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Experimental Studies on Periplocin of Cortex Periplocae and siRNA Targeted to STAT3 Induce Apoptosis in Human Hepatocellular Carcinoma Celline SMMC7721

Author: ZhangLiJie
Tutor: DanBaoEn
School: Hebei Medical University
Course: Immunology
Keywords: Periplocin of Cortex Periplocae RNAi STAT3 signal transduction hepatocellular carcinoma apoptosis
CLC: R735.7
Type: PhD thesis
Year: 2008
Downloads: 366
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Abstract


Objective: Malignant tumor is one of the serious diseases threatening human life and health. According to the report of the World Health Organization, there are about 9 million new cases each year, and there are 7 million people died of tumor. This figure is in an increasing trend each year. Therefore, overcoming cancer is still a major issue facing medical workers. Chemotherapy play an important role in three major treatments of antitumor. Cancer chemotherapy has made great progress after nearly 50 years of research and development of anticancer drugs. In particular, it prolongs survival time of patients with hematological malignancies significantly. However, treatment of more than 90% of malignant solid tumor has not yet reached satisfactory curative effect. There are still half patients without response or resistance in the treatment of cancer and eventually lead to treatment failure. Meanwhile, Synthetic drugs exist severe side effects. Therefore, the discovery and development of new anticancer drugs is still a very difficult and long-term mission and challenges for the medical profession.At present, the emergence of a new generation of anticancer drugs specificly targeted to the abnormal signal pathway in tumor cells makes up for the shortcomings of traditional chemotherapy drugs. Such drugs with high selectivity for tumor cells, and with low toxicity for normal cells is targeted to the difference between tumor cells and normal cells. This treatment targeted to cell receptor, key genes and regulatory moleculars is also known as targeted therapy. It is necessary to find the possiblely inhibited target in tumor cells for targeted therapy. Otherwise it would become no aimless.Fortunately, with the rapid development of molecular oncology and molecular pharmacology, the molecular mechanism of tumorigenesis and development has been gradually articulated and revealed. A number of hot targets in the abnormal signal pathway of tumor cells have been found. Among them, STAT3(Signal transducers and activators of transcription 3, STAT3)signaling pathway has been identified as an effective molecular target for the treatment intervention to a variety of human tumor.Periplocin of Cortex Periplocae(CPP)is the natural active component found in the drug discovery research work started by our laboratory in cooperation with North China Pharmaceutical Corporation. It has antitumor activity. Compared with the chemical synthetic drugs, natural active substances often have the characteristics of novel structure, high activity and small adverse reactions. They are attracted the attention of pharmaceutical industry. They are the important research and development resources of new drug and the main sources of drugs which we developed possesing our own intellectual property rights in China. The antitumor mechanism of Chinese medicine active ingredients is complex. They are often involved more links, multi-channel, multi-target. It is worthy of attention and exploration whether CPP impacts STAT3 signaling pathway which is abnormal expression and activation in tumor cells during CPP producing a marked antitumor effect. It is of great significance to clarify this issue for systematically revealing highly complex antitumor mechanism of Chinese medicine active components and further developing antitumor effect of CPP. It will provide theoretical and experimental basis for further development and clinical application periplocin of Cortex Periplocae.RNA interference(RNAi) means sequence-specific gene silencing caused by the exogenous or endogenous double-stranded RNA(dsRNA) in the organism’s cells. In this process, long double-stranded RNA was recognized and digested by a kind of RNAaseⅢendonucleases named Dicer enzyme. It becomes short double-stranded RNA only with 21~25nt, that is, small interfering RNA(siRNA). siRNA is the effector molecule in the RNAi process. siRNA was unwinded into sense strand and antisense strand. Then the antisense strand combines endonuclease, exonuclease and helicase to form a complex named RNA induced silencing complex(RISC). RISC recognizes and digests the mRNA with homologous sequence through siRNA antisense strand and then the mRNA was degradated immediately. This process results in a specific gene silencing. Theoretically, if we known some oncogene specific to some tumor cells, we can transfect specific siRNA targeted to the oncogene into target cell, and achieve the purpose of treating cancer by gene silencing. The siRNA research on cancer therapy indicates that using siRNA specific to oncogenes or anti-apoptotic genes which are overexpression in tumor cells can inhibit tumor growth, kill tumor cells, induce apoptosis of tumor cells, increase sensitivity of radiotherapy and chemotherapy. RNAi is expected to make significant breakthroughs in the treatment of malignant tumors. If CPP combined with RNAi treats the tumor cells, after RNAi downregulating excessive activation of STAT3, can CPP improve the antitumor effect? Can CPP also suppress STAT3 signaling pathway? Based on the consideration of the above issues, this study will investigates:①analyzing the expression of STAT3 and tyrosine phosphorylation of STAT3, identifing the excessive activation of STAT3 in SMMC7721 cells;②construction of RNA interference recombinant plasmid targeted to STAT3, silencing STAT3 gene expression of SMMC7721 cells, meanwhile, combined CPP intervention, through in vivo and in vitro experiments, observing the inhibiting proliferation and inducing apoptosis effects of CPP and RNAi on SMMC7721 cells, observing the impact on STAT3 signaling of CPP and RNAi, profoundly revealing the antitumor molecular mechanism of CPP and RNAi;③observing whther the method of CPP combined RNAi can produce a more significant antitumor effect, so as to provide theoretical and experimental basis for establishing new cancer therapy strategy.Methods:1 Analysis of STAT3 expression in SMMC-7721 cellls Western blot and flow cytometry were used to detected the expression of STAT3 protein and phospho-STAT3(P-STAT3).2 Construction and identification of RNA interference expression vector of targeting STAT3 Three specific siRNAs targeted to STAT3 and unrelated sequence HK-shRNA were designed and synthesized. Ligate the template with linearized plasmid. The ligation was transformed into the competence bacteria. The recombinant plasmid was then identified by enzyme digestion and DNA sequence analysis. After comfirming the inserted sequence, the recombinant plasmid was transfected into SMMC7721 cellls by lipofectamineTM 2000. Transfection efficiency was identificated by laser confocal microscopy after transfection for 48h. The most effective plasmid expression vector of inhibiting STAT3 expression was screened out by semi-quantitive RT-PCR and Western blot and used for the follow-up experiments.3 Effects of CPP and STAT3 RNAi on apoptosis in SMMC7721 celllsFive experimental groups were divided.A group: the blank control group, not taking any intervention, add the RPMI1640 medium without bovine serum in the experiments;B group: HK plasmid control group, transfected by Pgenesil-1-HK recombinant plasmid into SMMC7721 cellls;C group: RNAi group, transfected by Pgenesil-1-STAT3-1 plasmid into SMMC7721 cellls;D group: CPP group, add CPP, its final concentration of 2.5μg/ml (this concentration is close to IC50 in pre-experiment), (ip, 1/d×20d);E group: RNAi combining CPP group, transfected by Pgenesil-1-STAT3-1 plasmid into SMMC7721 cellls, at the same time, add CPP, its final concentration of 2.5μg/ml, (ip, 1/d×20d);After transfection for 48h, cell proliferation activity was assayed by MTT; tumor cell cycle and apoptosis rate were detected by flow cytometry; the ultrastructural changes of SMMC7721 cells treated with CPP and RNAi were observed by transmission electron microscopy.4 Effects of CPP and RNAi on STAT3 signaling pathway in SMMC7721 celllsThe changes of molecules related to STAT3 signaling pathway, including upstream moleculars such as JAK1, P-JAK1, STAT3, P-STAT3 and downstream moleculars such as Survivin, Bcl-2 and Bax, were detected by Western blot and RT-PCR.5 Antitumor effect and impact on STAT3 signaling pathway of CPP and RNAi in tumor-bearing nude miceSMMC7721 cells were washed and adjusted to 1×107/ml cell density with serum-free RPMI1640 solution. Each BALB/C-nu nude mouse was inoculated subcutaneously in the right back with 2×106 (0.2ml) SMMC7721 tumor cells to establish animal model of tumor-bearing nude mice. Five groups were divided, each group has five nude mice.A group: model control group, saline was injected to tumor-bearing nude mice for 20 days;B group: HK plasmid control group, HK plasmid was injected to tumor-bearing nude mice for 20 days, 1/d×20d;C group: RNAi group, Pgenesil-1-STAT3-1 plasmid was administered to the tumor-bearing mice (20μg, multi-point injection in the tumor, 1/2d×20d);D group: CPP group, CPP was administered to the tumor-bearing mices (10mg/kg, ip, 1/d×20d);E: RNAi combining CPP group, Pgenesil-1-STAT3-1 plasmid was administered to the tumor-bearing mice (20μg, multi-point injection in the tumor, 1/2d×20d), meanwhile, CPP was administered to the tumor-bearing mices (10mg/kg, ip, 1/d×20d).The kinetics of tumor formation and tumor growth were measured, tumor growth inhibition rate(IR) was calculated. The tumor samples were taken. Plasmid transfection and expression were observed by laser confocal microscopy. The effects of CPP and RNAi on cell cycle and cell apoptosis were determined by flow cytometry. Changes of JAK1, P-JAK1, STAT3, P-STAT3 were detected by Western blot and immunohistochemistry. CD34 expression was analyzed by immunohistochemistry, using MV(microvessel, MV) count to show its expression level.Results: 1 There was expression and activation of STAT3 in SMMC-7721 cells. These results suggested that the expression and activation of STAT3 may play an important role in the development and pathogenesis of human hepatocellular carcinoma.2 The enzyme digestion analysis and DNA sequencing showed that RNA interference expression vectors of targeting STAT3 and unrelated gene HK expression vector were constructed successfully.The recombinant plasmid was transfected into SMMC7721 cells. After transfection for 48h, if observed green fluorescence under laser confocal microscope, SMMC7721 cells were sucessfully transfected with siRNA and pEGFP plasmid, while green fluorescence was not observed in SMMC7721 cells which were not transfected. SMMC7721 cells emiting green fluorescence of four experimental groups were accounted for 68%, 70%, 66%, 65%, respectively. There was no significant difference in the fluorescence intensity among the four experimental groups(P>0.05).STAT3 mRNA expression levels of five experimental groups: non-transfected group, transfected by HK, transfected by STAT3-1, transfected by STAT3-2, transfected by STAT3-3 were 0.906±0.081, 0.891±0.101, 0.302±0.083, 0.596±0.112, 0.603±0.057, respectively. STAT3 mRNA expression levels of transfected by STAT3-1, transfected by STAT3-2 and transfected by STAT3-3 were significantly lower than non-transfected and transfected by HK group (P<0.05). STAT3 mRNA expression level in transfected STAT3-1 group was the minimum. There was no significant difference between non-transfected group and transfected by HK(P>0.05). STAT3 mRNA expression inhibitory rates in the three groups of transfected by STAT3-1, transfected by STAT3-2 and transfected by STAT3-3 were 66.10%, 26.37%, 21.21%, respectively.STAT3 protein expression levels of five experimental groups were 1.116±0.081, 1.108±0.101, 0.383±0.093, 0.682±0.120, 0.742±0.151, respectively. STAT3 protein expression levels of transfected by STAT3-1, transfected by STAT3-2 and transfected by STAT3-3 were significantly lower than non-transfected and transfected by HK group (P<0.05). STAT3 protein expression in transfected by STAT3-1 group was the minimum. There was no significant difference between non-transfected group and transfected by HK(P>0.05). STAT3 protein expression inhibitory rates in the three groups of transfected by STAT3-1, transfected by STAT3-2 and transfected by STAT3-3 were 65.43%, 38.45%, 33.03%, respectively.The results of RT-PCR and Western blot suggest that Pgenesil-1-STAT3-1, Pgenesil-1-STAT3-2, Pgenesil-1-STAT3-3 recombinant plasmids can all knockdown STAT3 mRNA and protein expression. Pgenesil-1-STAT3-1 has the most strongest interference effect. Pgenesil-1-STAT3-1 screened out will be used for the follow-up related experimental studies.3 A concentration-dependent and time-dependent proliferation inhibition of CPP was demonstrated in SMMC7721 with IC50 value(for 48h) of (4.05±0.21)μg/ml.SMMC7721 cell proliferation in RNAi, CPP, RNAi combining CPP groups was inhibited to varying degrees in different time (24h, 48h, 72h ). Proliferation inhibitory rates in RNAi, CPP, RNAi combining CPP groups increased with the extension of time and increased significantly compared to blank control group(P<0.05). There was no statistical significance between blank control group and HK group(P>0.05). Proliferation inhibitory rate in RNAi combining CPP group increased significantly compared to RNAi group and CPP group(P<0.05). The results suggested that RNAi can significantly enhance the inhibition of CPP on cell proliferation, there was certain synergistic effect between the two treatment. SMMC7721 cell proliferation inhibition was more obvious if joint using the two methods.Compared to control group, the number of G0/G1 phase cells increased markedly, but which of S and G2/M phase cells decreased, PI values decreased in RNAi, CPP, RNAi combining CPP groups, the difference was significant(P<0.05). The changes of RNAi combining CPP group were the most obvious, the proportion of tumor cells in the G0/G1 phase was increased from (39.5±3.6)% to (80.6±3.3)%, the proportion of S and G2/M phase cells was decreased from (27.7±2.4)% and (32.8±2.5)% to (13.3±1.9)% and (6.1±2.0)%, suggesting that CPP and RNAi could hinder the cell cycle of SMMC7721 cells at G0/G1 phase. The blockage effect of CPP combining RNAi was the strongest. There was no significant difference between blank control group and HK group(P>0.05).The results of flow cytometry showed that the apoptosis rate of SMMC7721 cells in RNAi, CPP, RNAi combining CPP groups increased significantly compared to A group(P<0.05). The apoptosis rate was highest in CPP combining RNAi group, reached (30.30±2.87)% from(4.10±1.50)%. Meanwhile, typical subdiploid peaks before G0/G1 phase was detected. There was no significant difference between blank control group group and HK group(P>0.05).The characteristic ultrastructural changes of apoptotic cells were observed in RNAi, CPP, RNAi combining CPP groups by transmission electron microscopy. The changes included cell shrinkage, smaller cell size, cell membrane integrity, microvilli and pseudo-foot of the membrane surface decreased or disappeared, cytoplasmic condensation, nuclear chromatin condensation, moon-shaped marginization along the nuclear membrane, density increased significantly, clear boundaries, and the endoplasmic reticulum expansion, nucleonic pycnosis, nucleus fragment, vacuolar degeneration of cytoplasm.4 CPP and RNAi can impact the expression of molecules related to STAT3 signaling pathway in SMMC7721 cells. Western blot results showed that JAK1 expression was no significant difference in the different treatment groups (P>0.05). P-JAK1 expression in CPP, RNAi combining CPP groups were decreased than that in blank control group (P<0.05), the IOD ratio decreased from blank control group (1.232±0.104) to CPP group (0.302±0.037) and RNAi combining CPP group(0.282±0.096), suggesting that CPP may downregulate the phosphorylation level of JAK1. STAT3 expression in RNAi, RNAi combining CPP groups were significantly lower than that in blank control group(P<0.05), the IOD ratio decreased from blank control group (1.114±0.154) to RNAi group (0.406±0.051) and RNAi combining CPP group (0.379±0.051), suggesting that specific RNAi targeted to STAT3 can significantly knockdown STAT3 expression. P-STAT3 in RNAi, CPP, RNAi combining CPP groups were significantly lower than that in blank control group (P<0.05), the IOD ratio decreased from blank control group (0.891±0.165) to RNAi group (0.307±0.032), CPP group (0.457±0.104) and RNAi combining CPP group (0.201±0.087), suggesting that RNAi combining CPP can make the decrease of STAT3 phosphorylation level more obvious. Expression level in HK group did not change significantly compared with blank control group (P>0.05), indicating that the inserted sequence of unrelated recombinant plasmid (HK) did not interfer STAT3 expression.RT-PCR and Western blot results showed that Survivin, Bcl-2 mRNA and protein expression in RNAi, CPP, RNAi combining CPP groups decreased significantly, especially in RNAi combining CPP group compared to blank control group(P<0.05); Bax mRNA and protein expression in RNAi, CPP, RNAi combining CPP groups increased, especially in RNAi combining CPP group(P<0.05). Survivin、Bax、Bcl-2 expression in HK group did not change significantly compared to blank control group (P>0.05). The results suggested that changes of Survivin, Bax and Bcl-2 gene and protein occurred after CPP or RNAi intervention and changes of mRNA were coincident with that of protein.5 The tumor in tumor-bearing nude mice of blank control group and HK group continued to grow, the growth speed is higher than other groups. The tumor in RNAi group and CPP group grew slowly. While the tumor in RNAi combining CPP group narrowed gradually than the original tumor. Weight and volume of tumor in blank control group and HK group were significantly larger than RNAi, CPP, RNAi combining CPP groups (P<0.01). Weight and volume of tumor in RNAi group and CPP group were significantly larger than RNAi combining CPP group (P<0.01). Inhibitory rates in RNAi, CPP, RNAi combining CPP groups were 49.6%, 53.7%, 85.7%, respectively. Inhibitory rate in RNAi combining CPP group is the highest.Green fluorescent was observed in SMMC7721 cells implanted into nude mice in HK, RNAi, RNAi combining CPP groups by laser confocal microscope. There was no green fluorescent in blank control group and HK group.Flow cytometry analysis showed that the number of G0/G1 phase cells in RNAi, CPP, RNAi combining CPP groups increased markedly, but which of S and G2/M phase cells decreased, PI values decreased in RNAi, CPP, RNAi combining CPP groups, the difference was significant compared to blank control group (P<0.05). The changes of RNAi combining CPP group were the most obvious.The above three treatments can hinder the cell cycle of SMMC7721 cells implanted into nude mice at G0/G1 phase. The apoptosis rate of SMMC7721 cells in RNAi, CPP, RNAi combining CPP groups increased significantly compared to blank control group (P<0.05). The changes of RNAi combining CPP group were the most obvious. Typical subdiploid peaks before G0/G1 phase was detected. Three treatment including CPP, RNAi, RNAi combining CPP can all induce apoptosis of SMMC7721 cells implanted into nude mice. The treatment of RNAi combining CPP induced apoptosis more effectively.Western blot results showed that JAK1 expression was no significant difference in the different treatment groups (P>0.05). P-JAK1 expression in CPP, RNAi combining CPP groups were decreased than that in blank control group(P<0.05). STAT3 expression in RNAi, RNAi combining CPP group were significantly lower than that in blank control group (P<0.05). P-STAT3 in RNAi, CPP, RNAi combining CPP groups were significantly lower than that in blank control group (P<0.05). Expression level of JAK1, P-JAK1, STAT3, P-STAT3 in HK group did not change significantly compared with blank control group (P>0.05).Immunocytochemical analysis showed that MVD of C, D and E group were (8.9±2.2)/HP, (9.1±2.5)/HP, (4.7±1.8)/HP, respectively. MVD of RNAi, CPP, RNAi combining CPP groups were decreased significantly compared to blank control group (14.6±4.8)/HP(P<0.05). The decrease in RNAi combining CPP group was the most obvious.Conclusion:1 There was expression and activation of STAT3 in human hepatocellular carcinoma cellline SMMC-7721. The result suggested that the expression and activation of STAT3 may play an important role in the development and the pathogenesis of human hepatocellular carcinoma.2 RNA interference expression vectors of targeting STAT3 were constructed successfully and the vector which is the best inhibitory effect on STAT3 expression was screened out. It will be used for the follow-up related experimental studies.3 CPP and siRNA targeted to STAT3 can inhibit SMMC7721 cell proliferation and induce apoptosis. Compared to only a single factor-treated group, the inhibition of CPP combining RNAi increased significantly, apoptosis rate was significantly increased.4 There was expression and activation of JAK1 in human hepatocellular carcinoma cellline SMMC-7721. siRNA targeted to STAT3 can not inhibit the expression and activation of JAK1. CPP can downregulate the expression of P-JAK1, P-STAT3, and has no effect on JAK1 and STAT3. RNAi and CPP can regulate downstream target genes such as Survivin、Bcl-2、Bax. Compared to only a single factor-treated group, the inhibitory effect on STAT3 molecular and the regulatory effect on STAT3 downstream moleculars of RNAi combining CPP increased significantly.5 CPP and RNAi have significant inhibitory effect on tumor growth in vivo, the treatment of RNAi combining CPP has more significant inhibitory effect. CPP and RNAi can block cell cycle and induce apoptosis. CPP can downregulate the expression of P-JAK1, P-STAT3. RNAi can downregulate the expression of STAT3、P-STAT3. Both of them can decrease the expression of CD34 which represents the ablity of angiogenesis. The treatment of RNAi combining CPP has more significant inhibitory effect on STAT3 signaling pathway.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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