RNA interference targeting CD147 inhibits the proliferation, invasiveness, and metastatic activity of thyroid carcinoma cells by down-regulating glycolysis

Abstract

A high rate of glycolytic flux, even in the presence of oxygen, is a key metabolic hallmark of cancer cells. Lactate, the end product of glycolysis, decreases the extracellular pH and contributes to the proliferation, invasiveness and metastasis of tumor cells. CD147 play a crucial role in tumorigenicity, invasion and metastasis; and CD147 also interacts strongly and specifically with monocarboxylate transporter1 (MCT1) that mediates the transport of lactate. The objective of this study was to determine whether CD147 is involved, via its association with MCT1 to transport lactate, in glycolysis, contributing to the progression of thyroid carcinoma. The expression levels of CD147 in surgical specimens of normal thyroid, nodular goiter (NG), well-differentiated thyroid carcinoma (WDTC), and undifferentiated thyroid carcinoma (UDTC) were determined using immunohistochemical techniques. The effects of CD147 silencing on cell proliferation, invasiveness, metastasis, co-localization with MCT1, glycolysis rate and extracellular pH of thyroid cancer cells (WRO and FRO cell lines) were measured after CD147 was knocked-down using siRNA targeting CD147. Immunohistochemical analysis of thyroid carcinoma (TC) tissues revealed significant increases in signal for CD147 compared with normal tissue or NG, while UDTC expressed remarkably higher levels of CD147 compared with WDTC. Furthermore, silencing of CD147 in TC cells clearly abrogated the expression of MCT1 and its co-localization with CD147 and dramatically decreased both the glycolysis rate and extracellular pH. Thus, cell proliferation, invasiveness, and metastasis were all significantly decreased by siRNA. These results demonstrate in vitro that the expression of CD147 correlates with the degree of dedifferentiation of thyroid cancer, and show that CD147 interacts with MCT1 to regulate tumor cell glycolysis, resulting in the progression of thyroid carcinoma.

Keywords: CD147; RNA interference; Thyroid cancer-experimental; genetics; glycolysis; pathology-thyroid.

Figure 1

Immunohistochemical staining demonstrating the correlations between CD147 expression and the pathological grading of TC tissues. A: CD147 expressed at various levels in normal thyroid tissues (a, ×200), NG (b, ×200), WDTC (c, papillary thyroid carcinoma (PTC), ×400; d, FTC, ×400) and UDTC (e, MTC, ×200; f, ATC, ×200). Staining is weak or absent in normal tissues and in NG specimens, whereas CD147 is strongly expressed in WDTC and UDTC samples. B: Comparison of the positive rate of CD147 expression. Normal thyroid tissues and NG compared with TC, *P < 0.005. C: Comparison of the positive rate of CD147 expression between WDTC and UDTC samples, *P < 0.005.

Figure 2

siRNA-mediated silencing of CD147 expression. A: Western blotting analysis of the CD147 (i) and MCT1 (ii) protein level after the transient transfection of WRO and FRO cells with pSUPER⁄CD147 siRNA (si-CD147). Expression of CD147 and MCT1 were both significantly decreased in siWRO and siFRO samples. B: Immunofluorescence confocal microscopy. WRO or FRO cells were co-labeled with anti-CD147 and anti-MCT1 antibodies. Co-localization of MCT1 with CD147 can be observed in the plasma membrane of WRO and FRO cells. The co-localized expression of CD147 and MCT1 was abrogated in siWRO and siFRO cells.

Figure 3

Effects of CD147 in the regulation of glycolysis. A: After CD147 silencing, extracellular pH was higher in siWRO or siFRO cells than in wild-type cells. *P < 0.005 in the comparison between siWRO or siFRO and the respective wild-type cells. B: The extracellular lactate concentration was significantly decreased in siWRO or siFRO cells compared with the respective wild-type cells after CD147 silencing. Compared to wild-type cells, in siWRO and siFRO it was markedly reduced to 68.7% and 41.5% respectively, *P < 0.005.

Figure 4

Knockdown of CD147 inhibits WRO and FRO cell proliferation. Effect of siRNA transfection of CD147 on WRO or FRO cell proliferation measured by CCK8 assay, *P < 0.005 in comparison between siWRO or siFRO and their respective wild-type cells.

Figure 5

Effects of specific siRNA transfection of CD147 on Matrigel™ invasion and metastasis. A: RNA interference targeting CD147 inhibits the invasiveness of TC cells: (a) WRO, (×400); (b) siWRO, (×400); (c) FRO, (×400); (d) siFRO, (×400). B: siRNA transfection of CD147 inhibits the metastatic activity of TC cells: (e) WRO, (×200); (f) siWRO, (×200); (g) FRO, (×200); (h) siFRO, (×200). C, D: Number of cells migrated or invaded was evaluated in three fields for each experimental group and the mean ± SD is shown. *P < 0.005 in comparison between siWRO or siFRO and their respective wild-type cells.