Role Of Pyruvate Kinase M2 In Human Thyroid Cancer Cell Growth

ObjectivesThis study aims to investigate the expression and fuction of PKM2 in thyroid cancer for the first time in the world. We used siRNA to knockdown endogenous PKM2 in thyroid cell lines 8505C, in order to explore the changes in human thyroid cancer cells proliferation.Materials and Methods1. SubjectsAnaplastic thyroid cancer cell line 8505C, papillary thyroid cancer cell lines IHH-4 and BPCAP, medullary thyroid cancer cell line TT, follicular thyroid cancer cell line ML-1 and FTC-133, normal thyroid cell line Nthy-ori 3-1.2. AgentsRabbit Anti-Human PKM2 Polyclonal Antibody, Goat Anti-Rabbit Antibody(FITC), real-time PCR Assay, immunohistochemical S-P Kit, BCA kit, pPKM2-U6-siRNA/Neo plasmid and negative control plasmid, Fugene HD transfective assay, DMSO, MTT, PI.3. MethodsWestern blot, Immunofluorescence and real-time PCR were used to analyze the expression of PKM2 in the thyroid cancer cell lines and the normal thyroid cell line. Building pPKM2-U6-siRNA/Neo and control plasmid, we transfected the plasmid in anaplastic thyroid cancer cell line 8505C. We found that the amount of PKM2 reduced. MTT method was used to explore the changes in the ability of proliferation in thyroid cancer cells.4. Statistical analysisAll statistical analysis was carried out using SPSS 17.0. Experiments were conducted in triplicates. The t-test was used to determine the differences of two groups of relative value. Measurement data of relative value of real-time PCR were expressed as mean±SD. Probability values less than 0.05 were considered significant.Results1. In real-time PCR test, PKM2 mRNA was overexpressed in thyroid cancer cells, whereas the amount of PKM2 was very low in normal thyroid cells. The amount of PKM2mRNA relative value in thyroid cancer cells is about 4.12-7.32 folds higher than normal thyroid cells.2. In Western blot test, PKM2 protein was detected in all thyroid cancer cell lines, but there was none in the normal thyroid cell line.3. In Immunofluorescence test, PKM2 was diffused located in the cell plasm of anaplastic thyroid cancer cell line 8505C, papillary thyroid cancer cell line BPCAP, medullary thyroid cancer cell line TT and follicular thyroid cancer cell line FTC-133. However, there was none in the normal thyroid cell line.4. The efficacy of transfection after 24h and 48h were81.1% and 100%, respectively. The efficacy of inhibiting growth after 48hours is 50.6%.Conclusion1. PKM2 was specific overexpressed in thyroid cancer cells, whereas there was almost little expression in normal thyroid cells. The amount of PKM2 expression is dependent on the pathological character.2. Using siRNA to knockdown PKM2 expression in thyroid cancer cell lines 8505C, we found that the cancer cell line 8505C growth was suppressed. These results demonstrated that PKM2 plays an important role in Warburg effect. PKM2 can be a new useful target for gene therapy in anaplastic thyroid cancer and other cancer overexpressing PKM2 in future.