Tumstatin Gene Expression In Renal Carcinoma And The Regulation Of Ornithine Decarboxylase Gene Expression On The Expression Of Tumstatin

Tumor is one of the most common and frequently encountered diseases. In particular, malignant tumor is the leading cause of influencing healthy. Nowadays, with the population aging, the morbidity and mortality of tumor continue to show an upward tendency in China. Despite having some progress, searching for highly sensitive, specific and efficient treatment for tumor remains difficult, because of the diversity of etiological factor and complexity of tumor cells. In 1971, Folkman proposed that tumor growth and progression relied on neovasculature. And then anti-agiogenic therapy became a hot research subject.Tumstatin is a novel endogenous inhibitor of angiogenesis, the same as angiostatin and endostatin, liberated from basement membrane. It is the bioactive NC I domain (28KD) of ColⅣα3, composed of 244 amino acids. Tumstatin can specifically suppress proliferation of endothelial cells (EC), induce apoptosis of EC and inhibit pathological angiogenesis and tumor growth. Among the type of endogenous angiogenesis inhibitors discovered recently such as arresten, endostatin, castatin; tumstatin is the most powerful inhibitor. Therefore, it is regarded as a promising therapeutic candidate in the control of tumor angiogenesis and growth.Renal carcinoma is one of the most common malignant tumors originating from renal parenchyma with high and growing incidence. The definite etiopathogenesis of renal cancer is unkown. At present, early detection, diagnosis and treatment with surgery (radical or partial nephrectomy) have yielded positive results. However, overall five year survival rate for this disease is around 40%. Radiotherapy and chemotherapy, adjunctive therapies for cancer treatment, can't cure tumor completely. And these traditionary therapies may also damage the normal tissues and cells. Nevertheless, anti-agiogenic therapy inhibits pathological angiogenesis specifically, while angiogenesis associated with development and tissue repair are unaffected. So, it becomes the potent treatment for cancer with good prospect. The distribution of tumstatin is limited to glomerular basement membrane, alveolar capillary basement membrane and the vascular basement membrane of several organs. So far, there is no information on the relationship between tumstatin and renal carcinoma. Our article revealed the tumstatin gene expression in renal cancer with mRNA and protein level to observe the relatioship between tumstatin gene expression and the pathogenesis of renal cancinoma. Moreover, we constructed a eukaryotic expression plasmid containing tumstatin gene to explore the feasibility for the utilization of gene therapy.Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of polyamine biosynthesis, catalyzing the decarboxylation of ornithine to produce putrescine. ODC, overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. In order to study whether ODC overexpression facilitate angiogenesis by inhibiting tumstatin expression, we first analyzed the relationship between ODC expression levels and tumstatin production in renal tissues and cells, and then established the ODC-overexpression HEK293 cells to examine the effect of ODC overexpression on tumstatin expression and vascular endothelial cell proliferation. Our results showed that ODC overexpression can supress the expression of tumstatin, which indicated that ODC promoting tumor angiogenesis by suppressing tumstatin expression in many cancers.In this study, we required anti-tumstatin antibody for sequential experiment, but there is no commercial antibody of tumstatin then. Therefore, we constructed the human tumstatin expression plasmid, purified and refolded the expressed fusion protein which was used to immunize the Balb/c mouse as the immunogen, and then prepared an anti-tumstatin polyclonal antibody. ELISA and western blot showed that the prepared antibody could combine the tumstatin protein specifically. The preparation of the polyclonal antibody facilitated the following study.PART ONE PROKARYOTIC EXPRESSION OF HUMAN TUMSTATIN GENE AND PREPARATION OF POLYCLONAL ANTI-TUMSTATIN ANTIBODYObjective:To construct the human tumstatin prokaryotic expression plasmid, purified the recombinant protein and prepared the anti-tumstatin polyclonal antibody.Methods:(1)The sequence encoding tumstatin was amplified by RT-PCR and then inserted into the prokaryotic expression vector pTriEx-4 by TA clone.(2) The positive plasmid constructs named pTriEx-tumstatin were finally transformed into E.coli JM109 (DE3) for expression which was induced by IPTG The expressed fusion protein was identified by SDS-PAGE and western blot with anti His.tag monoclonal antibody.(3) The recombinant protein was purified by nickel chelate affinity column, refolded by dialyzing and concentrated by PEG8000. The concentrated solution was analyzed through SDS-PAGE and western blot.(4) Tumstatin/Adjuvant mixture was used to immunize Balb/c mice and collected murine antiserum. The antibodies in the serum were titrated by ELISA and tested by western blot.Results:(1) The cDNA encoding human tumstatin was amplified.(2) The prokaryotic expression plasmid pTriEx-tumstatin was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing. (3) Tumstatin was expressed significantly in E.coli, and then purified, refolded and concentrated effectively. SDS-PAGE and western blot confirmed the identity of the fusion protein.(4) The anti-tumstatin polyclonal antibody was prepared successfully. ELISA and western blot showed that the antibody could immunobind to recombinant human tumstatin protein.Conclusions:The anti-tumstatin polyclonal antibody was prepared successfully using the recombinant tumstatin protein as the immunogen. It was the necessary agent for subsequent research.PART TWO THE RELATIONSHIP BETWEEN TUMSTATIN GENE EXPRESSION AND RENAL CARCINOMAObjective:To detect the expression of tumstatin in renal carcinoma and construct a human tumstatin eukaryotic expression plasmid to examine its effect on the proliferation of vascular endothelial cells and renal cancer cells.Methods:(1) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression level of tumstatin in renal cancer tissues at the mRNA and protein level.(2) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression levels of tumstatin mRNA and protein in renal cancer cells.(3) Constructed eukaryotic expression plasmid pcDNA-tumstatin and identified by restriction enzyme digestion and DNA sequencing.(4) Transfected plasmid pcDNA-tumstatin into human umbilical vein endothelial cell line ECV304 and human renal carcinoma cell line ACHN. The expression of tumstatin in the two cell lines by RT-PCR and western blot.(5) CCK-8 assay was used to analyze the effect of pcDNA-tumstatin on the growth of ECV304 and ACHN cells.(6) Cell cycle distribution of ECV304 was detected by flow cytometiy analysis. The effect of tumstatin expression on cell cycle regulated protein cyclin D1 was measured by western blot analysisResults:(1) The tumstatin mRNA levels in renal carcinoma tissues (0.57±0.75) were significantly lower than in normal tissues (1.08±1.08) (P<0.05). Western blot also showed that tumstatin protein is expressed at low levels in most of the tumor tissues (1.41±0.34) when compared with the corresponding normal tissues (1.82±0.48) (P<0.05).(2) RT-PCR and western blot showed that tumstatin mRNA and protein levels in ACHN cells were significantly lower than in HEK293 cells.(3) Restriction endonuclease digestion result showed that the direction and length of inserted tumstatin fragment in pcDNA-tumstatin was right, further confirmed by DNA sequencing.(4) The results of RT-PCR and western blot showed that tumstatin was highly expressed in both ECV304 and ACHN cells transfected with pcDNA-tumstatin.(5) The proliferation trend was significantly decreased in ECV304 cells transfected with pcDNA-tumstatin, compared with cells transfected with empty vector and the untransfected cells. However, there is no remarkable effect on ACHN cells.(6) Tumstatin expression induced ECV304 cells to arrest at G1 phase and decreased the expression of cyclin D1.Conclusions:The expression of tumstatin is down-regulated in renal carcinoma, indicating that changes in tumstatin gene expression are related to the development of renal cancer. pcDNA3.1 (+)-mediated overexpression of tumstatin inhibits endothelial cell proliferation specifically by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1. Administration of tumstatin using a gene therapy approach might represent a promising treatment option for renal cancer treatment.PART THREE ORNITHINE DECARBOXYLASE GENE EXPRESSION REGULATES THE EXPRESSION OF TUMSTATINObjective:To investigate the relationship between ODC gene expression and tumstatin expression.Methods:(1) Semiquantitative RT-PCR and western blot analysis were used to detect the expression levels of ODC and tumstatin in renal cancer tissues.(2) The expression levels of ODC and tumstatin in cancer cells (human renal cancer cell ACHN and human lung cancer cell A549) were detected by semiquantitative RT-PCR and western blot analysis.(3) Constructed ODC-overexpressing plasmid pcDNA-ODC and identified by restriction enzyme digestion and DNA sequencing.(4) The antisense ODC expressing vector pcDNA-ODCr was transformed into E.coli to amplify, confirmed by restriction enzyme digestion and DNA sequencing.(5) Transfected plasmid pcDNA3.1 and pcDNA-ODC into HEK293 cells with LipofectamineTM 2000, used G418 to select positive cells.3-4 weeks later, picked out the clone, digested with trypsinase and continued culturing to establish the mock transfectants and ODC transfectants.(6) The expression levels of ODC and tumstatin protein were examined by RT-PCR and western blot analysis using total cell RNA and cell lysates from HEK293 cells treated by PBS,pcDNA3.1,pcDNA-ODC,pcDNA-ODC and pcDNA-ODCr, putrescine.(7) Semiquantitative RT-PCR was used to detect the expression levels of ODC and tumstatin mRNA in HEK293 cells treated like above. (8) Two luciferase reporter plasmids of tumstatin gene promotor pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed. Cotransfected pGL-tumstatin2.2kb and pRL-TK into HEK293 cells treated like above to observe the effect of ODC overexpression on tumstatin promoter activity by Dual Luciferas Reporter gene assay.(9) CCK-8 assay was used to analyze the effect of conditioned media from HEK293 cells treated like above on the growth of ECV304.Results:(1) RT-PCR showed that thirty-two of thirty-eight cancer tissues overexpressed ODC mRNA compared with the normal renal tissues. And twenty-four of thirty-two ODC-overexpressing human renal cancers showed suppressed tumstatin mRNA expression. The result indicated that there was a correlation between ODC gene expression and tumstatin expression.(2) RT-PCR showed that the expression of tumstatin was remarkably suppressed in the ODC-overexpressing cell lines ACHN and A549, compared with the corresponding normal cells (HEK293 and HELF), as determined by western blot.(3) The ODC-overexpressing plasmid pcDNA-ODC was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing.(4) Restriction endonuclease digestion showed that the length of inserted ODC antisense RNA fragment is right. DNA sequencing further confirmed that the pcDNA-ODCr plasmid delivered a 120bp fragment complementary to the initiation codon of ODC gene.(5) The results of RT-PCR and western bolt showed that the positive ODC-overexpressing cells selected by G418 highly expressed ODC mRNA and protein.(6) ODC mRNA and protein were overexpressed in ODC transfectants, and the elevated ODC expression level returns to that of mock transfectants, while there were no changes in putrescine treated group, as comared with PBS treated group and mock transfectants. The expression of tumstatin in ODC transfectants and putrescine treated group at both the mRNA and protein levels was significantly suppressed. The suppression of tumstatin expression was rescued after pcDNA-ODCr transfected. VEGF mRNA expression levels in all HEK293 treated remained unchanged.(7) The tumstatin promoter luciferase reporter plasmids pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed successfully. Both of them have promoter activity and pGL-tumstatin2.2kb presented stronger promoter activity than pGL-tumstatin0.5kb. Moreover, the activity of pGL-tumstatin2.2kb decreased about 45.8%,45.2% by ODC overexpression and putrescine respectively.(8) Conditioned media from ODC transfectants and putrescine treated group induced 1.41±0.07 and 1.46±0.07 fold ECV304 cell proliferation respectively, compared with media from the mock transfectants (P<0.05).Conclusions:ODC overexpression and administration of putrescine could inhibite tumstatin expression which maybe the underlying mechanism that the elevated ODC expression levels facilitate endothelial cell proliferation (angiogenesis).