A review on the role of long non-coding RNA and microRNA network in clear cell renal cell carcinoma and its tumor microenvironment.

Renal cell carcinoma (RCC) is the second lethal urogenital malignancy with the increasing incidence and mortality in the world. Clear cell renal cell carcinoma (ccRCC) is one major subtype of RCC, which accounts for about 70 to 80% of all RCC cases. Although many innovative therapeutic options have emerged during the last few decades, the efficacy of these treatments for ccRCC patients is very limited. To date, the prognosis of patients with advanced or metastatic ccRCC is still poor. The 5-year survival rate of these patients remains less than 10%, which mainly attributes to the complexity and heterogeneity of the tumor microenvironment (TME). It has been demonstrated that long non-coding RNAs (lncRNAs) perform an indispensable role in the initiation and progression of various tumors. They mostly function as sponges for microRNAs (miRNAs) to regulate the expression of target genes, finally influence the growth, metastasis, apoptosis, drug resistance and TME of tumor cells. However, the role of lncRNA/miRNA/mRNA axis in the TME of ccRCC remains poorly understood. In this review, we summarized the biological function of lncRNA/miRNA/mRNA axis in the pathogenesis of ccRCC, then discussed how lncRNA/miRNA/mRNA axis regulate the TME, finally highlighted their potential application as novel biomarkers and therapeutic targets for ccRCC.

Immunological characteristics of immunogenic cell death genes and malignant progression driving roles of TLR4 in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) was a rare malignancy featured with the weak immunotherapeutic response. So far, disorders of immunogenic cell death genes (ICDGs) were identified as the driving factors in cancer progression, while their roles in ATC remained poorly clear. Datasets analysis identified that most ICDGs were high expressed in ATC, while DE-ICDGs were located in module c1_112, which was mainly enriched in Toll-like receptor signalings. Subsequently, the ICD score was established to classify ATC samples into the high and low ICD score groups, and function analysis indicated that high ICD score was associated with the immune characteristics. The high ICD score group had higher proportions of specific immune and stromal cells, as well as increased expression of immune checkpoints. Additionally, TLR4, ENTPD1, LY96, CASP1 and PDIA3 were identified as the dynamic signature in the malignant progression of ATC. Notably, TLR4 was significantly upregulated in ATC tissues, associated with poor prognosis. Silence of TLR4 inhibited the proliferation, metastasis and clone formation of ATC cells. Eventually, silence of TLR4 synergistically enhanced paclitaxel-induced proliferation inhibition, apoptosis, CALR exposure and release of ATP. Our findings highlighted that the aberrant expression of TLR4 drove the malignant progression of ATC, which contributed to our understanding of the roles of ICDGs in ATC.

Single-cell sequencing analysis fibrosis provides insights into the pathobiological cell types and cytokines of radiation-induced pulmonary fibrosis.

BACKGROUND: Radiotherapy is an essential treatment for chest cancer. Radiation-induced pulmonary fibrosis (RIPF) is an almost irreversible interstitial lung disease; however, its pathogenesis remains unclear. METHODS: We analyzed specific changes in cell populations and potential markers by using single-cell sequencing datasets from the Sequence Read Archive database, PERFORMED from control (0 Gy) and thoracic irradiated (20 Gy) mouse lungs at day 150 post-radiation. We performed IHC and ELISA on lung tissue and cells to validate the potential marker cytokines identified by the analysis on rat thoracic irradiated molds (30 Gy). RESULTS: Single-cell sequencing analysis showed changes in abundance across cell types and at the single-cell level, with B and T cells showing the most significant changes in abundance. And four cytokines, CCL5, ICAM1, PF4, and TNF, were significantly upregulated in lung tissues of RIPF rats and cell supernatants after ionizing radiation. CONCLUSION: Cytokines CCL5, ICAM1, PF4, and TNF may play essential roles in radiation pulmonary fibrosis. They are potential targets for the treatment of radiation pulmonary fibrosis.

CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment.

Anaplastic thyroid carcinoma (ATC) is an extremely malignant type of endocrine cancer frequently accompanied by extrathyroidal extension or metastasis through mechanisms that remain elusive. We screened for the CREB3 transcription-factor family in a large cohort, consisting of four microarray datasets. This revealed that CREB3L1 was specifically up regulated in ATC tissues and negatively associated with overall survival of patients with thyroid cancer. Consistently, high expression of CREB3L1 was negatively correlated with progression-free survival in an independent cohort. CREB3L1 knockdown dramatically attenuated invasion of ATC cells, whereas overexpression of CREB3L1 facilitated the invasion of papillary thyroid carcinoma (PTC) cells. Loss of CREB3L1 inhibited metastasis and tumor growth of ATC xenografts in zebrafish and nude mouse model. Single-cell RNA-sequencing analysis revealed that CREB3L1 expression gradually increased during the neoplastic progression of a thyroid follicular epithelial cell to an ATC cell, accompanied by the activation of the extracellular matrix (ECM) signaling. CREB3L1 knockdown significantly decreased the expression of collagen subtypes in ATC cells and the fibrillar collagen in xenografts. Due to the loss of CREB3L1, ATC cells were unable to activate alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs). After CREB3L1 knockdown, the presence of CAFs inhibited the growth of ATC spheroids and the metastasis of ATC cells. Further cytokine array screening showed that ATC cells activated α-SMA-positive CAFs through CREB3L1-mediated IL-1α production. Moreover, KPNA2 mediated the nuclear translocation of CREB3L1, thus allowing it to activate downstream ECM signaling. These results demonstrate that CREB3L1 maintains the CAF-like property of ATC cells by activating the ECM signaling, which remodels the tumor stromal microenvironment and drives the malignancy of ATC.

Efficacy of Qingfei oral liquid for idiopathic pulmonary fibrosis in rats and related network pharmacology study.

To investigate the therapeutic effect and mechanism of Qingfei oral liquid in idiopathic pulmonary fibrosis. Seventy-two male SD rats were divided into control group, model group, pirofenidone group and Qingfei group with 18 animals in each group. The idiopathic pulmonary fibrosis was induced in last three groups by intratracheal injection of bleomycin; pirofenidone group was given oral administration of pirofenidone b.i.d for 21 d, and Qingfei group was given Qingfei oral liquid 3.6 mL/kg q.d for Lung tissues were obtained for HE staining, Masson staining and transforming growth factor (TGF)-ß immunohistochemical staining. Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were detected in tissue homogenates. The BATMAN-TCM database was used to retrieve the chemical components and their corresponding targets of Qingfei oral solution by network pharmacology method, and then the component-target-disease network diagram was constructed. Finally, the pathway enrichment analysis was carried out to explore the molecular mechanism of Qingfei oral liquid against idiopathic fibrosis. Histopathology results showed that Qingfei oral liquid had a similar relieving effect on pulmonary fibrosis as the positive drug pirfenidone; TGF-ß secretion had a significant reduction in lung tissues of Qingfei group; and Qingfei oral liquid had better regulatory effect on SOD, MDA and GSH than pirfenidone. The results of component-target-disease network and pathway enrichment analysis showed that the related molecular pathways were concentrated in inflammation, extracellular matrix and cytokines. Qingfei oral liquid has a good therapeutic effect on idiopathic pulmonary fibrosis in rats via regulation of inflammation, extracellular matrix and cytokines.

Identification of novel key genes associated with the metastasis of prostate cancer based on bioinformatics prediction and validation.

BACKGROUND: Metastatic prostate cancer (PCa) is a lethal tumor. However, the molecular mechanisms underlying PCa progression have not been fully elucidated. METHODS: Transcriptome expression profiling and clinical information on primary and metastatic PCa samples were obtained from TCGA. R software was used to screen the DEGs, and LASSO logistical regression method was utilized to identify the pivotal PCa metastasis-related DEGs. The transcriptional expression levels of the key genes were analyzed using the UALCAN database, and the corresponding protein expression were validated by Immunohistochemistry (IHC). Survival analysis of the key genes was performed using the GEPIA database. Wound healing assay and Transwell assay were conducted to determine whether knockdown of the key genes influence the migration and invasion abilities of PCa cells (22Rv1 and PC3). GSEA was performed to predict key genes-mediated signaling pathways for the development of PCa. Western blotting was used to evaluate the expression changes of E-cadherin, Twist1, and Vimentin in PCa cells with the key genes silencing. An in vivo mouse metastatic model for PCa was also generated to verify the important role of ISG15 and CST2 in PCa metastasis. RESULTS: A comparison between primary and metastatic PCa tissues was conducted, and 19 DEGs were screened. Among these, three key genes were identified that might be closely associated with PCa progression according to the LASSO logistical analysis, namely ISG15, DNAH8, and CST2. Further functional experiments revealed that knockdown of ISG15 and CST2 suppressed wound healing, migration, and invasion of PCa cells. To explore the molecular mechanism of ISG15 and CST2 in the development of PCa, GSEA was performed, and it was found that both genes play crucial roles in cell adhesion molecules, extracellular matrix-receptor interaction, and focal adhesion. Western blotting results exhibited that inhibiting ISG15 and CST2 led to increase the expression of E-cadherin and decrease the expression of Twist1 and Vimentin. Additionally, the metastatic in vivo study demonstrated that both PC3 and 22Rv1 cells expressing with luciferase-shISG15 and luciferase-shCST2 had significantly lower detectable bioluminescence than that in the control PCa cells. CONCLUSION: ISG15 and CST2 may participate in PCa metastasis by regulating the epithelial-mesenchymal transition (EMT) signaling pathway. These findings may help to better understand the pathogenetic mechanisms governing PCa and provide promising therapeutic targets for metastatic PCa therapy.

Resveratrol inhibits the migration, invasion and epithelial-mesenchymal transition in liver cancer cells through up- miR-186-5p expression.

To investigate the molecular mechanism of resveratrol inhibiting the metastasis of liver cancer . HepG2 and Huh7 cells were treated with different concentrations of resveratrol, and the cell viability was determined by CCK-8 assay to determine the optimal concentration of resveratrol for subsequent experiments. The expressions of miR-186-5p in liver cancer tissues and liver cancer cells were determined by quantitative real-time RT-PCR. The migration and invasion of HepG2 and Huh7 cells were detected by wound healing assay and Transwell assay, and the expression levels of epithelial-mesenchymal transition (EMT) related proteins were determined by Western blotting. Resveratrol with concentration of had no effect on the viability of HepG2 and Huh7 cells, so the concentration of resveratrol in subsequent experiments was 6.25 µmol/L. Resveratrol inhibited the wound healing and invasion of liver cancer cells; increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. The expression of miR-186-5p was significantly down-regulated in liver cancer tissues and cells compared with the adjacent tissues and normal liver cells (both <0.05). Furthermore, resveratrol induced the expression of miR-186-5p in liver cancer cells (both <0.01). Overexpression of miR-186-5p suppressed the migration, invasion and EMT of liver cancer cells. Knockdown of miR-186-5p blocked the inhibition effects of resveratrol on the migration, invasion and EMT of liver cancer cells. Resveratrol could inhibit the metastasis of liver cancer , which might be associated with up-regulating miR-186-5p.

Identification of key microRNAs and hub genes in non-small-cell lung cancer using integrative bioinformatics and functional analyses.

Non-small-cell lung cancer (NSCLC) is an extremely debilitating respiratory malignancy. However, the pathogenesis of NSCLC has not been fully clarified. The main objective of our study was to identify potential microRNAs (miRNAs) and their regulatory mechanism in NSCLC. Using a systematic review, two NSCLC-associated miRNA data sets (GSE102286 and GSE56036) were obtained from Gene Expression Omnibus, and the differentially expressed miRNAs (DE-miRNAs) were accessed by GEO2R. Survival analysis of candidate DE-miRNAs was conducted using the Kaplan-Meier plotter database. To further illustrate the roles of DE-miRNAs in NSCLC, their potential target genes were predicted by miRNet and were annotated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) program. Moreover, the protein-protein interaction (PPI) and miRNA-hub gene regulatory network were established using the STRING database and Cytoscape software. The function of DE-miRNAs in NSCLC cells was evaluated by transwell assay. Compared with normal tissues, a total of eight DE-miRNAs was commonly changed in two data sets. The survival analysis showed that six miRNAs (miR-21-5p, miR-31-5p, miR-708-5p, miR-30a-5p, miR-451a, and miR-126-3p) were significantly correlated with overall survival. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that target genes of upregulated miRNAs were enriched in pathways in cancer, microRNAs in cancer and proteoglycans in cancer, while the target genes of downregulated miRNAs were mainly associated with pathways in cancer, the PI3K-Akt signaling pathway and HTLV-I infection. Based on the miRNA-hub gene network and expression analysis, PTEN, EGFR, STAT3, RHOA, VEGFA, TP53, CTNNB1, and KRAS were identified as potential target genes. Furthermore, all six miRNAs exhibited significant effects on NSCLC cell invasion. These findings indicate that six DE-miRNAs and their target genes may play important roles in the pathogenesis of NSCLC, which will provide novel information for NSCLC treatments.

SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides To Enhance the Nucleotide-Binding Oligomerization Domain-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages.

There is increasing evidence that proton-coupled oligopeptide transporters (POTs) can transport bacterially derived chemotactic peptides and therefore reside at the critical interface of innate immune responses and regulation. However, there is substantial contention regarding how these bacterial peptides access the cytosol to exert their effects and which POTs are involved in facilitating this process. Thus, the current study proposed to determine the (sub)cellular expression and functional activity of POTs in macrophages derived from mouse bone marrow and to evaluate the effect of specific POT deletion on the production of inflammatory cytokines in wild-type, Pept2 knockout and Pht1 knockout mice. We found that PEPT2 and PHT1 were highly expressed and functionally active in mouse macrophages, but PEPT1 was absent. The fluorescent imaging of muramyl dipeptide-rhodamine clearly demonstrated that PEPT2 was expressed on the plasma membrane of macrophages, whereas PHT1 was expressed on endosomal membranes. Moreover, both transporters could significantly influence the effect of bacterially derived peptide ligands on cytokine stimulation, as shown by the reduced responses in Pept2 knockout and Pht1 knockout mice as compared with wild-type animals. Taken as a whole, our results point to PEPT2 (at plasma membranes) and PHT1 (at endosomal membranes) working in concert to optimize the uptake of bacterial ligands into the cytosol of macrophages, thereby enhancing the production of proinflammatory cytokines. This new paradigm offers significant insight into potential drug development strategies along with transporter-targeted therapies for endocrine, inflammatory, and autoimmune diseases.

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells.

The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d3-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d3-l-histidine and low affinity for GlySar, with Km values of 16.3 ± 1.9 µM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants.

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 µM) > mouse (15.6 µM) ≈ rat (11.9 µM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans.

Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3.

1. Organic cation transporters (OCTs) play an important role in drug safety and efficacy. Protoberberine alkaloids are ubiquitous organic cations or weak bases with remarkable biological actives. This study was to elucidate the potential interaction of alkaloids (coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline) with OCTs using Madin-Darby canine kidney (MDCK) cells stably expressing human OCT1, OCT2 and OCT3. 2. All the tested alkaloids significantly inhibited the uptake of MPP(+), a model OCT substrate, in MDCK-hOCTs cells with the IC50 of 0.931-9.65 µM. Additionally, coptisine, jatrorrhizine and epiberberine were substrates of all the hOCTs with the Km of 0.273-5.80 µM, whereas berberrubine was a substrate for hOCT1 and hOCT2, but not for hOCT3, the Km values were 1.27 and 1.66 µM, respectively. The transport capacity of coptisine in MDCK cells expressing the variants of hOCT1-P341L or hOCT2-A270S was significantly higher than that in wild-type (WT) cells with the Clint (Vmax/Km) of 379 ± 7.4 and 433 ± 5.7 µl/mg protein/min, respectively. 3. The above data indicate that the tested alkaloids are potent inhibitors, and coptisine, jatrorrhizine, epiberberine and berberrubine are substrates of hOCT1, hOCT2 and/or hOCT3 with high affinity. In addition, the variants (OCT1-P341L and OCT2-A270S) possess higher transport capacity to coptisine than WT hOCTs.

[Establishment and application of cell models with stable expression of hOCTN1/2].

Human carnitine/organic cation transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1 (+) plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K(m) and V(max) of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 µmol·L-1 and 1 427 ± 49 pmol·mg(-1)(protein)·min(-1); while K(m) and V(max) of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 µmol·L(-1) and 2 454 ± 64 pmol·mg(-1)(protein)·min(-1). Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

Expression and regulation of the proton-coupled oligopeptide transporter PhT2 by LPS in macrophages and mouse spleen.

Membrane transporter PhT2 (SLC15A3), which belongs to the proton-coupled oligopeptide transporter family, mediates the transport of di/tripeptides and histidine utilizing an inwardly directed proton gradient and negative membrane potential. The aim of this study was to elucidate the molecular expression of PhT2 in macrophages and mouse tissues and to explore the regulation of PhT2 by lipopolysaccharide (LPS). The results showed relatively high expression of PhT2 in J774A.1 and THP-1 macrophage cells, mouse spleen, and lung. Using an LPS-induced inflammatory cell model, we found that hPhT2 mRNA expression was up-regulated in THP-1 cells and that the up-regulation was suppressed by pyrrolidine dithiocarbamate, a specific inhibitor of NF-κB. Similar results were observed in mouse spleen during LPS-induced acute inflammation. Using dual-labeling immunofluorescence and confocal laser scanning microscopy, we confirmed that mPhT2 was colocalizing with lysosome-associated membrane protein 1 in transfected HEK293 cells. These results suggested that PhT2, a lysosomal membrane transporter, was up-regulated by LPS via the NF-κB signaling pathway.

Role of sodium taurocholate cotransporting polypeptide (NTCP) in HBV-induced hepatitis: Opportunities for developing novel therapeutics.

Hepatitis B is an infectious disease caused by the HBV virus. It presents a significant challenge for treatment due to its chronic nature and the potential for developing severe complications, including hepatocirrhosis and hepatocellular carcinoma. These complications not only cause physical and psychological distress to patients but also impose substantial economic and social burdens on both individuals and society as a whole. The internalization of HBV relies on endocytosis and necessitates the involvement of various proteins, including heparin sulfate proteoglycans, epidermal growth factor receptors, and NTCP. Among these proteins, NTCP is pivotal in HBV internalization and is primarily located in the liver's basement membrane. As a transporter of bile acids, NTCP also serves as a receptor facilitating HBV entry into cells. Numerous molecules have been identified to thwart HBV infection by stifling NTCP activity, although only a handful exhibit low IC50 values. In this systematic review, our primary focus dwells on the structure and regulation of NTCP, as well as the mechanism involved in HBV internalization. We underscore recent drug breakthroughs that specifically target NTCP to combat HBV infection. By shedding light on these advances, this review contributes novel insights into developing effective anti-HBV medications.

Peptide Transporter 1-Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis.

Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

Rab11-FIP4 interacts with ARF5 to promote cancer stemness in hepatocellular carcinoma.

Recent studies suggest that Rab11-family interacting proteins (Rab11-FIPs) play an important role in tumorigenesis and progression. Among the Rab11-FIPs, Rab11-FIP4 has been reported to be significantly upregulated in various cancers, including hepatocellular carcinoma (HCC). However, the possible effect on HCC stemness and the underlying mechanism has never been characterized. Here, we found that Rab11-FIP4 was dramatically increased in HCC cell lines and tissues, and had a positive correlation with cancer stemness. Functional studies revealed that elevated expression of Rab11-FIP4 in HCC cells significantly promoted sphere formation, and enhanced the mRNA and protein levels of stemness-associated markers, ALDH1A1, CD133, NANOG, and OCT4. Conversely, the knockdown of Rab11-FIP4 suppressed the cancer stem cell (CSC)-like characteristics of HCC cells. Moreover, silencing of Rab11-FIP4 obviously increased the sensitivity of HCC cells to sorafenib. Mechanistically, Rab11-FIP4 was shown to interact with ADP-ribosylation factor 5 (ARF5) to influence cell cycle-related proteins, CDK1/cyclin B, thereby promoting HCC stemness. Taken together, our results uncovered an essential role for Rab11-FIP4 in regulating CSC-like features of HCC cells and identified Rab11-FIP4 as a potential target for HCC therapy.

Effects of the CYP3A inhibitors, voriconazole, itraconazole, and fluconazole on the pharmacokinetics of osimertinib in rats.

Background: Osimertinib, as third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is the first-line treatment approved to treat advanced T790M mutation-positive tumors. Triazole antifungals are therapeutic drugs for cancer patients to reduce the risk of opportunistic fungal infections. Our objective was to investigate whether three triazole antifungals (voriconazole, itraconazole, and fluconazole) could change the pharmacokinetics of osimertinib in rats. Methods: The adult male Sprague-Dawley rats were randomly divided into four groups (n = 6): control (0.3% CMC-Na), and voriconazole (20 mg/kg), itraconazole (20 mg/kg), or fluconazole (20 mg/kg) combined with osimertinib (10 mg/kg) group. Tail vein blood samples were collected into heparin tubes at various time points within 0-48 h after osimertinib administration. Osimrtinib's plasma concentration was detected using HPLC-MS/MS system equipped with a Waters XBridge C18 column, with the mobile phase consisting of acetonitrile and 0.2% formic acid water at a flow rate of 0.5 mL/min. Results: Co-administration with voriconazole or fluconazole increased the Cmax of osimertinib by 58.04% and 53.45%, respectively; the AUC0-t increased by 62.56% and 100.98%, respectively. However, when co-administered with itraconazole, the Cmax and AUC0-t of osimertinib only increased by 13.91% and 34.80%, respectively. Conclusions: Our results revealed that the pharmacokinetics of osimertinib were significantly changed by voriconazole and fluconazole in rats, whereas it was slightly affected by itraconazole. This work will contribute to a more comprehensive understanding of the pharmacokinetic properties of osimertinib when co-administered with triazole antifungals.

Identification of potential pseudogenes for predicting the prognosis of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) remains a highly deadly malignant tumor with high recurrence and metastasis rates. Cancer stem cells (CSCs) are involved in tumor metastasis, recurrence, and resistance to drugs, which have attracted widespread attention in recent years. Research has shown that pseudogenes may regulate stemness to promote the progression of HCC, but its specific mechanisms and impact on prognosis remain unclear. METHODS: In this study, clinical prognosis information of HCC was first downloaded from The Cancer Genome Atlas (TCGA) database. Then we calculated the mRNA expression-based stemness index (mRNAsi) of HCC. We also screened the differentially expressed pseudogene (DEPs) and conducted univariate Cox regression analysis to investigate their effect on the prognosis of HCC. Further, genomic mutation frequency analysis and weighted gene co-expression network analysis (WGCNA) were performed to compare the role of pseudogenes and stemness in promoting the progression of HCC. Finally, we conducted the correlation analysis to examine the potential mechanism of pseudogenes regulating stemness to promote the progression of HCC and detected the possible pathways through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS: Herein, we revealed that the high stemness of HCC correlated with an unfavorable prognosis. We obtained 31 up-regulated and 8 down-regulated DEPs in HCC and screened CTB-63M22.1, a poor prognostic indicator of HCC. In addition, CTB-63M22.1 had a mutation frequency similar to mRNAsi and acted in a module similar to that of mRNAsi on HCC. We then screened two RNA-binding proteins (RBPs) LIN28B and NOP56 with the highest correlation with stemness. We also discovered that they were primarily enriched in the biological process as examples of mitotic nuclear division and cell cycle. CONCLUSIONS: Collectively, these results revealed that pseudogenes CTB-63M22.1 may regulate cancer stemness by regulating RBPs, suggesting that CTB-63M22.1 may serve as an innovative therapeutic target and a reliable prognostic marker for HCC.

Research progress on non-protein-targeted drugs for cancer therapy.

Non-protein target drugs, especially RNA-based gene therapies for treating hereditary diseases, have been recognized worldwide. As cancer is an insurmountable challenge, no miracle drug is currently available. With the advancements in the field of biopharmaceuticals, research on cancer therapy has gradually focused on non-protein target-targeted drugs, especially RNA therapeutics, including oligonucleotide drugs and mRNA vaccines. This review mainly summarizes the clinical research progress in RNA therapeutics and highlights that appropriate target selection and optimized delivery vehicles are key factors in increasing the effectiveness of cancer treatment in vivo.