| Primary hepatic carcinoma(PHC) is the tumor derived from hepatic cells and ductal cells. Hepatocellular carcinoma(HCC) is the predominant part,which mortality rate is the second in our country.Its clinical treating methods mostly includes surgical operation,radiotherapy and chemotherapy.Although surgical operation is the most effective, great majority patients are midadvanced and have missed the chance for surgery.However, HCC is not very sensitive to radiotherapy and chemotherapy.So researching and developing new natural, harmfulless and high-efficiency medicine,and the search of new gene targets of HCC are hotspots of science studies.Apigenin is a kind of flavonoid compound natrurally exists in many fruits,vegetables(including celery),beans, mandarin orange,tea,wheat and several condiments.Previous studies have shown that apigenin could inhibit malignant tumor (esophageal cancer, pancreatic cancer, ovarian cancer, colon cancer and so on) cell growth by blocking cell cycle at G2/M phase and promoting apoptosis.The latest research demonstrated that apigenin could inhibit hepatoma cell growth through blocking cell cycle at G2/M phase,but the molecular mechanism is not very clear.The strategy of this study is as follows:we chose hepatoma cell Huh-7 as the research object.Firstly,we detected the anti-HCC effect of apigenin by Colormetric MTT-assay, Flat plate clone formation test,FACS and tumorigenicity test of nude mice.Secondly,we compared the differentially expressed genes between cells treated and untreated with apigenin by cDNA microarray.Finally, we validated the result of cDNA microarray selectively by qRT-PCR and Western-blot and provided theoretic basis for investigating the molecular mechanism of the cytotoxic effect of apigenin on Huh-7.Part I The inhibitory effect of apigenin on growth of Huh-7 in vitro and in vivoObjective: 1. To detect the influence of apigenin on biological activity(cell proliferation,cell clone,cell cycle and cell apoptosis) of Huh-7 cell.2. To investigate the inhibiting effect of apigenin on the growth of Huh-7 in vivo.Methods:1. Colormetric MTT-assay was used to measure the inhibition ability of apigenin on Huh-7 cell proliferation.2. Detected the ability of cell clone by flat plate clone formation test.3. Detected the cell cycle distribution by PI dyeing methods and flow cytometry.4. Detected the cell apoptosis by Annexin V-FITC/PI dyeing methods and flow cytometry.5. Influence of apigenin on tumorigenicity of Huh7 in nude mice was detected by animal mode,and observed the pathological changes under microscope through HE dyeing.Results:1. The growth curves showed that apigenin could significantly inhibited the growth of Huh-7 cells,and we also found that the inhibitory effect would strengthened when the dose increased.The differences between each group all have statistical significance. (P<0.05)2. Flat plate clone formation test showed that when treated with apigenin,either the size or the number of clones was decreased.The diferences were significantly (P<0.001)3. When Huh-7 cells treated with apigenin,cell cycle distribution was changed. G0/G1 phase rate decreased and G2/M phase rate increased,and the cell cycle was blocked at G2/M phase. Additionally, we found that the blocking effect would strengthened when the dose increased.The differences between each group all have statistical significance. (P<0.05)4. Cell apoptosis test have shown that apigenin could promote apoptosis,and presented dose-dependent. The differences between each group all have statistical significance. (P<0.05)5. When treated with apigenin,the size of tumors in nude mice were smaller than untreated ones (P<0.05) And we could observe obvious necrosis in tumor tissue of nude mice treated with apigenin by HE dyeing.Conclusion: Apigenin partially inhibited Huh-7 growth in vitro and in vivo by blocking cellcycle at G2/M phase and promoting apoptosis.Part II The molecular mechanism of inhibitory effect of apigenin on hepatoma cell Huh-7Objective: To preliminarily investigate the molecular mechanism of inhibitory effect ofapigenin on hepatoma cell Huh-7.Methods:1. Compared the differentially expressed genes between cells treated and untreated with apigenin by cDNA microarray.2. Validated the result of cDNA microarray selectively by qRT-PCR and Western-blot.Results:In the 41,000 genes,we found that apigenin could dramatically change the expression of 1,764 genes in Huh7 cells by cDNA microarray.1,336 genes have been upregulated (≥2 times)and 428 genens have been downregulated(≤0.5).In these differentially expressed genes, most are involved in nucleic acid binding, transporter, catalytic or enzyme regulator activity, transcriptional regulation, cytoskeletal structure and/or adhesion, signal transduction, metabolism, apoptosis or the immune response. For examples:metabolism related gene SLC27A3 has been upregulated to 27.69 times, transcription related genes OVOL1 and OTP have been upregulated to 6.23 and 5.53 times, antioncogene ZDHHC2 has been upregulated to 5.10 times,signal transduction related gene BMPR1A has been upregulated to 5.09 times, immune response related gene IL-4R has been downregulated 81.1%, ubiquity-dependent protein catabolism related gene USP18 has been downregulated 80.4%.And the results of qRT-PCR and Western-blot were accordance with the result of cDNA microarray.We further analysed relevanted functional genes of cell cycle and cell apoptosis and found that apigenin have upregulated some cell-cycle inhibited genes,and simultaneously downregulated several cell-cycle induced genes.The overall trend presented the inhibiting of cell cycle process.Conclusion:Apigenin may inhibit Huh-7 cell growth by regulating multi-genes.For example: metabolism related gene SLC27A3, transcription related genes OVOL1 and OTP, signal transduction related gene BMPR1A, immune response related gene IL-4R,ubiquity-dependent protein catabolism related gene USP18and several related functional genes of cell cycle and cell apoptosis. |
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