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. 2018 Jan 9;8(1):204.
doi: 10.1038/s41598-017-18509-1.

Chemokine receptor 7 overexpression promotes mesenchymal stem cell migration and proliferation via secreting Chemokine ligand 12

Ling Liu  1 , Jian-Xiao Chen  1 , Xi-Wen Zhang  1 , Qin Sun  1 , Lan Yang  1 , Airan Liu  1 , Shuling Hu  1 , Fengmei Guo  1 , Songqiao Liu  1 , Yingzi Huang  1 , Yi Yang  1 , Hai-Bo Qiu  2
Affiliations

Chemokine receptor 7 overexpression promotes mesenchymal stem cell migration and proliferation via secreting Chemokine ligand 12

Ling Liu et al. Sci Rep. .

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Abstract

Great interest has been shown in mesenchymal stem cell (MSC) therapy in a wide variety of clinical domains. However, the therapeutic efficiency depends on the proliferation and migration of MSCs. Chemokine receptors are involved in regulating the proliferation and migration to the specific organs of MSCs in different microenvironments. CXC receptor seven (CXCR7), a newly discovered Chemokine ligand 12 (CXCL12) receptor, has organ specificity for tumour migration. We hypothesized that CXCR7 expression affects proliferation and migration of MSCs. In present study, we constructed long-term and stable mMSCs lines overexpressing and suppressing CXCR7 modifications with lentiviral vectors. The transduction efficiencies, mRNA and protein expression of CXCR7 were significantly regulated. CXCR7 gene overexpression promoted mMSCs proliferation and migration, whereas suppressing CXCR7 had the opposite effect. Additional CXCL12 improved the vertical migration of mMSCs. The overexpression of CXCR7 increased the MSC-secreted CXCL12, VCAM-1, CD44 and MMP2 levels, which contributed to the improvement of mMSC proliferation and migration. Therefore, overexpressing CXCR7 improved the proliferation and migration of mMSCs, which may be attributable to the CXCL12 secreted by MSCs, leading to a positive feedback loop for CXCL12/CXCR7 axis. Our results may provide a potential method for improving the treatment effectiveness of mMSCs by overexpressing CXCR7.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Long-term transgene expression efficiency in mMSCs after lentiviral vector transduction. (a) The mMSCs were transduced separately with pHBLV-CMVIE-CXCR7-ZsGreen-T2A-Puromycin (MSC-OE-CXCR7), pHBLV-CMVIE-ZsGreen-T2A-Puromycin (MSC-OENC-CXCR7), pHBLV-U6-ShRNA-ZsGreen-Puromycin (MSC-Sh-CXCR7) and pHBLV-U6-ZsGreen-Puromycin (MSC-ShNC-CXCR7) lentiviral vectors were cultured for 20 passages and observed with light microscopy (top) and fluorescence microscopy with green fluorescent protein (middle), 100× and 250×; the percentage of ZsGreen-positive cells were analysed by flow cytometry (bottom) at passage 20 after transduction. (b) Quantitative real-time PCR analysis shows CXCR7 mRNA expression in mMSCs after pHBLV-CMVIE-CXCR7-ZsGreen-T2A-Puromycin and pHBLV-U6-ShRNA-ZsGreen-Puromycin transduction. (n = 4; *p < 0.05 vs. MSC-OENC-CXCR7, # p < 0.05 vs. MSC-Sh-CXCR7). (c,d) FCM analysis shows CXCR7 overexpression in mMSCs after transduction. (n = 3; *p < 0.05 vs. MSC-OENC-CXCR7). (e,f) FCM analysis shows CXCR7 suppression in mMSCs after transduction. (n = 3; # p < 0.05 vs. MSC-ShNC-CXCR7).
Figure 2
Figure 2
The effect of CXCR7 on mMSC proliferation. Cell growth curves after transduction for 7 days were evaluated by the CCK-8 assay. (a) The proliferation rate of the MSC-OE-CXCR7 group was significantly higher than the MSC-OENC-CXCR7 group from days 3 to 7. (n = 8; *p < 0.001 vs. MSC-OENC-CXCR7). (b) The proliferation rates of the MSC-Sh-CXCR7 groups were significantly lower than the MSC-ShNC-CXCR7 group from days 5 to 7. (n = 8; # p < 0.05 vs. MSC-ShNC-CXCR7).
Figure 3
Figure 3
The effect of CXCR7 on the horizontal migration of mMSCs. The ability of mMSCs to horizontally migrate after transduction was examined using the scratch assay. The wound sites (area cleared of cells in the centre of the scratched area) were observed and photographed at 0, 12 h and 18 h, 100×. A bar graph shows the quantitative results of scratch healing. (n = 3; *p < 0.05 vs. MSC-OENC-CXCR7, # p < 0.05 vs. MSC-ShNC-CXCR7).
Figure 4
Figure 4
The effect of CXCR7 on the vertical migration of mMSCs. Cells that migrated to the lower surface of Transwell inserts were stained with crystal violet and observed under a microscope at 12 h (a) and 18 h (b), 200×. A bar graph shows the quantitative results of cell migration. (n = 3; *p < 0.05 vs. MSC-OENC-CXCR7, Δ p < 0.05 vs. MSC-OE-CXCR7, # p < 0.05 vs. MSC-ShNC-CXCR7, & p < 0.05 vs. MSC-Sh-CXCR7).
Figure 5
Figure 5
The concentration of CXCL12 in the supernatant of transwell chambers was examined using ELISA ruling out the interference of exogenous CXCL12 protein. (n = 3; *p < 0.001 vs. MSC-OENC-CXCR7, Δ p < 0.05 vs. MSC-OE-CXCR7, # p < 0.05 vs. MSC-ShNC-CXCR7, & p < 0.05 vs. MSC-Sh-CXCR7).
Figure 6
Figure 6
The expression of VCAM-1, CD44 and MMP2 was examined using ELISA. (n = 3; *p < 0.05 vs. MSC-OENC-CXCR7, Δ p < 0.001 vs. MSC-OE-CXCR7, # p < 0.05 vs. MSC-ShNC-CXCR7, & p < 0.05 vs. MSC-Sh-CXCR7).
Figure 7
Figure 7
The concentration of Collagen-I in the supernatant of transwell chambers was measured using ELISA. (n = 3).
Figure 8
Figure 8
The concentration of TNF-α and IL-10 in the supernatant of transwell chambers was measured using ELISA. (n = 3).
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