2.Apoptosis-inducing effects of AgLA2 on SPC-A-1 cells and its mechanism in vitro
Huagang LIU ; Sanhai QIN ; Limin LIU ; Bolong WANG ; Yancheng LIU ; Zhenfeng CHEN
Chinese Pharmacological Bulletin 2003;0(11):-
Aim To investigate the apoptosis-inducing effects of AgLA2 on lung carcinoma cells SPC-A-1 and its mechanism in vitro.Methods The MTT assay was used to assess the proliferation of SPC-A-1 cells treated with AgLA2 in vitro.Apoptosis-inducing effects was investigated by DNA agarose gel electrophoresis,cell morphology and Elisa.RT-PCR was used to measure the expression of bcl-2 and bax mRNA,and immunocytochemistry was used to measure the expression of bcl-2 and bax protein.Results The IC50 of AgLA2 to SPC-A-1 cells was(3.447?0.436)mg?L-1.Treated with AgLA2,typical nuclear chromatine condensation and fragmentation were observed.The concentration of Caspase-3 in the group treated with AgLA2 was higher than that of the control group.Treated with AgLA2,bcl-2 mRNA,protein expression decreased while bax mRNA,protein expression increased.Conclusions AgLA2 can inhibit proliferation and induce apoptosis of SPC-A-1 cells.Its mechanism of action may be related to changing the ratio of bax/bcl-2 and the set-point of apoptosis,making the apoptosis power hold dominance.
3.Establishment of Model of Dynamic Change of Immune Status of ACLF Induced by ConA Repeated Administration in Mice
Nannan ZHANG ; Shuyin YANG ; Liuying CHEN ; Shan YIN ; Shijin WANG ; Sanhai LIU ; Beibei WANG ; Zheng WANG ; Hai LI
Chinese Journal of Gastroenterology 2016;21(6):326-330
Background:Acute-on-chronic liver failure( ACLF)is a commonly seen liver failure in China,and lacking an animal model that can effectively simulate the dynamic change of immune status of ACLF. Aims:To establish an animal model that can simulate dynamic change of immune status of ACLF by repeated administration of concanavalin A(ConA). Methods:Mice were randomly divided into normal control group and ConA repeated administration group. Mice in ConA repeated administration group were injected with ConA 15 mg/ kg through retrobulbar angular vein every 48 hours for 5 times,and mice in control group were injected with same volume of 0. 9% NaCl solution. Serum levels of IL-6,IL-10,IL- 12,TNF-α,IFN-γ,MCP-1 in peripheral blood were assessed by CBA assay,and the ratio of IL-10/ TNF-α was calculated. The expression of HLA-DR,number and proportion of CD4+ T cells and the expression of PD-1 of monocytes in peripheral blood were detected by flow cytometry. Results:Peripheral blood cytokines changed from predominated proinflammatory cytokines into predominated anti-inflammatory cytokines with the increasing in time of administration in ConA repeated administration group. Compared with control group,HLA-DR expression of monocytes in peripheral blood was significantly decreased(P <0. 05),number and proportion of CD4+ T cells were significantly decreased(P <0. 05), and PD-1 expression was significantly increased( P < 0. 05)in ConA repeated administration group. Conclusions:An animal model of ACLF immune status from systemic inflammatory response syndrome( SIRS) to compensatory antiinflammatory response syndrome(CARS)induced by repeated ConA stimulation is successfully established.
4.Clinical significance of NFAT5 expression in hepatocellular carcinoma and its regulatory effect on the expression of inflammatory related factors IL-6 and TNF-α
Liang CHEN ; Sanhai LIU ; Qiang HUANG
Chinese Journal of Endocrine Surgery 2023;17(6):720-723
Objective:To explore the clinical significance of nuclear factor of activated T cells 5 (NFAT5) expression in hepatocellular carcinoma and its regulatory effect on the expression of inflammatory related factors IL-6 and TNF-α.Methods:Immunohistochemistry was used to detect the expression of NFAT5 in the tissues of 76 patients with liver cancer and their adjacent tissues. HuH-7 cells were divided into experimental group and control group. The cells in the experimental group were transfected with NFAT5-siRNA plasmid, and the cells in the control group were transfected with NC-siRNA plasmid. The content of NFAT5 mRNA in the cells of each group was detected by fluorescence quantitative PCR, and the content of NFAT5 mRNA in each group was detected by Western blotting. The expression of NFAT5, IL6 and TNF-α in the cells, and the proliferation ability of the cells in each group was detected by CCK8.Results:The positive rate of NFAT5 in liver cancer tissues was 81.58% (62/76) , and the expression rate in adjacent tissues was 6.58% (5/76) . The expression rate of NFAT5 in hepatocellular carcinoma tissues was significantly higher than that in adjacent tissues, and the difference was statistically significant. Academic significance ( P<0.001) . After transfection of siRNA, the expression levels of NFAT5 mRNA in HuH-7 cells in experimental group and control group were 1.03±0.19 and 0.33±0.11, respectively. The protein expression levels of NFAT5 were 0.94±0.14 and 0.26±0.07, respectively, and the expression levels of IL6 were 1.09±0.16 and 0.49±0.10, respectively. The expression levels of TNF-α were 1.21±0.13 and 0.28±0.08, respectively, and the expressions of IL6 and TNF-α were significantly decreased (all P<0.05) . The absorbance of HuH-7 cells in the experimental group and the control group at 120 h was 2.21±0.12 and 1.12±0.11, respectively, and the cell proliferation ability of the experimental group was significantly decreased ( P<0.05) . Conclusion:The expression of NFAT5 is significantly increased in hepatocellular carcinoma, which may be related to the malignancy of hepatocellular carcinoma. And NFAT5 affects the proliferation of hepatocellular carcinoma cells by regulating the expression of IL6 and TNF-α.
5.Effects of microRNA-26a targeted high-mobility group A2 protein antibody on the proliferation and migration of hepatoma cells
Ruojue JIN ; Sanhai LIU ; Qiang HUANG ; Chunming WU ; Guangwei ZHOU
Chinese Journal of Primary Medicine and Pharmacy 2021;28(10):1441-1447
Objective:To investigate the effects of miRNA-26a (miR-26a) on the target gene HMGA2 on the proliferation and migration of hepatoma cells and the underlying mechanism. Methods:Liver cancer tissue samples ( n = 30) and adjacent normal tissue samples ( n = 30) pathologically confirmed by Wenzhou Hospital of Traditional Chinese Medicine between September 2018 and September 2019 were collected. MiR-26a mimics, control mimics (miR-Control), high-mobility group A2 protein (HMGA2) siRNA or negative control siRNA (Control) were transfected into human hepatoma cell lines HepG2 or Huh-7 cells. The expression of miR-26a in hepatocellular carcinoma tissue was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). MTT assay and scratch test were performed to determine the ability of cell proliferation and migration. RT-qPCR and western blotting were performed to detect miR-26a and HMGA2 mRNA expression. The relationship between miR-26a and HMGA2 mRNA was analyzed using Bioinformatics and luciferase reporter gene assay. Results:RT-qPCR results showed that the expression level of miR-26a in hepatocellular carcinoma tissue was 0.11 ± 0.02, which was significantly lower than that in normal tissues (0.25 ± 0.03, t = 21.268, P < 0.05). The expression level of miR-26a in stage III + IV was 0.05 ± 0.01, which was significantly lower than that in stage I + II (0.09 ± 0.01, t = 15.491, P < 0.05). Cell experiment showed that in the miR-26a group, the proliferation ability of Huh-7 cells was (3.10 ± 0.30) and (4.10 ± 0.40), and the proliferation ability of HepG2 cells was (3.08 ± 0.31) and (4.11 ± 0.40), which was significantly lower than that in the control group [(3.90 ± 0.40), (5.50 ± 0.60), (3.92 ± 0.41), (5.49 ± 0.58), t = 8.764, 10.634, 11.148, 10.728, all P < 0.05]. In the miR-26a group, the migration ability was (0.50 ± 0.06), (0.65 ± 0.07), which was significantly lower than that in the control group [(1.00 ± 0.10), (0.96 ± 0.10), t = 23.483, 13.910, both P < 0.05]. Bioinformatics and in vitro experiments showed that HMGA2 was a direct target of miR-26a. Restoring the expression of HMGA2 in miR-26a mimics-transfected cells, compared with that in the miR-26a group [(0.24 ± 0.02), (0.31 ± 0.03);(0.45 ± 0.05)], could significantly reverse the inhibitory effect of miR-26a on tumor cell proliferation and migration [(0.31 ± 0.03), (0.40 ± 0.04);(0.93 ± 0.08), t = 10.634, 9.859, 27.868, all P < 0.05). Conclusion:miR-26a inhibits the proliferation and migration of hepatoma cells by directly targeting HMGA2. The abnormal decrease of miR-26a and the increase of HMGA2 may be the important factors that participate in the occurrence and development of liver cancer.