The Experimental Study On Silencing CXCR4 Gene By RNA Interference Inhibits The Laryngeal Carcinoma Invasion And Metastasis In Vitro

Laryngeal carcinoma , a malignant tumor of epithelial origin, represents 10% ENT of malignant tumor in china. surgery operation and radiotherapy are often adopted for the Laryngeal carcinoma in early stage.In the cases of middle and advanced stage ,surgery is used as the predominant therapy of comprehensive treatment.Since 1980s the treatment of the laryngeal carcinoma has made rapid progress. Operation,radiotherapy,chemotherapy and the application of the immunization therapy have greatly raised the survival rate of laryngeal carcinoma patients by five years.However,those therapies can cause high rate of disability and complications as well as many serious toxic and side effects.Recurrence and metastasis are still the major death cause of laryngeal carcinoma.In recent years, the tumor foundation research has obtained significantly progression and gene therapy displays good perspective to the patients with tumors. RNA interference(RNAi)is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double stranded RNA.It has rapidly developed into one of the most widely applied technologies with therapeutic potential in molecular and cellular research.The chemokines are soluble, small molecular weight (8?14 kDa) proteins, Chemokine receptors belong to the G protein-coupled receptor (GPCR) superfamily,Chemokines bingding with chemokine receptors can cause many biological events such as embryogenesis, wound healing, angiogenesis, leukocyte homeostasis and lymphoid organ development, inflammatory diseases, pro and anti-tumor responses.More than 50 different chemokines and 20 different chemo-kine receptors have been cloned. Chemokines usually bind to multiple receptors, and the same receptor may bind to more than one chemokine. However, there is one exception to this rule: the SDF-1, which binds exclusively to CXCR4 , has CXCR4 as its only receptor. This fact alone suggests that the SDF-1/CXCR4 axis plays a uniquely important biological role. Many studies disclosed that the biological axis of SDF-1/CXCR4 play important roles in proliferation,apoptosis ,adhesion ,angiogenesis,invasion and metastasis in tumer.So, from the gene level,employing the RNA interference technique to silence CXCR4 gene to block the biological axis of SDF-1/CXCR4 is recent study focus .We employed the RNA interference technique to silence gene expression of CXCR4 in laryngeal carcinoma cell line Hep-2 to study the effect of the proliferation apoptosis adhesion and invasion in vitro .To Study invasion and metastasis molecular mechanisms of laryngeal carcinoma. We suggest that CXCR4 is a novel target for gene therapy of laryngeal carcinoma.PART ONEExpression and significance of CXCR4 in human laryngeal carcinomaObjective: To observe the expression and significance of CXCR4 in human laryngeal carcinoma tissues .Methods: Immuohistochemistry was used to detect the expressions of CXCR4 in 65 samples of human laryngeal carcinoma. The relationship CXCR4 and the clinicpathological diameters was statistically analyzed.Results:①CXCR4 were positively expressed in laryngeal carcinoma tissues while they were negative in the control group. Their expressions were significantly higher than those of control group( P < 0. 01);②The expression of CXCR4 in laryngeal carcinoma declined with the rise of the different grade of laryngeal carcinoma( P < 0. 01),The expression of CXCR4 were significantly higher in the group with lymph node metastasis than those without lymph node metastasis( P < 0. 05).Conclusion:Chemokine Receptor CXCR4 are highly expressed in laryngeal carcinoma,and their expression were associated with differentiation grade of laryngeal carcinoma and neck lymph node metastasis.PART TWOThe construction of eukaryotic expression Plasmid of shRNAmir for CXCR4Objective: To construct the shRNAmir eukaryotic expression Plasmid specific for human CXCR4 gene and to observe its silencing effects on CXCR4 gene.Methods: Getting the number NM-003467 of CXCR4 in the database of GenBank to design single-stranded DNA oligos.using Invitrogen's RNAi Designer, an online tool of Co. Invitrogen to design miR RNAi. Electing three sites 262, 713 and 961 because of high scores. Annealing was operated in the single-stranded oligos to generate a ds oligo. Cloning this ds oligo into pcDNA?6.2-GW/EmGFPmiR with T4 DNA Ligase ,Restriction Enzyme Digestion and sequencing were used to evaluate the recombinant,The expression Plasmids of (pCXCR4miR/GFP-262,pCXCR4miR/GFP-713and pCXCR4miR/GFP-961) and the control plasmid (pcDNA?1.2/V5-GW/lac),the negative control plasmid (EmGFPpcDNA6.2 ?GW/EmGFP ) were transfected by Lipofectimine 2000 into the Hep-2 cells, The expression of CXCR4 in transfected Hep-2 cells was evaluated by immuohistochemistry,Fluorescence microscopy and RT-PCR.Results:Compared with the control groups,Protein and mRNA expression were significantly decreased in Hep-2 cells transfected with expression Plasmid, pCXCR4miR/GFP- 713 expression Plasmid had the most eficient inhibitory efect, followed by pCXCR4miR/ GFP-262 and pCXCR4miR/GFP-961.Conclusion: The constructed CXCR4 shRNAmir Eukaryotic expression Plasmid can block the expression of CXCR4 in laryngeal carcinoma cells, and it can be used to study the effects of CXCR4 on the invasion, metastasis of laryngeal carcinoma cells.PART THREEThe effects of silencing CXCR4 gene by RNA interference on the proliferation,apoptosis, adhesion and invasion in transfected Hep-2 cells.Objective: To study the effects of expression Plasmid pCXCR4miR/GFP-713 on the proliferation,apoptosis, adhesion and invasion in transfected Hep-2 cells.Methods: By using MTT assay ,the proliferation capability of Hep-2 cells was tested.Apoptosis rate was evaluated by flow cytometric analysis.The abilities of invasion and adhesion were detected by Transwell chamber assay and cell ashedion assay.RT-PCR was used to detect mRNA of VEGF ,E-CD gene.Results:Compared with blank control group and Negative control group,Cell proliferation curve revealed that cell growth was significantly inhibited in experimental group (P<0.01) .FCM demonstrated that experimental group enhanced apoptosis of cells Compared with blank control group Negative control group, (P<0.01).The inhibitory rate on adhesion in experimental group was 27.7% at 30 min,28.9% at 60 min, 25.6% at 90 min and 31.1% at 120min, Compared with blank control group Negative control group, (P<0.01),The inhibition of invasion was observed by fewer penetrating cells by Transwell chamber assay(72±4.535. 69±6.437 and 30±4.119,respectively). (P<0.01) .RT-PCR revealed that The VEGF and E-CD mRNA expression were 0.752±0.008, 0.815±0.010 in experimental group respectively, compared with blank control group and Negative control group (P<0.05).Conclusion: By transfecting with the expression Plasmid pCXCR4miR/GFP-713,the cell proliferation, invasion ability of human Laryngeal carcinoma Hep-2 cells can be inhibited obviously,apoptosis was enhanced , the expression of VEGF mRNA can be down-expressed , cell-matrix adhesion can be enhanced but cell-cell adhesion be reduced significantly.