Activation of 5-HT6 Receptors in the Ventrolateral Orbital Cortex Produces Anti-Anxiodepressive Effects in a Rat Model of Neuropathic Pain.

The comorbidity of anxiety and depression frequently occurs in patients with neuropathic pain. The ventrolateral orbital cortex (VLO) plays a critical role in mediating neuropathic pain and anxiodepression in rodents. Previous studies suggested that 5-HT6 receptors in the VLO are involved in neuropathic pain. Strong evidence supports a close link between 5-HT6 receptors and affective disorders such as depression and anxiety disorders. However, it remains unclear whether the 5-HT6 receptors in the VLO are involved in neuropathic pain-induced anxiodepression. Using a rat neuropathic pain model of spared nerve injury (SNI), we demonstrated that rats exhibited significant anxiodepression-like behaviors and the expression of VLO 5-HT6 receptors obviously decreased four weeks after SNI surgery. Microinjection of the 5-HT6 receptor agonist EMD-386088 into the VLO or overexpression of VLO 5-HT6 receptors alleviated anxiodepression-like behaviors. These effects were blocked by pre-microinjection of a selective 5-HT6 receptor antagonist (SB-258585) or inhibitors of AC (SQ-22536), PKA (H89), and MEK1/2 (U0126) respectively. Meanwhile, the expression of p-ERK, p-CREB, and BDNF in the VLO decreased four weeks after SNI surgery. Furthermore, administration of EMD-386088 upregulated the expression of BDNF, p-ERK, and p-CREB in the VLO of SNI rats, which were reversed by pre-injection of SB-258585. These findings suggest that activating 5-HT6 receptors in the VLO has anti-anxiodepressive effects in rats with neuropathic pain via activating AC-cAMP-PKA-MERK-CREB-BDNF signaling pathway. Accordingly, 5-HT6 receptor in the VLO could be a potential target for the treatment of the comorbidity of neuropathic pain and anxiodepression.

Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain.

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

Spinal interleukin-16 mediates inflammatory pain via promoting glial activation.

Proinflammatory cytokines are crucial contributors to neuroinflammation in the development of chronic pain. Here, we identified il16, which encodes interleukin-16 (IL-16), as a differentially expressed gene in spinal dorsal horn of a complete Freund's Adjuvant (CFA) inflammatory pain model in mice by RNA sequencing. We further investigated whether and how IL-16 regulates pain transmission in the spinal cord and contributes to the development of inflammatory pain hypersensitivity. RNA sequencing and bioinformatics analysis revealed elevated IL-16 transcript levels in the spinal dorsal horn after CFA injection. This increase was further confirmed by qPCR, immunofluorescence, and western blotting. Knockdown of IL-16 by intrathecal injection of IL-16 siRNA not only attenuated CFA-induced mechanical and thermal pain hypersensitivity, but also inhibited enhanced c-fos expression and glial activation in the spinal dorsal horn in male mice injected with CFA. Moreover, exogenous IL-16 induced nociceptive responses and increased c-fos expression and glial activation in spinal dorsal horn. This effect was largely impaired when CD4, the binding receptor for IL-16, was inhibited. In addition, CD4 expression was upregulated in the spinal dorsal horn after CFA injection and CD4 was present in microglia and in contact with astrocytes and activated spinal neurons. Taken together, these results suggest that enhanced IL-16-CD4 signaling triggers pain and activates microglia and astrocytes in the spinal dorsal horn, thus contributing to inflammatory pain. IL-16 may serve as a promising target for the treatment of inflammatory pain.

Spinal apolipoprotein E is involved in inflammatory pain via regulating lipid metabolism and glial activation in the spinal dorsal horn.

INTRODUCTION: Inflammation and nerve injury promote astrocyte activation, which regulates the development and resolution of pain, in the spinal dorsal horn. APOE regulates lipid metabolism and is predominantly expressed in the astrocytes. However, the effect of astrocytic APOE and lipid metabolism on spinal cellular function is unclear. This study aimed to investigate the effect of spinal Apoe on spinal cellular functions using the complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model. METHODS: After intraplantar injection of CFA, we assessed pain behaviors in C57BL6 and Apoe knockout (Apoe-/-) mice using von Frey and Hargreaves' tests and analyzed dorsal horn samples (L4-5) using western blotting, immunofluorescence, quantitative real-time polymerase chain reaction, and RNA sequencing. RESULTS: The Apoe levels were markedly upregulated at 2 h and on days 1 and 3 post-CFA treatment. Apoe was exclusively expressed in the astrocytes. Apoe-/- mice exhibited decreased pain on day 1, but not at 2 h, post-CFA treatment. Apoe-/- mice also showed decreased spinal neuron excitability and paw edema on day 1 post-CFA treatment. Global transcriptomic analysis of the dorsal horn on day 1 post-CFA treatment revealed that the differentially expressed mRNAs in Apoe-/- mice were associated with lipid metabolism and the immune system. Astrocyte activation was impaired in Apoe-/- mice on day 1 post-CFA treatment. The intrathecal injection of Apoe antisense oligonucleotide mitigated CFA-induced pain hypersensitivity. CONCLUSIONS: Apoe deficiency altered lipid metabolism in astrocytes, exerting regulatory effects on immune response, astrocyte activation, and neuronal activity and consequently disrupting the maintenance of inflammatory pain after peripheral inflammation. Targeting APOE is a potential anti-nociception and anti-inflammatory strategy.

Crosstalk between m6A modification and alternative splicing during cancer progression.

Background N6-methyladenosine (m6A), the most prevalent internal mRNA modification in eukaryotes, is added by m6A methyltransferases, removed by m6A demethylases and recognised by m6A-binding proteins. This modification significantly influences carious facets of RNA metabolism and plays a pivotal role in cellular and physiological processes. Main body Pre-mRNA alternative splicing, a process that generates multiple splice isoforms from multi-exon genes, contributes significantly to the protein diversity in mammals. Moreover, the presence of crosstalk between m6A modification and alternative splicing, with m6A modifications on pre-mRNAs exerting regulatory control, has been established. The m6A modification modulates alternative splicing patterns by recruiting specific RNA-binding proteins (RBPs) that regulate alternative splicing or by directly influencing the interaction between RBPs and their target RNAs. Conversely, alternative splicing can impact the deposition or recognition of m6A modification on mRNAs. The integration of m6A modifications has expanded the scope of therapeutic strategies for cancer treatment, while alternative splicing offers novel insights into the mechanistic role of m6A methylation in cancer initiation and progression. Conclusion This review aims to highlight the biological functions of alternative splicing of m6A modification machinery and its implications in tumourigenesis. Furthermore, we discuss the clinical relevance of understanding m6A-dependent alternative splicing in tumour therapies.

CoREST1 in primary sensory neurons regulates neuropathic pain in male mice.

AIMS: Epigenetic regulation is implicated in the neurogenesis of neuropathic pain. The repressor element 1 (RE1) silencing transcription factor (REST) corepressor (CoREST) proteins function as corepressors in the REST complex and/or LSD1 epigenetic complex. In the current study, we aimed to find the expression profile of CoREST1 in the dorsal root ganglion (DRG) and investigate whether it plays a role in neuropathic pain. MAIN METHODS: The evoked pain behaviors in mice were examined by the von Frey test and thermal test in a spinal nerve ligation (SNL)-induced neuropathic pain mice model. CoREST1 siRNA or virus was administered by DRG microinjection or intrathecal injection. The CoREST1 expression in DRGs was examined by immunofluorescence, quantitative PCR, Western blotting, and co-immunoprecipitation. KEY FINDINGS: CoREST1 was non-selectively expressed in large, medium, and small DRG neurons, and it exclusively colocalized with LSD1. In neuropathic pain models, peripheral nerve injury induced the upregulation of CoREST1 and increased binding of CoREST1 with LSD1 in injured DRGs in male mice. Furthermore, CoREST1 siRNA prevented the development of SNL-induced pain hypersensitivity as well as led to the reduction of established pain hypersensitivity during the maintenance period in SNL mice. Conversely, the overexpression of CoREST1 in DRGs by in vivo transfection of virus-induced pain hypersensitivity in naive mice. SIGNIFICANCE: Our study demonstrated that CoREST1, along with LSD1, was expressed in primary sensory neurons specifically in response to nerve injury, and promoted nociceptive pain hypersensitivity in mice. Thus, CoREST1 might serve as a potential target for treating neuropathic pain.

HPV E7-drived ALKBH5 promotes cervical cancer progression by modulating m6A modification of PAK5.

Human papillomavirus (HPV) infection is a causative agent of cervical cancer (CC). N6-methyladenosine (m6A) modification is implicated in carcinogenesis and tumor progression. However, the involvement of m6A modification in HPV-involved CC remains unclear. Here we showed that HPV E6/7 oncoproteins affected the global m6A modification and E7 specifically promoted the expression of ALKBH5. We found that ALKBH5 was significantly upregulated in CC and might serve as a valuable prognostic marker. Forced expression of ALKBH5 enhanced the malignant phenotypes of CC cells. Mechanistically, we discovered that E7 increased ALKBH5 expression through E2F1-mediated activation of the H3K27Ac and H3K4Me3 histone modifications, as well as post-translational modification mediated by DDX3. ALKBH5-mediated m6A demethylation enhanced the expression of PAK5. The m6A reader YTHDF2 bound to PAK5 mRNA and regulated its stability in an m6A-dependent manner. Moreover, ALKBH5 promoted tumorigenesis and metastasis of CC by regulating PAK5. Overall, our findings herein demonstrate a significant role of ALKBH5 in CC progression in HPV-positive cells. Thus, we propose that ALKBH5 may serve as a prognostic biomarker and therapeutic target for CC patients.

Phosphorylation of Neurofilament Light Chain in the VLO Is Correlated with Morphine-Induced Behavioral Sensitization in Rats.

Neurofilament light chain (NF-L) plays critical roles in synapses that are relevant to neuropsychiatric diseases. Despite postmortem evidence that NF-L is decreased in opiate abusers, its role and underlying mechanisms remain largely unknown. We found that the microinjection of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) into the ventrolateral orbital cortex (VLO) attenuated chronic morphine-induced behavioral sensitization. The microinjection of TSA blocked the chronic morphine-induced decrease of NF-L. However, our chromatin immunoprecipitation (ChIP)-qPCR results indicated that this effect was not due to the acetylation of histone H3-Lysine 9 and 14 binding to the NF-L promotor. In line with the behavioral phenotype, the microinjection of TSA also blocked the chronic morphine-induced increase of p-ERK/p-CREB/p-NF-L. Finally, we compared chronic and acute morphine-induced behavioral sensitization. We found that although both chronic and acute morphine-induced behavioral sensitization were accompanied by an increase of p-CREB/p-NF-L, TSA exhibited opposing effects on behavioral phenotype and molecular changes at different addiction contexts. Thus, our findings revealed a novel role of NF-L in morphine-induced behavioral sensitization, and therefore provided some correlational evidence of the involvement of NF-L in opiate addiction.

Nerve injury-induced upregulation of apolipoprotein E in dorsal root ganglion participates in neuropathic pain in male mice.

Apolipoprotein E (ApoE) is an apolipoprotein involved in lipid metabolism and is primarily responsible for lipid transport and cholesterol homeostasis in the central nervous system (CNS). The aim of this study is to explore the role of ApoE in the pathological development of neuropathic pain. First, we examined the location of ApoE in the dorsal root ganglion (DRG) and spinal cord in male mice using immunohistochemistry, and found that ApoE was predominantly expressed in DRG satellite glial cells (SGCs) and macrophages and spinal cord astrocytes. Using a spinal nerve ligation (SNL)-induced neuropathic pain mouse model, we found that nerve injury caused an increase in ApoE expression in the injured DRGs, but not in the spinal cord after SNL surgery. Furthermore, we observed reduced SNL-induced pain hypersensitivity in ApoE knockout mice compared to wild-type mice. Moreover, an antisense oligonucleotide (ASO) targeting the Apoe gene sequence, which was microinjected into the DRG or administered intrathecally, not only reduced ApoE expression in DRG but also attenuated SNL-induced pain hypersensitivity. Finally, we found that a tyrosine kinase receptor AXL, which was previously demonstrated to contribute to neuropathic pain, may mediate ApoE function under neuropathic pain condition. In conclusion, our data suggest that ApoE in DRG promote pain hypersensitivity via the DRG membrane receptor AXL in neurons under neuropathic pain conditions. This study revealed a novel mechanism between lipid homeostasis and neuropathic pain.

Alternative splicing of HSPA12A pre-RNA by SRSF11 contributes to metastasis potential of colorectal cancer.

BACKGROUND: Dysregulation of alternative splicing (AS) induced by serine/arginine-rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine-rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown. METHODS: The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA-seq technology was used to analyze and screen SRSF11-triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini-gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co-IP) and Phospho-tag SDS-PAGE experiments were used to investigate PAK5-mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction. RESULTS: In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro-metastatic role in vitro and in vivo. By screening SRSF11-regulated AS events, we identified the binding motif of SRSF11-triggered splicing-switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N-cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation. CONCLUSIONS: SRSF11 exerts pro-metastatic effects in CRC by inhibiting the AS of HSPA12A pre-RNA. Our findings point to SRSF11-regulated HSPA12A splicing as a novel relationship between SRSF11-regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC.

Lysine-specific demethylase 1 in primary sensory neurons participates in chronic compression of dorsal root ganglion-induced neuropathic pain.

Low back and radicular pain syndromes, usually caused by local inflammation and irritation to the nerve root and dorsal root ganglion (DRG), are common throughout medical practice, but sufficient pain relief is scarce. In this study, we employed a chronic compression of DRG (CCD)-induced radicular pain model in rats to explore whether lysine-specific demethylase 1 (LSD1), a histone demethylase and transcriptional co-repressor, is involved in the pathological process of radicular pain. We found that LSD1 was expressed in various-sized DRG neurons by immunohistochemistry. CCD induced the upregulation of LSD1 in compressed L4-L5 DRGs. Moreover, either LSD1 small interfering RNAs or LSD1 inhibitor attenuated CCD-induced pain hypersensitivities. LSD1 was also upregulated in the injured lumbar 4 (L4) DRG in a spinal nerve ligation (SNL)-induced neuropathic pain mouse model. Nevertheless, LSD1 was not altered in L3-L5 DRGs in complete Freund's adjuvant-induced inflammatory pain mouse model, paclitaxel- or streptozotocin-induced neuropathic pain models. Furthermore, knockdown of LSD1 in the injured L4 DRG reversed SNL-induced pain hypersensitivities in mice. Therefore, we speculate that nerve injury induced the upregulation of LSD1 in the injured DRGs, which contributes to neuropathic pain hypersensitivities; thus, LSD1 may serve as a potential target for the treatment of radicular pain and neuropathic pain.

METTL3 potentiates progression of cervical cancer by suppressing ER stress via regulating m6A modification of TXNDC5 mRNA.

N6-methyladenosine (m6A) is the most abundant chemical modification on mRNA and plays significant roles in many bioprocesses. However, the functions of m6A on cervical cancer (CC) tumorigenesis remain unclear. Here we found methyltransferase-like 3 (METTL3), a core member of the m6A methyltransferase family, was greatly upregulated as an independent prognostic factor in CC. Mechanistically, the transcription factor ETS1 recruited P300 and WDR5 which separately mediated H3K27ac and H3K4me3 histone modification in the promoter of METTL3 and induced METTL3 transcription activation. Furthermore, we identified TXNDC5 as a target of METTL3-mediated m6A modification through MeRIP-seq, and revealed that METTL3-mediated TXNDC5 expression relied on the m6A reader-dependent manner. Functionally, we verified that METTL3 promoted proliferation and metastasis of CC cells by regulating of TXNDC5 expression through in vitro and in vivo experiments. In addition, our study verified the effect of METTL3/TXNDC5 axis on ER stress. Taken together, METTL3 facilitates the malignant progression of CC, suggesting that METTL3 might be a potential prognostic biomarker and therapeutic target for CC.

SHMT2 promotes the tumorigenesis of renal cell carcinoma by regulating the m6A modification of PPAT.

OBJECTIVE: Serine hydroxymethyltransferase 2 (SHMT2) is the first rate-limiting enzyme for serine/glycine biosynthesis and one carbon metabolism. Here, we explore the underlying mechanism of how SHMT2 functions in renal cell carcinoma (RCC) initiation. METHODS: In this study, SHMT2 expression was assessed in RCC tissues. In vitro experiments were performed to investigate the functional role of SHMT2. The detailed mechanisms of SHMT2-mediated PPAT were addressed. RESULTS: Increased SHMT2 facilitated RCC cell proliferation by inducing the G1/S phase transition. And SHMT2 promoted the expression of PPAT. Mechanism dissection revealed that SHMT2 enhanced the m6A modification through the endogenous methyl donor SAM mediated by SHMT2 via serine/glycine one carbon metabolic networks. SHMT2-catalyzed serine/glycine conversion regulated PPAT expression in an m6A-IGF2BP2-dependent manner. SHMT2 promoted RCC cell proliferation by upregulating PPAT expression. CONCLUSIONS: SHMT2 promotes RCC tumorigenesis by increasing PPAT expression. Thus, SHMT2 may be a novel potential therapeutic target for RCC.

Genome-Wide Identification and Transcriptional Expression Profiles of PP2C in the Barley (Hordeum vulgare L.) Pan-Genome.

The gene family protein phosphatase 2C (PP2C) is related to developmental processes and stress responses in plants. Barley (Hordeum vulgare L.) is a popular cereal crop that is primarily utilized for human consumption and nutrition. However, there is little knowledge regarding the PP2C gene family in barley. In this study, a total of 1635 PP2C genes were identified in 20 barley pan-genome accessions. Then, chromosome localization, physical and chemical feature predictions and subcellular localization were systematically analyzed. One wild barley accession (B1K-04-12) and one cultivated barley (Morex) were chosen as representatives to further analyze and compare the differences in HvPP2Cs between wild and cultivated barley. Phylogenetic analysis showed that these HvPP2Cs were divided into 12 subgroups. Additionally, gene structure, conserved domain and motif, gene duplication event detection, interaction networks and gene expression profiles were analyzed in accessions Morex and B1K-04-12. In addition, qRT-PCR experiments in Morex indicated that seven HvMorexPP2C genes were involved in the response to aluminum and low pH stresses. Finally, a series of positively selected homologous genes were identified between wild accession B1K-04-12 and another 14 cultivated materials, indicating that these genes are important during barley domestication. This work provides a global overview of the putative physiological and biological functions of PP2C genes in barley. We provide a broad framework for understanding the domestication- and evolutionary-induced changes in PP2C genes between wild and cultivated barley.

Spinal 5-HT2A receptor is involved in electroacupuncture inhibition of chronic pain.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, and it is a typical disease which can develop chronic pain. Our previous study has proved that endocannabinoid (2-AG)-CB1R-GABA-5-HT pathway is involved in electroacupuncture (EA) mediated inhibition of chronic pain. However, it is still unclear which among the 5-HT receptor subtype is involved in EA evoked 5-HT mediated inhibition of chronic pain in the dorsal spinal cord. 5-HT2A is a G protein-coupled receptor and it is involved in 5-HT descending pain modulation system. We found that EA treatment at frequency of 2 Hz +1 mA significantly increased the expression of 5-HT2A receptor in the dorsal spinal cord and intrathecal injection of 5-HT2A receptor antagonist or agonist reversed or mimicked the analgesic effect of EA in each case respectively. Intrathecal injection of a selective GABAA receptor antagonist Bicuculline also reversed the EA effect on pain hypersensitivity. Additionally, EA treatment reversed the reduced expression of GABAA receptor and KCC2 in the dorsal spinal cord of KOA mice. Furthermore, we demonstrated that intrathecal 5-HT2A receptor antagonist/agonist reversed or mimicked the effect of EA up-regulate of KCC2 expression, respectively. Similarly, intrathecal injection of PLC and PKC inhibitors prevented both anti-allodynic effect and up-regulation of KCC2 expression by EA treatment. Our data suggest that EA treatment up-regulated KCC2 expression through activating 5-HT2A-Gq-PLC-PKC pathway and enhanced the inhibitory function of GABAA receptor, thereby inhibiting chronic pain in a mouse model of KOA.

5-HT7 Receptor Is Involved in Electroacupuncture Inhibition of Chronic Pain in the Spinal Cord.

Knee osteoarthritis (KOA) is a common and disabling condition characterized by attacks of pain around the joints, and it is a typical disease that develops chronic pain. Previous studies have proved that 5-HT1, 5-HT2, and 5-HT3 receptors in the spinal cord are involved in electroacupuncture (EA) analgesia. The 5-HT7 receptor plays antinociceptive role in the spinal cord. However, it is unclear whether the 5-HT7 receptor is involved in EA analgesia. The 5-HT7 receptor is a stimulatory G-protein (Gs)-coupled receptor that activates adenylyl cyclase (AC) to stimulate cyclic adenosine monophosphate (cAMP) formation, which in turn activates protein kinase A (PKA). In the present study, we found that EA significantly increased the tactile threshold and the expression of the 5-HT7 receptor in the dorsal spinal cord. Intrathecal injection of 5-HT7 receptor agonist AS-19 mimicked the analgesic effect of EA, while a selective 5-HT7 receptor antagonist reversed this effect. Moreover, intrathecal injection of AC and PKA antagonists prior to EA intervention prevented its anti-allodynic effect. In addition, GABAA receptor antagonist bicuculline administered (intrathecal, i.t.) prior to EA intervention blocked the EA effect on pain hypersensitivity. Our data suggest that the spinal 5-HT7 receptor activates GABAergic neurons through the Gs-cAMP-PKA pathway and participates in EA-mediated inhibition of chronic pain in a mouse model of KOA.

MicroRNA-138-1-3p sensitizes sorafenib to hepatocellular carcinoma by targeting PAK5 mediated ß-catenin/ABCB1 signaling pathway.

BACKGROUND: Sorafenib is a kinase inhibitor that is used as a first-line therapy in advanced hepatocellular carcinoma (HCC) patients. However, the existence of sorafenib resistance has limited its therapeutic effect. Through RNA sequencing, we demonstrated that miR-138-1-3p was downregulated in sorafenib resistant HCC cell lines. This study aimed to investigate the role of miR-138-1-3p in sorafenib resistance of HCC. METHODS: In this study, quantitative real-time PCR (qPCR) and Western Blot were utilized to detect the levels of PAK5 in sorafenib-resistant HCC cells and parental cells. The biological functions of miR-138-1-3p and PAK5 in sorafenib-resistant cells and their parental cells were explored by cell viability assays and flow cytometric analyses. The mechanisms for the involvement of PAK5 were examined via co-immunoprecipitation (co-IP), immunofluorescence, dual luciferase reporter assay and chromatin immunoprecipitation (ChIP). The effects of miR-138-1-3p and PAK5 on HCC sorafenib resistant characteristics were investigated by a xenotransplantation model. RESULTS: We detected significant down-regulation of miR-138-1-3p and up-regulation of PAK5 in sorafenib-resistance HCC cell lines. Mechanistic studies revealed that miR-138-1-3p reduced the protein expression of PAK5 by directly targeting the 3'-UTR of PAK5 mRNA. In addition, we verified that PAK5 enhanced the phosphorylation and nuclear translocation of ß-catenin that increased the transcriptional activity of a multidrug resistance protein ABCB1. CONCLUSIONS: PAK5 contributed to the sorafenib resistant characteristics of HCC via ß-catenin/ABCB1 signaling pathway. Our findings identified the correlation between miR-138-1-3p and PAK5 and the molecular mechanisms of PAK5-mediated sorafenib resistance in HCC, which provided a potential therapeutic target in advanced HCC patients.

METTL3-mediated m6A methylation of SPHK2 promotes gastric cancer progression by targeting KLF2.

N6-methyladenosine (m6A) RNA methylation is profoundly involved in epigenetic regulation, especially for carcinogenesis and tumor progression. Mounting evidence suggests that methyltransferase METTL3 regulates malignant behaviors of gastric cancer (GC). However, the clinical significance and biological implication of SPHK2 and its related m6A modification in GC remain unclear. In this study, quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were utilized to detect the expression profiles and prognostic significance of SPHK2 in GC. Here, we showed that increased SPHK2 was signified a poor prognosis of GC patients. Phosphorylation and ubiquitination assays were used to investigate the possible mechanisms of SPHK2-mediated KLF2 expression. SPHK2 can promote the phosphorylation of KLF2, which triggers the ubiquitination and degradation of KLF2 protein in GC. Methylated RNA immunoprecipitation (MeRIP) was performed to uncover the m6A modification of SPHK2 mRNA. METTL3 promotes translation of SPHK2 mRNA via an m6A-YTHDF1-dependent manner. Functionally, SPHK2 facilitates GC cell proliferation, migration and invasion by inhibiting KLF2 expression. SPHK2/KLF2 regulates the cell proliferation, migration, and invasion induced by METTL3 in GC. Overall, our findings reveal that METTL3-mediated m6A modification of SPHK2 contributes to GC progression, which extends the understanding of the importance m6A methylation in GC and represents a potential target for GC therapy.