Study On Mechanism Of Raddeanin A And Drug Combination On Human Hepatoma Cells QGY-7703

Cancer is a major threat to human health.Every year,8 million people die from cancer all over the world.The development of anticancer drugs has always been a hot topic in pharmaceutical research.Traditional chemical anticancer drugs have many side effects and are prone to drug resistance,which seriously reduces the quality of life of patients.The monomeric active components extracted from traditional Chinese medicine not only have good anticancer activity,but also have an unparalleled advantage of chemical anticancer drugs.It is of great practical significance to develop monomeric anticancer drugs.Chinese medicine Anemone raddeana Regel,also known as"bamboo knot","Cyperus rosea"and"black beak",are the dried roots and stems of anemones,Anemone raddeana Regel.Anemone raddeana Regel as drug was first written by Liu Wentai of the Ming Dynasty"Bencao pinhui recorded"in essence.Raddeanin A?Raddeanin A RDA?is one of the main active Anemone raddeana Regel chemical composition.Previous studies showed that RDA has good antitumor activity,and has a good inhibitory effect on liver cancer,ovarian cancer,lung cancer and so on.However,the anti-cancer mechanism of RDA is not yet clear.In this paper,the effect of RDA on human hepatoma cell QGY-7703 was studied,and the inhibitory effect of RDA on the migration and invasion of QGY-7703 cells,the inhibitory effect of DNA methylation on intracellular DNA and the inhibition of the abnormal activation of NF-?B signal pathway in cell and its mechanism were studied.In addition,the inhibitory effects of RDA and cisplatin?DDP?on the growth of QGY-7703 cells,the effect on intracellular reactive oxygen species?ROS?and the expression of apoptosis genes were also studied.Part 1:the inhibitory mechanism of RDA on NF-?B signaling pathway.We used siRNA gene silencing technology and RT-PCR,W-B technology to study the inhibitory effect of RDA on NF-?B signaling pathway.Immunofluorescence was used to study the effect of RDA on nuclear transport of NF-?B p65 subunit.We used double luciferase reporter gene method to verify the effect of RDA on NF-?B signaling pathway.The experimental results show that siRNA-homo-1716 has the best silencing effect.After transfection of siRNA,the relative gene expression of NF-?B in the positive control group was 0.16,while that in the blank group was 0.87,and the RDA group was 0.53.In immunofluorescence assay,the nuclear transport process of NF-?B p65 subunit in RDA group was significantly inhibited,and the number of dyed cells was significantly reduced.In the double luciferase reporter gene experiment,the average ratio of fluorescein to the sea kidney fluorescein in RDA group was 2.2268,the control group was 1.2363,and the blank group was 0.8087.These studies indicate that RDA can strongly inhibit the abnormal activation of NF-?B signaling pathway.The mechanism is that RDA can inhibit the transport of p65 protein into the nucleus of NF-?B family.Double luciferase reporter gene test confirmed that RDA could inhibit NF-kappa B signaling pathway.Part2:Study on the mechanism of inhibition of DNA methyl A third part of Anemone raddeanaWe used the CCK-8 method to study the half inhibitory concentration of RDA and QGY-7703.The content of 5-methylcytosine in QGY-7703 cells was detected by molecular fluorescence spectrometry with bisulfite method.RT-PCR and W-B were used to study the specific mechanism of RDA inhibiting the DNA methylation of QGY-7703 cells.The results showed that the IC₅₀ of QGY-7703 cells after RDA administration was 6.20 48h mol/L.In the DNA methylation test,the relative content of 5-methylcytosine in RDA group was 9.6%,which was lower than 14.8%in blank group,but higher than 4.2%in positive control group.In the RT-PCR experiment,the relative expression of DNMT1 gene in RDA group was 5.86,8.03 in blank group and 2.07 in positive control group.The relative expression level of DNMT3a gene in group RDA was 3.4,4.46 in blank group and 1.5 in positive control group.The relative expression level of DNMT3b gene in group RDA was 5.6,6.35 in blank group and 3.1 in positive control group.Studies have shown that RDA inhibits invasion and migration of QGY-7703 cells.RDA can significantly reduce the content of 5-methylcytosine in QGY-7703 cells.The mechanism is that RDA inhibits the activity of DNA methyltransferase DNMT1 and DNMT3a,thereby inhibiting its DNA methylation.Part3:Study on the anticancer mechanism of second part RDA combined with cisplatinWe used Transwell cell and Matrimex matrix glue to study the inhibitory effect of RDA on invasion and migration of QGY-7703 cells.We used CCK-8 and SRB to study the inhibitory effect of RDA and its combination with cisplatin?DDP?on the proliferation of hepatocellular carcinoma cell QGY-7703.V-FITC/PI double staining and PI single staining were used to study the effect of RDA and its combination with DDP on the apoptosis and cell cycle of QGY-7703 cells.In the cell migration experiment,the average number of cells in the blank group was 61 under the fluorescence inverted microscope,the average number of cells in the positive control group was 43,and the average number of cells in the RDA group was22.In the cell invasion experiment,the average number of cells in the blank group was 67under the fluorescence inverted microscope,the average number of cells in the positive control group was 53,and the average number of cells in the RDA group was 21.The effects of RDA and its combination with DDP on reactive oxygen species?ROS?in cells were studied by molecular fluorescence spectrometry.The effects of RDA and DDP combined with DDP on the expression of p53,Bax,Bcl-2 and Survivin four genes related to apoptosis in QGY-7703 cells were studied by RT-PCR and W-B.The experimental results show that the correlation between RDA and DDP measured by two methods of SRB and CCK-8 is good for IC50 of QGY-7703 cells,and the fitting coefficient of two is 0.97.The combination of RDA and DDP has good synergistic effect when the final concentration is 5?mol/mL and 10?mol/L respectively.The results of flow cytometry showed that when RDA was used alone,the G1 phase cells accounted for 60.6%of the total,while S phase accounted for 30.35%of the total,while G2/M phase was 9.05%.After RDA combined with DDP,G1 phase was69.12%,S stage was 18.76%,G2 stage was 12.12%.In the positive control group,the apoptosis rate of QGY-7703 cells was 14.26%,the apoptosis rate of RDA group was 22.51%,while that of the two groups was 59.11%.When the concentration of RDA is 1 mol/L,the fluorescence intensity of DCF is 11.763.When the concentration is 5?mol/L,the fluorescence intensity of DCF is 15.075,and the fluorescence intensity is 18.645 when the concentration is 10?mol/L.When the DDP of RDA 1?mol/L and DDP 5?mol/L are combined with DCF,the fluorescence intensity is 19.462,while the RDA of 5 mol/L mol/L and DDP of 10?mol/L is 26.295.When the concentrations of RDA were 1,5,and 10?mol/L.the relative expression of p53 gene was 0.91,1.28,1.64,and the relative expression of the Bax gene was 0.95,1.62,1.94,and the relative expression of the Bcl-2 gene was 2.03,1.68,1.41,and the relative expression of survivin gene was 2.15,1.53,and survivin.When the concentration of DDP is 10?mol/L,the relative expression of the above four genes is 1.93,2.19,1.19 and 1.21,respectively.The relative expression of the above four genes was 1.75,2.01,1.29,1.26 in the combination group?5?mol/L RDAand 10?mol/L DDP?.Studies have shown that the combined application of RDA and DDP can inhibit the proliferation of QGY-7703 cells.The combination of the two can also accelerate the apoptosis of QGY-7703cells.The combination of RDA and DDP can arrest QGY-7703 cell cycle in G1 phase,and significantly increase cell apoptosis rate.In addition,the combination of the two will also increase the content of reactive oxygen species in QGY-7703 cells and accelerate cell death.The combination of RDA and DDP significantly increased the expression of p53 and Bax genes,and down regulated the expression of Bcl-2 and Survivin genes.