The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.
Background Continual expression of PD-L1 in tumor cells is critical for tumor immune escape and host T cell exhaustion, however, knowledge on its clinical benefits through inhibition is limited in breast cancer. N6-methyladenosine (m6A) plays a crucial role in multiple biological activities. Our study aimed to investigate the regulatory role of the m6A modification in PD-L1 expression and immune surveillance in breast cancer. Methods MeRIP-seq and epitranscriptomic microarray identified that PD-L1 is the downstream target of METTL3. MeRIP-qPCR, absolute quantification of m6A modification assay, and RIP-qPCR were used to examine the molecular mechanism underlying METTL3/m6A/IGF2BP3 signaling axis in PD-L1 expression. B-NDG and BALB/c mice were used to construct xenograft tumor models to verify the phenotypes upon METTL3 and IGF2BP3 silencing. In addition, breast cancer tissue microarray was used to analyze the correlation between PD-L1 and METTL3 or IGF2BP3 expression. Results We identified that PD-L1 was a downstream target of METTL3-mediated m6A modification in breast cancer cells. METTL3 knockdown significantly abolished m6A modification and reduced stabilization of PD-L1 mRNA. Additionally, METTL3-mediated PD-L1 mRNA activation was m6A-IGF2BP3-dependent. Moreover, inhibition of METTL3 or IGF2BP3 enhanced anti-tumor immunity through PD-L1-mediated T cell activation, exhaustion, and infiltration both in vitro and in vivo. PD-L1 expression was also positively correlated with METTL3 and IGF2BP3 expression in breast cancer tissues. Conclusion Our study suggested that METTL3 could post-transcriptionally upregulate PD-L1 expression in an m6A-IGF2BP3-dependent manner to further promote stabilization of PD-L1 mRNA, which may have important implications for new and efficient therapeutic strategies in the tumor immunotherapy.
Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of interferon-alfa-1b (IFN-␣-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-␣ at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 ؋ 10 6 U/kg/day, 1.5 ؋ 10 7 U/kg/day for treated groups, and 3 ؋ 10 7 U/kg/day; tumor volume was 8,475 mm 3 ؎ 2,636 mm 3 , 7,963 mm 3 ؎ 3,214 mm 3 , 769 mm 3 ؎ 287 mm 3 , and 13 mm 3 ؎ 9 mm 3 ; incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 ؎ 4 days, 53 ؎ 8 days, 81 ؎ 6 days, and 105 ؎ 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 ؋ 10 5 U/kg/day, 1 ؋ 10 6 U/kg/day, 4 ؋ 10 6 U/kg/day, 7.5 ؋ 10 6 U/kg/day, 1.5 ؋ 10 7 U/kg/day, and 3 ؋ 10 7 U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-␣ inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. Hepatocellular carcinoma (HCC) is one of the most frequent fatal malignancies occurring in Asia and Africa. 1 In China, it is the second major cause of cancer death in males and the third in females. 2,3 Surgical resection is the most important treatment. Unfortunately, the postoperative recurrence rate remains high. 4,5 HCC is generally not sensitive to chemotherapy. HCC is a typical hypervascular tumor. Intrahepatic and lung metastases suggest its hematogenous dissemination. Recently, angiogenesis was proved to be an important factor influencing HCC metastases and recurrence. 6 Therefore, agents that inhibit angiogenesis maybe provide a new approach to suppress the tumor growth and recurrence of HCC. In addition to the well-documented, antiproliferative, and immunomodulatory effects of interferon (IFN), the antiangiogenic properties of IFN have been proven. [7][8][9][10][11] The systemic administration of IFN-␣ decreased the expression of angiogenic factors, such as bFGF, 12 inhibited the angiogenesis, and resulted in regression of human tumors. 13,14 This study evaluates whether IFN-␣-1b can suppress tumor growth, prolong animal life-span, and inhibit recurrence after resection by inhibition of angiogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.