Expression And Clinical Significance Of Both PTEN And VEGF In Hepatocellular Cancer

PART-1 Expression and Clinical Significance of both PTEN and VEGF in Hepatocellular CancerSection A Expression and relation with clinically pathological character of PTEN in HCC tissuesOBJECTIVE: To investigate expressions and significance of the tumor supressor gene phosphatase and tensin homlog deleted on chromosome ten protein (PTEN) in hepatocellular carcinoma (HCC) and pericarcinomatous tissues, and analyze relationship and clinical signification between its expression and occurring, development, invasion and metastasis of HCC.METHODS: Immunohistochemical staining with S-P methods and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect protein and mRNA expressions of PTEN in 60 cases of HCC and their pericarcinomatous tissue; furthermore it was compared with difference of expressions of PTEN protein and mRNA between them.RESULT: 1. It was significantly weaker of PTEN protein positive expression intensity of HCC tissue than that of pericarcinomatous tissue, there was significance of difference between two groups(t=3.29, P<0.05).2. Positive expression of PTEN mRNA in pericarcinomatous tissue was significantly higher than that of carcinoma tissue, there was significance of difference between two groups (t=25.034, P<0.01) .3. Expression of PTEN protein in I and II grade HCC tissue whose differentiation was better was significantly stronger than that of III and IV grade carcinoma tissue, there was significance of difference between two groups (t=26.009, P<0.01).Though Expression of PTEN protein was obviously decreased (P<0.01) along with tumor invasion and clinic development raising, it had not relativity with sex, tumor size and AFP level of patient(P>0.05) . CONCLUSIONS:The result of this experiment showed that mRNA and protein expression of PTEN of carcinoma tissue was significantly lower than that of pericarcinomatous tissue, and that their positive expression level was obviously decreased along with pathology differentiation reducing and invasion raising of HCC tissue, therefore it was presumed that it might be related with genesis and prognosis of HCC for height and Iowness of PTEN gene expression rate, expression level of PTEN could reflect to differentiation and invasion of HCC tissue at a certain extent , they took on negative correlation, and imperfection of PTEN high expression hinted that prognosis was bad.Section B Expression and relation with clinically pathological character of VEGF in HCC tissueOBJECTIVE:To investigate expressions and significance of vascular endothelial growth factor(VEGF) in hepatocellular carcinoma (HCC) and pericarcinomatous tissue, and analyze relationship and clinical signification between its expression and occurring, development, invasion and metastasis of HCC.METHODS:Immunohistochemical staining with S-P methods and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect protein and mRNA expressions of VEGF in 60 cases of HCC and their pericarcinomatous tissue,it was compared with difference of expressions of VEGF protein and mRNA between them. RESULTS: 1. It was significantly stronger of VEGF protein positive expression intensity of HCC tissue than that of pericarcinomatous tissue, there was significance of difference between two groups(t=27.843, P<0.05).2.Positive expression rate of VEGF mRNA was 76.67% In HCC tissue, but it was 26.77% in pericarcinomatous tissue , positive expression of VEGF mRNA in carcinoma tissue was significantly higher than that in pericarcinomatous tissue, there was significance of difference between two groups (t=27.372, P<0.01) . 3.Expression of protein and mRNA of VEGF was obviously increased (P<0.01) along with tumor invasion and clinic development raising, but it had not relativity with sex, tumor size and AFP level of patient (P>0.05) .CONCLUSIONS: 1.In this study, it was shown that positive expression of mRNA and protein of VEGF in tumor tissue was obviously higher than that of pericarcinomatous tissue(P<0.01) ,but was increased (P<0.01) along with tumor invasion and clinic development raising(P<0.01), it might be that it wanted a lot of neo-vessel to provide oxygen and nutrition on process of HCC development ,as a result that transcription and translation and expression of VEGF all enhanced for those tumor which invasion was strong and clinic malignant degree was high, therefore expression level of VEGF could reflect to differentiation and invasion of HCC tissue at a certain extent , they took on positive correlation.2. In this study, it was found that VEGF mostly expressed on hepatoma cell and weakly on vascular endotheliocyt, thereby it was presumed that VEGF might act on self tumor cell by autocrine and (or) vascular endotheliocyt by paracrine, its detailed mechanism wanted more experiment to approve.Section C Correlation of expression and relation with clinically pathological character and invasion activity of PTEN, VEGF and MVDin HCCOBJECTIVE:HCC is solid malignant tumor who has rich blood stream. Vascularization took an important rule on occurring, development, invasion and metastasis of HCC. The prophase experiment of this study showed that expression of PTEN and VEGF took on definite action in occurring, development, invasion and metastasis of HCC, their level could reflect to differentiation and invasion of HCC tissue at a certain extent. The experiment would observe correlation of expression and relation with vascularization of PTEN and VEGF in HCC.METHODS: Irnmunohistochemical staining with S-P methods and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect MVD and expressions of protein and mRNA of VEGF in 60 cases of HCC and their pericarcinomatous tissue.RESULTS: 1. MVD in HCC tissue was 49.74±12.10 and obviously higher than that in pericarcinomatous tissue .By statistics analysis, MVD expression increased with increasing of tumor size, invasion and clinic malignant degree, there was significance of difference (P<0.05) ,but not significantly different of organization-pathology grade and AFP level (P>0.05) .2.Pearson correlation analysis showed that it took on obviously negative correlation for positive expression intensity of mRNA and protein of PTEN and VEGF in HCC tissue, correlation quotiety was respectively -0.613 and -0.572 (P<0.01), it may be said that two gene both took on negative correlation on transcription and translation level and there was significance of difference.3. It took on negative correlation for MVD and PTEN protein in HCC tissue, correlation quotiety was -0.439(P<0.01), but it took on positive correlation for expression of MVD and VEGF, correlation quotiety was 0.645 (P<0.01), there was significance of difference.CONCLUSIONS: 1. This study found that it took on negative correlation for PTEN and VEGF protein(r=-0.572, P <0.01), thus showed that it might exist affinity with VEGF exceptionally being activated and expression imperfection of PTEN gene. PTEN might be provided with action of regulating mRNA and protein expression of VEGF, which had certain clinic significance that it could be estimated course of diseases and intrahepatic metastasis and prognosis of HCC patient according to positive rate of PTEN protein. 2. In HCC, MVD took on positive correlation with expression of VEGF protein, negative correlation with expression of PTEN protein. Distributing of MVD was most on tumor margin which may be high invasion area of HCC tumor usually located at its peripheral area where cell secreted more cytokine like VEGF, those hinted that VEGF acted the important function on neo-vascularization, that neonate vessel number was more abundant and tumor cell was easier to leave original part, enter vessel, occurring, invasion and metastasis, that HCC who had high expression of MVD was easier to relapse, metastasis and having bad prognosis than that of low expression.Part-2 The change of VEGF expression and morphology in Vector PTEN transfected hepG2 cellsOBJECTIVE: to investigate the change of VEGF expression in hepG2 cells after PTEN vector trandfection. It is shown that PTEN play an important role in the pathogenesis of hepatocellular carcinoma and regulate the Angiogenesis by VEGF in the development of cancer.METHODS: vector PTEN transfected cultured hepG2 cells.then PTEN and VEGF was dected at mRNA level by RT-PCR and at protein level by immunohistochemistry, Westen Blot and ELISA.morphological change of tumor cells was observed in mormoxic and hypoxic conditions respectively.that the activity of proliferation of tumors cells decreased for the PTEN transfection.RESULT: after vector PTEN transfection succeeded in hepG2 cells.PTEN expression was detected in hepG2 cells at mRNA and protein level. The VEGF expression decreased than that before transfection at mRNA and protein level respectively, the proliferation activity of tumor cells was also down regulated for the transfection.CONCLUSION: PTEN plays an important role in the pathogenesis of hepatocellular carcinoma. PTEN can down regulate the VEGF expression which is associated with the Angiogenesis in the process of cancer. PTEN also suppress the activity of proliferation of tumor cells in hepatocellular carcinoma.Part-3 The effect of VEGF oligonucleotide tranfected hepG2 cells on PTEN expression and morphological change.OBJECTIVE: To investigate the effect of VEGF oligonucleotide transfected hepG2 cells on PTEN expression and morphological change, proliferation activity of tumor cells.METHODS: artificial VEGF oligonucleotide tranfected the hepG2 cells. Then the efficiency of transfection was decteted by VEGF expression investigation. VEGF and PTEN expression are decteted in trandected tumor cells by FCS, RT-PCR and Westen Blot.Activity of proliferation of tumor cells is also decteted by FCS. Morphological change after transfection is observed by LESMRESULTS: VEGF expression is decteted at mRNA and protein level after VEGF oligonucleotide transfection in hepG2 cells. PTEN expression decreased in tranfected tumors cells compared with that before transfection.the proliferation activity of tumor cells is down regulated after transfection.CONCLUSION: VEGF oligonucleotide tranfection could be a therapic new method to the hepatocellular carcimona for its suppression activity of PTEN expression and proliferation in tumor cells.