Luciferase Reporter Research Articles

LINC01133 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells by upregulating CTNNB1 by acting as a sponge for miR-214-3p

BackgroundOsteoporosis results from decreased bone mass and disturbed bone structure. Human bone marrow mesenchymal stem cells (hBMSCs) demonstrate robust osteogenic differentiation, a critical process for bone formation. This research was designed to examine the functions of LINC01133 in osteogenic differentiation.MethodsDifferentially expressed lncRNAs affecting osteogenic differentiation in hBMSCs were identified from the GEO database. A total of 74 osteoporosis patients and 70 controls were enrolled. hBMSCs were stimulated to undergo osteogenic differentiation using an osteogenic differentiation medium (OM). RT-qPCR was performed to evaluate LINC01133 levels and osteogenesis-related genes such as osteocalcin, osteopontin, and RUNX2. An alkaline phosphates (ALP) activity assay was conducted to assess osteogenic differentiation. Cell apoptosis was detected using flow cytometry. Dual luciferase reporter assay and RIP assay were employed to investigate the association between miR-214-3p and LINC01133 or CTNNB1. Loss or gain of function assays were conducted to elucidate the impact of LINC01133 and miR-214-3p on osteogenic differentiation of hBMSCs.ResultsLINC01133 and CTNNB1 expression decreased in osteoporotic patients but increased in OM-cultured hBMSCs, whereas miR-214-3p showed an opposite trend. Depletion of LINC01133 suppressed the expression of genes associated with bone formation and ALP activity triggered by OM in hBMSCs, leading to increased cell apoptosis. Nevertheless, this suppression was partially counteracted by the reduced miR-214-3p levels. Mechanistically, LINC01133 and CTNNB1 were identified as direct targets of miR-214-3p.ConclusionsOur study highlights the role of LINC01133 in positively regulating CTNNB1 expression by inhibiting miR-214-3p, thereby promoting osteogenic differentiation of BMSCs. These findings may provide valuable insights into bone regeneration in osteoporosis.

Identification of Tumor Suppressive miR-144-5p Targets: FAM111B Expression Accelerates the Malignant Phenotypes of Lung Adenocarcinoma.

Accumulating evidence suggests that the passenger strands microRNAs (miRNAs) derived from pre-miRNAs are closely involved in cancer pathogenesis. Analysis of our miRNA expression signature of lung adenocarcinoma (LUAD) and The Cancer Genome Atlas (TCGA) data revealed that miR-144-5p (the passenger strand derived from pre-miR-144) was significantly downregulated in LUAD tissues. The aim of this study was to identify therapeutic target molecules controlled by miR-144-5p in LUAD cells. Ectopic expression assays demonstrated that miR-144-5p attenuated LUAD cell aggressiveness, e.g., inhibited cell proliferation, migration and invasion abilities, and induced cell cycle arrest and apoptotic cells. A total of 18 genes were identified as putative cancer-promoting genes controlled by miR-144-5p in LUAD cells based on our in silico analysis. We focused on a family with sequence similarity 111 member B (FAM111B) and investigated its cancer-promoting functions in LUAD cells. Luciferase reporter assay showed that expression of FAM111B was directly regulated by miR-144-5p in LUAD cells. FAM111B knockdown assays showed that LUAD cells significantly suppressed malignant phenotypes, e.g., inhibited cell proliferation, migration and invasion abilities, and induced cell cycle arrest and apoptotic cells. Furthermore, we investigated the FAM111B-mediated molecular networks in LUAD cells. Identifying target genes regulated by passenger strands of miRNAs may aid in the discovery of diagnostic markers and therapeutic targets for LUAD.

Abstract C055: Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element

Abstract Genome-wide association studies (GWAS) have identified independent signals at 5p15.33 across numerous cancers, with protective alleles for one cancer often conferring risk for another. Many of these associations are thought to act via allele-specific alterations in the cis- regulation of target genes. Transcriptomic analyses in multiple tissue types implicate two plausible target genes: TERT and CLPTM1L. However, the mechanisms linking cancer susceptibility alleles to their presumed target genes, along with the observed antagonistic pleiotropy between different cancers remain elusive. With this in mind, we employed a comprehensive approach, integrating statistical fine-mapping of GWAS summary statistics with massively parallel reporter assays (MPRA) and a densely tiled CRISPRi screen performed across eight cell lines, representative of four distinct cancers associated with 5p15.33 alleles – pancreatic, melanoma, lung, and bladder cancer. Integrative screening identified up to 30 variants across multiple signals in the locus with putative allele-specific transcriptional activity. Furthermore, a variable number tandem repeat (VNTR) in intron 9 of CLPTM1L emerged as a potent transcriptional enhancer in CRISPRi screening. Leveraging PacBio sequencing, VNTR alleles of European ancestry samples from the 1000 Genomes Project were genotyped and found to be highly polymorphic, with longer VNTRs linked to the haplotype tagged by rs31490-A (risk increasing for pancreatic cancer). Subsequent imputation into a pancreatic cancer GWAS of 9,040 cases and 12,496 controls revealed an association between longer VNTRs and risk of pancreatic cancer (OR for VNTR in the 75th percentile = 1.17, 95% CI = 1.13-1.22, P = 2.21x10-12). The significance of the association for the lead SNP rs31490 after conditioning on the VNTR genotype dropped to P= 2.20x10-5, suggesting that VNTR alleles merit consideration as part of the credible variant set for pancreatic cancer. Targeted CRISPRi assays suggested VNTR-mediated cis-regulation of both TERT and CLPTM1L in PANC-1 and MIA PaCa-2 pancreatic cancer cell lines, while dual luciferase reporter assays in the same cell lines revealed that VNTR enhancer activity is mediated by Hippo pathway transcription factors. In ongoing experiments, we aim to expand on luciferase reporter assays and DNA-protein binding assays in relevant cell lines with the aim of not only deciphering how longer VNTRs differentially exert their transcriptional effects compared to shorter alleles but also characterizing the divergent directions of effect between different cancer types. These findings underscore the proposition that pancreatic cancer susceptibility at the 5p15.33 locus may be mediated by both SNPs and an uncharted class of variants, yet to be characterized in the post-GWAS landscape. Citation Format: Aidan O'Brien, Hyunkyung Kong, Minal Patel, Katelyn E Connelly, Mai Xu, Irene Collins, Jun Zhong, Jason Hoskins, Michelle Ho, Brenen Papenburg, Mark M Iles, Matthew H Law, Maria-Teresa Landi, Rachael Stolzenber- Solomon, Brian M Wolpin, Alison P Klein, Nick Orr, Stephen J Chanock, Sara Lindström, Marc-Henri Stern, Ludmila Prokunina-Olsson, Jieyon Choi, Kevin M Brown, Laufey T Amundadottir. Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C055.

LncRNA HAND2-AS1 Inhibited Colon Cancer Progression By Regulating miR-3118/ZG16 Axis.

LncRNA HAND2-AS1 is a novel cancer regulator, but the role and mechanisms of HAND2-AS1 involved with colon cancer (CC) progression remains unknown. The purpose of this research was to figure out how HAND2-AS1 regulates the progression of CC. Using qRT-PCR, we studied expression levels of miR-3118, HAND2-AS1, and ZG16 in CC tissues and cells. Protein levels of apoptosis-related proteins (Bax and Bcl-2) and ZG16 were quantified by western blotting. In vitro function analysis referred to western blotting, wound healing assay and CCK-8. The binding association among miR-3118, HAND2-AS1, and ZG16 was investigated using luciferase reporter and RIP assays. The functional role of HAND2-AS1 was analyzed using xenograft tumor models in vivo. In tissues and cells of CC, HAND2-AS1 was downregulated. We observed that HAND2-AS1 overexpression declined CC cell proliferation and migration while facilitating apoptosis. We further verified that when HAND2-AS1 is overexpressed it reduced CC tumor development in vivo. In CC cells and tissues, miR-3118 competed with HAND2-AS1 and was elevated. Further it was noted that the HAND2-AS1 when overexpressed, lessened the survival of CC cells, however overexpression of miR-3118 restored these changes. ZG16 was shown to be a target of miR-3118, it was found that ZG16 was downregulated in CC tissue and cells. We observed, high expression of ZG16 partially restored the enhanced malignant phenotype caused by miR-3118 overexpression. HAND2-AS1 inhibited CC progression by upregulating ZG16 expression through sponging miR-3118. Hence, HAND2-AS1/miR-3118/ZG16 axis could be a possible new target for CC treatment.

The transcription factors NR5A1 and JUNB cooperate to activate the Axl promoter in mouse Sertoli cell lines.

The Axl gene is a receptor tyrosine kinase essential for male fertility. With other Tyro3 family members, it regulates cell apoptosis and preserves the organization of seminiferous tubules. However, the regulation of the expression of Axl in testicular Sertoli cells is not entirely understood. The transcription factors NR5A1 and JUNB are involved in several male fertility mechanisms such as sex development and steroidogenesis. We hypothesize that Axl promoter activity is regulated by cooperation between JUNB and NR5A1 in Sertoli cells. Following transfections of TM4 Sertoli cells with DsiRNA interference against Axl, our results show that cell morphology may be regulated by AXL. Using transfections of expression plasmids and reporter plasmids containing the Axl promoter, we report that Axl expression is highly activated by cooperation between NR5A1 and JUNB in TM4 and 15P-1 Sertoli cells. Chromatin immunoprecipitation and luciferase reporter assays with 5' promoter deletions demonstrate that JUNB and NR5A1 are being recruited to DNA regulatory elements in the proximal region of the Axl promoter. The fourth intronic region of Axl also participates in the recruitment of JUNB. Thus, Axl expression is regulated by a cooperation between the transcription factors JUNB and NR5A1 and influences the morphology of TM4 Sertoli cells.

Identification and functional analysis of a new cold induced LncRNA44154

With the development of high-throughput sequencing technology, numerous unknown long noncoding RNAs (lncRNAs) have been discovered. Preliminary research found that after being subjected to low-temperature enviroment, lncRNA44154 expression in the subcutaneous fat of pigs significantly increased. In order to analyze its biological function and regulatory mechanism, qRT-PCR was used to detect its spatiotemporal expression pattern. The nucleoplasmic isolation and FISH were used to locate it subcellular. Through overexpression or interference assays discussed the effect of lncRNA44154 on adipocyte proliferation and differentiation. RNA-seq was used to screen the biological pathways that lncRNA44154 may be involved in. RNA-pull down was used to find the interaction protein and double luciferase reporter gene detection experiment was used to validate the target gene. It was found that the expression of lncRNA44154 had tissue specificity and played a role in adipocyte proliferation. It was expressed in both cytoplasm and nucleus. After overexpression of lncRNA44154 in adipocytes, the expression level of Cyclins B, D, and E were all up-regulated and the cell proliferation ability was significantly enhanced. The number of positive cells increased significantly and the DNA replication activity was significantly enhanced. LncRNA44154 can promote the proliferation of porcine preadipocytes by encouraging cells to enter the S phase of the cell cycle. The result of interfering with lncRNA44154 is the opposite. Further analysis revealed that lncRNA44154 regulates intracellular lipid metabolism, inhibits the production of intracellular lipid droplets, and inhibits preadipocyte differentiation. RNA-seq results indicate that lncRNA44154 may be involved in biological pathways such as AMPK, PPAR and cAMP signaling pathway. It may work by regulating the RPS4X gene. This research may provide a new perspective for investigating the regulation of adipose metabolism by lncRNAs.

Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

Abnormal SUMOylation is implicated in non-alcoholic fatty liver disease (NAFLD) progression. Forkhead box protein A1 (FoxA1) has been shown to protect liver from steatosis, which was down-regulated in NAFLD. This study elucidated the role of FoxA1 deSUMOylation in NAFLD. NAFLD models were established in high-fat diet (HFD)-induced mice and palmitate acid (PAL)-treated hepatocytes. Hepatic steatosis was evaluated by biochemical and histological methods. Lipid droplet formation was determined by BODIPY and Oil red O staining. Target molecule levels were analyzed by RT-qPCR, Western blotting, and immunohistochemistry staining. SUMOylation of FoxA1 was determined by Ni-NTA pull-down assay and SUMOylation assay Ultra Kit. Protein interaction and ubiquitination were detected by Co-IP. Gene transcription was assessed by ChIP and dual luciferase reporter assays. Liver FoxA1 knockout mice developed severe liver steatosis, which could be ameliorated by sirtuin 6 (Sirt6) overexpression. Nutritional stresses reduced Sumo2/3-mediated FoxA1 SUMOylation at lysine residue K6, which promoted lipid droplet formation by repressing fatty acid β-oxidation. Moreover, Sirt6 was a target gene of FoxA1, and Sirt6 transcription activity was restrained by deSUMOylation of FoxA1 at site K6. Furthermore, nutritional stresses-induced deSUMOylation of FoxA1 promoted the ubiquitination and degradation of FoxA1 with assistance of murine double minute 2 (Mdm2). Finally, activating FoxA1 SUMOylation delayed the progression of NAFLD in mice. DeSUMOylation of FoxA1 at K6 promotes FoxA1 degradation and then inhibits Sirt6 transcription, thereby suppressing fatty acid β-oxidation and facilitating NAFLD development. Our findings suggest that FoxA1 SUMOylation activation might be a promising therapeutic strategy for NAFLD.

Discovery of vitexin as a novel VDR agonist that mitigates the transition from chronic intestinal inflammation to colorectal cancer

Colitis-associated colorectal cancer (CAC) frequently develops in patients with inflammatory bowel disease (IBD) who have been exposed to a prolonged state of chronic inflammation. The investigation of pharmacological agents and their mechanisms to prevent precancerous lesions and inhibit their progression remains a significant focus and challenge in CAC research. Previous studies have demonstrated that vitexin effectively mitigates CAC, however, its precise mechanism of action warrants further exploration. This study reveals that the absence of the Vitamin D receptor (VDR) accelerates the progression from chronic colitis to colorectal cancer. Our findings indicate that vitexin can specifically target the VDR protein, facilitating its translocation into the cell nucleus to exert transcriptional activity. Additionally, through a co-culture model of macrophages and cancer cells, we observed that vitexin promotes the polarization of macrophages towards the M1 phenotype, a process that is dependent on VDR. Furthermore, ChIP-seq analysis revealed that vitexin regulates the transcriptional activation of phenazine biosynthesis-like domain protein (PBLD) via VDR. ChIP assays and dual luciferase reporter assays were employed to identify the functional PBLD regulatory region, confirming that the VDR/PBLD pathway is critical for vitexin-mediated regulation of macrophage polarization. Finally, in a mouse model with myeloid VDR gene knockout, we found that the protective effects of vitexin were abolished in mid-stage CAC. In summary, our study establishes that vitexin targets VDR and modulates macrophage polarization through the VDR/PBLD pathway, thereby alleviating the transition from chronic colitis to colorectal cancer.

Long non-coding RNA LNC-POTEM-4 promotes HCC progression via the LNC-POTEM-4/miR-149-5p/Wnt4 signaling axis

Information on the potential role of the long non-coding RNA LNC-POTEM-4 in cancer progression is limited. Our preliminary study found that LNC-POTEM-4 was overexpressed in hepatocellular carcinoma (HCC) tissues, which led us to further investigate the biological function and molecular mechanism of LNC-POTEM-4 in HCC development. LNC-POTEM-4 expression in HCC tissues was examined using transcriptome sequencing and quantitative reverse transcription PCR. The relationships between LNC-POTEM-4 and the stage and prognosis of HCC in patient data from the TCGA database were analyzed. The effects of LNC-POTEM-4 on proliferation, invasion/migration, and epithelial-mesenchymal transition marker expression in HCC cells were evaluated in vitro using gain- and loss-of-function assays, while its effects on tumor growth and metastasis were explored through animal experiments. A LNC-POTEM-4/microRNA (miR)-149-5p/Wnt4 regulatory signaling axis was identified using bioinformatics analysis, and dual luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Co-transfection of LNC-POTEM-4 and Wnt4 expression plasmids was employed to confirm the new signaling pathway. We found that LNC-POTEM-4 was overexpressed in HCC tissues and was linked to poor staging and prognosis. LNC-POTEM-4 promoted proliferation, invasion, migration, and the epithelial-mesenchymal transition of HCC cells in vitro. Silencing of LNC-POTEM-4 inhibited HCC growth and distant metastasis in vivo. Mechanically, LNC-POTEM-4 was found to function as a competitive endogenous RNA, upregulating Wnt4 by sponging miR-149-5p to promote HCC progression. Wnt4 overexpression may have counteracted the tumor-inhibition effect of LNC-POTEM-4 silencing. In conclusion, LNC-POTEM-4 upregulated Wnt4 to activate the Wnt signaling pathway and stimulate the malignancy tendency of HCC by sponging miR-149-5p, providing a prospective target for the detection and therapy of HCC. However, the effects of LNC-POTEM-4 on the miR-149-5p/Wnt4 signaling axis should be further studied in animal experiments.

Empagliflozin Attenuates Pulmonary Arterial Remodeling Through Peroxisome Proliferator-Activated Receptor Gamma Activation.

The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.

C-Myc alone is enough to reprogram fibroblasts into functional macrophages

BackgroundMacrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases.MethodsThe composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac.ResultsHere we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models.ConclusionsOur findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

Negative regulation of CcPAL2 gene expression by the repressor transcription factor CcMYB4-12 modulates lignin and capsaicin biosynthesis in Capsicum chinense fruits

Peppers globally renowned for their distinctive spicy flavor, have attracted significant research attention, particularly in understanding spiciness regulation. While the activator MYB's role in spiciness regulation is well-established, the involvement of repressor MYB factors remains unexplored. This study identified the MYB4 transcription factor through RNA-seq and genome-wide analysis as being associated with spiciness. Consequently, CcMYB4-2 and CcMYB4-12 were cloned from Hainan Huangdenglong peppers, both exhibiting nuclear subcellular localization. qRT-PCR analysis revealed that CcMYB4-2/4-12 had high expression levels during the accumulation period of capsaicin, but there were differences in their peak expression levels, which may be related to the formation of pepper spiciness. Heterologous expression in Arabidopsis thaliana resulted in significantly elevated CcMYB4-2/4-12 expression levels and reduced lignin content. In CcMYB4-2 silenced plants, PAL expression remained unchanged, while PAL expression significantly increased in CcMYB4-12 silenced plants, leading to elevated lignin content and reduced capsaicin content. Yeast one-hybrid (Y1H) and dual luciferase reporter assays (DLR) demonstrated that CcMYB4-2/4-12 inhibited the transcription of CcPAL2 by binding to its promoter. Notably, CcMYB4-12 exhibited more pronounced inhibition. Therefore, it is hypothesized that CcMYB4-12 plays a pivotal role in regulating lignin and capsaicin biosynthesis. This study elucidates the molecular mechanism of MYB4 binding to the PAL promoter, providing a foundational understanding for analyzing phenylpropanoid metabolism and its diverse branches. Key messageThrough functional verification analysis of the repressor CcMYB4, transcriptional regulation experiments revealed that CcMYB4 can bind to the CcPAL2 promoter, negatively regulating the capsaicin biosynthesis in Capsicum chinense fruits.

The ERF transcription factor ZbERF3 promotes ethylene-induced anthocyanin biosynthesis in Zanthoxylum bungeanum

Ethylene regulates fruit ripening, and in Zanthoxylum bungeanum, fruit color deepened with increasing of ethylene during fruit ripening. However, the molecular mechanism of this physiological process was still unclear. In this study, through the combined analysis of transcriptome and metabolome, it was found that ethylene release was consistent with anthocyanin synthesis, and ethylene response factors (ERFs) were significantly related to anthocyanin biosynthesis during the fruit ripening of Z. bungeanum. Ethylene treatment significantly induced fruit coloration and promoted anthocyanin synthesis and the expression of ZbERF3. Furthermore, Yeast one-hybrid assays and Luciferase reporter assays demonstrated that ZbERF3 directly bound to the promoter of ZbMYB17 and transcriptionally activated its expression. What's more, it was demonstrated that ZbMYB17 directly bound to the promoter of ZbANS, promoting anthocyanin biosynthesis. Overall, this study revealed the mechanism of ERF and MYB synergistically regulating the coloration of Z. bungeanum fruit.

JARID2 activation by NFYA promotes stemness of triple-negative breast cancer cells through the PI3K/AKT pathway

ABSTRACT Background This study aimed to investigate the role of Jumonji AT Rich Interacting Domain 2 (JARID2) in regulating triple-negative breast cancer (TNBC) stemness and its mechanism. Research design and methods Bioinformatics analysis examined JARID2 expression, prognosis, and transcription factors. Quantitative polymerase chain reaction, western blot, and immunohistochemistry detected expression. Dual luciferase reporter gene and chromatin immunoprecipitation assays verified binding. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay detected viability and proliferation. Sphere formation assay detected the sphere formation efficiency. Flow cytometry detected CD44+/CD24− -marked stem cells. A xenograft tumor model verified the effect of JARID2 in vivo. Results JARID2 and nuclear transcription factor Y subunit α (NFYA) were upregulated in TNBC tissues and positively correlated. Knockdown of JARID2 or NFYA inhibited cell stemness by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. Enforced JARID2 expression rescued the suppressive effect of NFYA knockdown on the PI3K/AKT signaling pathway and cell stemness. Knockdown of JARID2 inhibited tumor growth and cell stemness in mice but was alleviated by concurrent overexpression of NFYA. Conclusions NFYA promotes TNBC cell stemness by upregulating JARID2 expression and regulating the PI3K/AKT signaling pathway, suggesting JARID2 as a potential target for innovating drugs that target TNBC stem cells.

Identification of zinc finger MIZ-type containing 2 as an oncoprotein enhancing NAD-dependent protein deacetylase sirtuin-1 deacetylase activity to regulate Wnt and Hippo pathways in non-small-cell lung cancer

BackgroundZinc finger MIZ-type containing 2 (ZMIZ2) can function as a coactivator and participate in the progression of certain malignant tumors; however, its expression and underlying molecular mechanism in non-small-cell lung cancer (NSCLC) remains unknown. In this study, we aim to analyze the expression of ZMIZ2 and its tumorigenic function in NSCLC, identifying its related factors.MethodsZMIZ2 expression in NSCLC tissue samples and cell lines was examined using immunohistochemistry and western blotting; its biological role was investigated using in vivo and in vitro assays. The association between ZMIZ2 and NAD-dependent protein deacetylase sirtuin-1 (SIRT1) was demonstrated using mass spectrometry and immunoprecipitation experiments. Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG)-based enrichment analysis, luciferase reporter assay, and real-time quantitative polymerase chain reaction (RT–qPCR) were conducted to verify the impact of ZMIZ2–SIRT1 combination on Hippo/Wnt pathways.ResultsZMIZ2 was highly expressed in NSCLC and positively associated with advanced pTNM staging, lymph node metastasis, and poor overall survival. Functional experiments revealed that ZMIZ2 promotes the proliferation, migration, and invasiveness of lung cancer cells—establishing its role as a promoter of oncogenes. Molecular mechanism studies identified SIRT1 as an assisted key factor interacting with ZMIZ2. KEGG enrichment analysis revealed that ZMIZ2 is closely related to Wnt/Hippo pathways; ZMIZ2–SIRT1 interaction enhanced SIRT1 deacetylase activity. Direct downregulation of intranuclear β-catenin and yes-associated protein (YAP) acetylation levels occurred independently of upstream proteins in Wnt/Hippo pathways; transcriptional activities of β-catenin-transcription factor 4 (TCF4) and YAP–TEA domain family transcription factors (TEADs) were amplified.ConclusionsZMIZ2 promotes the malignant phenotype of lung cancer by regulating Wnt/Hippo pathways through SIRT1, providing an experimental basis for discovering novel biomarkers and developing tumor-targeted drugs.Graphical

ABRACL upregulated by transcription factor CBX4 promotes proliferation and migration and inhibits the apoptosis of gastric cancer cells.

Gastric cancer (GC) is a predominant health concern in many countries. Actin-binding Rho activating C-terminal-like (ABRACL) belongs to a new family of low molecular weight proteins and has been implicated in cancers. This study was implemented to elucidate the role and mechanism of ABRACL in GC. mRNA and protein expression of ABRACL and CBX4 in human gastric epithelium cell line GES-1 and GC cell lines were assessed with RT-qPCR and western blot. The transfection efficacy of sh-ABRACL, oe-CBX4, and sh-CBX4 was examined with RT-qPCR and western blot. AGS cell proliferation, migration, and invasion were evaluated using CCK-8, colony formation assay, wound healing, and Transwell assays, respectively. With western blot analysis, flow cytometry, and caspase-3 assay kits, the expressions of MMP2 and MMP9, cell apoptosis, and caspase-3 activity were estimated. Western blot was adopted to estimate the contents of apoptosis-related proteins. Luciferase reporter and chromatin immunoprecipitation (ChIP) were applied to verify the interaction between ABRACL and CBX4. The expression of ABRACL and CBX4 was increased in GC tissues and cells. After interfering with ABRACL, the proliferation, migration, and invasion of GC cells were inhibited while apoptosis was promoted. We also discovered that CBX4 could bind to ABRACL and transcriptionally regulate ABRACL expression in AGS cells. Rescue experiments revealed that CBX4 overexpression partially reversed the regulatory effects of ABRACL silencing on the proliferation, migration, invasion, and apoptosis of GC cells. Collectively, ABRACL transcriptionally upregulated by CBX4 promoted the malignant progression of GC.

Overexpression of ZFP69B promotes hepatocellular carcinoma growth by upregulating the expression of TLX1 and TRAPPC9

BackgroundT-cell leukemia homeobox protein 1 (TLX1) has been revealed as a hub transcription factor in leukemia, while its function in hepatocellular carcinoma (HCC) has not been well described. Here, we investigated the regulation and function of TLX1 in HCC.MethodsTLX1 and its possible upstream and downstream molecules in HCC were identified using bioinformatics tools, which were then verified by RT-qPCR assay. CCK-8, wound healing, and Transwell invasion assays were performed to detect the effects of TLX1 knockdown on HCC cells. The interactions between TLX1 and trafficking protein particle complex subunit 9 (TRAPPC9) or Zinc finger protein 69 homolog B (ZFP69B) were further probed by ChIP and luciferase reporter assays. Rescue experiments were finally conducted in vitro and in vivo.ResultsTLX1 was highly expressed in HCC cells, and the knockdown of TLX1 led to reduced malignant biological behavior of HCC cells. TLX1 bound to the promoter region of TRAPPC9, thereby promoting TRAPPC9 expression. Overexpression of TRAPPC9 attenuated the effect of TLX1 reduction on suppressing malignant behavior of HCC cells. ZFP69B was also highly expressed in HCC cells and bound to the promoter region of TLX1 to induce TLX1 expression. Knockdown of ZFP69B inhibited the viability and mobility of HCC cells in vitro and tumor growth in vivo, and overexpression of TLX1 rescued this inhibition.ConclusionThese findings suggest that ZFP69B promotes the proliferation of HCC cells by directly upregulating the expression of TLX1 and the ensuing TRAPPC9.

Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3'-untranslated region (3'-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3' end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1's third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

Temperature Sensitive PNIPAm-g-PEI/Gold Nanotriangle for Gene Delivery Promotion.

As a two-dimensional material, gold nanotriangles (GNTs) are rarely studied in the field of gene delivery. In this study, a temperature-responsive GNTs was developed as a novel carrier for gene delivery. The temperature-sensitive copolymer PNIPAm-g-PEI was grafted onto the surface of GNTs to construct a PNIPAm-g-PEI/GNTs composite controllable release platform. The lower critical solution temperature (LCST) of PNIPAm-g-PEI/GNTs was found to be 42°C, and the particle size of PNIPAm-g-PEI/GNTs was 150nm at this temperature. Gel electrophoresis experiments showed that PNIPAm-g-PEI/GNTs completely condensed DNA at 20μg/mL, and PNIPAm-g-PEI/GNTs promoted the release of DNA under laser irradiation. Luciferase and green fluorescent protein reporter gene assays demonstrated that the transfection efficiency of PNIPAm-g-PEI/GNTs was 1.5 and 7.2 times that of PEI, respectively. These results demonstrated the promising potential of temperature-responsive GNTs as effective and safe gene delivery vectors.

Exosome-mediated transfer of lncRNA RP3-340B19.3 promotes the progression of breast cancer by sponging miR-4510/MORC4 axis

BackgroundThis study aims to explore the molecular mechanism of lncRNA RP3-340B19.3 on breast cancer cell proliferation and metastasis and clinical significance of lncRNA RP3-340B19.3 for breast cancer.MethodsThe subcellular localization of lncRNA RP3-340B19.3 was identified using RNA fluorescence in situ hybridization (FISH). The expression of lncRNA RP3-340B19.3 in breast cancer cells, breast cancer tissues, as well as the serum and serum exosomes of breast cancer patients, was measured through quantitative RT-PCR. In the in vitro setting, we conducted experiments to observe the effects of RP3-340B19.3 on both cell migration and proliferation. This was achieved through the utilization of transwell migration assays as well as clone formation assays. Meanwhile, transwell migration assays and clone formation assays were used to observe the effects of MDA-MB-231-exosomes enriched in RP3-340B19.3 on breast cancer microenvironment cells MCF7 and BMMSCs. Additionally, western blotting techniques were used to assess the expression levels of proteins associated with essential cellular processes such as proliferation, apoptosis, and metastasis. In vivo, the impact of RP3-340B19.3 knockdown on tumour weight and volume was observed within a nude mice model. We aimed to delve into the intricate molecular mechanisms involving RP3-340B19.3 by using bioinformatics analysis, dual luciferase reporter gene experiments and western blotting. Moreover, the potential correlations between RP3-340B19.3 expression and various clinical pathological characteristics were analyzed.ResultsOur investigation revealed that RP3-340B19.3 was expressed in both the cytoplasm and nucleus, with a noteworthy increase in breast cancer cells. Notably, we found that RP3-340B19.3 exerted a promoting influence on the proliferation and migration of breast cancer cells, both in vitro and in vivo. MDA-MB-231-exosomes enriched in RP3-340B19.3 promoted the proliferation and migration of MCF7 and BMMSCs in vitro. Mechanistically, RP3-340B19.3 demonstrated the capability to modulate the expression of MORC4 by forming a complex with miR-4510. This interaction subsequently triggered the activation of the NF-κB and Wnt-β-catenin signaling pathways. Furthermore, our study highlighted the potential diagnostic utility of RP3-340B19.3. We discovered its presence in the serum and exosomes of breast cancer patients, showing promising efficacy as a diagnostic marker. Notably, the diagnostic potential of RP3-340B19.3 was particularly significant in relation to distinguishing between different pathological types of breast cancer and correlating with tumour diameter.ConclusionOur findings establish that RP3-340B19.3 plays a pivotal role in driving the proliferation and metastasis of breast cancer. Additionally, exosomes enriched in RP3-340B19.3 could influence MCF7 and BMMSCs in tumour microenvironment, promoting the progression of breast cancer. This discovery positions RP3-340B19.3 as a prospective novel candidate for a tumour marker, offering substantial potential in the realms of breast cancer diagnosis and treatment strategies.