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. 2019 Nov 28;105(12):2813–2823. doi: 10.3324/haematol.2019.227579

Figure 2.

Figure 2.

CD47 is a direct target of miR-155 in multiple myeloma (MM) cells. (A) Correlation analysis of endogenous miR-155 with CD47 expression in patient dataset (GSE17498, n=38 MM patients) presented as scatter plots. Linear regression with Pearson’s correlation coefficients (r) and P-value were presented in the graph. (B) Box whisker plot showing miR-155 expression in GSE17498 dataset. Normal donor (ND): n=3, stage I (Durie-Salmon): n=9, stage II (Durie-Salmon): n=9 and stage III (Durie-Salmon): n=16. (C) The total RNA including miRNA was isolated from MM resistant and sensitive cell lines and the level of miR-155 was assessed by qualitative polymerase chain reaction (qPCR). The values were normalized to SNORD72. (D) The 8226-R5 and MM.1R cells were transfected with 20 mM of either miR-155 mimics or scramble control using Hiperfect transfection reagent for 48 hours (h), and the level of miR-155 was assessed by qPCR. (E) The 8226-R5 and MM.1R cells were transfected with 20 mM of either miR-155 mimics or scramble control using Hiperfect transfection reagent for 48 h, and the whole cell lysate was subjected to Western blot with indicated antibodies. (F) The miR-155 binding site in the CD47 3′-UTR. Putative conserved target sites in the CD47 3′-UTR were identified using the TargetScan algorithm. Matched nucleic acid–base pairs were linked as “-” (G) The 3’ UTR sequence of human CD47 was cloned into the luciferaseexpressing vector pEZX-MT01 to the downstream of the firefly luciferase gene. For construction a mutant 3’UTR reporter clone, the miR-155 binding site in the 3’UTR of CD47 was inactivated by several mutations. The cells were transiently co-transfected with reporter plasmids (pEZX-MT-Control, pEZX-wt-CD47-3′UTR or pEZX-mut- CD47-3′UTR) and miR-155 mimics or control miRNA. Cells were harvested 48 h after transfection and luciferase activities were analyzed as the relative activity of firefly to Renilla. Readings from the empty plasmid (pEZX-MT-control) were used for normalization. *P<0.05, **P<0.01, ***P<0.001 were considered as significant at 95% confidence interval. NS: not significant.