Necdin inhibits the proliferation of osteosarcoma cells by regulating the expression of VEGFA

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Necdin inhibits the proliferation of osteosarcoma cells by regulating the expression of VEGFA

https://doi.org/10.21203/rs.3.rs-1542547/v1

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Necdin (NDN) acts as a tumor suppressor gene in some malignant tumors, but it is unclear about the role of NDN in osteosarcoma (Osteosarcoma, OS). The results show that the expression level of NDN is low, while that of vascular endothelial growth factor A (VEGFA) is significantly up-regulated in OS cells and tissues. Overexpression of NDN can accelerate apoptosis of OS cells making them less invasiveness, and reduce the tubular shape of human umbilical vein endothelial cells. Meanwhile, the overexpression of NDN can inhibit the expression of VEGFA. Bioinformatics analysis demonstrates that there is no noticeable relationship between NDN and VEGFA and no statistical significance. The studies in vivo further confirmed that NDN could inhibit tumor growth and angiogenesis in xenograft nude mice. Our results show that NDN is a potential tumor suppressor in OS, and it can inhibit tumor angiogenesis by regulating VEGFA. Therefore, NDN may be a new biomarker for the prognosis and a new target for treatment of OS.

Osteosarcoma

Necdin

VEGFA

Proliferation

Osteosarcoma (OS) is the most common primary malignant bone tumor, which often occurs in adolescents with incidence rate was about 3 to 4 cases per 1 million people per year. It is characterized by high malignancy, aggressiveness, recurrence and metastasis rate ^1–3, which seriously damages the health of adolescents. The current standard treatment for OS includes preoperative chemotherapy, surgical resection, and postoperative chemotherapy. Although the 5-year survival rate is about 62% ⁴, the therapeutic effect is still poor for patients with relapse and lung metastasis, mainly because of the resistance to chemotherapeutics. In addition, the existing chemotherapeutic drugs cause many serious side effects due to its poor targeting. Therefore, the development of new therapeutic targets is of great significance for the treatment of OS patients.

The angiogenesis is affected by the pro-angiogenesis and anti-angiogenic factors ^5,6. Vascular endothelial growth factor A (VEGFA) is a polypeptide encoded by several genes, which is one of the platelet-derived growth factor (PDGF) family. The expression of VEGF in cancer cells can help angiogenesis resulting in tumor growth ⁷. Therefore, inhibition of angiogenesis by blocking the expression of VEGF has become an effective method to treat cancer. Several researches have demonstrated that anti-angiogenesis therapy targeting VEGF could inhibit the progression of advanced cancer ^8,9. VEGF has a significant role in the angiogenesis and progression of OS.

Necdin (NDN) is a member of the melanoma antigen (MAGE) family, which is composed of more than 60 genes shared the highly conserved MAGE homology domain (MHD) ^10,11. Previous studies have shown that overexpression of NDN could inhibit the growth of ovarian cancer ¹², breast cancer ¹³, and melanoma ¹⁴. As a multifunctional protein, NDN can directly connect to DNA and behave like a transcription factor ¹⁵. In addition, researches have shown that NDN could also regulate transcriptional activity and transcription indirectly by affecting famous transcription factors such as E2F1 and p53 ^16,17. Moon et al. found that NDN could inhibit the angiogenesis of human umbilical vein endothelial cells under hypoxic conditions and regulate the transcriptional activation of VEGF ^18,19. However, whether NDN can affects the angiogenesis of bone flesh by regulating the expression of VEGF in OS remains unclear.

In this work, we tested the relative expression levels of NDN and VEGFA in OS and bone tissues and OS cells. Besides, we studied the influence of overexpression of NDN on the apoptosis, invasion and angiogenesis of Saos-2 and U2OS cells, and explored the mechanism of action in vitro. The results show that the expression level of NDN is negatively correlated with the expression of VEGFA in OS. Additionally, NDN inhibits the proliferation and angiogenesis of OS by interacting with VEGFA. Therefore, NDN is a tumor suppressor and can inhibit the proliferation of OS by reducing angiogenesis.

Clinical tissue specimens

Tissue specimens were obtained from the tumor tissues of 6 cases of OS patients and 3 cases of normal bone tissues obtained during the diagnosis and treatment of the Orthopedics Department of the Second Hospital of Shanxi Medical University (Taiyuan, China), and then qRT-PCR and Western blot were carried out. In the pathology department, 30 paraffin-embedded specimens of osteosarcoma patients and 6 paratumoral bone tissue specimens were selected for immunohistochemistry. All patients obtained informed consent. The research protocols were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University, (2021) YX No. (098).

Cell line and culture

The human osteosarcoma cell lines Saos-2, U2OS, Mg63 and human chondrogenic cell line C28 were used in this work. Saos-2, U2OS, Mg63and C28 cells were incubated in complete culture medium with supplementary 10% fetal bovine serum, 1% penicillin and 89% DMEM/F-12 (Gibco, USA), and placed in a 37-degree cell incubator with 95%humidity and 5% CO2.

RNA extraction and qRT-PCR

Total RNA was extracted used TRIzol reagent (Thermo Fisher Scientific). PrimeScript RT Master Mix kit (Takara) was applied to reverse transcription. Amplify cDNA we use SYBR Green Premix Ex TaqII kit (Takara, Japan). Use the QuantStudio™ 6 Flex real-time fluorescence quantification system (Thermo Fisher Scientific) to evaluate the relative expression and the internal reference 18S. The sequences of NDN are 5'-CGAGCTCATGTGGTACGT-3' and 3'-CGATGACATCTTTCACCATGTC-5'. The sequences of 18S are 5'-AAACGGCTACCACATCCA-3' and 3'-CACCAGACTTGCCCCTCCA-5'. The sequence of VEGFA is 5'-GTTTGACAAGACCACCAAACT-3' and 3'-CCGCATAATCTGCATGGTGAT -5'. The qPCR reaction conditions for NDN, VEGFA, 18S are 95℃ 15 min, 95℃ 30 s, 55℃ 5 s, 72℃ 5 s, A total of 40 cycles.

Immunohistochemistry

Tumor tissue, normal bone tissue adjacent to the tumor, and xenograft tumor tissue of patients with osteosarcoma were fixed (4% paraformaldehyde), dehydrated, embedded in paraffin, sliced (5mm), then deparaffinization, rehydration and other standard immunohistochemical staining procedure were performed. The sections were incubated with anti-NDN/VEGFA/CD31 (Santa Cruz Biotechnology, Dallas, TX, USA) primary antibody one night at 4°C. After washed, biotin-labeled goat anti-mouse/rabbit IgG (PV-9000, Beijing Zhongshan Bridge) was incubated at 37°C for 1 h, and then stained with hematoxylin. A slide scanner and software (purchased from 3DHISTECH, Hungary) were used for image acquisition, and ImageJ image analysis system was used for positive result determination.

Cell transfection

Saos-2 and U2OS cells were cultured in a six-well plate (1x10⁵cells per well). When the cells reached 30% confluence, use lentivirus UP-NDN (LV-NDN, Genechem, Shanghai, China) and negative control lentiviruses (LV-Control) were transfected into Saos-2 at MOI = 5, and U2OS cells at MOI = 10 for 16 hours.

Western blot analysis

The levels of target protein were analyzed by western blot and H2b and orglyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression act as the control. Cells and tissue were disintegrated in RIPA buffer containing PMSF, phosphatase and protease inhibitors (Keygen, Nanjing, China) on ice. Sample were separated in 10% SDS/PAGE gels, then transferred to nitromethyl cellulose membra ((BOSTER, Wuhan, China). The membranes were probed with anti-NDN (FLT-1; sc-101224,1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-VEGF (sc-7269,1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-H2B (Abcam USA ab6877). GAPDH (cat. 5174, 1:800 dilution; Cell Signaling Technology). ECL kit (BOSTER, Wuhan China) were utilized to develop the signals. The quantification of protein levels in the tumor were carried out via normalizing with H2B and GAPDH by IMAGEJ software.

Transwell invasion assay

Matrigel Basement Membrane Matrix (Cat.354248, Corning, England) was diluted with DMEM/F12 without FBS at a dilution of 1:7 and was added to the upper Transwell chambers 70µl/well. After incubated in 37℃ for 2 hours, each upper chamber was added with 40000 cells suspended in 150 µL serum-free medium, while 650µl complete medium for each lower chamber. After 36 h, the cells on the upper surface are erased, and the cells on the lower surface was dyed with 1% crystal violet for 30 min, photographed under a microscope.

Apoptosis detection (flow cytometry)

Take the transfected Saos-2 and U2OS cells in the logarithmic growth phase, digest the cells with 0.25% trypsin without EDTA, wash the cells twice with PBS (centrifuged at 1000 rpm for 5 min), and take 100 µl containing 5×10⁵ Add 5µl Annexin V-PE (4A Biotech, China) to the cell suspension in a flow tube. After mixing, incubate for 5 minutes in the dark, add 400µl PBS, and then add 10µl Annexin V-7AAD by pipetting and mixing, and immediately perform streaming detection.

Angiogenesis experiment

After mixing Matrigel (Cat.354248, Corning, England) with DMEM/F-12 serum-free medium at a ratio of 1:1, add 50µl of diluted Matrigel to each well of a 96-well plate. When the Matrigel was solidified, add 1×10⁴ cells/100µl HUVEC cells per well to 96-well plate, and add 100µl culture supernatant of saos-2 cells transfected with LV-NDN and LV-Control, and incubate for 24h. A positive control group was set up, and 10 ng/mL human VEGF 165 recombinant protein was added to each well. Observe the angiogenesis in the hole under a microscope, and use Image J software to calculate the total length of blood vessel branches in each field of view.

Mouse model and bioimaging analysis

Animal welfare and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006), and approved by the animal ethics committee of Shanxi Medical University. BALB/c female nude mice aged 4 to 6 weeks were randomized and implanted subcutaneously with 1X10⁶ Saos-2/LV-UP-NDN and Saos-2/LV-CON-NDN cells in 200 µl Matrigel (Cat.354248, Corning, England) in their back (n = 6 per group). The implanted tumors were monitored weekly. After inoculation, individual tumor-bearing mice were anesthetized by being given an i.p. injection of ketamine (75 mg.kg^-1) and dexmedetomidine (0.5 mg.kg^-1) after being injected i.v. with 2nM of MMPSense^TM680 for 24 h. Then bioimaging was conducted and the vascularization was determined by FMT using TRUEQUANT software (Perkin Elmer, Waltham, MA, USA). The tumors were dissected to analyze their volumes and weights after experiment. Tumor tissues were saved in 4% paraformaldehyde, or RNAlater solution or lysis buffer.

Co-immunoprecipitation assay

In this experiment, FLAG+ cell samples were used as the control group, and FLAG+Ndn as the experimental group. Flag-Necdin (bait protein) COIP was performed with flag magnetic beads. In the flag antibody test results, no bait protein was detected in the control group. The bait protein can be detected in the experimental group, indicating that the COIP experiment was successful.

1.1 The expression of NDN in osteosarcoma tissues and osteosarcoma cell lines is lower expressed

NDN expression in the Saos2, U2OS, Mg63 were lower than that in the C28 (Figures 1A, B) and the represents quantified data of the Western blot (Figures 1C). The qRT-PCR and immunohistochemistry results and quantified of immunohistochemistry result indicated that the NDN expression was higher in normal bone than that in human OS samples (Figures 1D, E, F). The Chi-square test found that the expression level of NDN has nothing to do with the patient’s gender, age, tumor size, Enneking stage and local recurrence, but is closely related to the occurrence of lung metastasis (Figures 1G) (p < 0.05; **p < 0.01; ***p < 0.001).

1.2 The expression of VEGFA is significantly increased in osteosarcoma tissues and osteosarcoma cell lines

VEGFA is up-regulated in Saos2、U2OS and Mg63 compared with C28 (Figure 1H). The expressions of protein in osteosarcoma tissues is up-regulated compared with normal bone tissues (Figure 1I), quantization of immunohistochemistry results (Figure 1J) (*p < 0.05; **p < 0.01; ***p < 0.001).

2.1 The expression of NDN and VEGFA after overexpressed NDN in Saos2 and U2OS cells

The relative levels of VEGFA-mRNA transcripts in the UP-NDN group was noticeably lower than those in the CON-NDN group (Figure 2A). Western blot results and the quantified result depicted that the relative protein expression levels of VEGFA in the UP-NDN group was also lower than CON-NDN group (Figure 2B, C). Similar results were obtained in U2OS (Figure 2D, E, F).

2.2 UP-NDN inhibit the invasion ability of Saos2 and U2OS cells

Compared with the control group, the number of cells reach the lower surface of the Transwell membrane was much lower in the UP-NDN group. The results suggest that high-expressing NDN can inhibit the invasion ability of Saos-2 and U2OS cells (Figure 2G).

2.3 UP-NDN promotes the apoptosis of Saos-2 and U2OS cells

The proportion of apoptotic and dead cells in SAOS-2 and U2OS cells increased after high expression of NDN (Figure 2H).

2.4 Overexpressing NDN inhibits angiogenesis in vitro

Compared with the Control group, adding LV-NDN conditioned medium can significantly inhibit angiogenesis. The angiogenesis ability of the human recombinant protein VEGF165 group was significantly enhanced than that of the Control group. It suggests that overexpression of NDN can inhibit angiogenesis (Figure 2I).

3.1 NDN inhibits xenograft tumor growth in vivo

As demonstrated in (Figure 3A, B and C), overexpression of NDN markedly inhibited tumor growth in nude mice volume and decreased tumor weight.

3.2 NDN inhibit angiogenesis of xenograft tumors in vivo

The fluorescence signal intensity and the quantified results of fluorescence intensity of tumor mice in UP-NDN group was significantly lower than that of tumor mice in CON-NDN group (P = 0.0013, Figure 3D, E), indicating that angiogenesis in UP-NDN group was less than that in CON-NDN group. Paraffin embedding, sectioning, HE staining, and immunohistochemical detection of tumor tissues in nude mice showed that the angiogenesis marker CD31, UP-NDN group was significantly reduced compared with CON-NDN group (Figure 3F).

3.3 NDN inhibits the expression of VEGFA in mouse OS cell xenograft tumors

qRT-PCR analysis demonstrated that the levels of VEGFA-mRNA transcripts in the NDN-UP group were evidently lower than that in NDN-CON group (Figure 3G). Western blot results and the quantified results of Western blot indicated that the protein expression levels of VEGFA in the NDN-UP group was lower than that in the NDN-CON group (Figure 3H, I).

4 The result of co-immunoprecipitation

There is no interaction between the two molecules of NDN and VEGFA as shown in co-immunoprecipitation (Figure 4).

NDN was first discovered in the nervous system, it has been shown to has the function in inhibiting tumorigenesis. A study published recently by Chapman and Knowles ²⁰ showed that NDN was down-regulated in both cancer cell lines and primary tumors, suggesting that NDN may have a tumor suppressor effect ²¹. Shama Virani et al. found that NDN was highly expressed in some tumors and had carcinogenic effects. NDN has different effects on different tumors, which may be related to the microenvironment and cell environment ²². Understanding its mechanisms is very important for the research of NDN in tumors.

Previous studies have shown that NDN could inhibit the growth of ovarian cancer, breast cancer, and melanoma ^12–14. However, there are few reports on the expression of NDN in OS. In this study, we found that the expression of NDN in the OS cell line (Saos2, U2OS, Mg63) and OS tissue was lower than that of the normal chondrocyte line C28 and bone tissue, while the expression of VEGFA was higher. Current studies have found that NDN played different roles in different tumors and VEGFA plays a vital role in the angiogenesis and progression of osteosarcoma ^23,24. The negative correlation between the expression of NDN and VEGFA indicates that NDN may act as a tumor suppressor to inhibit the progression of OS.

Angiogenesis is necessary for aggressive tumor growth and metastasis, and helps control tumor progression. Researches have shown that various angiogenesis inhibitors were effective in tumor treatment ^25,26. With the increase of tumor size and metastatic potential and the formation of new blood vessels, the "angiogenesis switch" can be adjusted by disturbing the local balance of pro-angiogenic and anti-angiogenic factors ²⁶. Generally, tumors secrete various angiogenic factors, including VEGF ²⁷. VEGF gene might be closely related to the apoptosis and proliferation of osteosarcoma cells, and appears to be crucial for formation of vasculogenic mimicry in osteosarcoma ²⁸. In order to further explore the role of NDN in OS, we used lentivirus to transfect OS cells (Saos-2, U2OS) to increase the expression of NDN in OS cells. The angiogenesis test suggests that NDN effectively inhibits the angiogenesis of human umbilical vein endothelial cells. At the same time, FMT further indicated that the fluorescence intensity of MMP at the tumor site was weakened, and the expression of the angiogenesis marker CD31 was down-regulated. In a word, NDN inhibits tumor growth by restraining the formation of blood vessels in OS.

Tumor growth requires sufficient blood vessels to supply nutrients. The VEGF and its receptor (VEGFR) regulatory pathways are the core factors of tumor angiogenesis, and their expression levels are closely related to the occurrence and development of OS ^10,11. Through the experiments in vivo and in vitro, we found that high expression of NDN could inhibit the expression of VEGFA. By searching the XENA biological information database, we found that the correlation between NDN and VEGFA gene expression was R=-0.029, P = 0.64 (P < 0.01), which shows no statistical difference. By analyzing the above results, we find that NDN can inhibit the expression of VEGFA, but not through its direct interaction. This may be the result of the expression changes of soluble growth factors (for example VEGF) mediated by NDN, instead of a direct effect of soluble NDN. It suggested that NDN may affect tumor cells in a paracrine manner by acting on tumor capillaries. Another possible explanation is that NDN acts as a transcription factor, or it interacts with famous transcription factors such as E2F1 and p53 to regulate its transcription activity and indirectly regulate transcription ^15–17. Therefore, the loss of NDN may promote tumor survival due to its effect on micro-vessels.

The disadvantage of this experiment is that the sample size is small, the mechanism of action between NDN and VEGFA has not been thoroughly explored, and NDN has not been used as a therapeutic target in clinical trials. This can be the direction of our future work. This study shows that the up-regulated expression of NDN in OS cells and tissues can inhibit the expression of VEGFA, thereby inhibiting the angiogenesis, proliferation and metastasis of OS. The low expression of NDN in OS may also be regulated by transcription factors, methylation, and microRNAs at the transcription and translation levels. This is a problem that needs to be studied in the future work. This study confirms that NDN is a potential tumor suppressor in OS, which will help to further study the pathogenesis of OS.

NDN, Necdin; OS, osteosarcoma; VEGFA, vascular endothelial growth factor A; FMT, fluorescence molecular tomography; HUVEC, human umbilical vein endothelial cell; GAPDH, orglyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real-time PCR; VEGFR, vascular endothelial growth factor receptor

Author Contributions

All authors contributed to the article and approved the submitted version.

Conflict of interest

All authors declare no conflict of interest.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (no. 81772867), the Youth Science Fund Project of China (no.81902273), the Youth Fund of the Second Hospital of Shanxi Medical University of China (no.202002-4) and the Key plan of Shanxi Province of China (no.201903D321156).

Ethics Statement

The animal study was reviewed and approved by the Ethics Committee of Shanxi Medical University (2017LL077).

Statistical Analysis Assay

All data are presented as the mean ± SEM, and the significance of differences among groups were analyzed by ANOVA test, and the difference in unpaired groups was analyzed using Student’s t-test. Mann–Whitney U test for comparisons between two groups and in multiple groups the Kruskal-Wallis-H test was used when data did not coincide with a normal distribution. Repeated measures ANOVA was used for repeated measurement data. Statistical significance was set at p < 0.05 and the analyses were performed using SPSS (version 21.0, SPSS Inc., Chicago, USA).

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No competing interests reported.

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Necdin inhibits the proliferation of osteosarcoma cells by regulating the expression of VEGFA

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