Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: Cellular metabolism in human myotubes after miR-136-3p transfection or NRDC silencing. Mitochondrial respiration in miR-136-3p-transfected or NRDC- silenced human myotubes was monitored using the Mitochondrial Stress Test. (A) OCR and (B) ECAR were measured using the Seahorse XFe24 Extracellular Flux Analyzer. The trace shows representative data. (C) Quantification of the mitochondrial respiration data for basal respiration, maximal respiration, ATP production, and spare respiratory capacity obtained from 3 independent experiments. Human myotubes were transfected with miR-136-3p or siRNA against NRDC before determination of (D) uptake of radiolabeled glucose, (E) rates of radiolabeled glucose oxidation, (F) conversion of radiolabeled glucose into glycogen, (G) rate of radiolabeled palmitic acid oxidation, (H) protein synthesis as assessed by incorporation of puromycin, and (I) lactate release into the media. Results are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.005 vs. control cells. ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; ns = no significance; OCR = oxygen consumption rate; OigoA = oligomycin A; Rot/AA = rotenone and antimycin A; si NRDC = small interfering RNA of NRDC; siRNA = small interfering RNA; scr = negative control for small interfering RNA.

Article Snippet: MiR-136-3p was labeled with Cy3 using Silencer small interfering RNA (siRNA) Labeling Kit with Cy3 Dye (Thermo Fisher Scientific) and loaded into exosome-enriched EVs with Exo-Fect siRNA/miRNA Transfection Reagent (System Biosciences, Palo Alto, CA, USA).

Techniques: Transfection, Control, Negative Control, Small Interfering RNA

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: lncRNA TDRG1 acts as a piRNA precursor to generate piRNAs A. RT‒qPCR analysis of TDRG1 overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal reference; empty vector transfection as the control (Ctrl). The relative expression was calculated relative to the expression level of Gapdh . Relative expression was normalized to Gapdh , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001). B. Expression levels of TDRG1 -related piRNAs after TDRG1 overexpression in GC-2 spd(ts), detected by RT‒qPCR. U6 snRNA was used as the internal reference; empty vector transfection as the control (Ctrl). Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequences overlap to 1700008K24Rik were used as negative controls. Relative expression was normalized to U6 , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: no significant difference).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: RNA-seq analysis of 1700008K24Rik -overexpressing GC-2 spd(ts) cells A . Volcano plot displaying differentially expressed genes (DEGs) in GC-2 spd(ts) cells overexpressing 1700008K24Rik versus control, based on RNA-seq data. The x-axis shows log 2 (fold change) the y-axis shows -log 10 ( P value). Red, green, and gray dots indicate up-regulated, down-regulated and non-DEGs, respectively. B-C . Gene Ontology (GO) enrichment analysis of up-regulated (B) and down-regulated (C) genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Selected biological processes that were potentially involved are listed on the right. D . KEGG pathway enrichment analysis of down-regulated genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Related pathways that were potentially involved are listed on the right. E-F . RT‒qPCR validation of RNA-seq results for up-regulated (E) and down-regulated (F) genes following 1700008K24Rik overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal control; empty vector transfectionas the experimental control (Ctrl). Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, NS: not significant). G . Predicted targets of the 1700008K24Rik-related piRNAs among the differentially expressed genes, analyzed using miRanda algorithm. Shown are eight spermatogenesis-related genes harboring potential piRNA targeting sites.

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: RNA Sequencing, Control, Over Expression, Biomarker Discovery, Plasmid Preparation, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: Knockdown of 1700008K24Rik impairs mouse spermatogenesis. A . Visualization of AAV9 virus delivery into mouse seminiferous tubules, with trypan blue indicating successful injection. B . RT‒qPCR analysis of 1700008K24Rik knockdown efficiency in mouse testes. Three-week-old mice were injected with AAV9-shRNA-GFP (shRNA) and analyzed after one month. The contralateral testis injected nontargeted shRNA (shCtrl) served as the control. Gapdh was used as the internal reference. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗∗ P < 0.01, NS: not significant). C . Representative images of testis size AAV9-shRNA-GFP (shRNA) and AAV9-shCtrl-GFP (shCtrl) injected mice. Viruses were delivered at 3 weeks of age; tissues were collected one month post-injection. n = 3. D . Testis weight quantification of shRNA and shCtrl groups. Relative testis weight was normalized to shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001), n = 3. E . H&E staining of paraffin-embedded testis sections from shRNA and shCtrl groups. Scale bars: 100 μm (overview) and 20 μm (magnified). F . Seminiferous epithelium thickness measurement in shRNA and shCtrl groups. Thickness was assessed from five H&E-stained sections per testis. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01). G . Epididymal sperm counts in shRNA and shCtrl groups. Relative sperm count was calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗ P < 0.01), n = 3. H . Expression levels of 1700008K24Rik -related piRNAs in shRNA or shCtrl testes, detected by RT‒qPCR. Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequence overlap to 1700008K24Rik were used as negative controls. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: not significant).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Knockdown, Virus, Injection, shRNA, Control, Expressing, Staining, Sequencing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell viability and proliferation (A–C) Western blot analysis of transfection efficiency for SPI1 overexpression and knockdown ( n = 3, n represents biological replicates). (D) CCK-8 assay assessing the effects of SPI1 overexpression and knockdown on NSCLC cell viability ( n = 6, n represents biological replicates). (E) Colony formation assay evaluating the impact of SPI1 overexpression and knockdown on NSCLC cell proliferation ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Western Blot, Transfection, Over Expression, Knockdown, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell migration, invasion, and EMT (A) Transwell assay assessing the effects of SPI1 on cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. (B) Western blot analysis of the impact of SPI1 on the expression levels of EMT-related proteins (N-cadherin, E-cadherin, and vimentin, n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Ⅰ, N-cadherin. Ⅱ, E-cadherin. Ⅲ, vimentin. Ⅳ, GAPDH.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Migration, Transwell Assay, Western Blot, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 promotes NSCLC cell invasion and migration by transcriptionally regulating miR-616-5p (A and B) RT-PCR analysis of miR-616-5p expression following SPI1 overexpression or knockdown ( n = 3, n represents biological replicates). (C–E) Transwell assay evaluating cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Knockdown, Transwell Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SF downregulates miR-616-5p activity by directly binding to and inhibiting SPI1 (A) Molecular docking analysis of SF with SPI1. (B) SPRi binding curve of SF to SPI1. (C and D) Western blot analysis of SPI1 expression in cells ( n = 3, n represents biological replicates). (E) RT-PCR analysis of miR-616-5p expression in cells ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: Activity Assay, Binding Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: In vivo targeting and anti-NSCLC efficacy of MSNs@SF-HA-FA (A) In vivo fluorescence imaging analysis ( n = 5, n represents the number of mice). (B) Ex vivo fluorescence imaging of major organs ( n = 5, n represents the number of mice). (C) Images of lung tumor tissues from mice ( n = 5, n represents the number of mice). (D) H&E-stained images and immunohistochemical analysis of SPI1 expression in xenograft tumors ( n = 5, n represents the number of mice), scale bars, 100 μm.

Article Snippet: Lentiviral shRNA targeting human SPI1 , GeneChem , N/A.

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Staining, Immunohistochemical staining, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell viability and proliferation (A–C) Western blot analysis of transfection efficiency for SPI1 overexpression and knockdown ( n = 3, n represents biological replicates). (D) CCK-8 assay assessing the effects of SPI1 overexpression and knockdown on NSCLC cell viability ( n = 6, n represents biological replicates). (E) Colony formation assay evaluating the impact of SPI1 overexpression and knockdown on NSCLC cell proliferation ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Western Blot, Transfection, Over Expression, Knockdown, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 regulates NSCLC cell migration, invasion, and EMT (A) Transwell assay assessing the effects of SPI1 on cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. (B) Western blot analysis of the impact of SPI1 on the expression levels of EMT-related proteins (N-cadherin, E-cadherin, and vimentin, n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Ⅰ, N-cadherin. Ⅱ, E-cadherin. Ⅲ, vimentin. Ⅳ, GAPDH.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Migration, Transwell Assay, Western Blot, Expressing

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SPI1 promotes NSCLC cell invasion and migration by transcriptionally regulating miR-616-5p (A and B) RT-PCR analysis of miR-616-5p expression following SPI1 overexpression or knockdown ( n = 3, n represents biological replicates). (C–E) Transwell assay evaluating cell migration and invasion ( n = 3, n represents biological replicates), scale bars, 200 μm. Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Knockdown, Transwell Assay

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: SF downregulates miR-616-5p activity by directly binding to and inhibiting SPI1 (A) Molecular docking analysis of SF with SPI1. (B) SPRi binding curve of SF to SPI1. (C and D) Western blot analysis of SPI1 expression in cells ( n = 3, n represents biological replicates). (E) RT-PCR analysis of miR-616-5p expression in cells ( n = 3, n represents biological replicates). Data are represented as mean ± SD. p values are based on a one-way ANOVA test. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: Hyaluronic acid and folic acid-modified mesoporous silica nanoparticles delivering sulforaphane suppress NSCLC via SPI1/miR-616-5p axis

doi: 10.1016/j.isci.2026.116293

Figure Lengend Snippet: In vivo targeting and anti-NSCLC efficacy of MSNs@SF-HA-FA (A) In vivo fluorescence imaging analysis ( n = 5, n represents the number of mice). (B) Ex vivo fluorescence imaging of major organs ( n = 5, n represents the number of mice). (C) Images of lung tumor tissues from mice ( n = 5, n represents the number of mice). (D) H&E-stained images and immunohistochemical analysis of SPI1 expression in xenograft tumors ( n = 5, n represents the number of mice), scale bars, 100 μm.

Article Snippet: Specific short hairpin RNA targeting SPI1 (sh-SPI1) and the SPI1 overexpression construct (OE-SPI1) were purchased from GeneChem (Shanghai, China).

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Staining, Immunohistochemical staining, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: Metastasis-related 12-gene signature, and identification of UPK1B and CGB5 as independent prognostic markers associated with advanced metastatic GC. (A) Venn diagram showing the intersection of genes upregulated in N1-3 stage and M1 stage GC tissues. (B) LASSO Cox regression analysis based on TCGA-STAD data identified a metastasis-associated prognostic risk signature. LASSO coefficient profiles of the signature genes in the merged dataset are shown (top) and the coefficient profile plot is generated against the log(λ) sequence (bottom). (C) The high-risk group of patients with GC exhibited a poor prognosis (overall survival). High expression of (D) CGB5 and (E) UPK1B was associated with poor overall survival in patients with GC based on data from the TCGA-STAD cohort. (F) Elevated UPK1B expression predicted poor prognosis (overall survival) of patients with GC in the Kaplan-Meier plotter database. UPK1B expression was increased in (G) M1 compared with M0 and in (H) N2 metastatic stage GC tissues compared with N0 stage. UPK1B, uroplakin 1B; CGB5, chorionic gonadotropin subunit β-5; GC, gastric cancer; LASSO, Least Absolute Shrinkage and Selection Operator; TCGA-STAD, The Cancer Genome Atlas Stomach Adenocarcinoma; HR, hazard ratio; TPM, transcripts per million.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Generated, Sequencing, Expressing, Selection

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B drives GC cell invasion and migration in a PI3K/AKT-dependent manner. (A) Gene set enrichment analysis indicated that genes upregulated in the UPK1B-high group were enriched in the PI3K/AKT pathway. (B) Protein levels of UPK1B in GC cell lines. (C) Knockdown of UPK1B reduced PI3K/AKT activation in MKN45 cells. Silencing UPK1B suppressed the (D) migration/invasion capacity and (E) wound closure rate of MKN45 cells. (F) Overexpression of UPK1B enhanced PI3K/AKT pathway activation in AGS cells, which was attenuated by the PI3K inhibitor LY294002. Inhibition of PI3K/AKT signaling reversed UPK1B-induced (G) migration/invasion capacity and (H) wound closure rate of AGS cells. UPK1B, uroplakin 1B; GC, gastric cancer; p-, phosphorylated; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Migration, Knockdown, Activation Assay, Over Expression, Inhibition, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: CDX2 acts as a transcriptional repressor of UPK1B and its high expression is associated with favorable prognosis of patients with GC. (A) Venn diagram showing overlapping predicted transcriptional regulators of UPK1B from ChEA and ChEA3 databases. (B) Knockdown of CDX2 in AGS cells resulted in increased UPK1B (C) mRNA and (D) protein expression. (E) Overexpression of CDX2 in MKN45 cells reduced UPK1B protein levels. Data from (F) The Cancer Genome Atlas Stomach Adenocarcinoma cohort and (G) the Kaplan-Meier plotter database indicated that high CDX2 expression was associated with improved prognosis of patients with GC. UPK1B, uroplakin 1B; GC, gastric cancer; si, small interfering RNA; NC, negative control; OE, overexpression; HR, hazard ratio; CDX2, caudal-related homeobox transcription factor 2; ChEA, ChIP-X Enrichment Analysis.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Expressing, Knockdown, Over Expression, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: For gene knockdown experiments, cells were transfected with a GV493 short hairpin RNA (sh) plasmid targeting UPK1B ( ) or a negative control scrambled plasmid (sh-NC), both obtained from GeneChem, Inc.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: LINC00184 regulates NDRG2 expression through DNMT1-mediated methylation of the NDRG2 promoter. (A) Expression level of LINC00184 in KYSE-150 cells following treatment with siRNA targeting LINC00184 (n=3). (B) Expression level of LINC00184 in TE-1 cells following treatment with siRNA targeting LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in KYSE-150 cells detected via MSP assay after overexpression or silencing of LINC00184. (D) Methylation level of the NDRG2 promoter in TE-1 cells detected via MSP assay after overexpression or silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2 promoter region detected by chromatin immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells with overexpression or silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in KYSE-150 cells after overexpression or silencing of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1 detected by RNA immunoprecipitation assay and quantified using RT-qPCR in TE-1 cells after overexpression or silencing of LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in KYSE-150 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (I) Methylation level of the NDRG2 promoter in TE-1 cells measured by MSP assay following LINC00184 overexpression combined with 5-AZA treatment. (J) Western blotting analysis of NDRG2 protein expression in KYSE-150 cells after LINC00184 overexpression and 5-AZA intervention. (K) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (J) (n=3). (L) Western blotting analysis of NDRG2 protein expression in TE-150 cells after LINC00184 overexpression and 5-AZA intervention. (M) Quantitative analysis of NDRG2 protein grayscale values obtained from the western blotting results in (L) (n=3). (N) Western blotting detection of NDRG2 protein levels under control conditions, single overexpression of LINC00184, and combined treatment with OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1 mRNA levels in control cells and LINC00184-overexpressing cells (n=6). (P) Western blotting analysis of total DNMT1 protein levels in control cells and LINC00184-overexpressing cells; GAPDH was used as the loading control (n=3). Data are presented as mean ± SEM (n=3). Comparisons between two groups were performed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA, long non-coding RNA; 5-AZA, 5-azacytidine; MSP, methylation-specific PCR. M, methylation; U, unmethylation.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Expressing, Methylation, MSP Assay, Over Expression, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Control, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: Inhibition of DNMT1 abrogates LINC00184-induced PI3K/AKT pathway activation and functional phenotypes. (A) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells following overexpression of LINC00184 and treatment with 5-AZA. (B) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (A). (C) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (A). (D) Western blotting analysis showing the protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells following overexpression of LINC00184 and treatment with 5-AZA. (E) Quantitative analysis of the pAKT/AKT protein expression level derived from the immunoblots in (D). (F) Quantitative analysis of the pPI3K/PI3K protein expression level derived from the immunoblots in (D). (G) Western blotting analysis of p-AKT and total AKT levels in esophageal squamous cell carcinoma cells under the following conditions: Control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein expression level corresponding to the immunoblots presented in (G). Data are presented as mean ± SEM (n=3). Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; NDRG2, N-Myc downstream regulated gene; RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Activation Assay, Functional Assay, Western Blot, Expressing, Over Expression, Derivative Assay, Control, Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Letters

Article Title: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1-mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

doi: 10.3892/ol.2026.15628

Figure Lengend Snippet: DNMT1 inhibition reverses LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell viability of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (B) Representative images of migrated KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (C) Representative flow cytometry dot plots showing the apoptosis rate of KYSE-150 cells following LINC00184 overexpression and treatment with 5-AZA. (D) Statistical counting of migrated KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative analysis of the apoptosis rate of KYSE-150 cells corresponding to (C) (n=3). (F) Cell viability of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA (n=3). (G) Representative images of migrated TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (H) Representative flow cytometry dot plots showing the apoptosis rate of TE-1 cells following LINC00184 overexpression and treatment with 5-AZA. (I) Statistical counting of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative analysis of the apoptosis rate of TE-1 cells corresponding to (H) (n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in ESCC cells under the conditions of control, OE-LINC00184 and OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3), Comparisons among multiple groups were analyzed by one-way analysis of variance. Statistical significance is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE, overexpression; NC, negative control; DNMT1, DNA methyltransferase 1; si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous cell carcinoma.

Article Snippet: The detection of knockdown efficiency by RT-qPCR adopted the identical reagent system, thermal cycling parameters and calculation method as aforementioned in the RT-qPCR section. siRNA #2, which produced the highest knock-down (>70% reduction) was selected for all subsequent loss-of-function assays. siRNA duplexes specifically targeting mouse DNMT1 transcript: Sense (S), 5′-GCUGGGAGAUGGCGUCAUA-3′; antisense (AS), 5′-CAGGGAGAUACCGCAGUAU-3′; or a random non-coding mRNA sequence: S, 5′-UUCUCCGAACGUGUCACGUTT-3′; AS, 5′-ACGUGACACGUUCGGAGAATT-3′ were synthesized and reconstituted in 1X siRNA buffer (Sangon Biotech Co., Ltd.).

Techniques: Inhibition, Over Expression, Flow Cytometry, Cell Counting, Control, Negative Control, Small Interfering RNA