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SMMC-7721 cells (the Chinese Academy of Sciences, China) and MHCC97-H cells (HangZhou Hibio Technology Co.,LTD, China) were maintained in the 1640 (Gibco, USA) and DMEM medium (Gibco, USA) respectively, with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin solution (Meilunbio, China) as the supplement. Cell lines were cultured in a humidified environment with 5% CO2 and 20% O2 (normoxic groups) or 1% O2 (hypoxic groups) at 37 °C. DEX (Yangtze River, China) with a concentration of 0.5 μg/mL was designated for subsequent experiments, and the α2-AR antagonist yohimbine (Aladdin, China) was used in this study.
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Animal experiments were carried out in accordance with the National Institutes of Health guidelines and were approved in advance by the Animal Ethical and Welfare Committee of Hibiotech Company (China). Experimental BALB/c nude mice (male, 4–6 weeks, 16–22 g) were purchased from Sleek Experimental Animal Center (China) and fed in the SPF-grade animal center of Hibiotech company. SMMC7721 cells were subcutaneously injected into the mice (5 × 106 cells per mouse). The tumor volume was monitored every three days until the 14th day. The subcutaneous tumor was cut into 1 mm3 sized tissue and inoculated under the left liver envelope of BALB/c nude mice[28]. The mice with successful modeling were randomly divided into low-dose, high-dose, and control groups (n = 8/per group) and intraperitoneally injected with an equal volume of 10 μg/kg, 25 μg/kg DEX, and normal saline respectively each day for consecutive 14 days .
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Individual wells of the tube formation slide (ibid, Germany) were coated with 250 µL Matrigel (Corning, USA) for 30 min before cell seeding. MHCC97-H and SMMC-7721 cells (2 × 104 cells/well) were seeded in coated wells and incubated for 12 h, 24 h, and 48 h. The tube shapes were photographed using an inverted microscope (Olympus, Japan).
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When the cell density reached 30%–50%, MHCC97-H and SMMC-7721 cells were cultured in Opti-MEM (Gibco, USA). The siRNA targeting HIF-1α and the negative control sequence were purchased from RiBobio (China) for transfection with ExFect2000, according to the manufacturer’s specifications. qPCR and western blotting were performed to detect HIF-1α expression. Optimal cell groups were determined for subsequent experiments. To mimic the effect of DEX on human hepatocellular carcinoma angiogenesis in vitro, cells were cultured with 0.5 μg/mL DEX for 12 h, 24 h, and 48 h in a medium containing a series of concentrations of yohimbine. Cultured cells were collected to analyze the expression of protein and RNA, and supernatants were harvested for ELISA.
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According to the extracted using TRIzol reagent (Generay Biotech, China), and HiScript® II qRT SuperMix (Vazyme Biotech, China) was used to extract total RNA and transcribe it into cDNA. The reaction was performed at 25 °C, 42 °C, and 70 °C for 10 min, 60 min, and 10 min, respectively. The ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech, China) was used to perform quantitative reverse transcriptase PCR (qRT-PCR). Actin was used as the internal reference gene listed in Table 1. The relative mRNA expression of the target genes was analyzed using the 2−ΔΔCt method[19]. The data were analyzed using the comparative Ct method. Primers (Sunny Biotech, China) for PCR were as follows:
Table 1. PCR primers sequences and length of fragments
Primer Sequence bp (s) Size of product (bp) Homo Actin F TGACGTGGACATCCGCAAAG 20 205 Homo Actin R CTGGAAGGTGGACAGCGAG 19 Homo HIF-1α F CACCACAGGACAGTACAGGAT 21 146 Homo HIF-1α R CGTGCTGAATAATACCACTCACA 23 -
First, cells were lysed in RIPA buffer (Beyotime, China) containing PMSF (Beyotime, China). Second, a bicinchoninic acid assay (Sigma, USA) was performed to quantify the protein concentrations. Third, the protein extracts (30 μg) were heated, denatured, loaded on 10% SDS-PAGE for electrophoresis, and then transferred to a PVDF membrane (Merck Millipore, Ireland). Thereafter, the membrane was blocked with 5% skim milk in TBS-T for 2 h at 37 °C. After that, the membrane was probed with primary and secondary antibodies at 4 °C overnight, aiming to detect the proteins of interest, and anti-β-actin (1:4,000, ab008), anti-HIF-1α (1:1,000, pA1-16601) were included. After four washes, the membrane was developed with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. HRP-conjugated secondary antibodies (Multi Sciences, China, 1:5,000). Staining was then observed using a micro ultraviolet spectrophotometer (Merinton, China) with ECL solution (Beyotime, China). Finally, the loading control was the constitutively expressed protein β-actin, and the blots were detected using an enhanced chemiluminescence system (Bio-Rad) and Image J.
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The concentration of VEGFA secreted by MHCC97-H and SMMC-7721 cells was determined by ELISA using the corresponding ELISA kit (Multi Sciences, China), following the manufacturer’s instructions. The samples collected from the supernatants and the standard products were placed into plates with the anti-VEGFA antibody (Invitrogen, USA) at room temperature for 2 h. Then, the substrate solution and stop solution were added to react gradually. The protein content was calculated according to the OD values at a wavelength of 450 nm.
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Paraffin-embedded mice hepatocellular carcinoma tissues were cut into slices. The slides were deparaffinized in xylene, hydrated in gradient alcohol, treated with 3% H2O2 to block endogenous peroxidase activity, and fixed with a citrate buffer for high-pressure thermal treatment. The tissues were cultured with mouse anti-CD31, anti-HIF-1α (Invitrogen, USA), and anti-VEGFA (Invitrogen, USA) antibodies at 4 ℃ overnight, followed by a setting with HRP-conjugated secondary antibodies (Multi Sciences, China) 37 ℃ for 30 min. The slices were successively stained with DAB, counterstained with hematoxylin, differentiated, turned blue, dehydrated, and sealed. The graphs were captured using a microscope under the same conditions.
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After 48-hour fixation in 4% paraformaldehyde and paraffin embedding, the tumor tissue was conventionally cut into 5 μm portions. Then, the portions were dewaxed and rehydrated, and the antigen was retrieved by PH 9.0. Slices were sealed at 37 ℃ for 30 min and hydrated for incubation after washing 3 times with TBS for 5 min each time. Then Rabbit anti-CD31 polyclonal antibody (Abcam, AB281583, 1:100 dilution) and anti-CD147 antibody (Proteinatech, 66443-1-IG, 1:100 dilution) were incubated overnight at 4 ℃. After washing 3 times in TBS-T, sections were incubated with diluted fluorescent-labeled secondary antibodies for 30 min at room temperature and then stained in darkness with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min to indicate the nuclei. Images were captured using a fluorescence microscope (U-25ND25; Olympus, Tokyo, Japan).
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Statistical analysis was performed using Graphpad Prism 9.0 (Graphpad, USA). Quantitative measurement data were displayed as the mean ± SEM. Two-way and one-way ANOVA procedures were used in the cell and animal models experiments respectively. Statistical significance was set at P < 0.05.
doi: 10.3967/bes2022.120
Dexmedetomidine Promotes Angiogenesis and Vasculogenic Mimicry in Human Hepatocellular Carcinoma through α2-AR/HIF-1α/VEGFA Pathway
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Abstract:
Objective Dexmedetomidine (DEX), the most specific α2-adrenergic receptor agonist widely used for its sedative and analgesic properties, has been reported to upregulate HIF-1α expression to protect hypoxic and ischemic tissues. However, it is largely unclear whether DEX can also upregulate Hypoxia-inducible factor-1 alpha (HIF-1α) expression and its downstream vascular endothelial growth factor-A (VEGFA) in cancer tissues with oxygen-deficient tumor microenvironment. Methods We used SMMC-7721 cells, MHCC97-H cells, and a mouse model of orthotopic hepatic carcinoma to explore the effect of DEX on angiogenesis and vasculogenic mimicry (VM) and its mechanism. Under normoxic (20% O2) and hypoxic (1% O2) conditions, DEX was used to intervene cells, and yohimbine was used to rescue them. Results The results showed that DEX promoted angiogenesis and VM in human liver cancer cells within a certain dose range, and the addition of yohimbine inhibited this effect. DEX could activate HIF-1α/VEGFA pathway, which was further verified by silencing HIF-1α. Consistently, in vivo results also showed that DEX can up-regulate HIF-1α/VEGFA expression, and enhance the number of VM channels and microvessel density (MVD). Conclusion We believe that HIF-1α/VEGFA might be an important signaling pathway by which DEX promotes angiogenesis and VM formation in human hepatocellular carcinoma, whereas α2-adrenergic receptor mediation might be the critical mechanisms. -
Key words:
- Hepatocellular carcinoma /
- Dexmedetomidine /
- Yohimbine /
- α2-adrenergic receptor /
- HIF-1a /
- VEGFA /
- Vascular mimicry /
- Angiogenesis
The authors declared no conflict of interest.
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: 2) CONFLICT OF INTEREST: -
Figure 1. Low-dose DEX promotes angiogenesis and VM in a mouse HCC orthotopic model. (A) CD147, CD31 and DAPI immunofluorescence staining showed the formation of angiogenesis and VM in tumor tissues. (A) White dotted circle indicated the VM lumen structures. Individual and merged images of CD147, CD31, and DAPI staining show that MVD and the number of VM in the low-dose group were significantly increased. But in the high-dose group, MVD and the number of VM was lower than in the control group. Scale bar: 50 μm. (B–C) Number of VM channels and MVD in each group. 0.01 < *P < 0.05, **P < 0.01. DEX: Dexmedetomidine; VM: vasculogenic mimicry; HCC: hepatocellular carcinoma.
Figure 2. VM formation in SMMC-7721 and MHCC97-H cell lines in vitro. (A) The tube formation assay in SMMC-7721 cells in each group. Scale bar, 50 μm. (B–D) The number of tubes formed by SMMC-7721 cells in each group. (E) The tube formation assay in MHCC97-H cells in each group. Scale bar, 100 μm. (F–H) The number of tubes formed by MHCC97-H cells in each group. Both under normoxia and hypoxia conditions, 0.5 μg/mL DEX promoted VM formation in SMMC-7721 cell and MHCC97-H lines in vitro. Yohimbine could inhibit this effect. 0.01 < *P < 0.05, **P < 0.01. NS means there is no statistically significant difference between groups.
Figure 3. Immunohistochemical staining of CD31, HIF-1α, and VEGFA. (A) Immunohistochemistry staining for CD31, HIF-1α, and VEGFA expression of HCC tissue. (B–D) The Optical Density Value of the immunohistochemistry staining for CD31, HIF-1α, and VEGFA. After 14 days of continuous intraperitoneal injection of low-dose and high doses of DEX, Immunohistochemistry staining demonstrated that CD31, HIF-1α, and VEGFA expression levels were enhanced remarkedly in the low dose groups but dropped in high dose group. Scale bar: 50 μm. 0.01 < *P < 0.05, **P < 0.01.
Figure 4. DEX upregulated the HIF-1α protein expression level in MHCC97-H and SMMC-7721 cells. (A–B) HIF-1α protein expression were analyzed with western blot in MHCC97-H and SMMC-7721 cells under 20% and 1% O2 conditions after treatment with DEX for 12 h, 24 h, and 48 h respectively, and β-actin was used as a protein loading control in western blot assay. (C–H) The gray value ratio of the western blot.
Figure 5. DEX upregulated the HIF-1α expression in MHCC97-H and SMMC-7721 cells at gene levels. (A–F) qRT-PCR analysis of mRNA levels of HIF-1α in MHCC97-H and SMMC-7721 cells. The mRNA levels of HIF-1α in the two cancer cell lines were substantially elevated after DEX treatment. Furthermore, hypoxia insult could aggravate, and Yohimbine could reverse it. The regulatory effect of DEX or Yohimbine was suppressed in HIF-1α si-RNA groups. Error bars represent SD. *P < 0.05, **P < 0.01. NS represent no statistically significant difference between the groups.
Figure 6. Determination of the VEGFA expression in the ELISA test. Two different conditions (20% O2 and 1% O2), three different time points, 12 h, 24 h, and 48 h in MHCC97-H (A–C) and SMMC-7721 cells (D–F), were tested in ELASA. The experiment was conducted with negative control. The VEGFA concentration (pg/mL) is shown on the horizontal axis. Error bars display the standard deviation of the mean of three experiments. *P < 0.05, **P < 0.01. NS represent there were no significant differences between groups.
Table 1. PCR primers sequences and length of fragments
Primer Sequence bp (s) Size of product (bp) Homo Actin F TGACGTGGACATCCGCAAAG 20 205 Homo Actin R CTGGAAGGTGGACAGCGAG 19 Homo HIF-1α F CACCACAGGACAGTACAGGAT 21 146 Homo HIF-1α R CGTGCTGAATAATACCACTCACA 23 -
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