The Expression And Function Of Prolyl Hydroxylase PHD2 In Hepatocellular Carcinoma

BackgroundProlyl hydroxylase protein family(PHDs) is an oxygen sensor protein located in the intracellular, can feel the direct oxygen partial pressure of molecular and exert its activity strictly dependent on intracellular oxygen partial pressure. PHD2 is the most important oxygen sensor in PHDs family. It is widely expressed in epithelial tissues,and is an important oxygen / redox sensitive proteases in intra-cellular. When the oxygen partial pressure is normal, PHD2 can make the hypoxia inducible factor-1alpha(inducible factor-1 hypoxia alpha, HIF-1?) being degraded rapidly through the pathway being dependent on oxygen. Under hypoxic conditions, PHD2 degrading HIF-1 ? is inhibited, which activate the hypoxia responsive regulatory pathway. A variety of hypoxia-related protein is produced to maintain the energy metabolism of cells.Hypoxia is a common characteristics which exist in variety of solid tumors,including hepatocellular carcinoma(Hepatocellular Carcinoma, HCC). Previous studies mainly focused on the transcriptional activation of multiple genes mediated by HIF-1 ?, and the corresponding regulatory mechanisms. In recent years the study found that the change of the activity of PHD2 is the initial step of tumor cells' hypoxia reaction. In many tumors, the abnormal expression of PHD2 can be detected,but the role of PHD2 in tumor progression has not yet been reached a consensus. A variety of related research often leads to contradictory conclusions, which is not only may play a role in tumor suppressor by degradation of HIF-1 alpha, may through other pathways to promote tumor cell proliferation, enhanced the invasion and metastasis ability, leading to tumor malignant progression. There are few reports about PHD2 in HCC, the relationship between the PHD2 expression of HCC and the prognosis of the patients, and the possible mechanism is not very clear.This paper intends to study from the following two parts, the first part is detection of PHD2 expression in HCC tissues and analysis of its relationship with the prognosis of patients with HCC combining with clinical data; the second part is the study of PHD2 impact on biological behavior of HCC by in vivo and in vitro experimental and to explore the molecular mechanism of the impact. The entire study is in order to provide a prognostic index for HCC patients and a potential target for clinical research of HCC.Part I: The expression of PHD2 in HCC tissue and the relationship between the expression and prognosis of patientsObjective : To investigate the expression of PHD2 in hepatocellular carcinoma and its relationship with prognosis of patient with HCC.Methods : We use immunohistochemistry(IHC) technique to detect PHD2 expression of 20 normal liver tissues and 20 cases of hepatocellular carcinoma. The hepatocellular carcinoma clinical samples were expanded to 220 cases. The expression of PHD2 was detected, and its correlation with clinicopathological features were then analyzed.. Follow-up of 220 cases of HCC patients survival time was recorded. And the statistically analysis was developed between the expression of PHD2 and prognosis of hepatic cell carcinoma patients. Survival curves were plotted,and the multivariate regression was analyzed. The correlation of the expression of PHD2 and the prognosis of hepatocellular carcinoma was revealed.Results:1The result of IHC detectionshowed thatthe expression of PHD2 was highed in hepatocellular carcinoma than that of normal liver tissue(P<0.05).2 The IHC technique was used to detect and grade the samples of 220 cases of HCC, Results suggested that the higher expression of PHD2 was significantly related to tumor size, degree of differentiation, TNM stage, and the higher level of alpha fetoprotein.3 Correlation analysis were cxcuted on the prognosis of patients with the erxpression of PHD2 in hepatocellular carcinoma. Results suggest that the shorter survival time was relatively with higher expression of PHD2. Compared with patients with lower expression of PHD2, the disease-free survival of the higher patients was significantly shorter(25.5 + 3.6 months vs. 6.7 + 3.4 months, log rank, P < 0.001),overall survival was shorter(37.5 + 3.6 months vs. 29.5 + 4.7 months, log rank, P <0.001). Analysis using stepwise regression method for Cox scale model of multifactors, results showed that the higher expression of PHD2, pathological stage, tumor size, serum AFP levels were independent prognostic overall survival in patients with HCC.Part?:The effect of PHD2 on the biological behavior of HCC and possible mechanismObjective: To investigate the effect of PHD2 on the biological behavior of HCC and to explore the related molecular mechanism.Methods : The expression of PHD2 was detected in three HCC cell lines(QGY7703, Bel7404, Hep3B) and normal liver cells group by Westen Blot. Using RNA interference technique, PHD2 RNAi and negative control si RNA were incubated with three strains of hepatocellular carcinoma cell respectively. A series of testing were executed, the silent effect of PHD2 was detected by Western Blot; the proliferation of hepatoma cell line was detected by MTT assay; HCC cell invasion and migration by was detected transwell assay; the apoptosis of hepatoma cells was detected by flow cytometry. Hepatocellular carcinoma cell line Hep3 B was selected to establish the transplantation tumor model of hepatocellular carcinoma in nude mice.After the model was established successfully, the experimental animals were divided into three groups, the cholesterol modified PHD2 RNAi was injected into the tumor in the PHD2 silence group. Blank group(untreated group)and normal saline group(normal saline was injected into the tumor in this group) were set up for control study.The injection was excuted every four days and nude mice tumor growth change was recorded. The nude mice were sacrificed sixteenth day after the start of the injection,followed by collecting tumor tissues. The expression of PHD2, p-Smad2/3 and oncogene c-myc was detected by Western Blot technique. IHC technique was used to detect the expression of c-Myc in 220 cases of HCC, and the correlation between the expression of PHD2 and and clinical pathological features of patients with HCC were analyzed.Results:1 The result showed that, compared with normal liver cell line, the expression of PHD2 in cancer cell line was significantly higher than that of control group in vitro cell trials. Compared with the negative control si RNA group, using interference PHD2 RNAi three hepatoma cell line proliferation, invasion and migration force has significantly decreased, and the apoptosis of hepatoma cells increased.2 In HCC nude mice transplanted tumor model, the tumor`s growth of P PHD2 silence group was slow(P<0.05). The tumor specimens using Western blot p-Smad2 /3 and the c-myc level. It was found PHD2 silence group, the level of p-Smad2 / 3 was significantly higher than that of the blank group and NS control group(P < 0.05) and c-myc level of PHD2 silence group, was significantly lower than that of the other two groups(P < 0.05).3 The expression of c-myc was detected in the 220 cases of clinical specimens by immunohistochemical technique. The result showed that the expression of c-myc was significantly related with the tumor size, degree of differentiation, TNM stage, higher level of alpha fetoprotein(AFP), and also is significantly correlated with the expression of PHD2(r = 0.557 P < 0.001).Conclusions1. The high expression of PHD2 is closely related to the poor prognosis of patients with HCC.2. The expression of PHD2 was higher in HCC cell lines than in normol cell lines; the over-expression of PHD2 could promote the proliferation of hepatoma cells,increases the migration and invasion of tumor cell, reduces the apoptosis of cancer cells.The role of PHD2 which promote the developing of cancer might is up-regulate the expression of c-myc oncogene through inhibit the expression of Smad2/3, which belonged to the signaling pathway of TGF beta 1-smad.