| Retinoblastoma is the most common intraocular malignancy in children, whichis initiated by the allelic loss of RB gene, and is reported to affect1in15,000livebirths. It represents almost four percent of all pediatric malignancies. Tumor hypoxiahas been recognized as a common feature of solid tumors and a negative prognosticfactor for response to treatment and survival of cancer patients. Hypoxia-induciblefactor-1(HIF-1), a molecular determinant of responses to hypoxia in mammaliancells, is a heterodimer consisting of two subunits, an oxygen-sensitive HIF-1α and aconstitutively expressed HIF-1β. HIF-1activity in tumors depends on availability ofthe HIF-1a subunit, the levels of which increase under hypoxic conditions andthrough activation of oncogenes and/or inactivation of tumor suppressor genes.Increased HIF-1has been correlated with increased angiogenesis, aggressive tumorgrowth, and poor patient prognosis. Currently there have been some studies on theimpact of hypoxia on intraocular RB which intensively focus on two aspects,angiogenesis and glucose metabolism. Little is known, however, about the hypoxicregulation of HIF-1α and the role of HIF-1a in intraocular RB. This research aims toinvestigate and evaluate the expression of HIF-1a in retinoblastoma specimens andcell lines, analyze the effect of silencing HIF-1a gene by RNA interference techniqueon the progression of human retinoblastoma in vitro and in vivo, providing theexperimental evidences for anti-tumor gene treatments of retinoblastoma. PartⅠ Expression and significance of HIF-1α in humanretinoblastomaObjectiveInvestigating and evaluating the expression of HIF-1αin retinoblastomaspecimens and cell lines; exploring the effect of hypoxia on the expression ofHIF-1αin retinoblastoma cell lines WERI-Rb-1and HOX-Rb44.Method1. The expression of HIF-1α in32cases human retinoblastoma paraffin-embedded tissues and11cases normal retina tissues was detected byimmunohistochemistry.2. The association between the expression of HIF-1α and clinicopathologicalcharacteristics of retinoblastoma including pathological types and clinical stages wereevaluated by Chi-square statistics.3. The effect of hypoxia on the expression of HIF-1α mRNA and protein inretinoblastoma cell lines WERI-Rb-1and HOX-Rb44were examined by RT-RCR andWestern blotting respectively.Result1. The expression rate of HIF-1α protein in tissue specimens of humanretinoblastoma was84.38%, which was higher than that in tissue of normal retinawith significant difference (P＜0.01).2. There was no significant difference in the expression rate of HIF-1α betweendifferentiated and undifferentiated type of retinoblastoma (P＞0.05); the expressionrate of HIF-1α was related to clinical stages of retinoblastoma (P＜0.01).3. The expression level of HIF-1α mRNA in retinoblastoma cell lines bothWERI-Rb-1and HOX-Rb44were positive respectively, and no obvious change wereobserved under hypoxia in contrast to that under normoxia (P＞0.05).4. The expression level of HIF-1α protein in retinoblastoma cell lines bothWERI-Rb-1and HOX-Rb44were up-regulated significantly under hypoxia, compared with that under normoxia with significant difference (P＜0.01).Conclusion1. There was a higher positive expression of HIF-1α in retinoblastoma tissue.The positive expression rate was related to the clinical stages of retinoblastoma.2. Hypoxia affected the expression of HIF-1α in retinoblastoma cell lines on theprotein level mainly.Part Ⅱ Construction of siRNA plasmids targeting HIF-1α gene andstudy of its inhibition effect in vitroObjectiveDesigning and constructing RNA interference plasmids targeting HIF-1α geneand testifying their effects and specificity in interfering HIF-1α gene expression, toprovide effective means for researching the function HIF-1α gene in humanretinoblastoma.Method1. Three RNA-interference plasmids targeting HIF-1α gene, pRNAT-CMV3.2/HIF-1αsiRNA Ⅰ~Ⅲ, were constructed and identified by using double enzymedigestion method.2. WERI-Rb-1cells were transfected by recombinant plasmids usingEntransterTMR and LipofectamineTM2000transfection reagents and the transfectionefficiency were examined by FCM.3. RT-PCR and western blotting were employed to detect the inhibition effect ofHIF-1αsiRNA Ⅰ~Ⅲ on the expression of HIF-1α mRNA and protein in WERI-Rb-1cells respectively.Result1. Three RNA-interference plasmids targeting HIF-1α gene, pRNAT-CMV3.2/HIF-1αsiRNA Ⅰ~Ⅲ, were constructed successfully. 2. The transfection efficiency of EntransterTMR transfection reagent was superiorto that of LipofectamineTM2000transfection reagent in human retinoblastoma cell lineWERI-Rb-1cells (P＜0.05).3. All three RNAi plasmids targeting HIF-1α gene, especially pRNAT-CMV3.2/HIF-1αsiRNA Ⅲ, showed significant inhibition effect in the expressionofHIF-1α gene in human retinoblastoma cell line WERI-Rb-1cells (P＜0.01).ConclusionConstructed RNA-interference plasmids targeting HIF-1αgene can effectivelyand specifically inhibit the expression of HIF-1α mRNA and protein in WERI-Rb-1cells, which will provide a useful tool to investigate the role of HIF-1αgene in RBdevelopment process.Part Ⅲ Effect of silencing HIF-1α gene by RNA interference on cellproliferation, apoptosis and expression of HIF-1α target genes inhuman retinoblastoma WERI-Rb-1cells in vitroObjectiveTo observe the effect of silencing HIF-1α gene by RNA interference on cellproliferation, cycle distribution, apoptosis and HIF-1α target genes expression inhuman retinoblastoma WERI-Rb-1cells in vitro.Method1. The cell proliferation inhibition was detected by MTT assay.2. The effect of silencing HIF-1α gene on cell apoptosis and cycle distributionwere examined by flow cytometry (FCM).3. The effect of silencing HIF-1α gene on the protein expression of P53, P21,bcl-2, Bax and VEGF genes were determined by Western blotting analysis. Result1. The siRNA-mediated HIF-1α gene silencing resulted in significant cell growthinhibition in HIF-1αsiRNA group under hypoxic condition.(P＜0.01, vs control).2. There was cell accumulation in the G1/G0phase and reduction in S and G2/Mphase in HIF-1αsiRNA group under hypoxic condition,(P＜0.01, vs control).3. The cell apoptosis rate was increased to around48.9%in HIF-1αsiRNA groupunder hypoxic condition (P＜0.01, vs control).4. The siRNA-mediated HIF-1α gene silencing led to slight reduction of bcl-2and p-p53protein (P＜0.05, vs control), significant increase of p21and Bax protein(P＜0.01, vs control), obvious down-regulation of VEGF protein (P＜0.01, vscontrol), as well as no change of p53protein (P＞0.05, vs control) in WERI-Rb-1cells in HIF-1αsiRNA group.ConclusionHIF-1α gene silencing could inhibit proliferation, increase apoptosis and inducecell cycle arrest in human retinoblastoma WERI-Rb-1cells by cutting off HIF-1αsignal pathways in vitro.Part Ⅳ Effect of silencing HIF-1α gene by RNA interference ontumor growth, survival and angiogenesis of retinoblastoma in vivoObjectiveTo investigate the effect of silencing HIF-1αgene on tumor growth, apoptosisand angiogenesis of the nude mouse retinoblastoma xenograft model in vivo.Method1. The nude mouse xenograft model of retinoblastoma was established bysubcutaneous injection. Different interventions were given to the mice byintratumoral injection to perform the HIF-1α RNA interference assay and the tumorgrowth was monitored, as well as the size and weight of tumor tissues were measured. 2. The inhibition effect of HIF-1αsiRNA interference on HIF-1α proteinexpression in the mice tumor tissues was detected by Western blotting.3. The microvessel density (MVD) of the mice tumor tissue was measured byCD34monoclonal antibody staining immunohistochemistry.4. The tumor cell apoptosis of the nude mice model of retinoblastoma wasexamined by TUNEL assayResult1. The nude mouse xenograft model of retinoblastoma was establishedsuccessfully.2. The tumor growth were suppressed obviously in HIF-1αsiRNA interferencegroup (P＜0.01, vs control).3. Silencing HIF-1α gene showed a significant inhibition effect of HIF-1αprotein expression in the tumor tissue of mouse models (P＜0.01, vs control).4. The MVD of tumor in HIF-1αsiRNA interference group was lower than thatin the three other control groups (P＜0.01).5. The amount of apoptotic tumor cells was significantly increased inHIF-1αsiRNA interference group (P＜0.01, vs control).ConclusionSilencing HIF-1α gene may reduce the MVD of tumor tissue, promote tumor cellapoptosis, suppress tumor growth by mediating its downstream genes in vivo, whichis of potential value in the gene therapy of human retinoblastoma... |
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