ABSTRACT
Objective To study the effect of human CCR4-NOT transcription complex subunit 7 (CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group, control group, and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy, and the expression level of CNOT7, transforming growth factor-β1 (TGF-β1), and nuclear factor-kappa B (NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer (NK) cells was detected by flow cytometry. Results Compared with the control group, the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group, and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However, in the CNOT7 overexpression group, the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells, and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However, in the CNOT7 overexpression group, the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.
ABSTRACT
Objective: To investigate the influence of gelatin sponge microparticles-TACE (GSMs-TACE) on myeloid-derived suppressor cells (MDSCs) in peripheral blood of patients with Barcelona clinic liver cancer (BCLC) classification stage B hepatocellular carcinoma (HCC). Methods: Five patients with clinically diagnosed BCLC B-stage HCC (HCC group) underwent GSMs-TACE. Flow cytometry was used to detect the frequency of MDSCs (the proportion of MDSCs clusters to HLA-DR-cell) in the peripheral blood of patients before GSMs-TACE and 10 days as well as 30 days after operation, respectively. Seven healthy volunteers (normal control group) were enrolled. The MDSCs frequency of normal control group was detected simultaneously with HCC group before GSMs-TACE. Statistical analysis was performed to compare the differences of the frequency of MDSCs in HCC patients before and after GSMs-TACE. And the frequency of MDSCs of HCC group was compared with that of normal control group. Results: The frequency of MDSCs in peripheral blood of patients with HCC before GSMs-TACE was (30.26±12.12)%, which decreased to (10.22±3.79)% after 10 days and decreased to (7.33±3.38)% after 30 days (P<0.001). Pairwise comparison showed that the frequency of MDSCs at 30 days (P<0.001) and 10 days (P=0.011) after GSMs-TACE was lower than that before operation,respectively. The frequency of preoperative MDSCs of HCC group was statistically higher than that of normal control group ([30.26±12.12]% vs [3.41±1.89]%, t=5.876, P<0.001). Conclusion: The frequency of MDSCs in peripheral blood of patients with BCLC B-stage HCC significantly reduced after GSMs-TACE treatment. GSMs-TACE treatment has positive regulation effect on the immune function of patients.
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Objective To explore the protective effect of ETaR siRNA on renal ischemia reperfusion injury (IRI) by changing the immuno-microenvironment in rats .Methods A total of 40 male Sprague-Dawley (SD) rats were randomized into four groups of sham ,IR ,negative siRNA and ETaR siRNA .A renal IRI model was generated by clamping left renal artery .ETaR siRNA was delivered into kidney through renal vein by a retrograde 'hydrodynamic' injection .Blood samples were collected for detecting renal function and kidney tissue harvested for Hematoxylin & Eosin (HE) staining , TdT-mediated dUTP Nick-End Labeling (TUNEL) staining ,polymerase chain reaction (PCR) and Western blot at 48 h post-reperfusion .Results Serum creatinine ,blood urea nitrogen and renal apoptotic cells increased and renal tissue was injured after IR . The changes were inhibited by ETaR siRNA . PCR showed that ETaR siRNA treatment significantly down-regulated the expressions of inflammatory factors TNF-α , IFN-γ and IL-6 and transcription factor NF-κB induced by IR .Conclusions ETaR siRNA can effectively improve the immunomicroenvironment and thereby alleviate renal ischemia reperfusion injury .