Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

Abstract

Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative (RT)PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions.

Keywords: Cell cycle; Cell proliferation; FABP3; Human mesenchymal stem cell; Hypoxia.

Figure 1

FABP3 expression pattern in MSCs. (A, a) FABP3 mRNA expression pattern in hypoxic human MSCs: Y axis: relative gene expression level, and X axis: cell group (n=12), * stands for p value, H vs. N: * p<0.05>0.01 and ** p=0.002; the circle symbolizes each individual sample, the red solid diamonds and the bar figure; (b) normal human MSC in cell cultures and (c) FABP3 overexpressed hMSCs in cell culture, the blue start represented a corresponding cell and RNA in Fig. A, a–c. (B) FABP3 protein expression and localization: the HIF1A (red, e, f) and FABP3 proteins (green, g, h) appeared in hypoxic MSC nucleus only, nucleus and cytoplasm, respectively; but HIF1A and FABP3 were all absent in the normoxic control cells (a, b, c, d). (C), Figure 1C FABP3 expression associated with oxygen condition. (D) No Relative regulation of FABP3 in no MSCs (Aorta endothelial cells, and human cancer cells).

Figure 1

FABP3 expression pattern in MSCs. (A, a) FABP3 mRNA expression pattern in hypoxic human MSCs: Y axis: relative gene expression level, and X axis: cell group (n=12), * stands for p value, H vs. N: * p<0.05>0.01 and ** p=0.002; the circle symbolizes each individual sample, the red solid diamonds and the bar figure; (b) normal human MSC in cell cultures and (c) FABP3 overexpressed hMSCs in cell culture, the blue start represented a corresponding cell and RNA in Fig. A, a–c. (B) FABP3 protein expression and localization: the HIF1A (red, e, f) and FABP3 proteins (green, g, h) appeared in hypoxic MSC nucleus only, nucleus and cytoplasm, respectively; but HIF1A and FABP3 were all absent in the normoxic control cells (a, b, c, d). (C), Figure 1C FABP3 expression associated with oxygen condition. (D) No Relative regulation of FABP3 in no MSCs (Aorta endothelial cells, and human cancer cells).

Figure 1

FABP3 expression pattern in MSCs. (A, a) FABP3 mRNA expression pattern in hypoxic human MSCs: Y axis: relative gene expression level, and X axis: cell group (n=12), * stands for p value, H vs. N: * p<0.05>0.01 and ** p=0.002; the circle symbolizes each individual sample, the red solid diamonds and the bar figure; (b) normal human MSC in cell cultures and (c) FABP3 overexpressed hMSCs in cell culture, the blue start represented a corresponding cell and RNA in Fig. A, a–c. (B) FABP3 protein expression and localization: the HIF1A (red, e, f) and FABP3 proteins (green, g, h) appeared in hypoxic MSC nucleus only, nucleus and cytoplasm, respectively; but HIF1A and FABP3 were all absent in the normoxic control cells (a, b, c, d). (C), Figure 1C FABP3 expression associated with oxygen condition. (D) No Relative regulation of FABP3 in no MSCs (Aorta endothelial cells, and human cancer cells).

Figure 2

(A) Evidence of FABP3 overexpression in MSC^FABP3LV(rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC^FABP3LV, but not in vector control MSCs (red, a) and (C) MSC^FABP3LV proteins were around the cellular nucleus under normoxic condition (green), but distributed to the cellular nucleus and cytoplasm under hypoxic condition (green).

Figure 2

(A) Evidence of FABP3 overexpression in MSC^FABP3LV(rtPCR). (B) Immunocytochemical images of FABP3 protein (green, b) present in MSC^FABP3LV, but not in vector control MSCs (red, a) and (C) MSC^FABP3LV proteins were around the cellular nucleus under normoxic condition (green), but distributed to the cellular nucleus and cytoplasm under hypoxic condition (green).

Figure 3

Overexpressing FABP3 inhibited cell growth and proliferation. (A) Cell growth image. (B) Western Blot result and (C), a summary of 7-days MSC^FABP3LV growth curve of normoxia and hypoxia. The cell growth curve indicates the cell proliferation rate. ** = p<0.01 and ^xx= f< 0.01.

Figure 3

Overexpressing FABP3 inhibited cell growth and proliferation. (A) Cell growth image. (B) Western Blot result and (C), a summary of 7-days MSC^FABP3LV growth curve of normoxia and hypoxia. The cell growth curve indicates the cell proliferation rate. ** = p<0.01 and ^xx= f< 0.01.

Figure 3

Overexpressing FABP3 inhibited cell growth and proliferation. (A) Cell growth image. (B) Western Blot result and (C), a summary of 7-days MSC^FABP3LV growth curve of normoxia and hypoxia. The cell growth curve indicates the cell proliferation rate. ** = p<0.01 and ^xx= f< 0.01.

Figure 4

(A & B) A summered ^rtPCR and q^rtPCR results * indicates the gene regulation was confirmed by both ^rtPCR and q^rtPCR. APC, JAG1, HSPA9 and FGF4 were down- or up-regulated in MSC^FABP3LV comparing to vector control cells, respectively. (C) Illustration of the FABP3 induced genes employed in the cell cycle pathway (KEGG database). The red and blue symbols represent a relative up- or down regulation of MSC^FABP3LV and MSC^CLV, respectively.

Figure 4

(A & B) A summered ^rtPCR and q^rtPCR results * indicates the gene regulation was confirmed by both ^rtPCR and q^rtPCR. APC, JAG1, HSPA9 and FGF4 were down- or up-regulated in MSC^FABP3LV comparing to vector control cells, respectively. (C) Illustration of the FABP3 induced genes employed in the cell cycle pathway (KEGG database). The red and blue symbols represent a relative up- or down regulation of MSC^FABP3LV and MSC^CLV, respectively.

Figure 4

(A & B) A summered ^rtPCR and q^rtPCR results * indicates the gene regulation was confirmed by both ^rtPCR and q^rtPCR. APC, JAG1, HSPA9 and FGF4 were down- or up-regulated in MSC^FABP3LV comparing to vector control cells, respectively. (C) Illustration of the FABP3 induced genes employed in the cell cycle pathway (KEGG database). The red and blue symbols represent a relative up- or down regulation of MSC^FABP3LV and MSC^CLV, respectively.