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Objective To investigate the inhibitory effect of cordycepin on immune function injury in lung cancer rats after radiation therapy through JAK2/STAT3 signaling pathway. Methods Fifty rats were used to establish tumor-bearing model and other 10 rats were taken as normal group. After successful modeling, the rats were randomly divided into model group, radiotherapy group, cordycepin group, agonist group and agonist+cordycepin group (10 rats in each group). We compared tumor weight, tumor volume, tumor inhibition rate, IL-6, TNF-α, spleen index and thymus index, the number of T lymphocyte subsets, JAK2, p-JAK2, STAT3 and p-STAT3 protein expression levels among all groups. Results Compared with normal group, IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 in model group were increased, while spleen index, thymus index, CD4+ and CD4+/CD8+ were decreased (P < 0.05). Compared with model group, tumor weight, tumor volume, spleen index, thymus index, CD4+ and CD4+/CD8+ were decreased in radiotherapy group, while IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 were increased (P < 0.05). Compared with radiotherapy group, tumor weight, tumor volume, IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 were decreased in cordycepin group, while tumor inhibition rate, spleen index thymus index, CD4+ and CD4+/CD8+ were increased; tumor weight, tumor volume, IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 protein expression were increased in agonist group, while tumor inhibition rate, spleen index, thymus index, CD4+ and CD4+/CD8+ were decreased (P < 0.05). Compared with agonist+cordycepin group, tumor weight, tumor volume, IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 were decreased in cordycepin group, while tumor inhibition rate, spleen index, thymus index, CD4+ and CD4+/CD8+ were increased; tumor weight, tumor volume, IL-6, TNF-α, CD8+, p-JAK2 and p-STAT3 were increased in agonist group, while tumor inhibition rate, spleen index, thymus index, CD4+ and CD4+/CD8+ were decreased (P < 0.05). Conclusion Cordycepin can effectively inhibit the immune function injury in lung cancer rats after radiation therapy, and may play a regulatory role by inhibiting the JAK2/STAT3 signal pathway.
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Objective To investigate the effect of microRNA361-5p on radiosensitivity of osteosarcoma cells and its downstream regulatory mechanisms.Methods The radioresistant osteosarcoma cell line HOS-R was constructed and the expression of microRNA-361-Sp in HOS and HOS-R cells was detected by RT-qPCR.The survival rate and apoptosis rate were detected by clone formation assay and flow cytometry in HOS-R cells treated with up-regulated or down-regulated of miR-361-Sp,or FOXM1 depletion.Dual fluorescent luciferase reporter and western blot assays were used to measure the relationship between miR-361-5p and FOXM1.he effects of miR-361-5p on radiosensitivity of osteosarcoma cells were determined by cloning formation assay and flow cytometry.Results RT-qPCR results showed that miR-361-5p was low expressed in HOS-R cells.Colony formation and flow cytometry assays demonstrated that overexpression of miR-361-5p significantly decreased the survival rate and increased the apoptosis rate of HOS-R cells.In contrast,FOXM1 downregulation inhibited cell survival rate and induced apoptosis.Moreover,miR-361-5p negatively regulated FOXM1 expression.Besides,FOXM1 overexpression attenuated the miR-361-5p upregulation-mediated promotion of radiosensitivity in HOS-R cells.Conclusion miR-361-5p was involved in the radiosensitivity of osteosarcoma cells by inhibiting FOXM1.
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Objective To investigate the effect of LncRNA HULC on radiosensitivity of osteosarcoma cells. Methods Osteosarcoma cells OS732 was infected by shRNA HULC lentivirus, and the interference effect was determined by qRT-PCR. Osteosarcoma cells infected with shRNA HULC lentivirus were irradiated with 8 Gy X-rays. MTT, PI monochrome staining and Annexin V-FITC/PI double staining were used to detect cell proliferation, cell cycle and apoptosis, respectively. Western blot was used to detect the protein levels of p21, Cyclin D1, C-Caspase-3 and Cyt-C in cytoplasm and mitochondria. Plate cloning assay was used to evaluate cell radiosensitivity. Results The expression of HULC in osteosarcoma cells was significantly down-regulated by shRNA HULC lentivirus infection. Down-regulation of HULC or irradiation inhibited osteosarcoma cell proliferation [(100. 00±9. 65)％ vs. (71. 36±5. 27)％, (63. 48± 5. 93)％, t=4. 512, 5. 585, P<0. 05 ] , blocked cell cycle [ ( 50. 15 ± 5. 14 )％ vs. ( 62. 35 ± 4. 22 )％, (66. 05±5. 23)％,t=3. 177,3. 756,P<0. 05], induced cell apoptosis [(2. 98±0. 23)％ vs. (22. 61± 3. 26)％, (26. 14±2. 81)％,t=8. 898,10. 498,P<0. 05], promoted the expressions of p21 and Cyclin D1 in cells, down-regulated the level of C-Caspase-3 protein, increased the level of Cyt-C protein in cytoplasm, and down-regulated the level of Cyt-C protein in mitochondria. Downregulation of HULC combined with irradiation yield much more effects on cell proliferation inhibition [ ( 71. 36 ± 5. 27 )％, (63.48±5.93)％ vs. (49.32±5.76)％, t=4.890, 2.967, P<0.05], cell cycle arrest [(62.35± 4. 22)％, (66. 05±5. 23)％ vs. (77. 17±7. 54)％, t=2. 983, 2. 106, P<0. 05], apoptosis induction [(22. 61±3. 26)％, (26. 14±2. 81)％ vs. (36. 21±3. 26) ％,t=6. 164, 4. 564, P<0. 05] and the expressions of p21, Cyclin D1, C-Caspase-3 and Cyt-C in osteosarcoma cells. The radiosensitization ratio of down-regulation of HULC was 1. 432. Conclusions Down-regulation of HULC enhances radiosensitivity of osteosarcoma cells, which may be related to cell cycle arrest and apoptosis induction.
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Objective To investigate the effect of CC chemokine receptor 4(CCR4) on sorafenib radiosensitivity and tumorigenesis of hepatocellular carcinoma cells in nude mouse models.Methods Western blot was used to detect the expression of CCR4 in hepatocellular carcinoma cell line.Lentivirus was utilized to construct PLC/PRF/5 and SMMC-7721 cell lines stably overexpressing and silencing CCR4,which were verified by Western blot.The influence of CCR4 on the radiosensitivity of hepatocellular carcinoma cells was assessed by plate clone formation assay.The effect of CCR4 on the tumorigenesis in hepatocellular carcinoma cells in vivo was evaluated by tumorigenesis assay in nude mice.Results CCR4 was highly expressed in highly-metastatic hepatocellular carcinoma cells and lowly expressed in hepatocellular carcinoma cells with low metastases.The PLC/PRF/5 and SMMC-7721 cells,which stably overexpressed and silenced CCR4,were successfully established.Overexpression of CCR4 reversed the inhibitory effect of sorafenib radiotherapy on PLC/PEF/5,whereas knockdown of CCR4 could increase the radiosensitivity of SMMC-7721 to sorafenib.Overexpressing CCR4 could promote the tumorigenicity of PLC/PEF/5,whereas knockdown of CCR4 could inhibit the tumorigenicity of SMMC-7721 in nude mice.Conclusion CCR4 overexpression significantly reduces the radiosensitivity of PLC/PRF/5 and increases the tumorigenicity in nude mice,whereas knockdown of CCR4 considerably increases the chemosensitivity and radiosensitivity of SMMC-7721 and suppresses the tumorigenicity in nude mice.
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Objective@#To investigate the effect of silencing GRAMD1A and inhibiting STAT5 signaling pathway on the radiosensitivity of Huh7 cells in order to provide new ideas for the clinical combined therapy of hepatocellular carcinoma.@*Methods@# The Huh7 cells silencing GRAMD1A was constructed by infecting lentivirus and verified by qPCR and Western blot. QPCR and luciferase reporter assays were used to detect the effect of silencing GRAMD1A on the expression of STAT5 and its downstream genes. Colony formation and apoptosis were detected to evaluate the effects of silencing GRAMD1A and STAT5 inhibitor SH-4-54 on cell radiosensitivity.@*Results@#After 2 Gy exposure of the constructed Huh7 cells, the colony formation ability of the silencing GRAMD1A combined irradiation group was significantly lower than that of the negative control combined irradiation group, and the difference was statistically significant (t=8. 494, P<0.05). Silence apoptosis in the GRAMD1A combined irradiation group was significantly increased compared with the negative control combined irradiation group (t=3.560, P<0.05). After silencing GRAMD1A, the radiosensitivity of Huh7 cells was significantly increased, and the expression of STAT5 and its downstream genes was significantly reduced in cells.The survival rate of the SH-4-54 inhibitor combined irradiation group was significantly lower than that of the dimethyl sulfoxide combined irradiation group, and the difference was statistically significant (t=8.660, P<0.05). SH-4-54 inhibited STAT5 after passage, the radiosensitivity of Huh7 cells was significantly increased.@*Conclusions@# Silencing GRAMD1A could significantly enhance the radiosensitivity of Huh7 cells via STAT5 signaling pathway, indicating that GRAMD1A plays an important role in the development and progression of HCC. This finding may provide a new target for HCC therapy.
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Objective To construct the KU80 inhibition cell model by RNAi in U2OS cell and to explore the relationship between the Ku80,telomeres and radiosensitivity in telomerase-negative tumor cells.Methods U2OS cells were transfected with the recombinant plasmids of pshRNA-K80 by the lipofectamine,and the stable transfected cell clones were selected by G418.After the selection,the cells were collected and analyzed by the flow cytometry.RT-PCR and Western blot were used to measure the expression of Ku80 and Real-time PCR was used to detect the length of telomeres.The radiosensitivity of U2OS was determined by clone formation array.Results The transfection efficiency of the positive cell clones detected by the flow cytometry was (83.23 ± 7.63) ％.The inhibition rate of the Ku80 gene transcription in the cell group with recombinant plasmid was(68.09 ± 1.16)％ and the inhibition rate of the Ku80 protein expression in the same group was (11.03 ± 2.45) ％.The results of Real-time PCR showed that the telomere length of the cell group with recombinant plasmid (1.07 ± 0.07) was significantly shorter than that of the control group (4.42 ± 1.30,F =38.58,P ＜ 0.05) and that of the empty plasmid group (4.11 ±0.84,F =38.58,P ＜ 0.05).Compared to the control group,the telomere length of the empty plasmid group did not changed(4.42 ±0.84 vs.4.11 ±0.84).U2OS cells with Ku80 expression suppressed had lower SF2 than that of the control cells (F =1089.61,P ＜0.05),and resulted in the SER of 1.47.Conclusions The Ku80 inhibition cell model in telomerase-negative U2OS cell line is successfully established which has the shorter telomere length,and is more sensitive to radiation.Telomere shortening caused by pshRNA-of Ku80 is likely to be one of the mechanisms of radiosensitization in this kind of cell model.
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To observe sublingual vein characteristics and the expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) proteins in sublingual tissues of Beagle dogs with cirrhotic portal hypertension.