incubated with serum-free medium or exosome-free FBS medium for 48 hours. The supernatant was collected, centrifuged, 300xg centrifuged 10min at 4 °C to remove cells, 2000xg centrifugation 20min to remove cell fragments, and exudates were stored at -80 °C. Exosome morphology was observed by transmission electron microscope (TEM). The exosome particle size was detected by PSS Nicomp 380Z3000 analyzer (USA).

Co-IP assay

Co-IP was performed in HEK 293T cells. The cells were collected and lysed in the whole cell extract buffer (400 mM KCl, 10 mM Na2HPO4, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.5% NP-40 with complete protease inhibitor cocktail). Appropriate antibodies and protein G Sepharose™ 4 fast flow beads were added to the extracts and mixtures were incubated overnight at 4 °C. After washing with the whole cell extract buffer, the beads were boiled in 1 × SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% glycerol, 0.1% bromophenol blue). Samples were loaded and separated by SDS-PAGE and transferred to Immun-Blot™ PVDF membrane (Bio-Rad). The membrane was then incubated with appropriate primary antibodies (1:1000 dilution) followed by HRP-conjugated secondary antibodies (Beyontime, 1:2000 dilution). Protein signals were visualized by Western blotting detection reagents.

Statistical analysis

All data is presented as a mean ± S.E.M. Statistical analysis was performed using Student's t-test or a one-way ANOVA.