| Objective:To investigate the difference of acetylation levels of histone H3K14,H3K18 and H3K27 in highly invasive human hepatoma LM3 cells and normal human liver LO2 cells,and to explore the relationship between the inhibitory effect of blueberry anthocyanins on highly invasive human hepatoma LM3 cells and the changes of acetylation levels of histone H3K14,H3K18 and H3K27.Methods:(1)Highly invasive human hepatocellular carcinoma LM3 cells and normal human liver LO2 cells were cultured in vitro.The cell morphology of LM3 cells and LO2 cells was observed under light microscope and the cell count was performed.The growth of highly invasive human hepatocellular carcinoma LM3 cells in logarithmic growth phase after 48 h was observed and counted using different concentration gradients(0,50,100,150,200μg/m L)of blueberry anthocyanins.(2)Immunofluorescence staining was used to observe the apoptosis of LM3 cells,LO2 cells and LM3 cells after blueberry anthocyanin intervention under fluorescence microscope.(3)RTCA method was used to dynamically monitor the growth and proliferation of LM3 cells and LO2 cells in real time.The proliferation time curves of the two groups were recorded,and the difference in proliferation between the two groups was analyzed.The proliferation of LM3 cells was monitored,and the cell indexes of LM3 cells treated with different concentrations of blueberry anthocyanin for 12 h,24h and34 h were recorded,and the optimal concentration and time of blueberry anthocyanin intervention were selected.(4)The nuclear proteins of LM3 cells and LO2 cells were obtained by nuclear plasma separation technique and the protein concentration was determined.The relative expression levels of ac H3K14,ac H3K18 and ac H3K27 in the nuclear proteins of the two groups were detected by Western Blot.Western Blot was used to detect the relative expression levels of ac H3K14,ac H3K18 and ac H3K27 modifications in LM3 nuclear proteins treated with 0μg/m L and the optimal concentration of blueberry anthocyanins.(5)Flow cytometry was used to detect the apoptosis level of LM3 cells treated with 0μg/m L and optimal concentration of blueberry anthocyanins.(6)After LM3 cells were treated with 0μg/m L and the optimal concentration of blueberry anthocyanin for a certain time,the migration and invasion ability of LM3 cells were detected by cell scratch test and Transwell invasion test.(7)Twenty-four healthy and clean male SD rats,200±20 g,aged 5-7 weeks,were randomly divided into control group,liver cancer group,saline intervention group and blueberry anthocyanin intervention group after 7 days of adaptive feeding.The rats in the liver cancer group,the saline intervention group and the blueberry anthocyanin intervention group were modeled by DEN solution.The control group was given the same amount of normal saline for intraperitoneal injection.The rats were weighed once every other week,and the dosage was adjusted according to the weight change.The mental status of the rats was closely observed,such as eating and activity.The blueberry anthocyanin intervention group was intervened by intraperitoneal injection of blueberry anthocyanin,and the saline intervention group was intervened by the same amount of saline.After the modeling,the rats were sacrificed by ether anesthesia,and the liver tissues were collected.The pathological conditions of liver tissues in the control group and the liver cancer group were observed and the liver weight index was calculated.The liver tissues at the same position of the right lobe of the liver of the four groups of rats were taken for HE staining and immunohistochemical Ki-67 staining to observe the pathological effects of blueberry anthocyanins on rat liver cancer tissues.(8)Western Blot was used to detect the relative expression levels of ac H3K14,ac H3K18 and ac H3K27 in the nuclear protein of liver cancer tissue in saline intervention group and blueberry anthocyanin intervention group.Results:(1)Under the same culture conditions,the adherent growth of normal human liver LO2 cells and highly invasive human liver cancer LM3 cells was observed under an optical microscope.The cells were transparent,highly refracted,and the outline was blurred.Compared with LO2 cells,LM3 cells have the characteristics of rapid proliferation,increased cell volume,large nucleus and pleomorphism(P<0.05).The results of Annexin V-FITC / PI staining showed that there was no significant difference between the two groups.The expression level of apoptosis signal in LO2 cells was slightly higher than that in LM3 cells.There was no significant difference in natural apoptosis between the two groups.RTCA results showed that with the increase of culture time,both LO2 cells and LM3 cells showed a proliferative state,and the proliferation of LM3 cells was better than that of LO2 cells,and LM3 cells had an infinite proliferation trend(P<0.05).(2)The highly invasive human hepatocellular carcinoma LM3 cells treated with0μg/m L blueberry anthocyanin(control group)grew vigorously,and there was no red-stained middle-late apoptosis signal.After intervention with different concentrations of blueberry anthocyanins(50,100,150,200μg/m L)for a certain period of time,the volume of LM3 cells decreased and the number of cells decreased.With the increase of blueberry anthocyanin concentration,the number of middle and late apoptotic cells gradually increased,and the morphology and number of cells changed significantly,that is,LM3 cells were smaller and fewer(all P<0.05).Compared with the control group,except for the intervention of 50μg/m L blueberry anthocyanin for12 h,the proliferation of LM3 cells after intervention with different concentrations of blueberry anthocyanin at the remaining time points decreased significantly in a dose-dependent manner(all P<0.05).When LM3 cells were treated with 100μg/m L blueberry anthocyanins for 24 h,the proliferation inhibition rate of LM3 cells was higher than 50%.Therefore,100μg/m L blueberry anthocyanins for 24 h could be selected as the optimal time and concentration for treating LM3 cells.(3)Compared with LO2 cells,the level of histone acetylation modification in LM3 cells was down-regulated.The protein expression of ac H3K14,ac H3K18 and ac H3K27 in LM3 cells was significantly lower than that in LO2 cells(P<0.05).Compared with the control group,the relative expression levels of ac H3K14,ac H3K18 and ac H3K27 in LM3 cells treated with 100μg/m L blueberry anthocyanins increased to varying degrees(all P<0.05).(4)Compared with the control group,the apoptosis rate of LM3 cells treated with 100μg/m L blueberry anthocyanins increased by about 26%,and the apoptosis rate of early apoptotic cells and late cells increased.The area of cell migration and the number of invasive cells were significantly reduced.Blueberry anthocyanins can promote LM3 cell apoptosis,inhibit migration and invasion.(5)During the period of liver cancer modeling,the rats in the liver cancer group,the saline intervention group and the blueberry anthocyanin intervention group all showed different degrees of loss of appetite,slow weight gain,partial weight loss,reduced stress response ability of exogenous stimulation,dry hair and partial shedding,and 1 rat died in the saline intervention group.Compared with the control group,the body weight of the rats in the liver cancer group decreased significantly,but the liver weight index increased significantly(P<0.05).(6)HE staining of pathological sections showed that the structure of hepatic lobules in the liver of rats in the control group was normal,the nucleus was clear,the cell size was uniform,and the central vein was the center.The liver cells were arranged radially in a cord around,and there was no hyperplasia of collagen fibers in the perisinusal space,and no hemorrhagic necrosis.The liver tissue cells of the rats in the liver cancer group were arranged in disorder,degeneration,necrosis,polymorphic,nest-like distribution,the nucleus was slightly larger and showed binuclear or multinuclear,the structure of the hepatic lobule was destroyed,and the regenerative nodules of the liver cells were wrapped by the widely proliferated fibrous tissue to form irregular liver cell clusters of different sizes.The liver tissue cells of rats in the saline intervention group had the characteristics of pleomorphism,nest-like distribution,enlarged nucleus,binuclear or multinuclear,fibrous tissue hyperplasia,pseudolobule formation and a small number of hemorrhagic lesions.The liver tissue cells of rats in the blueberry anthocyanin intervention group had the same characteristics as those in the liver cancer group and the saline intervention group,that is,they had the same atypia.Compared with the saline intervention group,the nuclear volume was slightly reduced,the degree of cell arrangement disorder and the proliferation of fibrous connective tissue were alleviated.The degree of hepatocellular carcinoma in the blueberry anthocyanin intervention group was lighter than that in the saline intervention group.The liver tissue structure of rats after blueberry anthocyanin intervention was improved,and some hepatic cords were arranged radially.(7)There were a large number of Ki-67 positive cells in the liver cancer tissues of rats in the saline intervention group,which were disordered and dense.The Ki-67 positive cells in the liver cancer tissues of the blueberry anthocyanin intervention group were significantly reduced compared with the saline intervention group,and the cells were arranged in a discrete manner.Compared with the saline intervention group,the number of cells with high expression of Ki-67 in the liver cancer tissue of the blueberry anthocyanin intervention group decreased,and the expression intensity of Ki-67 in the liver cancer tissue of the blueberry anthocyanin intervention group decreased.The relative expression levels of histone ac H3K14,ac H3K18 and ac H3K27 site modification in liver cancer tissues of rats in blueberry anthocyanin intervention group were significantly increased(P<0.05).Conclusions:(1)The unlimited proliferation of highly invasive human hepatocellular carcinoma LM3 cells may be related to the change of histone acetylation modification level;the occurrence and development of LM3 cells may be related to the down-regulation of histone ac H3K14,ac H3K18 and ac H3K27 modification levels.(2)Blueberry anthocyanins can inhibit the proliferation of highly invasive human liver cancer LM3 cells,promote apoptosis,reduce cell migration and invasion,and inhibit the progression of rat liver cancer.This may be related to the up-regulation of histone ac H3K14,ac H3K18,and ac H3K27 site modification levels in liver cancer cells. |
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