Effects Of Hypoxia And Its Mechanism On Invasion And Metastasis Of Esophageal Carcinoma Eca109Cells In Vitro And In Vivo

Esophageal carcinoma is one of the most common malignant disease, withhigh incidence, and poor curative effect. Currently the main treatments foresophageal carcinoma included surgery and radiotherapy. Although in recentyears, the advance of diagnosis and treatment technology makes esophagealcarcinoma treatments continuingly improved, and the survival rate enhanced,the effect is still unsatisfactory for its5-year survival is only about15%. Themain impact factors for prognosis of patients with esophageal carcinoma wasinvasion and metastasis, therefore, to explore its mechanism is of greatimportance in the esophageal carcinoma therapy.In recent years, a mounting body of evidence has demonstrated that ahypoxic microenvironment is common phenomena in many tumor types. As astimulating factor, hypoxia changes the transcription activity of many geneswhich induce many biological responses. Strong evidence has accumulatedthat hypoxia plays a pivotal role in tumor angiogenesis, acquired treatmentresistance and progression. In the process, hypoxia inducible factor-l (HIF-1)plays a key role in response to hypoxia, but the mechanism which may becomplicated are not fully understood. HIF-l, as a key determinant in hypoxicmicroenvironment, has become one of the most popular subjects in recentyears.HIF-1is a heterologous dimmers which composed of two subunits of HIF-lα and HIF-l β. Under normoxia, HIF-l α is degradated throughubiquitin-proteasome compound quite easily. However, its degradated processis blocked under hypoxia, which induces accumulation of HIF-lα, thencombines with HIF-lβ to form activation. HIF-l has effect on expression of its target genes. All target genetic structurecontains a low oxygen response component (HRE). HRE is a nucleotidesequence which is contained in promoter or enhancer of target genes, and itscore sequence is5,-RCGTG-3,. HIF-1α plays a crucial role in tumor celladaptation to the hypoxic microenvironment through transcriptional regulationof its target genes. Therefore, it is of great importance to study the effects ofHIF-lα in tumor growth, invasion and metastasis.RNA interference (RNAi) is a technology that was first described by Fire etal, and has subsequently been elucidated as a multistep mechanism.Double-stranded RNAs are processed by the enzyme dicer into shortinterfering RNAs (siRNA). These siRNAs are bound to RNA-inducedsilencing complexes and mediate the degradation of their complementaryRNA. In this study, firstly, expression of HIF-1α, E-cadherin and MMP-2under hypoxia induced by CoCl₂was detected. Migration and invasion ofesophageal carcinoma cells were detected after rapamycin intervention invitro, in order to explore the effect and its mechanism of hypoxia on migrationand invasion. Secondly, short hairpin RNA (shRNA)-plasmid vector of HIF-1α gene were transfected into esophageal carcinoma cells with lipofectaminereagent, effect on migration and protein expression of HIF-1α, E-cadherin,MMP-2and Snail was detected by western blot, further to discuss the specificmolecular mechanism. Finally, in order to further explore the effect of HIF-1α on invasion and metastasis in vivo, through establishment of transplantedtumor model in nude mice.Part â…  Effects of Hypoxia on migration and invasion of esophagealcarcinoma Eca109cells in vitroObjective: To explore the effect of hypoxia on migration and invasion ofesophageal carcinoma and its mechanism.Methods: CoCl₂was used as chemical hypoxia-inducing reagent tomimic tumor hypoxic microenvironment. mRNA and protein levels of HIF-1α, E-cadherin and MMP-2at different hypoxic culture phases were detectedby semi-quantitative RT-PCR and immunohistochemistrym, respectively. After being treated with rapamycin combined with hypoxia, the proteinexpression of HIF-1α, E-cadherin and MMP-2was assayed by western blot.The effect of rapamycin combined with hypoxia on migration and invasionwere tested by cell scratch assay and transwell chambers.Results: Under hypoxia, mRNA level of HIF-1α had no significantchange, while its protein level increased obviously; mRNA and protein levelsof E-cadherin were down-regulated, but the mRNA and protein levels ofMMP-2were up-regulated. Under hypoxia, rapamycin inhibited theexpression of HIF-1αand MMP-2, but E-cadherin was up-regulated. Themigration and the number of invading cells decreased (P<0.05) after thetreatment of rapamycin under hypoxia.Conclusion: Hypoxia can increase protein level of HIF-1α inesophageal carcinoma, which promote migration and invasion possiblythrough down-regulating E-cadherin and up-regulating MMP-2.Part â…¡ Construction and identification of recombined HIF-1α-shRNAlentivirus and establishment of stabilized transfected esophagealcarcinoma cell linesObjective: To construct HIF-1αRNAi lentiviral vectors and HIF-1αgene specific silenced Eca109cells, then the change of migratory ability andexpression of E-cadherin, MMP-2, Snail were detected after Eca109cellstransfected with pGCSIL-HIF-1α-shRNA, to further explore its specialmolecular mechanism.Methods: Three pairs of anti-sense oligonucleotide fragments and a pairof non-sense sequence was designed and annealed, and then introduced intoRNA interfering expression vector pGCSIL, and the recombinant expressionvector pGCSIL-HIF-1α-shRNA was successfully constructed; Eca109cellsstably expressing pGCSIL-HIF-1α-shRNA were screened using G418; ThemRNA and protein expression of the HIF-1α gene were investigated aftertransfection with pGCSIL-HIF-1α-shRNA by RT-PCR and Western blot,respectively; Characteristics of cell migration of the Eca109cells transfectedwith pGCSIL-HIF-1α-shRNA were determined using transwell chamber extraneous migratory experiment. Expression of E-cadherin, MMP-2andSnail was detected by Western blot.Results: Three siRNA expression vector pGCSIL-HIF-1α-shRNA1,pGCSIL-HIF-1α-shRNA2and pGCSIL-HIF-1α-shRNA3weresuccessfully constructed using gene recombination technology, in whichpGCSIL-HIF-1α-shRNA2was screened as most inhibitory effect;Expression levels of the HIF-1αgene in Eca109cells transfected withpGCSIL-HIF-1α-shRNA2was significantly lower than those in control anduntransfected groups; The results of Transwell chamber extraneous migratoryexperiment showed that the number of Eca109cells transfected with pGCSIL-HIF-1α-shRNA2notably decreased in through pore compared with controland untransfected groups (P<0.05), there was no significant difference incontrol and untransfected groups (P>0.05). E-cadherin expression increased,and MMP-2decreased once HIF-1αinhibited effectively. Expression of Snaillevel enhanced under hypoxia, whereas it decreased after Eca109cellstransfected by pGCSIL-HIF-1α-shRNA2. There was a positive correlationbetween HIF-1α and Snail (r=0.840,P<0.05).Conclusion: Vector recombined with HIF-1α-shRNA is successfullyconstructed, and identified the most inhibitory effect one. Expression of HIF-1α mRNA and protein decreases after Eca109cells transfected with HIF-1αshRNA, so have established the HIF-1α knocking down cell linesuccessfully. Migratory ability weakens in siRNA group. HIF-1α candecrease E-cadherin through up-regulating Snail. Meanwhile, expression ofMMP-2decrease after Eca109cells transfected with HIF-1α shRNA, whosemechanism needs to further explore.Part â…¢ Effect and its mechanism of HIF-1α by RNAi on invasion andmetastasis of xenografted human esophageal carcinomaObjective: To establish a proper model of human esophageal carcinomain nude mice and study the ablity of tumor formation. To study the effects ofHIF-1α silencing on invasion and metastasis in vivo.Methods: Transplantable tumors were produced using Eca109cells and the transfected Eca109cells with pGCSIL-HIF-1α-shRNA and vacancyplasmids, respectively, in nude mice. Tumor volumes in every group weremeasured. All lymph nodes were processed with hematoxylin-eosin staining.The metastatic rate and the positive rate were compared in each group. Tumortissues were collected from the three groups, and then HIF-1α, E-cadherin,MMP-2mRNA and protein were detected by RT-PCR and Western blotrespectively.Results: As indicated in the growth curve of tumors,pGCSIL-HIF-1α-shRNA obviously inhibited the growth of transplantable tumors comparedwith control and untranfected groups (P<0.05). Compared with control anduntranfected groups, the metastatic rate and positive rate of lymph nodes inRNAi group decreased. Expression levels of mRNA and protein of HIF-1αand MMP-2genes were markedly lower, while E-cadherin higher in Eca109cells transfected with pGCSIL-HIF-1α-shRNA compared with control anduntransfected groups (P<0.05).Conclusion: HIF-1α-siRNA obviously inhibites the growth oftransplantable tumors and effectively inhibit the invasive and matastasisability of Eca109cells in vivo, which have correlation with its effects onE-cadherin and MMP-2, and it will provide the new idea and the theoreticalbasis for target gene therapy of esophageal carcinoma.