| Background:Prostaglandin E2(PGE2) is the main product of arachidonic acid after cyclooxygenase (COX) catalytic reaction. As an important lipid signal molecule in human body, PGE2is involved in the occurrence and development of various tumors, including hepatocellular carcinoma (HCC). After binding with its four kinds of E prostanoid receptors in the cell membrane, named EP1receptor, EP2receptor, EP3receptor and EP4receptor, PGE2exerts diverse biological effects by triggering cell signal transduction and regulating gene expression. Previous results from our laboratory showed that PGE2markedly promotes the cell growth, migration and invasion of hepatocellular carcinoma cells, yet the molecule mechanisms underlying these are not very clear.c-myc is a kind of cellular proto-oncogene, and its product is the nuclear transcriptional factor c-Myc protein. c-Myc protein can upregulate the transcriptional expression of numerous target genes by binding to the gene sequence and recruiting some related factors, thus facilitate cell proliferation and tumorigenesis. Activiation or overexpression of c-myc proto-oncogene is also involved in the occurrence and development of hepatocellular carcinoma.c-myc itself can be regulated by growth promoting signal. Many extracellular messenger molecules can upregulate or activate the c-myc expression via binging to their specific receptors and transmiting intracellular signal, and finally accelerate cell growth, proliferation and so on. Some data showed that, cyclooxygenase-2(the rate-limiting enzyme for PGE2synthesis) was positive correlated with the expression of c-Myc protein in hepatocellular carcinoma tissues. Yet till now, there is no study on the effect and its mechanism of PGE2on the expression of c-Myc in HCC, and also no data about its role in PGE2induced HCC cell growth and invasion.Aims:1. To study the effect of PGE2on the mRNA transcription and protein expression of c-Myc in HCC cells2. To study the role of c-Myc protein in PGE2induced HCC cell growth and invasion3. To investigate the role of EP4receptor in the PGE2’s effect on the expression of c-Myc in HCC cells4. To elucidate the signal pathway and underlying mechanisms of EP4receptor signal on the expression of c-Myc in HCC cellsMethods:1. Human hepatocellular carcinoma cell line Huh7was cultured in conventional conditions.2. WST assay was used to detect the growth of HCC cells induced by PGE2and other treatments.3. Transwell method was apply to measure the invasiveness of HCC cells induced by PGE2and other treatments. 4. Using PGE2, EP4receptor agonist or them combined with inhibitor, activator or siRNA interference specific to the important targets of signal pathway to treat HCC cells, the mRNA transcription of c-Myc was assayed by Real-time PCR method and the expression of c-Myc protein was detected by western blot method.5. The EIA kit was used to determine the level of cAMP in HCC cells after EP4receptor agonist stimulation.Results:1. Huh7cells were exposed to3μM PGE2for Oh to2h, and the expression level of c-Myc protein in HCC cells increased steadily, and at2h reached its maximum which was183%fold of Oh. Moreover, the mRNA transcription of c-Myc in HCC was also upregulated by3μM PGE2treatment, and quickly reached the peak value at1h, which was5.16fold of Oh.2. After treated with OμM to10μM PGE2for2h, the expression of c-Myc protein in HCC cells was detected by western blot. The results showed that0.1μM PGE2treatment could increase its expression to172%fold of control, and the levels of0.3μM to10μM PGE2treatment were all50%higher than that of control. Real-time PCR experiment revealed that the mRNA transcription levels of c-myc in HCC cells when exposed to0.1μM to10μM PGE2for1h were notably upregulated and were all more than3fold of control.3. WST assay showed that PGE2treatment for24h increased the cell growth of HCC cells to112%of control, when combined with c-Myc siRNA interference the cell growth decreased to98%of control, meanwhile the c-Myc siRNA interference downregulated the cell growth to93%of control. 4. Transwell invasion assay showed that PGE2treatment for24h increased the invasion ability of HCC cells to132%of control, when combined with c-Myc siRNA interference it decreased to91%of control.5. Huh7cells were exposed to the EP4receptor selective agonist PGE1alcohol(3μM) for Oh to2h, and the expression level of c-Myc protein in HCC cells increased steadily from100%at Oh to161.5%at2h. Moreover, the expression level of c-Myc protein in HCC cells treated with OμM to1OμM PGE1alcohol for2h also rised gradually along with the increase of dose of PGE1alcohol, and it was128.5%by0.1μM PGE1alcohol treatment and reached190%by10μM PGE1alcohol treatment.6. The results of Real-time PCR assay showed that the transcription levels of c-Myc mRNA in HCC cells was quickly upreguiated by the EP4receptor selective agonist PGE1alcohol(3μM) treatment from Oh to2h, and reached maximum value at1h which was4.71fold of Oh, then declined to1.32at2h. In addition, the transcription level of c-myc mRNA in HCC cells showed a dose-dependent increase by lh’s OμM to10μM PGE1alcohol treatment, and peaked at3μM tolOμM with4.06and4.05fold of control, respectively.7. HCC cells were pretreated with EP4receptor antagonist GW627368X (4μM) for1h followed by sitmulation with PGE2or PGE1alcohol for2h. The protein expression of c-Myc in HCC cells showed a decrease from192.3%or191.7%by PGE2or PGE1alcohol treatment respectively to105.3%or78.3%by PGE2or PGE1alcohol treatment with GW627368X, and also GW627368X treatment lowered the protein expression of c-Myc to69%of control. Real-time PCR assay revealed that, like its effect on c-Myc protein, pretreatment with GW627368X significantly suppressed the upregulation of mRNA transcription of c-myc in HCC cells induced by PGE2or PGE1alcohol. 8. HCC cells were transfected with siRNA specific to EP4receptor followed by PGE2or PGE1alcohol treatment, and the protein expression of c-Myc in HCC cells was examined by western blot. The results showed that the protein expression of c-Myc declined from173.1%in "N.C.siRNA+PGE2" group and160.9%in "N.C.siRNA+PGE1alcohol" group to82.3%in "EP4siRNA+PGE2" group and65.5%in "EP4siRNA+PGE1alcohol"group, and the protein expression of c-Myc also decreased to55%of control with single EP4siRNA treatment.9. HCC cells were pretreated for1h with Act D (5μg/ml) or CHX (50μg/ml), inhibitors of RNA synthesis and protein synthesis respectively, followed by sitmulation with PGE1alcohol for2h, and the protein expression of c-Myc was determined by western blot. The results showed that the protein expression level of c-Myc decreased from194%in "PGE1alcohol" group to59.5in "PGE1alcohol+Act D" and36.5%in "PGE1alcohol+CHX" group, additionally the Act D or CHX treatment markedly downregulated the expression of c-Myc protein with the value53%and34%respectively.10. HCC cells were treated with the EP4receptor agonist PGE1alcohol for0min to60min, and the intracellular cAMP level was determined by EIA method. The results showed that the cAMP level increased to433.3fold of control after5min’s PGE1alcohol treatment, peaked at30min to45min whose values were1555.9%and1554.9%respectively, and it decreased a little at60min whose value was1247.1%.11. Treatment of HCC cells with0μM to20μM forskolin, an activator of Adenylate Cyclase for2h increased the protein expression of c-Myc in a dose-dependent manner, and the protein expression of c-Myc reached maximum295.7%of control when10μM forskolin was used. Real-time PCR experiment revealed that, like its effect on c-Myc protein expression, forskolin also upregulated the mRNA transcription of c-myc in HCC cells by one hour’s treatment, and the value was2.651fold of control when0.5μM foskolin was used, then it increased gradually along with the rise of forskolin’s dose used, and peaked at20μM foskolin, whose value was6.054fold of control.12. Pretreatment of HCC cells with200μM adenylate cyclase inhibitor SQ22536effectively blocked the upregulation of c-Myc protein induced by PGE1alcohol. The protein expression of c-Myc decreased from212.5%in "PGE1alcohol" group to109.5%in "PGE1alcohol+SQ22536" group, and the sole SQ22536treatment downregulated the protein expression of c-Myc, whose value was66.5%. In transcription level, the results showed that SQ22536also partly inhibited the PGE1alcohol induced c-myc transcription, and the level decreased from4.65in "PGE1alcohol" group to3.93in "PGE1alcohol+SQ22536" group.13. Pretreatment of HCC cells with10μM PKA inhibitor H89effectively blocked the upregulation of c-Myc protein induced by PGE1alcohol. The protein expression of c-Myc decreased from258%in "PGE1alcohol" group to128%in "PGE1alcohol+H89" group that was close to the baseline level. Likewise, in transcription level H89also inhibited the PGE1alcohol induced c-myc transcription completely, and the level decreased from4.11in "PGE1alcohol" group to1.11in "PGE1alcohol+H89" group.14. Stimulation of HCC cells with the EP4receptor agonit PGE1alcohol (3μM) for Omin to60min led to a significant increase in phosphorylation of CREB protein at the site Ser133, and at5min the phosphorylation level of CREB was515%fold of control, at15min it reached maximum827%, and up to60min it was very high. 15. CREB siRNA not only lowered the baseline level of c-Myc protein expression in HCC cells, but also effectively reduced the EP4receptor signal mediated c-Myc protein expression. The expression level of c-Myc protein decreased from175.5%in "N.C.siRNA+PGE1alcohol" group to97%in "CREB siRNA+PGE1alcohol" group.16. Pretreatment of HCC cells with10μM to40μM PI3K inhibitor LY294002suppressed PGE1alcohol induced c-Myc protein expression. The expression level of c-Myc protein decreased from245%of PGE1alcohol group to157.5%,119.5%,121%of PGE1alcohol together with10μM,20μM,40μM LY294002respectively, and LY294002also downregulated the baseline level of c-Myc protein expression in HCC cells.Conclusion:By binding to EP4receptor, PGE2activates the Gs/AC/cAMP/PKA/CREB and Akt signal pathway, and then upregulates the mRNA transcription and protein expression of c-Myc in hepatocellular carcinoma cells, eventually promotes the cell growth and invasion of hepatocellular carcinoma cells. |
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