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. 2018 Sep 12;9:2071. doi: 10.3389/fimmu.2018.02071

Figure 1.

Figure 1

Suppression of breast cancer cell metastasis and shrimp virus infection by shrimp mja-miR-35. (A) The upregulated and downregulated miRNAs in shrimp in response to WSSV infection. (B) The prediction of miRNAs targeting human CHI3L1 gene. As predicted, the CHI3L1 3′UTR was targeted by mja-miR-35 from 38 upregulated miRNAs. The seed sequence of mja-miR-35 was underlined. (C) Effects of mja-miR-35 on CHI3L1 expression in M2 macrophages. M2 macrophages were transfected with the synthesized mja-miR-35 or mja-miR-35-scrambled. At 24 h after transfection, the CHI3L1 transcript level was determined using quantitative real-time PCR. The secreted CHI3L1 protein level of M2 macrophages was examined using Western blotting. GAPDH and β-actin served as loading controls. (D) The effects of mja-miR-35 expression in M2 macrophages on breast cancer cell migration. M2 macrophages were transfected with the synthesized mja-miR-35 and co-cultured with breast cancer cells MDA-MB-231. As a control, mja-miR-35-scrambled was used. A linear wound was created in the confluent monolayer of MDA-MB-231 cells. At 24 h after co-culture, the cancer cells were examined using microscopy. (E) Expression of mja-miR-35 in shrimp in response to WSSV infection. At different time points post-infection, mja-miR-35 was detected in hemocytes of WSSV-infected shrimp by Northern blotting. U6 was used as a loading control. (F) The influence of mja-miR-35 on WSSV infection. The synthesized mja-miR-35 was injected into WSSV-infected shrimp. At different time post-infection, the shrimp were subjected to quantitative real-time PCR to quantify the WSSV copies. As controls, WSSV alone and mja-miR-35-scrambled were included in the injections. (G) The effects of mja-miR-35 on the mortality of WSSV-infected shrimp. The shrimp were simultaneously injected with WSSV and mja-miR-35. WSSV alone and mja-miR-35-scrambled were included in the injections as controls. At different time after injection, the cumulative mortality of shrimp was monitored. Data presented were representatives of triplicate assays (*p < 0.05; **p < 0.01).