Inhibiting S-palmitoylation arrests metastasis by relocating Rap2b from plasma membrane in colorectal cancer.

Rap2b, a proto-oncogene upregulated in colorectal cancer (CRC), undergoes protein S-palmitoylation at specific C-terminus sites (C176/C177). These palmitoylation sites are crucial for Rap2b localization on the plasma membrane (PM), as mutation of C176 or C177 results in cytosolic relocation of Rap2b. Our study demonstrates that Rap2b influences cell migration and invasion in CRC cells, independent of proliferation, and this activity relies on its palmitoylation. We identify ABHD17a as the depalmitoylating enzyme for Rap2b, altering PM localization and inhibiting cell migration and invasion. EGFR/PI3K signaling regulates Rap2b palmitoylation, with PI3K phosphorylating ABHD17a to modulate its activity. These findings highlight the potential of targeting Rap2b palmitoylation as an intervention strategy. Blocking the C176/C177 sites using an interacting peptide attenuates Rap2b palmitoylation, disrupting PM localization, and suppressing CRC metastasis. This study offers insights into therapeutic approaches targeting Rap2b palmitoylation for the treatment of metastatic CRC, presenting opportunities to improve patient outcomes.

High expression of GPR50 promotes the proliferation, migration and autophagy of hepatocellular carcinoma cells in vitro.

G protein-coupled receptors (GPCRs) play important roles in tumorigenesis and the development of hepatocellular carcinoma (HCC). GPR50 is an orphan GPCR. Previous studies have indicated that GPR50 could protect against breast cancer development and decrease tumor growth in a xenograft mouse model. However, its role in HCC remains indistinct. To detect the role and the regulation mechanism of GPR50 in HCC, GPR50 expression was analyzed in HCC patients (gene expression omnibus database (GEO) (GSE45436)) and detected in HCC cell line CBRH-7919, and the results showed that GPR50 was significantly up-regulated in HCC patients and CBRH-7919 cell line compared to the corresponding normal control. Gpr50 cDNA was transfected into HCC cell line CBRH-7919, and we found that Gpr50 promoted the proliferation, migration, and autophagy of CBRH-7919. The regulation mechanism of GPR50 in HCC was detected by isobaric tags for relative and absolute quantification (iTRAQ) analysis, and we found that GPR50 promoted HCC was closely related to CCT6A and PGK1. Taken together, GPR50 may promote HCC progression via CCT6A-induced proliferation and PGK1-induced migration and autophagy, and GPR50 could be an important target for HCC.

The orphan GPR50 receptor interacting with TßRI induces G1/S-phase cell cycle arrest via Smad3-p27/p21 in BRL-3A cells.

The liver has the powerful capacity to regenerate after injury or resection. In one of our previous studies, GPR50 was observed to be significantly upregulated at 6 h, following a partial hepatectomy (PH) in rat liver regeneration (LR) via gene expression profile. However, little research has been done on the regulation and mechanism of GPR50 in the liver. Herein, we observed that the overexpression of GPR50 inhibited the proliferation of BRL-3A cells. To further explore the molecular mechanisms of GPR50 in the regulation of BRL-3A cell proliferation, interaction between GPR50 and transforming growth factor-beta I (TßRI) and iTRAQTM differential proteomic analysis were elucidated, which suggested that GPR50 may interact with TßRI to activate the TGF-ß signaling pathway and arrest BRL-3A cell cycle G1/S transition. Subsequently, the potential mechanism underlying the role of GPR50 in hepatocyte growth was also explored through the addition of a signaling pathway inhibitor. These data suggested that interaction between the orphan GPR50 receptor and TßRI induced the G1/S-phase cell cycle arrest of BRL-3A cells via the Smad3-p27/p21 pathway.

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p.

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.

Serine peptidase inhibitor Kazal type III (SPINK3) promotes BRL-3A cell proliferation by targeting the PI3K-AKT signaling pathway.

The serine protease inhibitor, Kazal type III (SPINK3), is a trypsin inhibitor associated with liver disease, which highly overexpresses in a variety of cancers. In one of our previous studies of our laboratory, Spink3 was observed to be significantly upregulated in rat liver regeneration (LR) via a gene expression profile. For the current study, rat hepatocyte BRL-3A cells were treated by gene addition/interference, and the addition of the exogenous rat recombinant protein SPINK3. It was revealed that both the overexpression of endogenous Spink3 and addition of exogenous rat recombinant SPINK3 (rrSPINK3) significantly promoted the cell proliferation of BRL-3A cells, whereas cell proliferation was inhibited when Spink3 was interfered. Furthermore, quantitative reverse transcription polymerase chain reaction and western blot results revealed that three signaling pathways, including extracellular-signal-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK)-signal transducer and activator of transcription (STAT), and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT), as well as their related genes, were altered following endogenous Spink3 addition/interference. Also, the PI3K-AKT and SRC-p38 pathways and their related genes were modified following exogenous SPINK3 treatment. Among them, the common signaling pathway was PI3K-AKT pathway. We concluded that SPINK3 could activate the PI3K-AKT pathway by enhancing the expression of AKT1 to regulate the proliferation of BRL-3A cells. This study may contribute to shedding light on the potential mechanisms of SPINK3 that regulate the proliferation of BRL-3A cells.

FAM134B promotes adipogenesis by increasing vesicular activity in porcine and 3T3-L1 adipocytes.

Family with sequence similarity 134, Member B (FAM134B), is a cis-Golgi transmembrane protein that is known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in autophagy-mediated turnover of endoplasmic reticulum (ER) membranes, tumor inhibition and lipid homeostasis. In this study, we provide mechanistic links between FAM134B and ARF-related protein 1 (ARFRP1) and further show that FAM134B resides in the Golgi apparatus. Here, we found that FAM134B increased lipid accumulation in adipocytes. Transport vehicle number and ADP-ribosylation factor (ARF) family gene expression were also increased by FAM134B overexpression, suggesting that vesicular transport activity enhanced lipid accumulation. ARF-related protein 1 (ARFRP1) is a GTPase that promotes protein trafficking. We show that FAM134B regulates the expression of ARFRP1, and the knockdown of ARFRP1 abolishes enhancement on lipid accumulation caused by FAM134B. In addition, FAM134B upregulates the PAT family protein (PAT), which associates with the lipid droplets (LDs) surface and promotes lipolysis by recruiting adipocyte triglyceride lipase (ATGL). These findings indicate that FAM134B promotes lipid accumulation and adipogenic differentiation by increasing vesicle transport activity in the Golgi apparatus and inhibiting the lipolysis of LDs.

Roles of Cyclin A, Myc, Jun and Ppm1l in tumourigenic transformation of NIH3T3 cell.

To analyse the mechanism of tumourigenic transformation of NIH3T3 cells at the transcriptional level, we used cancerogen 3-methylcholanthrene (3-MCA) and cancerogenic substance phorbol-12-myristate-13-acetate (PMA) to transform NIH3T3 cells and the assessment of transformation was performed using Giemsa staining and methylcellulose colony formation assay. Changes in gene expression profile were detected by Mouse Genome 430 2.0 microarray; and quantitative real-time polymerase chain reaction and Western blotting were used to verify the expression changes of mRNAs and proteins, respectively. With the aid of bioinformatics method, five signalling pathways were identified to participate in different stages of NIH3T3 cell transformation. Further, our study suggested that oncogenes Cyclin A, Myc, Jun and the tumour suppressor gene Ppm1l may play important roles in these pathways.

In situ fabrication of cobalt nanoflowers on sulfonated and fluorinated poly (arylene ether ketone-benzimidazole) template film for the electrocatalytic oxidation of glucose.

Using sulfonated and fluorinated poly (arylene ether ketone) comprising functional strong coordination group benzimidazole (SPAEK-F-BI) as a template film, a novel fabrication method of cobalt nanoflowers (CoNFs) and non-enzymatic glucose electrochemical sensor was developed in this work. After the precursors Co2+ ions were cooperatively bound by sulfonate and imidazole functionalities contained in SPAEK-F-BI film through ion exchange and strong coordination action, cobalt colloid nuclei were formed and grew to flower-like nanostructures by subsequent in-situ electrochemical reduction on SPAEK-F-BI film modified GCE. Characterization of SPAEK-F-BI film and CoNFs/SPAEK-F-BI film on GCE was performed in detail by FT-IR spectroscopy and scanning electron microscopy (SEM) attached with energy dispersive spectroscopy (EDS), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The results of SEM showed that beautiful CoNFs constructed by Co colloid nanosheets with just a few nanometers thickness were well dispersed on uniform SPAEK-F-BI film modified GCE, and the density of CoNFs was mainly influenced by the concentration of the precursor solution CoSO4. The CoNFs/SPAEK-F-BI composite modified electrode exhibited good electrocatalytic activity toward glucose oxidation in 0.1M NaOH solution, and the kinetic parameters of glucose oxidation were determined using chronoamperometry. When it was applied for the determination of glucose by amperometry at a potential of 0.6V versus Ag/AgCl, the linear range from 5µM to 1.14mM and the detection limit of 800nM (S/N = 3) were obtained. Finally, it was successfully employed to detect the glucose in human serum real samples, and the results were agreed closely with those measured in hospital.

Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways.

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G2 /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.