Targeting the “undruggable” cancer driver genes: <i>Ras</i>, <i>myc</i>, and <i>tp53</i>

The term “undruggable” is to describe molecules that are not targetable or at least hard to target pharmacologically. Unfortunately, some targets with potent oncogenic activity fall into this category, and currently little is known about how to solve this problem, which largely hampered drug research on human cancers. Ras, as one of the most common oncogenes, was previously considered “undruggable”, but in recent years, a few small molecules like Sotorasib (AMG-510) have emerged and proved their targeted anti-cancer effects. Further, myc, as one of the most studied oncogenes, and tp53, being the most common tumor suppressor genes, are both considered “undruggable”. Many attempts have been made to target these “undruggable” targets, but little progress has been made yet. This article summarizes the current progress of direct and indirect targeting approaches for ras, myc, two oncogenes, and tp53, a tumor suppressor gene. These are potential therapeutic targets but are considered “undruggable”. We conclude with some emerging research approaches like proteolysis targeting chimeras (PROTACs), cancer vaccines, and artificial intelligence (AI)-based drug discovery, which might provide new cues for cancer intervention. Therefore, this review sets out to clarify the current status of targeted anti-cancer drug research, and the insights gained from this review may be of assistance to learn from experience and find new ideas in developing new chemicals that directly target such “undruggable” molecules.     

The chloroplast genome comparative characteristic of artificial breeding tree,a case about <i>Broussonetia kazinoki</i> × <i>Broussonetia papyrifera</i>

Broussonetia kazinoki × Broussonetia papyrifera (ZJGS) is a hybrid species in Moraceae family, which has a very complicated hybrid origin. The excellent characteristics of fast growth, strong soil and water conservation ability, high leaf protein content and stem fiber content in ZJGS make it both ecological benefits in the mining area and economically valuable. This study aims to further understand ZJGS and other Moraceae taxa through the ZJGS chloroplast (cp) genome structure and the comparison with 12 closely related Moraceae species. Among the 13 Moraceae species, the cp genome length of seven Broussonetia species (ranges from 160,239 bp to 162,594 bp) is larger than that of six Morus species (ranges from 158,459 bp to 159,265 bp). Among the 77 shared protein-coding genes (PCGs) in Moraceae species, the obvious positive selection of Ka/Ks ratios acted on petD and rpl16 genes of B. kazinoki and B. papyrifera, respectively. Phylogenetic analysis based on shared PCGs from 28 species shows that ZJGS is closely related to maternal B. kazinoki. These findings provide data support for the origin of ZJGS hybridization and provide genomic resources for future ZJGS resource development and molecular breeding.     

Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of <i>Serratia ureilytica</i> DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.     

Genome-wide identification of <i>U-box</i> gene family and expression analysis under abiotic stresses in <i>Salvia miltiorrhiza</i>

Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and in response to different abiotic stresses, but little is known about U-box genes in Danshen (root of Salvia miltiorrhiza Bunge). Here, we identified and characterized 70 SmPUB genes based on its genome sequence. Phylogenetic analysis of U-box genes from S. miltiorrhiza and Arabidopsis suggested that they can be clustered into seven subgroups (I–VII). Typical U-box domains were found in all identified SmPUB genes through the analysis of conserved motifs. Moreover, qRT-PCR was applied to analyze the relative expression levels of U-box genes in S. miltiorrhiza roots and leaves under PEG-induced water deficit and salt stresses. Results revealed that the SmPUB genes exhibited stronger response to drought than to salt stress. To the best of our knowledge, this report is the first to perform genome-wide identification and analysis of the U-box gene family in S. miltiorrhiza, and the results provide valuable information for better understanding of the function of U-box in S. miltiorrhiza.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Detection of new antibiotic resistance gene profile in <i>Escherichia coli</i> associated with avian leukosis virus infection from broiler chickens

The Escherichia coli (E. coli) is prevailing worldwide, but the epidemiology of E. coli infections feature regional distribution characteristics to some extent. E. coli, as a zoonotic pathogen, can be transferred from animals to humans through food chain or via contact with wounds, causing a public health risk. We reported the swelling of proventriculus and tracheal bleeding following the death in two broiler chickens (Gallus gallus domesticus) from Beijing, China. To investigate whether a virus was involved in the infection, Madin Darby Bovine Kidney (MDCK) cells were co-cultured with supernatants of proventriculus, trachea and spleen homogenates. The avian leucosis virus was detected in the samples of proventriculus and trachea, but the avian influenza virus, the Newcastle disease virus and the avian infectious laryngotracheitis virus were not detected. E. coli isolates were resistant to almost all the antimicrobial as tested except for the combinations of amoxicillin/clavulanic acid and sulfamethoxazole/trimethoprim. PCR tests demonstrated the presence of antibiotic resistance genes in these E. coli isolates and further research revealed a novel gene profile with the presence of CTX-M-1, gyrA, gyrB, oqxA, oqxB, parC and Sul2 antibiotic resistance genes in a strain isolated from a proventriculus sample. These results demonstrated that the presence of antibiotic resistant E. coli would not necessarily cause outbreak of large-scale disease. However, when the bacteria carrying new antibiotic resistance genes enter the environment, it may result in the development of more virulent strains which will potentially impact human and animal health.     

Response of <i>MaHMA2</i> gene expression and stress tolerance to zinc stress in mulberry (<i>Morus alba</i> L.)

HMA2 (heavy metal ATPase 2) plays a crucial role in extracellular and intracellular Zn²⁺ transport across biomembranes, maintaining ion homeostasis, and playing an important role in the normal physiological metabolism, growth, and development of plants. In our study, a novel HMA2 gene, named MaHMA2, was isolated and cloned from white mulberry (Morus alba L.). The gene sequence obtained was 1,342 bp long, with an open reading frame of 1,194 bp, encoding a protein of 397 amino acids, with a predicted molecular mass of 42.852 kD and an isoelectric point of 7.53. This protein belonged to the PIB-type ATPase transport protein family. We analyzed the expression of the MaHMA2 gene by quantitative real-time PCR. The results showed that the level of MaHMA2 gene expression decreased to a Zn concentration of 800 mg/kg. Malondialdehyde and proline levels increased and responded to increasing Zn when the MaHMA2 gene was silenced, whereas the activities of peroxidase and superoxide dismutase tended to increase in response to increasing Zn²⁺ ion stress concentrations but were lower in the gene-silenced plants. These findings suggested that the MaHMA2 gene played an active role in the tolerance response of mulberry to Zn stress.     

Structural characterization of four <i>Rhododendron</i> spp. chloroplast genomes and comparative analyses with other azaleas

Azalea is a general designation of Rhododendron in the Ericaceae family. Rhododendron not only has high ornamental value but also has application value in ecological protection, medicine, and scientific research. In this study, we used Illumina and PacBio sequencing to assemble and annotate the entire chloroplast genomes (cp genomes) of four Rhododendron species. The chloroplast genomes of R. concinnum, R. henanense subsp. lingbaoense, R. micranthum, and R. simsii were assembled into 207,236, 208,015, 207,233, and 206,912 bp, respectively. All chloroplast genomes contain eight rRNA genes, with either 88 or 89 protein-coding genes. The four cp genomes were compared and analyzed by bioinformatics, and the phylogenetic analysis based on chloroplast genomes of 26 species of Ericaceae, Actinidiaceae, and Primulaceae under Ericales was conducted. A comparison of the linear structure of cp genomes of four Rhododendron showed that there were substantial sequence similarities in coding regions, but high differences in non-coding regions. A phylogenetic analysis, based on chloroplast whole genome sequences, showed that all Rhododendron species are in the clade Ericaceae. This study provides valuable genetic information for the study of population genetics and evolutionary relationships in Rhododendron and other azalea species.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

Increased <i>MAD2L2</i> expression predicts poor clinical outcome in Colon Adenocarcinoma

Background: Colon adenocarcinoma (COAD) is the second leading cause of cancer death worldwide thus, identification of COAD biomarkers is critical. Mitotic Arrest Deficient 2 Like 2 (MAD2L2) is a key factor in mammalian DNA damage repair and is highly expressed in many malignant tumors. This is a comprehensive study of MAD2L2 expression, its diagnostic value, prognostic analysis, potential biological function, and impact on the immune system of patients with COAD. Methods: Gene expression, clinical relevance, prognostic analysis, diagnostic value, GO/KEGG cluster analysis, data obtained from TCGA, and bioinformatics statistical analysis were performed using the R package. Immune responses to MAD2L2 expression in COAD were analyzed using TIMER. The expression of MAD2L2 in HCT116 cells induced by the inflammatory factor TNF-α was detected using Western blot. Results: Our results underscore the clinical diagnostic value and potential biological significance of MAD2L2 in patients with COAD. A high level of MAD2L2 expression has been found in COAD and correlated with tumor status and colon polyps. ROC curve analysis showed that MAD2L2 expression has high diagnostic value in COAD. Analysis of immune infiltration results showed that MAD2L2 expression was positively correlated with neutrophil levels. The western blot results demonstrated that MAD2L2 was dose-dependently present with TNF-α. GO/KEGG revealed that MAD2L2 overexpressed and coexpressed genes were mostly involved in biological functions, including hypoxia response, response to reduced oxygen levels, mitochondrial translation elongation, and other processes. Conclusion: MAD2L2 as a new COAD biomarker contributes to our understanding of how alterations in gene expression and the immunological environment contribute to the development of colon cancer. Following further investigation, MAD2L2 may prove to be a viable target factor for clinical diagnosis and therapy of COAD.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.