Study On CD147via IGFBP2Regulation Apoptosis Of Malignant Melanoma In Vitro And In Vivo

Background Malignant melanoma(MM), one of the most malignant cutaneous tumors, is characterized by its high potential for invasiveness and metastasis. And apoptosis of the tumor is the key to the inhibition of tumor growth, reversal of tumor drug resistance, and treatment of tumor. Studies have discovered that the human cell-surface molecule CD147, an integral plasma membrane glycoprotein belonging to the Ig superfamily, is highly expressed on the surface of various tumor cells. It stimulates the production of multiple matrix metalloproteinases (MMPs) by tumor cells and fibroblasts, serving as a key regulator of tumor cell invasiveness and metastasis. In addition, there is growing evidence that CD147may regulate the apoptosis of tumour cells, but the mechanism is still unknown. In our study, We successfully established a stable CD147shRNA A375cell line to explore the mechanism.Methods and Results1) Using stable RNA interference, we succeeded in establishing two CD147knock-down A375cell lines (A375-sh-CD147-C1and A375-sh-CD147-C2).Compared to A375-sh-ctrl cells, CD147expression was down-regulated by0.86±0.08,0.30±0.06(P<0.01) in A375-sh-CD147-C1and A375-sh-CD147-C2, respectively.We chose the better inhibitory effect, A375-sh-CD147-C1, for the following experiment, and renamed it for A375-sh-CD147.2)The apoptosis:Western Blot detected A375-sh-CD147cells expressed apoptosis related protein, cle-PARP and PTEN at significantly higher levels than A375-sh-ctrl cells(3.12±0.22-fold,3.42±0.28-fold, respectively). Flow Cytometer detected that the average rate of apoptosis in A375-sh-CD147were also higher than the control cells (4.23±0.25- fold).Electron microscopic observation comfirmed the morphology changes of apoptosis related to CD147expression.(P<0.01)3) Human Apoptosis Antibody Array tested the total protein of these two cell lines, and found9apoptosis related protein, such as IGFBP2(Insulin-like Growth Factor Binding Protein2) etc.4) Western Blot and Real Time-PCR confirmed that CD147could increase of IGFBP2at protein and mRNA level.(P<0.01)5) Correlation between CD147and IGFBP2:Immunoprecipitation demonstrated that there was no directly correlation with the two proteins. Further study, we found that the signal pathway protein, p-Akt and p-mTOR, were significantly higher lower in A375-sh-CD147cells than the control cells. Meanwhile, the pathway inhibitor could reduced the expression of IGFBP2(3.69±0.74-fold, P<0.01).6) Animal models:We used immunodeficient nude mice to determine the effect of CD147knock-down on the apoptosis potential of A375cells. Immunohistochemistry detected that the expression of CD147and IGFBP2in A375-sh-CD147mouse models, were all significantly lower than A375-sh-ctrl mouse models. And TUNEL found the apoptosis rate were in accordance with the expression of CD147. Cle-PARP protein expression also demonstrated the result (P<0.01)7)Malignant melanoma tissue microarray also demonstrate there is positive correlation between CD147and IGFBP2, and these proteins expression increased from primary MM to metastatic MM.(P<0.05)ConclusionWe propose that CD147could up-regulated the expression of IGFBP2through Akt/mTOR signal pathway, and together mediate the apoptosis of MM. Our results also suggest that CD147may represent a novel therapeutic target to treat MM clinically.