GFI1 protein and mRNA levels are upregulated upon p53 knockdown.

<p>(A) MO7e and Molt3 cells were infected with the lentivirus containing a p53 shRNA. The expression of GFI1 and p53 proteins was examined by Western blot analysis. GFI1 mRNA levels were examined by RT-PCR. Subsequently, control (Ctr) and p53 knocked down (KD) MO7e (B) and Molt3 (D) cells were treated with Doxo (25 ng/ml) for times as indicated and examined for expression of p53 and GFI1. The results shown in B and D were quantitated using ImageJ and normalized based on the levels of β-actin protein and GAPDH mRNA for MO7e (C) and Molt3 (E) cells. (F) Human umbilical cord blood CD34+ cells were treated with Doxo prior to evaluation of the expression of p53 and GFI1.</p

Mapping of the <i>GFI1</i> promoter region required for p53-mediated repression.

<p>(A and B) Schematic diagrams of <i>GFI1</i> promoter fragments cloned into pGL3-basic vector (A) or inserted upstream of the <i>SV40</i> promoter of the pGL3-promoter vector. (C and D) p53^−/− HCT116 cells were transfected with the indicated <i>GFI1</i> promoter luciferase reporter constructs without or with p53. Luciferase activities were measured 36 hrs after transfection.</p

Repression of <i>GFI1</i> by p53 is independent of HDACs and p21^Cip1.

<p>(A) <i>p53</i>^−/− HCT116 cells were transfected with <i>GFI1</i> promoter (−1933/+468 bp) luciferase reporter construct without or with p53 and treated with TSA (0.15 µM) 8 hrs later. Luciferase activities were measured 24 hrs after TSA treatment. (B) <i>p21^Cip1</i>^+/+ and <i>p21^Cip1</i>^−/− HCT116 cells were transfected with <i>GFI1</i> promoter (−1933/+468 bp) reporter construct and treated with Doxo (400 ng/ml) 8 hrs later. Luciferase activities were measured 16 hrs after Doxo treatment.</p

Knockdown of GFI1 increases cell death in response to DNA damage.

<p>U937 (A–C) and HL-60 (D–F) cells were transduced with empty lentivirus (Ctr) or lentiviruses containing two different shRNAs against <i>GFI1</i> and examined for GFI1 expression by Western blot analysis (A and D). Cells were left untreated or treated with Doxo at 200 ng/ml for 10 hrs prior to evaluation of living cell numbers by trypan blue exclusion (B and E) and MTS assays (C and F).</p

Identification of the repressive p53 RE in <i>GFI1</i> core promoter.

<p>(A) Nucleotide sequences of wild type (WT) and mutated <i>GFI1</i> core promoter fragments as compared with conserved repressive p53 RE. Potential p53 RE in <i>GFI1</i> core promoter are underlined. Mutated nucleotides are in bold. (B) <i>p53</i>^−/− HCT116 cells were transfected with WT or M1 <i>GFI1</i> promoter (−1933/+468 bp; left panel), or WT or M2 <i>GFI1</i> promoter (−460/+6 bp; right panel) luciferase reporter construct without or with p53. Luciferase activity was measured 36 hrs after transfection. (C) <i>p53</i>^−/− HCT116 cells were transfected with pGL3-basic plasmid containing WT or M1 <i>GFI1</i> promoter fragment (−4840/+184 bp) along with p53. Binding of p53 to the <i>GFI1</i> promoter fragments was examined by ChIP assays (left panel). Expression of p53 was confirmed by Western blot analysis (right panels).</p

p53 binds to and represses <i>GFI1</i>.

<p>(A) <i>p53</i>^−/− HCT116 cells were transfected with the <i>GFI1</i> promoter (−1933/+468 bp) luciferase reporter construct along with the wild type (WT) or W248 mutant p53. Luciferase activities were measured 36 hrs later and normalized for β-Gal activities. Data are shown as mean ± SD. (B) <i>p53</i>^+/+ and <i>p53</i>^−/− HCT116 cells were transfected with <i>GFI1</i> promoter luciferase reporter construct and treated with Doxo (400 ng/ml) 8 hrs later. Luciferase activities were measured 16 hrs after Doxo treatment. (C) <i>p53</i>^−/− HCT116 cells were transfected with pGL3-basic plasmid containing <i>GFI1</i> promoter fragment (−4840/+184 bp) either alone or together with p53. ChIP assays were carried out using the anti human p53 or an irrelevant species-matched antibody. The indicated regions of <i>GFI1</i> promoter were amplified by PCR. (D) ChIP experiment was carried out on Molt3 cells using the anti human p53 and control antibodies.</p

Gfi1 has no significant effect on the expression of Bax, Bak and Bcl-2.

<p>(A) Ba/F3 and Ramos cells transduced with the inducible Gfi1-expressing lentiviral construct were preincubated with Doxy (1 µg/ml) for 24 hrs and then treated with Doxo (100 ng/ml) for times as indicated. The expression of Gfi1 and the Bcl-2 family members as indicated was examined by Western blot analysis. (B) The levels of the Bcl-2 family members in control and GFI1 knocked down U937 and HL-60 cells were examined by Western blot analysis.</p

Overexpression of Gfi1 inhibits DNA damage-induced cell death.

<p>Ba/F3 (A–C) and Ramos (D–F) cells were transduced with the inducible lentiviral expression construct for Gfi1 and examined for Gfi1 expression by Western blot analysis after incubation with Doxy (1 µg/ml) for 24 hrs (A and D). Cells were then exposed to Doxo (100 ng/ml for Ba/F3 cells and 2 mg/ml for Ramos cells) for 18 hrs with or without preincubation with Doxy (1 µg/ml) for 24 hrs. Cell viabilities were determined by exclusion of trypan blue staining (B and E), percentages of apoptotic (annexin V-positive) cells were assessed by flow cytometry after staining with annexin V and 7-AAD (C), and the numbers of living cells were quantitated by MTS assay (F).</p

Engineering Translational Activators with CRISPR-Cas System

RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility

Table_2_Hepatitis C virus infection is associated with high risk of breast cancer: a pooled analysis of 68,014 participants.doc

IntroductionBreast cancer is the most common malignancy among women. Previous studies had shown that hepatitis C virus (HCV) infection might serve as a risk factor for breast cancer, while some studies failed to find such an association.MethodsIn this study, we presented a first attempt to capture and clarify this clinical debate via a cumulative analysis (registration ID: CRD42023445888). ResultsAfter systematically searching and excluding the irrelevant publications, five case-control or cohort studies were finally included. The synthetic effect from the eligible studies showed that patients with HCV infection had a significantly higher prevalence of breast cancer than non-HCV infected general population (combined HR= 1.382, 95%CI: 1.129 to 1.692, P=0.002). There was no evidence of statistical heterogeneity during this pooled analysis (I2 = 13.2%, P=0.33). The sensitivity analyses confirmed the above findings. No significant publication bias was observed among the included studies. The underlying pathophysiological mechanisms for this relationship might be associated with persistent infection/inflammation, host immune response, and the modulation of HCV-associated gene expression. DiscussionThough the causal association between HCV infection and breast cancer did not seem quite as strong, screening for HCV might enable the early detection of breast cancer and help to prevent the progression of the disease. Since the topic of this study remains a matter of clinical debate, further studies are still warranted to validate this potential association.Systematic review registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023445888</p