| Background: Hepatocellular carcinoma is a very common malignant tumor which is associated with significant mortality.The treatment strategy for patients with hepatocellular carcinoma is limited.Tumor-removal operation can only be used in the early stage of cancer and antineoplastic drugs may be limited efficacy and have significant toxicity.The pathogenesis of malignant tumor is very complex and gene mutations play an important role in the process of its occurrence and development.Gene targeting treatment may be a promising way for tumor treatment which is expected to prolong the survival time of patients with hepatocellular carcinoma.The DEK gene was discovered in acute myeloid leukemia in 1992.It encodes a structural protein that is closely related to cell proliferation and apoptosis which is involved in various biological processes such as DNA damage repair,m RNA splicing,transcription regulation and cell differentiation.Studies have found that overexpression of DEK gene wound promote the occurrence and development of cancer and DEK gene expression in malignant tumor is significantly higher than normal tissue.So far,the role of DEK gene in the the occurrence and development of primary hepatocellular carcinoma is mostly unclear.Our previous study has confirmed that DEK gene is highly expressed in the hepatocellular carcinoma tissue.This research would focus on the biological mechanism of DEK in the proliferation,apoptosis,and migration of hepatocellular carcinoma cells and reveal the pathogenesis of hepatocellular carcinoma in vivo and in vitro which may provide a theoretical basis for further exploring of new treatment method of hepatocellular carcinoma.Methods: 1.We successfully constructed and verificated the DEK knockdown and overexpression recombinant plasmids.2.We successfully packaged lentiviruses with DEK knockdown and overexpression of recombinant plasmids.3.Real-time PCR and Western blot were used to detect the expression of DEK m RNA and DEK protein from the following four hepatocellular carcinoma cells Bel-7402,Huh7,Hep G2 and SMMC-7721.We selected two hepatocellular carcinoma cells with high DEK expression.4.DEK knockdown and overexpression of recombinant lentivirus infected hepatocellular carcinoma cells.After two weeks of purinamycin screening,Real-time PCR and Western blot were used to detect the expression level of DEK m RNA and protein respectively,to construct a stable knockdown and overexpression cell line.5.Effects of DEK knockdown on the biological behavior of hepatocellular carcinoma cells: We selected the stable transfected cell lines with higher DEK knockdown efficiency,respectively used CCK8 cell proliferation experiment,flow cytometry technology to detect the proliferation,apoptosis and cell cycle distribution of hepatocellular carcinoma cells,and cell scratch repair experiment was used to detect the migration ability of hepatocellular carcinoma cells.6.To study the subcutaneous tumorigenesis of nude mice after DEK gene knockdown and overexpression: 30 BALB/C nude mice of 4-6 weeks were divided into 6 groups: OE group,OE-Control group,NC group,sh-Control group,KD1 group and KD3 group.Equal amount of cell suspension in logarithmic phase was injected into the right armpit of each group.The growth rate,survival,body weight,tumorigenesis time,tumor growth rate and volume of nude mice were observed.After the growth of the tumor,the longest and shortest diameter of the tumor were measured every two days,and the tumor volume was calculated.After 5 weeks,the nude mice were killed,the tumor was taken out,weighed and its volume was measured,at the same time,the nude mice were dissected to observe whether there was metastasis.Results:1.The Enzymatic digestion and sequencing confirmed that we successfully constructed DEK knockdown and overexpression recombinant plasmids.2.Fluorescent expression in the control group confirmed that we successfully packaged the lentivirus.3.The results of Real-time PCR showed that the expression levels of DEK m RNA in Bel-7402 and Huh7 hepatocellular carcinoma cells were higher than that in SMMC-7721 and Hep G2 cells.4.Western blot results showed that the expression levels of DEK protein in Bel-7402 and Huh7 hepatocellular carcinoma cells were higher than that in SMMC-7721 and Hep G2 cells.5.Real-time PCR and Western blot results showed that compared with the negative control,the transferred recombinant knockdown and overexpression plasmids could effectively increase or decrease the DEK expression.We selected stable transfected cells with higher DEK knockdown efficiency for subsequent experiments.6.The CCK8 experiment obtained the absorbance values of cells in each group at 12,24,48 and 72 hours,which showed that the absorbance values of Bel-7402 and Huh7 knockdown groups at 24,48,and 72 hours were significantly lower than those of the blank control and negative controls(p<0.05).7.The results of flow cytometry showed that the apoptosis rate of Bel-7402 and Huh7 knockdown groups were significantly higher than those of the blank and negative control groups(p<0.05).8.The results of the scratch test showed that the scratch healing rate of Bel-7402 and Huh7 knockdown groups were significantly lower than those of the blank and negative control groups(p<0.05).9.The results of flow cytometry detection of cell cycle showed that compared with the blank and negative control groups,the proportion of cells increased at the G0-G1 phase and decreased at the S phase in the two knockdown groups,but in Bel-7402 group the proportion of cells at the G2-M phase also decreased(p<0.05).10.The results of subcutaneous tumor formation experiments in nude mice showed that the average volume of the transplanted tumors in the DEK overexpression group was larger than that in the DEK knockdown group.Conclusions: 1.In this experiment,the recombinant DEK knockdown and overexpression plasmids were successfully constructed,and the lentivirus was successfully packaged.2.Two hepatocellular carcinoma cell lines Bel-7402 and Huh7 with high expression of DEK were screened.3.Two hepatocellular carcinoma cell lines with stable knockdown and overexpression of DEK were successfully constructed.4.In vitro cell function experiments confirmed that targeted silencing of DEK expression in hepatocellular carcinoma cells could inhibit cell proliferation,migration ability,induce apoptosis,and arrest cell cycle at the G0 / G1 phase.5.At the level of animal experiments in vivo,subcutaneous tumorigenesis in nude mice show that targeted silencing of the DEK gene inhibited the growth of hepatocyte transplantation tumor. |
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