The Crosstalk Between HIF-1 And TLRs/NF-κB Pathways In The Microenvironment Of Oral Squamous Cell Carcinoma

Neoplastic tumors are one of the leading causes of human death. OSCC have an incidence of more than 300,000 new cases worldwide each year. Despite advances in surgery, radiotherapy and chemotherapy, five-year survival rate has remained unchanged in recent years(50% to 60% with good treatment). To develop new treatment strategies, many efforts have been attempted to understand the microenvironment during tumor development, as tumor microenvironment provides conditions for growth and progression of malignant cells. In solid tumor like OSCC, hypoxia and inflammation are two key features of tumor microenvironment during tumor process.Hypoxia is the result of insufficient blood supply to support proliferating tumor cells. Tumor hypoxia is associated with tumor progression and poor clinical prognosis, which is known to induce genes involved in the regulation of cell proliferation as we reported before.Another aspect of tumor microenvironment that has a significant role in neoplastic progression is inflammation. NF-κB is a key transcription factor during inflammation response, which is thought to be a critical link between inflammation and cancer. NF-κB activation usually results in up-regulation of anti-apoptotic genes thereby providing cell survival mechanism. Thus suppression of NF-κB should have therapeutic potential. However in solid tumor, targeting NF-κB couldn’t get a well treatment result. This may be caused by tumor microenvironment. Once inflammation signaling pathway is inhibited, there existed other pathway stimulating the expression of NF-κB. Recently, hypoxia in solid tumor microenvironment has been proved as an important factor of activating NF-κB. However, the mechanism of how hypoxia regulating NF-κB is still ambiguous.The study reveal that in OSCC HIF-1 and TLRs/NF-κB have a positive regulation loop which further connects hypoxia and inflammation in tumor together. This finding could help us better understand the relationship between hypoxia and inflammation in tumor microenvironment and provide us novel method for treatment of OSCC.Part OneExpression of HIF-1,TLR3,TLR4 in OSCC and their relationship with clinic pathological status and prognosis of OSCC patientsObjective To study the expression of HIF-1,TLR3,TLR4 in OSCC patients and their relationship with clinic pathological status and prognosis.Methods 90 cases of OSCC patients were collected from Nanjing Stomatology hospital. Immunohistochemistry was performed to observe the expression of HIF-1,TLR3,TLR4.Results were evaluated and clinical data was analyzed by x2.Kaplan-Meier survival curves were plotted and log-rank was performed to study the relationship between expression of HIF-1,TLRs and prognosis of OSCC patients.Results HIF-1,TLR3,TLR4 expression in OSCC is associated with patient survival and histological grade. Part TwoEffect of HIF-1 expression on proliferation, migration and apoptosis of OSCC under hypoxia microenvironmentObjective To detect the effect of hypoxia microenvironment in OSCC cell lines and roles of HIF-1.Methods After construction of recombined HIF-1α shRNA lentivirus and establishment of stabilized transfected oral squamous cell carcinoma,we used CCK-8 assay, Flow Cytometry assay, Cell migration assay to detect the effect of hypoxia on OSCC cell lines.Results Hypoxia could help increase the expression of HIF-1α in OSCC.HIF-1 could help OSCC cell lines survival, proliferation, migration and avoid apoptosis in hypoxia environment.Part ThreeEffect of TLRs/NF-κB pathway on expression of HIF-1 in OSCC microenvironmentObjective To study whether HIF-1 could be up-regulated through TLRs/NF-κB pathway in oral squamous cell carcinoma.Methods Expression of TLR3 and TLR4 in OSCC cell lines was detected.TLR3 and TLR4 pathway were activated through LPS and Poly Ⅰ:C. Then we proved if TLR3 and TLR4 increasing HIF-1 through NF-κB.HIF-1 report plasmid was constructed to confirm this result. Wild type of promoter sequence in HIF-1 was cloned into pGL-3.Cell transfected with this report system were treated by LPS or Poly Ⅰ:C.We then measured the expression of luciferase.Results TLR3 and TLR4 were expressed in OSCC.TLR3 and TLR4 could increase the expression of HIF-1 through activating NF-κB in OSCC. Part Four Effect of HIF-1 Pathway on Expression of TLRs/NF-κB in Oscc Microenvironment Objective to Study the Expression Changes of TLRs/NF-κB in Hypoxia and Confirm the Role of HIF-1Methods We Detected the Expression of TLRs/NF-κB in Hypoxia and Role of HIF-1. ChIp Assay Was Used to Check Which Site Is the Functional Binding Site for HIF-1 in Promoter Sequence of TLR3 and TLR4.Results Expression of TLRs/NF-κB Was Highly Increased by HIR-1 in Hypoxia Condition. HIF-1 Could Bind to the Sequence in Promoter Region of TLR3 and TLR4.Part FiveEffect of Combination Inhibition of HIF-1 and NF-κB on Oral Squamous Cell Carcinoma Proliferation in VivoObjective to Study the Combination Inhibition of HIF-1 and NF-κB in Oral Squamous Cell Carcinoma Proliferation in VivoMethods 40 Nude Mice Were Separated into Four Groups:Control Group,Inhibition of HIF-1,inhibition of NF-κB and Combination Inhibition of HIF-1 andNF-κB. After Treatment, Tumor Tissue Was Collected. the Weights of Tumor Tissue WereMeasured. Further IHC Experiments Were Operated and Detected by Several Markers:HIF-1α,TLR3,TLR4,VEGF, Ki67 and NF-κB.Results Combinated Inhibition of HIF-1 and NF-κB Could Help Increase the Treatment Outcome of Oscc,Which Could Also Reduce the Expression Level of Several Makers.