| BackgroundMalignant melanoma (MM) is one of the most life-threatening tumors and is characterized by high invasiveness, frequent metastasis, and poor prognosis, as well as disordered energy metabolism to support rapid growth and high proliferation, including disordered oxidative phosphorylation (OXPHOS) process. OXPHOS, which is largely carried out in mitochondrial complexes, increases in metastatic MM.CD147is a high glycosylated transmembrane protein mainly expressed on cell surface, which is abundant in many metabolic active cells, including cancer cells. CD147acts as a chaperone to associate with varies of proteins, to influence different cellular functions. In the previous studies, CD147is closely related to the growth, invasiveness, angiogenesis and metastasis of MM. Knocking down CD147with siRNA could down-regulate glycolysis and inhibit the malignant behaviors of melanoma. The aim of this study was to determine that whether a potential binding partner of CD147exists in mitochondria and whether the interaction has an effect on mitochondrial function of malignant melanoma, to further influence the OXPHOS and participate in cancer energy metabolism.Objective1. To find out the location of CD147in primary and metastatic melanoma specimens.2. To discover the expression of mitochondrial CD147in human malignant melanoma cells (A375cell line), and investigate the potential binding partner of CD147in mitochondria. 3. To investigate the effects of CD147interacting with NDUFS6in regulating Complex Ⅰ activity of A375cells.4. To investigate the effects of CD147in regulating the cell viability and apoptosis through the mitochondrial-dependent pathway in A375cells.Methods1. The expression of CD147in10primary MM and10metastatic MM specimens was detected by immunohistochemistry, and the difference of its location was analyzed.2. Mitochondrial and cytoplasmic fractions of A375cells were isolated, western blot was used to determine the expression of CD147in each fraction.3. The location of CD147on A375cells mitochondria was detected by immonofluorescence.4. The yeast two hybrid screening was used to find out the potential candidates interacting with CD147in mitochondria. NDUFS6was a positive target. Then the CD147-Myc and Flag-NDUFS6plasmids were built and co-transfected into HEK293T cells. The interaction was confirmed using immunoprecipitation and immunofluorescence.5. CD147with different domain(s) deleted plasmids were built, that is, delete Ig domain Ⅰ (34-89), delete Ig domain Ⅱ (105-199), transmembrane domain (207-230), cytoplasmic domain (231-end), and both transmembrane and cytoplasmic domains (207-end), also with the Myc-tagged. The specific binding sites with NDUFS6were detected by immunoprecipitation and immunofluorescence.6. A375cells were transfected with normal control siRNA and CD147siRNAs. The effects of siRNA was detected by western blot. The A375cell with normal control siRNA was named A375-siNC, while CD147siRNA named A375-siCD147or A375-siBSG.7. The Complex Ⅰ acitvity of A375-siNC/siCD147was tested by dipstick assay kit.8. Berberine was used as a tool drug to induce mitochondria damage. Alamar Blue assay helped to detemine the viability of A375-siNC and A375-siCD147cells.9. A375-siNC and A375-siCD147cells were treated with berberine, cells apoptosis was tested and assessed by the expression of cleaved-PARP by western blot.10. Exogenous Bcl-2was added to A375-siNC and A375-siCD147cells, and treated with berberine. Cells apoptosis was tested as described above.Results1. CD147expressed on the membrane of all specimens, showed in cytoplasm in2primary MM and8metastatic MM.2. CD147was highly expressed in A375cells mitochondria, and co-localized with ATP5B on mitochondria.3. NDUFS6was confirmed to be a potential interacting protein with CD147by yeast two hybrid screening.4. The interaction between NDUFS6and CD147was detected by co-immunoprecipitation, and NDUFS6was co-localized with CD147in A375cells in immunofluorescence experiment.5. Flag-antibody was used to immunoprecipitate CD147-Myc with different domains deleted. The band disappeared when we deleted the Ig domain I and transmembrane domain of CD147, indicating these2domains were the binding sites of the association. This result was in accordance with the following immunofluorescence experiment.6. The2targets of CD147siRNA transfection decreased the activity of Complex I in A375cells by25.1%and27.8%seperately (P<0.05).7. CD147siRNA transfection significantly decreased the A375cells viability when they were treated with berberine (P<0.01).8. CD147siRNA increased the apoptosis of A375cells induced by berberine. The exogenous anti-apoptotic protein Bcl-2was introduced to both A375cells, protected them from apoptosis induced by berberine successfully.Conclusions1.CD147tended to translocate to cytoplasm in metastatic melanoma;2.CD147exists in the mitochondria of A375cells, interacts with NDUFS6. And CD147siRNA decreased Complex Ⅰ activity, probably regulating OXPHOS of MM through this interaction.3. CD147protects A375cells viability and apoptosis probably through the mitochondrial pathway. |
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