Identification of an angiogenesis-related risk score model for survival prediction and immunosubtype screening in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable B-cell malignancy, but with the emergence of immunotherapy, a potential cure is hopeful. The individualized interaction between the tumor and bone marrow (BM) microenvironment determines the response to immunotherapy. Angiogenesis is a constant hallmark of the BM microenvironment in MM. However, little is known about the potency ability of angiogenesis-associated genes (AAGs) to regulate the immune microenvironment of MM patients. METHODS: We comprehensively dissected the associations between angiogenesis and genomic landscapes, prognosis, and the immune microenvironment by integrating 36 AAGs. Immunohistochemistry was performed to verify the correlation between angiogenic factor expression and patient prognosis. Single-sample gene set enrichment analysis was applied to quantify the relative abundance of 28 infiltrating cells. The AAG score was constructed using the least absolute shrinkage and selection operator Cox regression model. RESULTS: Angiogenesis was closely correlated with MM patient prognosis, and the mutation intensity of the AAGs was low. Immunohistochemistry confirmed that high microvessel density predicted poor prognosis. Three AAG clusters and two gene clusters with distinct clinical outcomes and immune characteristics were identified. The established AAG_score model performed well in predicting patient prognosis and active immunotherapy response. The high-AAG_score subgroup was characterized by reduced immune cell infiltration, poor prognosis, and inactive immunotherapy response. Multivariate analyses indicated that the AAG_score was strongly robust and independent among the prognostic variables. CONCLUSION: This study revealed that angiogenesis is significantly related to MM patient prognosis and immune phenotype. Evaluating the AAG signature was conducive to predicting patient response to immunotherapy and guiding more efficacious immunotherapy strategies.

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.

Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

Paeoniflorin Ameliorates BiPN by Reducing IL6 Levels and Regulating PARKIN-Mediated Mitochondrial Autophagy.

Background: Bortezomib-induced peripheral neuropathy (BiPN) is a common complication of multiple myeloma (MM) treatment that seriously affects the quality of life of patients. The purpose of the present study was to explore the therapeutic effect of paeoniflorin on BiPN and its possible mechanism. Methods: ELISA was used to measure the level of interleukin-6 (IL6) in the plasma of MM patients, and bioinformatics analysis was used to predict the mechanism underlying the effect of paeoniflorin on peripheral neuropathy. Cell and animal models of BiPN were constructed to evaluate mitochondrial function by measuring cell viability and mitochondrial quality and labeling mitochondria with MitoTracker Green. Nerve injury in mice with BiPN was assessed by behavioral tests, evaluation of motor nerve conduction velocity, hematoxylin-eosin (HE) staining, electron microscopy and analysis of the levels of reactive oxygen species (ROS). Western blotting and immunohistochemistry (IHC) were used to assess the expression of autophagy-related proteins. Results: In MM patients, IL6 levels were positively correlated with the degree of PN. The results of bioinformatics analysis suggested that paeoniflorin ameliorated PN by altering inflammation levels and mitochondrial autophagy. Paeoniflorin increased PC12 cell viability and mitochondrial autophagy levels, alleviated mitochondrial damage, and reduced IL6 levels. In addition, paeoniflorin effectively improved the behavior of mice with BiPN, relieved sciatic nerve injury in mice, increased the expression of LC3II/I, beclin-1, and Parkin in sciatic nerve cells, and increased the expression of LC3B and Parkin in the nerve tissue. Conclusion: The present study confirmed that paeoniflorin significantly ameliorated peripheral neuropathy (PN) caused by bortezomib, possibly by reducing IL6 levels to regulate PARKIN-mediated mitochondrial autophagy and mitochondrial damage.

[Research Progress of Non-coding RNAs in the Regulation of Multiple Myeloma Bone Disease--Review].

Multiple myeloma bone disease is the most common complication of multiple myeloma, which mutually promotes the progression of multiple myeloma, severely affects patients' survival quality and prognosis. Recently, many studies revealed that non-coding RNAs play an important role in the imbalance of bone remodeling by regulating gene expression and participating in various signaling pathways. Additionally, most bone lesions fail to heal even when myeloma patients are in complete remission due to the sustained suppression of osteoblast activity, while non-coding RNAs may become a novel research field and clinical intervention targets. In this review, the latest research advances of non-coding RNAs which affect the occurrence and progress of multiple myeloma bone disease are summarized briefly.

Exosomal circ-CACNG2 promotes cardiomyocyte apoptosis in multiple myeloma via modulating miR-197-3p/caspase3 axis.

Multiple myeloma (MM) secreted exosomes are essential in MM-related complications such as osteolytic bone lesions and renal failure, but their role and underlying mechanism in cardiac complications has not yet been clarified. Here, we investigated the effects of U266 (a MM cell line) exosomes (U266-exo) on regulating the viability, cell cycle, oxidative stress and apoptosis of H9C2 cells and the role of circ-CACNG2 in these effects. We found that U266-exo coculture significantly inhibited viability and promoted apoptosis of H9C2 cells, and serum exosomes of MM patients harbored high level of circ-CACNG2. The clinical data analyses indicated that circ-CACNG2 was an independent prognostic and diagnostic indicator of MM-related cardiac complications. Also, in vitro experiments showed that circ-CACNG2 inhibited viability and promoted apoptosis of H9C2 cells. RIPA, pull-down assays, dual-luciferase reporter assays, and RNA FISH assays revealed that miR-197-3p could bind to circ-CACNG2 and caspase3 directly. Rescue experiments proved that circ-CACNG2 can increase the expression of caspase3 by binding to and decreasing the expression of miR-197-3p. In conclusion, MM-exosomes could inhibit cardiomyocyte viability and promote apoptosis partially through circ-CACNG2/miR-197-3p/caspase3 axis.

A novel circRNA-miRNA-mRNA network revealed exosomal circ-ATP10A as a biomarker for multiple myeloma angiogenesis.

The importance of angiogenesis in multiple myeloma (MM) is unquestionable; however, to date, the success of antiangiogenic therapies has been fairly limited. Exosomal circular RNAs (circRNAs) have been proven to be pivotal players in angiogenesis in various cancers. Nevertheless, their role in MM remains unknown. Therefore, we aimed to identify differentially expressed circRNAs in peripheral blood exosomes from MM patients and explore their diagnostic and prognostic values. We screened 2,052 circRNAs with significant differential expression between MM patients and healthy controls via high-throughput sequencing. qRT-PCR confirmed that the expression of circ-ATP10A was significantly increased in MM patients. The bioinformatics analyses suggested that circ-ATP10A can act as a microRNA (miRNA) sponge and regulate the expression of downstream vascular endothelial growth factor-B (VEGFB), hypoxia-inducible factor-1alpha (HIF1A), platelet-derived growth factor subunit A (PDGFA), and fibroblast growth factor (FGF). The immunohistochemical results indicated that the circ-ATP10A level was positively correlated with the protein levels of VEGFB and marrow microvessel density (MVD) in MM patients, and the receiver operating characteristic (ROC) curve, area under the ROC curve (AUC) and Kaplan-Meier survival curve analyses confirmed it as a prognostic biomarker. Collectively, our study indicates that exosomal circ-ATP10A is a valuable prognostic biomarker in MM and may promote MM angiogenesis by targeting hsa-miR-6758-3p/hsa-miR-3977/hsa-miR-6804-3p/hsa-miR-1266-3p/hsa-miR-3620-3p and modulating their downstream mRNAs, such as VEGFB, HIF1A, PDGF, and FGF.

[Research Progress of Non-coding RNA in Multiple Myeloma with Heart Disease---Review].

Some non-coding RNAs (ncRNA), as functional RNA molecules, lack potential to encode proteins, but can affect gene expression and disease progression through a variety of mechanisms. In multiple myeloma (MM), cardiovascular disease is one of the most common complications, which may be related to a variety of factors, including patient's own factors, disease-related factors, drug factors, etc. Non-coding RNA is considered to be an important regulator of cardiovascular event risk factors and cell function, and an important candidate target for improving the condition and prognostic assessment. This article briefly summarized the role of non-coding RNA in cardiac amyloidosis caused by MM, damage to the heart by inflammatory factors, and heart disease caused by chemotherapy drugs in recent years.