MicroRNA-383 Regulates the Apoptosis of Tumor Cells through Targeting Gadd45g

<div><p><i>Background</i></p><p>MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNA molecules that inhibit gene expression at post-transcriptional level. Gadd45g (growth arrest and DNA-damage-inducible 45 gamma) is a stress-response protein, which has been implicated in several biological processes, including DNA repair, the cell cycle and cell differentiation.</p><p><i>Results</i></p><p>In this work, we found that miR-383 is a negative regulator of Gadd45g. Forced expression of miR-383 decreased the expression of Gadd45g through binding to the 3′ untranslated region (3′-UTR), whereas inhibition of miR-383 increased Gadd45g expression. The presence of miR-383 increased the cellular sensitivity to DNA damage in breast cancer cells, which was rescued by ectopic expression of Gadd45g without the 3′-UTR. miR-383 also regulates the expression of Gadd45g in embryonic stem (ES) cells, but not their apoptosis under genotoxic stress. miR-383 was further showed to negatively regulate ES cell differentiation via targeting Gadd45g, which subsequently modulates the pluripotency-associated genes. Taken together, our study demonstrates that miR-383 is a negative regulator of Gadd45g in both tumor cells and ES cells, however, has distinct function in regulating cell apoptosis. miR-383 may be used as antineoplastic agents in cancer chemotherapy.</p><p><i>Conclusion</i></p><p>We demonstrate for the first time that miR-383 can specifically regulates the expression of Gadd45g by directly targeting to the 3-UTR region of Gadd45g mRNA, a regulatory process conserved in human tumor cells and mouse embryonic stem cells. These two compotents can be potentially used as antineoplastic agents in cancer chemotherapy.</p></div

miR-383 modulates ES cell differentiation through Gadd45g.

<p>(A and B) Quantitative RT-PCR analysis for differentiation (A) and pluripotency (B) marker genes in miR-383 mimic or control transfected ES cells cultured with LIF or with RA for 3 days. The data are shown as relative expression compared with the control cells cultured in the presence of LIF (set as 1.0). Values are means ± SD. (C) Protein levels of Gadd45g were detected in R1 cells transfected with Gadd45g siRNAs. (D) Quantitative RT-PCR was performed to analyze the expression of Nestin, Nanog, Sox2, Dppa4, Gdf3, and Isl1 between control and Gadd45g siRNA transfected ES cells cultured with LIF or RA. The mRNA levels at control siRNA transfected cells were set at 1.0. Values are means ± SD. (E and F) The protein levels of Isl1, Dppa4 and Gdf3 were examined by western blotting after miR-383 overexpression (E)or Gadd45g siRNA (F) in the conditions of RA treatment.</p

miR-383 represses Gadd45g expression by directly targeting Gadd45g 3′-UTR.

<p>(A) Schematic representation of miR-383 binding site on the Gadd45g 3′-UTR. Shaded texts indicate the conserved sequences among human, mouse, rat, rhesus monkey and horse. (B) Gadd45g 3′-UTR sequence containing the predicted target sites was inserted into the pMIR reporter vector, immediately downstream the luciferase gene. The mutant reporter construct was generated by introducing four-mismatch mutation. (C) Relative luciferase activities of Gadd45g 3′-UTR reporter or mutated Gadd45g 3′-UTR reporter in MCF-7 cells with or without miR-383 mimic. Firefly luciferase reading was normalized to that of the Renilla luciferase. Values are means ± SD. (D) MCF-7 cells were co-transfected with the Gadd45g 3′-UTR reporter construct, and anti-miR-383 or anti-control, supplemented by pRL vector, and luciferase activities were analyzed after 48 h. Values are means ± SD. (E) The effect of miR-383 mimic or anti-miR-383 on Gad4d45g protein levels. Protein expression of Gadd45g was determined by western blotting in MCF-7 and MDA-MB-231 cells at 48 h after transfection. β-actin was used as a loading control. (F) Relative Gadd45g mRNA expression was measured by qRT-PCR in MCF-7 and MDA-MB-231 cells transfected with miR-383 mimic or control. Levels were normalized to GAPDH expression. Values are means ± SD.</p

The expression of miR-383 and Gadd45g is negatively correlated post UV irradiation.

<p>(A) The mRNA level of Gadd45g was measured by qRT-PCR post UV irradiation (60 J/m²) at indicated time in MCF-7 cells, and normalized to those of GAPDH (top). The mRNA level at 0 h was set as 1.0. The protein level of Gadd45g was measured by western blotting, and β-actin was used as a loading control (bottom). (B) Relative Gadd45g mRNA expression was measured by qRT-PCR 12 h post UV irradiation at different doses (top). The data at 0 J/m² was set as 1.0. Western blotting was used to analyze Gadd45g protein levels. β-actin was used as a loading control (bottom). (C and D) The expression of miR-383 was measured by qRT-PCR post UV irradiation (60 J/m²) at indicated time (C) or different doses (D) in MCF-7 cells, and normalized to the level of U6. The data at 0 h or 0 J/m² was set as 1.0. Values are means ± SD. (E and F) A significant negative correlation was found between miR-383 and Gadd45g expression under UV irradiation post different time or at different doses.</p

Additional file 1: of Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood

Figure S1. FACS staining of live/dead cells. A Representative images of FACS staining of live/dead cells of PB MNCs by four PB MNC isolation methods at day 0 or after 8 days. B Representative images of FACS staining of live/dead cells of PB MNCs at indicated time points. PB MNCs isolated with Ficoll method. (PPTX 99 kb

Additional file 2: of Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood

Figure S2. Differentiated PB iPSC clones did not express pluripotency markers OCT4, NANOG, TRA-1-60, and SSEA4. Representative images captured using Leica confocal microscope. (PPTX 292 kb

Additional file 3: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S1. showing sequencing results of parental and inserted iPSCs. a Parental iPSCs have the known F9 gene mutation c.676C > T, p.Arg226Trp. b Inserted iPSCs (colony 5) have a heterozygous mutation of c.676C > T, p.Arg226Trp. (DOCX 393 kb

Additional file 7: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S4. showing characterization of hepatocytic functions. Differentiated cells had functions of glycogen storage (a) and ICG uptake (b), and also expressed LDL-receptor (c) and had ability for LDL uptake (d). All scale bars represent 100 Îźm. (DOCX 1747 kb

Additional file 5: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S2 showing characterization of iPSC colony 5. a Karyotype of iPSC colony 5 was normal. b qRT-PCR analysis showed expression of OCT4, SOX2, and NANOG of iPSC colony 5. PBMNCs of patient used as negative control, H1 embryonic stem cells used as positive control. c Immunofluorescence staining showed expression of TRA-1-60, SSEA4, OCT4, and NANOG. d Sections of teratomas stained with H&E (endoderm: pancreas; mesoderm: muscle; ectoderm: nerve fibers). All scale bars represent 100 Îźm. (DOCX 1261 kb

Additional file 6: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S3. showing off-target effects detection in successful inserted iPSCs. Using Cas-OFFinder, 1799 potential off-target sites that differed from the sgRNA sequence by up to five nucleotides in the genome were found. We found 97,968 indels, 3084 SVs, 51,628 SNPs, and 2225 CNVs unique to the inserted iPSCs compared to that in the parental iPSCs. Since indels and SVs comprise virtually all of the mutations introduced by CRISPR-Cas9, we focused solely on indels and SVs. Through comparison of potential off-target sites, and indels and SVs unique to the inserted iPSCs, we found no overlapping mutation between them. (DOCX 302 kb