Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.

RNA activation (RNAa) is an uncharacterized mechanism of transcriptional activation mediated by small RNAs, such as microRNAs (miRNAs). A critical issue in RNAa research is that it is difficult to distinguish between changes in gene expression caused indirectly by post-transcriptional regulation and direct induction of gene expression by RNAa. Therefore, in this study, we seek to identify a key factor involved in RNAa, using the induction of ZMYND10 by miR-34a as a system to evaluate RNAa. We identify the positive transcription elongation factors CDK9 and DDX21, which form a complex with nuclear AGO and TNRC6A, as important transcriptional activators of RNAa. In addition, we find that inhibition of DDX21 suppresses RNAa by miR-34a and other miRNAs without inhibiting post-transcriptional regulation. Our findings reveal a strong connection between RNAa and release of paused Pol II, facilitating RNAa research by making it possible to separately analyze post-transcriptional regulation and RNAa.

Expression of L-type amino acid transporter 1 is a poor prognostic factor for Non-Hodgkin's lymphoma.

L-type neutral amino acid transporter 1 (LAT1) is a heterodimeric membrane transport protein involved in neutral amino acid transport. LAT1 is highly expressed in various malignant solid tumors and plays an essential role in cell proliferation. However, its role in malignant lymphoma remains unknown. Here, we evaluated LAT1 expression level in tissues from 138 patients with Non-Hodgkin lymphoma (NHL). Overexpression of LAT1 was confirmed in all types of NHL and we found that there is a significant correlation between the level of LAT1 expression and lymphoma grade. The LAT1 expression was higher in aggressive types of lymphomas when compared with static types of lymphomas, suggesting that active tumor proliferation requires nutrient uptake via LAT1. The expression level of LAT1 was inversely correlated with patients' survival span. Furthermore, pharmacological inhibition of LAT1 by a specific inhibitor JPH203 inhibits lymphoma cell growth. In conclusion, our study demonstrated that LAT1 expression can be used as a prognostic marker for patients with NHL and targeting LAT1 by JPH203 can be a novel therapeutic modality for NHL.

WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7.

Since cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.

rAAV6-mediated miR-29b delivery suppresses renal fibrosis.

BACKGROUND: Previous studies showed that microRNA-29b (miR-29b) inhibits renal fibrosis. Therefore, miR-29b replacement therapy represents a promising approach for treating renal fibrosis. However, an efficient method of kidney-targeted miRNA delivery has yet to be established. Recombinant adeno-associated virus (rAAV) vectors have great potential for clinical application. For kidney-targeted gene delivery, the most suitable AAV serotype has yet to be established. Here, we identified the most suitable AAV serotype for kidney-targeted gene delivery and determined that AAV-mediated miR-29b delivery can suppress renal fibrosis in vivo. METHOD: To determine which AAV serotype is suitable for kidney cells, GFP-positive cells were identified by flow cytometry after the infection of rAAV serotype 1-9 vectors containing the EGFP gene. Next, we injected rAAV vectors into the renal pelvis to determine transduction efficiency in vivo. GFP expression was measured seven days after injecting rAAV serotype 1-9 vectors carrying the EGFP gene. Finally, we investigated whether rAAV6-mediated miR-29b delivery can suppress renal fibrosis in UUO mouse model. RESULTS: We found that rAAV6 vector is the most suitable for targeting kidney cells regardless of animal species in vitro and rAAV6 is the most suitable vector for kidney-targeted in vivo gene delivery in mice. Intra-renal pelvic injection of rAAV vectors can transduce genes into kidney TECs. Furthermore, rAAV6-mediated miR-29b delivery attenuated renal fibrosis in UUO model by suppressing Snail1 expression. CONCLUSION: Our study has revealed that rAAV6 is the most suitable serotype for kidney-targeted gene delivery and rAAV6-mediated miR-29b delivery into kidney TECs can suppress established renal fibrosis.

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer.

Triple negative breast cancer (TNBC) is responsible for significant number of breast cancer-associated deaths because of lacking of successful molecular-targeted therapy. To explore a therapeutic target for TNBC, we performed a siRNA-mediated knockdown screening and identified Polo-like kinase 1 (PLK1) as a potential therapeutic target for TNBC. Knockdown of PLK1 as well as a small compound inhibitor for PLK1, BI-2536, induced G2/M arrest and created polyploid cell population, shown by increased DNA content and nuclear size. Inhibition of PLK1 eventually triggered apoptosis in multiple TNBC cell lines. In addition, we confirmed that PLK1 was significantly overexpressed in the tissues from TNBC patients compared with the tissues of normal mammary glands and benign breast tumors. Our data indicated that PLK1 plays a pivotal role in the regulation of mitosis of TNBC cells. Although future in vivo studies are warranted, targeting PLK1 by a selective inhibitor such as BI-2536 can be an attractive molecular-targeted therapy for TNBC.

Development of Novel Small Hairpin RNAs That do not Require Processing by Dicer or AGO2.

The innate cytokine response to nucleic acid is the most challenging problem confronting the practical use of nucleic acid medicine. The degree of stimulation of the innate cytokine response strongly depends on the length of the nucleic acid. In this study, we developed a 30-nucleotide single-strand RNA, termed "guide hairpin RNA (ghRNA, ghR)", that has a physiological function similar to that of miRNA and siRNA. The ghR caused no innate cytokine response either in vitro or in vivo. In addition, its structure does not contain a passenger strand seed sequence, reducing the unwanted gene repression relative to existing short RNA reagents. Systemic and local injection of ghR-form miR-34a (ghR-34a) suppressed tumor growth in a mouse model of RAS-induced lung cancer. Furthermore, Dicer and AGO2 are not required for ghR-34a function. This novel RNA interference (RNAi) technology may provide a novel, safe, and effective nucleic acid drug platform that will increase the clinical usefulness of nucleic acid therapy.