| Objective: The hepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country, which is associated with high lever of malignancy, poor prognosis. Therefore, to explore the pathogenesy of HCC remain a challenge to modern medical science. The HCC grow quickly, causing tissue oxygen deficiency. In this state, the Multiplication and infiltration of HCC are mainly because of the existing of HIF-1. Hypoxia inducible factor-1α(HIF-1α) is an unique active transcription factor in the specific oxygen deficiency, which expresses commonly in malignant tumors. HIF-1αis essential in activating angiogenesis, anaerobic metabolism, for the proliferation and migration of tumor cells.At present , the investigation on bcl-2,bax,survivin relative the proliferation and migration of HCC is so deep and concrete,but the way did not appear in literature about regulation and influence between bcl-2,bax,survivin and HIF-1.In this research, we explored whether HIF-1 can be up-regulated in hepatocellular carcinoma cells SMMC-7721 treated with YC-1(the suppressor of HIF-1α)and the ASODN of HIF-1αrespectively. The aim of this expriment is to explore the effects of YC-1 and the ASODN of HIF-1αon the cell proliferation and the growth of human hepatocellular carcinoma cell line SMMC-7721.By detecting the expression of bcl-2,bax,survivin mRNA and protein in SMMC-7721 cultured in normal and treated conditions by RT-PCR and Western blot, we identify the relation between HIF-1 and bcl-2, bax, survivin. It will provide a theoretical foundation for clarifying the mechanism of invasion and metastasis of HCC and exploring a new therapeutic way.Methods: SMMC-7721 cells were divided as control group and experiment group. The former were cultured with normal condition, the later were treated by YC-1 or ASODN with various concentration and time. All of the cells were collected on time. Effects of YC-1 or ASODN on the proliferation of SMMC-7721 cells was measured by MTT colorimetric method.Changes of cell cycle and apoptosis rate after treatment with YC-1 or ASODN with various concentration and time were detected by FCM. The expression of bcl-2,bax,survivin mRNA level in cultured SMMC-7721 was evaluated by RT-PCR. The bcl-2, bax, survivin protein level in cytoplasm was evaluated by western blot. The OD value of electrophoresis strip of DNA product or that of bcl-2, bax, survivin protein was analyzed by gel scanner. Beta-actin was used as an internal standard. The relative expression level of bcl-2, bax, survivin was represented with the ratio between produces of bcl-2, bax, survivin and that of beta-actin. Data were analyzed with GLM using SPSS13.0 statistical software. A level of P<0.05 was considered statistically significant.Results1 The results of MTT1.1 MTT colorimetric method showed that the proliferation of SMMC-7721 cells can be inhibited by YC-1. After treated with YC-1, the OD values of treated groups decreased compared with control group, and there was statistically significant difference between control group and every treatment group (P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). Furthermore, with the increasing concentration of YC-1 and prolonging of treating time, the OD values decreased gradually, which indicate that the inhabitation of the proliferation of SMMC-7721 cells by YC-1 displays a dose-and-time-dependent manner (Fig 2,Table 1) (P<0.05).1.2 MTT colorimetric method showed that the proliferation of SMMC-7721 cells can be inhibited by ASODN. After treated with ASODN, the OD values of ASODN treated groups decreased compared with control group, and there was statistically significant difference between control group and every treatment group (P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). Furthermore, with the increasing concentration of ASODN and prolonging of treating time, the OD values decreased gradually, which indicate that the inhabitation of the proliferation of SMMC-7721 cells by ASODN displays a dose-and-time- dependent manner (Fig 2,Table 1) (P<0.05).2 The results of cell cycle and apoptosis2.1 The analysis results of distribution of cell cycle by flow cytometry indicates that after treated with YC-1, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase decreased grudually. But G2/M phase were not changed obviously. That was, YC-1 could induce an inhibition of cell cycle in G1 phase by a dose-and-time-dependent manner. Furthermore, with the increasing concentration of YC-1 and prolonging treatment time, the apoptotic percentage was increased gradually (Fig6,Table7),(P<0.05), which was in a dose-and-time- dependent manner.2.2 The analysis results of distribution of cell cycle by flow cytometry indicates that after treated with ASODN,the number of cells in G0/G1 phase increased gradually (P<0.05), while the number of cells in S phase decreased grudually(P<0.05). But G2/M phase were not changed obviously(P>0.05). Among every treatment group, there was also statistically significant difference (P<0.05). That was, ASODN could induce an inhibition of cell cycle in G0/G1 phase by a dose-and-time-dependent manner(Fig.7,11, Table 5,6). Furthermore, with the increasing concentration of ASODN and prolonging treatment time, the apoptotic percentage was increased gradually (Fig.4,Table 8) ,(P<0.05) ; (Fig.8,Table 10) , (P<0.05), which was in a dose-and-time-dependent manner.3 The results of PCR3.1 Following the increasing of the concentration of YC-1, expression of bcl-2 mRNA (Fig 12, 14) (Table 11)and survivin mRNA (Fig 20, 22) (Table 13) decreased gradually compared with control group, and there was statistically significant difference between control group and every treatment group (P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). But the change of expression of bax mRNA was not obviously observed. With the increasing concentration of YC-1 and prolonging treatment time, expression of bcl-2 mRNA(Fig.12,14,Table 11) and survivin mRNA (Fig.20, 22,Table 13)were decreased gradually(P<0.05), which were in a dose-and-time-dependent manner. But there is no obviors change of the bax mRNA(P>0.05).3.2 Following the increasing of the concentration of ASODN, expression of bcl-2 mRNA and survivin mRNA decreased gradually,compared with control group, and there was statistically significant difference between control group and every treatment group (P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). But the change of expression of bax mRNA was not obviously observed. Furthermore, with the increasing concentration of ASODN and prolonging treatment time, expression of bcl-2 mRNA (Fig.13,15, Table 14) and survivin mRNA (Fig.21,23, Table 16) were increased gradually (P<0.05), which were in a dose-and-time-dependent manner. But there is no obviors change of the bax mRNA(Fig.17, 19,Table 15)(P>0.05).4 The results of Western4.1 Following the increasing of the concentration of YC-1, expression of bcl-2 protein and survivin protein decreased gradually,but bax protein was increased gradually, and there was statistically significant difference between control group and every treatment group (P<0.05). Among every treatment group, there was also statistically significant difference (P<0.05). By increasing of the concentration of YC-1 and prolonging of treatment time, expression of bcl-2 protein (Fig.24, 26, Table 14) and survivin protein (Fig.33, 35, Table 22)were decreased gradually,but bax protein (Fig 29, 31, Table 18) was increased gradually, all of effects were dose-dependent and time-dependent pattern (P<0.05). 4.2 By increasing of the concentration of ASODN, expression of bcl-2 protein (Fig24, 26,Table 14) and survivin protein (Fig33,35, Table 22) were decreased gradually,but bax protein (Fig 29, 31, Table 18) was increased gradually, Among every treatment group, there was also statistically significant difference (P<0.05). By increasing of the concentration of ASODN and prolonging of treatment time, expression of bcl-2 protein (Fig.24,26, Table 14) and survivin protein (Fig.33, 35, Table 22)were decreased gradually,but bax protein (Fig 29,31, Table 18) was increased gradually, all of effects were dose-dependent and time-dependent pattern (P<0.05).Conclusion1 YC-1 and ASODN could inhibit the proliferation and induce the apoptosis of human hepatocellular carcinoma cell line SMMC-7721 in vitro with a dose-dependent and time-dependent pattern,which provide us a new way to cure the HCC.2 The analysis results of distribution of cell cycle demonstrate that the YC-1 and ASODN can inhibit the proliferation and affect the cell cycle by inhibiting the G1 period of SMMC-7721 cell. All of effects were dose-dependent and time-dependent pattern.3 The YC-1 and ASODN can induce the Apoptosis of HCC by evaluating the Apoptosis ratio, the Apoptosis ratio increase with the dug concentration and the prolonging of treatment time. All of effects were dose-dependent and time-dependent pattern4 HIF-1αcan promote the proliferation and induce the apoptosis of human hepatocellular carcinoma cell line SMMC-7721 in vitro. The mechanisms were supposed to be that HIF-1αup-regulates the expression of Survivin and bcl-2 and down-regulates the expression of bax. The effects were in time-dependent pattern.5 The application of the HIF-1αinhibitor and ASODN offer a new way for the treatment of the HCC.
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