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Objective:To investigate the mechanism that how Musashi RNA-binding protein 2 (MSI2) regulates HCC growth.Methods:Short hairpin RNA (shRNA) was transfected to inhibit MSI2 expression, and cells were divided into transfection control plasmid (sh-Ctrl) group and sh-MSI2 group. In the MSI2 overexpression experiment, cells were divided into control group (Vector group, transfected with blank plasmid Vector) and overexpression group (MSI2 group, transfected with MSI2 recombinant plasmid). Cell proliferation was detected by CCK-8 and plate cloning assays. Western blotting was used to detect the expressions of β-catenin, transcription factor 7 (TCF7) and lymphoid enhancer factor 1 (LEF1) after MSI2 intervention. In the Rescue recovery experiment, cells were divided into MSI2+ sh-Ctrl group (transfected with MSI2 recombinant plasmid and sh-Ctrl plasmid at the same time) and MSI2+ sh-β-catenin group (transfected with MSI2 recombinant plasmid and sh-β-catenin plasmid at the same time). On the basis of overexpression of MSI2, β-catenin was knocked down to detect the proliferation ability of HCC cells.Results:The proliferation rates of HepG2 and MHCC97H cells in the sh-MSI2 group were lower than those in the sh-Ctrl group, and the difference was statistically significant ( P<0.05). The proliferation rates of SMMC-7721 cells and MHCC97L cells in the MSI2 group were higher than those in the Vector group, and the difference was statistically significant ( P<0.05). The results of the clone formation experiment showed that compared with the sh-Ctrl group, the number of HepG2 cell clones in the sh-MSI2 group [(129.7±6.5) vs. (286.0±12.8)] and the number of MHCC97H cell clones [(134.0±6.7) vs. (248.0±14.1)] were reduced, and the differences were statistically significant ( P<0.05); compared with the Vector group, the number of SMMC-7721 cell clones [(242.0±5.6) vs. (135.3±8.7)] and MHCC97L cell clones [(308.0±9.0) vs. (149.7±5.9)] in the MIS2 group were increased, and the difference was statistically significant ( P<0.05). The mRNA and protein expression of β-catenin, TCF7 and LEF1 in HepG2 and MHCC97H cells in the sh-MSI2 group were lower than those in the sh-Ctrl group, and the differences between groups were statistically significant ( P<0.05). In contrast, compared with the Vector group, the mRNA and protein expression levels of β-catenin, TCF7 and LEF1 in SMMC-7721 and MHCC97L cells in the MSI2 group were all increased, and the differences were statistically significant ( P<0.05). Compared with the MSI2+ sh-Ctrl group, the cell proliferation ability of the MSI2+ sh-β-catenin group was decreased, and the difference was statistically significant ( P<0.05). Plate cloning experiments showed that the number of cell clones in the MSI2+ sh-β-catenin group was less than that in the MSI2+ sh-Ctrl group [(138.3±7.0) vs. (246.3±8.0), P=0.028]. Conclusion:MSI2 promotes HCC cell proliferation through Wnt/β-catenin signaling pathway, leading to tumor progression.
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Objective To analyze tumor immune microenvironment and related mechanisms in liver cancer.Methods We included 10 cases of hepatocellular carcinoma,hepatitis B patients and healthy volunteers from January 2015 to December 2017 in Shanxi Grand Hospital.We first detected the peripheral and local GM-CSF level in each group,detected myeloid-derived suppressor cells (MDSCs) GM-CSF and pathway-related protein expression.from liver cancer,tumor margin and normal liver tissue through flow cytometry and immunohistochemistry,Finally,we transfected the CCR4-NOT transcriptional complex subunit 7 (CNOT7) recombinant plasmid in the hepatoma cell line,and then detected the related protein expression.Results There was no significant difference for peripheral blood GM-CSF level between liver cancer group,hepatitis group and control group (P＞0.05).The level of local GM-CSF was (32.2±8.9) ng/L,which was higher than that of hepatocellular carcinoma (9.7±2.7) ng/L and normal liver tissue (11.6±2.9) ng/L.The difference was statistically significant (P＜0.05).The proportion of MDSCs at the edge of the tumor was (9.9 ±3.6) ％,which was higher than that of liver cancer (4.0± 1.5) ％ and normal liver tissue (6.3±2.3) ％,and the difference was statistically significant (P＜0.05).Immunohistochemistrydata was consistent with previous data.Compared with normal liver tissue,CNOT7 and STAT3 were highly expressed in liver cancer tissues,while STAT1 was lowly expressed.HepG2 human hepatoma cells were selected for transfection.Compared with the empty plasmid group,CNOT7 expression was decreased in the knocking out group at the same time STAT1 expression was increased,STAT3 and GM-CSF expression was decreased.Conclusion In hepatocellular carcinoma,the secretion of GM-CSF increased and the number of MDSCs increased.Knocking out CNOT7 reduced GM-CSF secretion and activate the JAK/STAT signaling pathway.
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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.
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Objective To study the regulation of dendritic cells by recombinant glycated polylysine-coupled MIP-3α-FL double-gene targeting expression vector in liver cancer immune microenvironment.Methods H22 hepatocarcinoma cells were transfected with recombinant plasmid of MIP-3α-FL (shMIP-3α-FL) and injected into hepatoma model mice.The survival time,tumor size were compared.Flow cytometry was used to measure the number and phenotype of tumor infiltrating DCs.Results Western blot and ELISA demonstrated that the secretion of MIP-3α and FL in H22 cells was significantly increased after transfection with MIP-3α-FL.The survival time of the mice in the experimental group was significantly prolonged,the tumor size decreased.Flow cytometry showed that the number of tumor-infiltrating DCs in the experimental group was significantly higher than that in the control group;the expression of CD80 and CD86 in the infiltrating dendritic cells (TIDCs) was significantly higher than that of the control group.Conclusions The co-action of MIP-3α and FL can significantly promote DC accumulation,maturation,and conjugate glycosylated polylysine carriers increase the precision of targeting and enhance the antigenpresentation of the DCs.
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Objective To investigate whether astragaloside Ⅳ can regulate the multidrug resistance of HepG2/GCS-resistant cell lines,restore the sensitivity of drag-resistant cell lines to adriamycin (ADM) and its mechanism.Methods We used recombinant GCS (shRNAS) and control recombinant plasmids and did the transfection with HepG2 cells.RT-PCR and Western blot were used to analyze the expression of GCS.Astragaloside Ⅳ cytotoxicity experiments and ADM were performed in experimental and control groups.Hoechst 33258 was detected in two groups,apoptosis was detected by flow cytometry,and protein expression of caspase 9,3,Bax,and bcl-2 were detected by Western blot.Results RT-PCR and fluorescence observation showed that GCS was highly expressed after the transfection.Western blot showed that compared with control group,and HepG2GCS group,GCS and MDR1 expression were higher than;Astragaloside Ⅳ cytotoxicity experiment showed tumor proliferation was not regulated by GCS(FHepG2GCS=0.308,FHepG2EV =0.216,FHepG2 =0.153,P＞ 0.05),ADM in vitro tumor cell inhibition experiments showed that HepG2GCS cells were resistant to ADM (50％ cell transplantation concentration were 7.5,7.5,15 μg/ml);Hoechst 33258 and flow cytometry showed that Astragaloside Ⅳ can restore ADM tumor inhibition;Western blot showed that compared to untreated HepG2EV and HepG2GCS cells,protein level of caspase 9,caspase 3 were increased in Ast+HepG2EV and Ast+HepG2GCS groups (t=7.17,P＜0.05).At the same time,Bax and Bcl-2 were significantly different in each group (P＜ 0.05).Conclusions Astragaloside Ⅳ reverses multidrug resistance in HepG2/GCS cell lines,restores its sensitivity to ADM,promotes apoptosis in tumor cells through the caspase pathway and Bax pathway,and thus plays an important role in cancer chemotherapy.