CJ-42794 Inhibits The Proliferation And Migration Of HCC Cells By Regulating C-myc And Snail Signals

Objective Hepatocellular carcinoma is one of the most malignant tumors in the world.Its incidence rate and mortality are high.Although surgery and drug therapy have improved,recurrence and metastasis lead to poor prognosis of HCC.Prostaglandin E2 is an important regulator in various cell growth processes.It mediates many physiological and pathological processes by participating in various cell metabolism activities.Prostaglandin E2 can induce the growth and proliferation of tumor cells,enhance the invasion and migration of tumor cells,inhibit the apoptosis of tumor cells,and promote tumor angiogenesis by binding with different prostaglandin E receptor subtypes.Prostaglandin E receptor includes EP1,EP2,EP3 and EP4 receptor subtypes.Among the four receptors,EP3 and EP4 have higher affinity than EP1 and EP2.In tumors,EP4 receptor is also the most widely and deeply studied EP receptor subtype.EP4 receptor is related to the proliferation and invasion of a variety of human cancer cells.It has been reported that EP4 receptor is the most closely related to human tumors.The results show that snail can inhibit the transcription of E-cadherin gene by binding with the promoter region of E-cadherin gene.Through influencing the expression of intercellular adhesion molecules,it can promote the transformation of epithelial stroma,promote the invasion and metastasis of tumor,and participate in the progress of tumor.Research shows that c-myc is abnormally expressed in many kinds of malignant tumors,which affects the proliferation and metastasis of tumors,and participates in the occurrence and progress of tumors.At present,there is no report in the hepatoma system.The purpose of this study is to study the expression,diagnosis and therapeutic significance of snail and c-myc in the pathogenesis of hepatoma through PGE2 receptor and its antagonists.Methods Hep G2 and Huh7 cells were cultured in DMEM containing 10%fetal bovine serum?FBS?and 1%penicillin/Streptomycin complete medium?high sugar?.The temperature of the medium was 37?,and 5%CO2 was added to the humidified incubator.Hep G2 and hum7 cells of logarithmic growth were collected.After routine digestion,the cells were resuspended in DMEM medium containing 10%FBS,and the cell concentration was adjusted to 5×10⁴m L^-1.100?L?3×10³?cells per well were inoculated into 96 well culture plate?repeat 3 pieces?,each group had 6 double holes,37?,5%CO₂overnight.The following drugs were added to the culture medium:control group?adding the same volume of vehicle?,PGE2 group?adding 1?M,2?m M,5?M PGE2 respectively?,cj-42794 group?5?M PGE2+5?M CJ-42794?,culture at 37?,5%CO2 for 24 hours and 48 hours respectively,and MTT method was used to detect the effect of cj-42794 on the proliferation of hepatoma cells.Drop 130?L DMEM into the upper chamber to make the membrane hydrophilic.The Hep G2 cells and Huh7 cells treated for 24 hours in different groups were digested and centrifuged.The cells were resuspended in 1ml serum-free DMEM medium and counted.2×10⁴cells were added into 600?L DMEM medium containing 30%FBS,the last cells in each group were the same.After absorbing the serum-free DMEM medium,the cell suspension containing 2×10⁴cells was evenly mixed,and then the cells were added into the cell culture chamber gradually and vertically,and then cultured in the cell culture chamber.During this period,the cell membrane penetration was observed dynamically.48 hours later,the effect of cj-42794 on the invasion of hepatoma cells was detected by Transwell method.After 24 hours of drug treatment,the protein was extracted from the collected cells by Ripa lysate,which was split at 4°for 30 min,centrifuged at 13200 rpm for 25 min,and the supernatant was obtained.Then BCA kit was used to detect the protein concentration.The protein was separated by 10%SDS polyacrylamide gelelectrophoresis.The protein was transferred to the PVDF membrane.The PVDF membrane was placed in the 5%skim milk powder at room temperature for 1h,then added a single antibody?c-myc1:1000,snail1:1000?and the mouse monoclonal anti-?-actin?1:500?4 degrees incubated overnight.Then the membrane and horseradish peroxidase labeled two antibody were incubated at room temperature for 1h.Western blot was used to detect the effect of cj-42794 on the expression of c-myc and snail in hepatoma cells.Results1. The effect of cj-42794 on the proliferation of hepatoma cells:24 hours after Hep G2stimulation,compared with the control group,the cell proliferation rate of 2?M PGE2,5?M PGE2 and cj-42794 group increased significantly,and 5?M PGE2 group increased most significantly?P<0.05?.Compared with 5?M PGE2 group,the cell increment rate of cj-42794 group decreased,but there was no significant difference?P>0.05?.After 48 hours of Hep G2 stimulation,compared with the control group,the cell proliferation rate of 2?M PGE2 and 5?M PGE2 groups increased significantly,and 5?M PGE2 group increased most significantly?P<0.05?.Compared with 2?M PGE2 group and 5?M PGE2 group,the cell proliferation rate of cj-42794 stimulation group was significantly lower?P<0.05?.After 24 hours of Huh7 stimulation,compared with the control group,the cell proliferation rate of 2?M PGE2,5?M PGE2 and cj-42794 groups increased significantly,and 5?M PGE2 group increased most significantly?P<0.05?.Compared with 5?M PGE2 group,the cell increment rate of cj-42794 group decreased significantly?P<0.05?.48 hours after Huh7stimulation,the cell proliferation rate of 5?M PGE2 group was significantly higher than that of the control group?P<0.05?.Compared with 2?M PGE2 and 5?M PGE2,the cell proliferation rate of cj-42794 stimulation group was significantly lower?P<0.05?.2. The effect of cj-42794 on the invasion of hepatoma cells:the results showed that the proliferation rate of Hep G2 cells was significantly higher than that of the control group?P<0.05?.Compared with PGE2?2?M,5?M?,the proliferation rate of cj-4279 cells was significantly inhibited?P<0.05?.Compared with the control group,the cell proliferation rate of 2?M PGE2 and 5?M PGE2 groups increased significantly?P<0.05?.Compared with PGE2?2?M,5?M?,cj-4279 significantly inhibited cell proliferation?P<0.05?.The results of the two kinds of cells are consistent,suggesting that PGE2 regulates the invasiveness of hepatoma cells through EP4 receptor.3. The effect of cj-42794 on the expression of c-myc in hepatoma cells:c-myc is one of the important proto oncogenes in the body,which can promote cell proliferation and tumorigenesis by up regulating the expression of many target genes.In order to explore whether cj-42794 mediates PGE2/EP4 signal in HCC,we used Western blot to detect the effect of cj-42794 treatment on the expression of c-myc in Hep G2 and Huh7 cells.The results showed that the protein expression level of c-myc in PGE2 group was significantly higher than that in the control group?P<0.05?.Cj-42794 significantly inhibited the effect of PGE2 on the expression of c-myc protein?P<0.05?.These results suggest that the effect of PGE2/EP4R on the biological behavior of hepatoma cells may be related to c-myc proto oncogene.The effect of cj-42794 on the expression of snail in hepatoma cells:snail is a transcription factor closely related to the development of tumor,which promotes the metastasis and invasion of tumor cells by influencing the expression of intercellular adhesion molecules.In order to elucidate the molecular mechanism of cj-42794affecting the biological behavior of hepatoma cells,we used Western blot to detect the effect of cj-42794 on the expression of snail in hepatoma cells.Hep G2 and Huh7 cells were treated with 1?M,2?M,5?M PGE2 and 5?M?PGE2+cj-42794?,respectively,for 24 hours.The results of Western blot showed that the expression level of snail protein in PGE2 group was significantly higher than that in the control group?P<0.05?;compared with PGE2 group,the expression level of snail protein in cj-42794 group was significantly lower?P<0.05?.These data indicate that the proliferation of HCC cells induced by PGE2 is regulated by snail.These results suggest that the effect of PGE2/EP4R on the biological behavior of hepatoma cells may be related to c-myc proto oncogene.Conclusions CJ-42794,a selective antagonist of EP4 receptor,inhibited the proliferation,invasion and migration of Hep G2 and Huh7 cells,and the expression of c-myc and snail in hepatoma cells,suggesting that PGE2 may affect the biological behavior of hepatoma cells through the c-myc and/or snail signals mediated by EP4receptor,and participate in the regulation of the occurrence and development of hepatoma,which is expected to provide experiments for the study of the mechanism of primary hepatoma The aim of this study is to provide a target for the study of drugs related to targeted treatment of liver cancer.