BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo . Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p , and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p , miR-29a-3p , PIK3R1 , and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1 , PIK3R1 , and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS The POU2F1 - miR-29b-3p / miR-29a-3p-PIK3R1 / PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.
MicroRNAs/metabolism* ; Humans ; Stomach Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/physiology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Animals ; Mice ; Octamer Transcription Factor-1/metabolism* ; Mice, Nude ; Class Ia Phosphatidylinositol 3-Kinase/metabolism* ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic/genetics* ; Male ; Immunohistochemistry ; Female

Humans ; Class I Phosphatidylinositol 3-Kinases/genetics* ; Breast Neoplasms/diagnosis* ; Mutation ; Female ; China ; DNA Mutational Analysis/methods* ; Genetic Testing/standards*

Objective To investigate whether miR-15b-5p can alleviate airway inflammation in asthma by negatively regulating ubiquitin specific peptidase 9X (USP9X) to down-regulate the expression of phosphatidylinositol 4, 5-diphosphate 3-kinase catalytic subunit α/AKT serine/threonine kinase 1 (PIK3CA/AKT1) pathway. Methods USP9X was predicted to be a direct target of miR-15b-5p by using an online database (miRWalk), and the luciferase reporter gene assay was performed to verify it. Co-immunoprecipitation (CO-IP) was used to verify the direct binding between USP9X and PIK3CA and the role of USP9X and its small molecule inhibitor WP1130 in the deubiquitination of PIK3CA. C57 mice were randomly divided into Control group, OVA group, OVA combined with NC group and miR-15b-5p agomir group, with 10 mice in each group. BEAS-2B cells were induced with interleukin 13 (IL-13) and treated with miR-15b-5p mimic. HE, Masson, PAS, immunohistochemistry, immunofluorescence staining, flow cytometry, Western blot and quantitative real-time PCR(qRT-PCR) were performed. Results It was found that the administration of miR-15b-5p agomir and mimic could reduce peribronchial inflammatory cells and improve airway inflammation, and miR-15b-5p could target negative regulation of USP9X. USP9X could directly bind to PIK3CA and regulate PIK3CA level in a proteasome-dependent manner, and USP9X could deubiquitinate K29-linked PIK3CA protein. Down-regulation of USP9X could increase PIK3CA ubiquitination level. WP1130, a small molecule inhibitor of USP9X, has the same effect as knockdown of USP9X, both of which could increase the ubiquitination level of PIK3CA and reduce the protein level of PIK3CA. Conclusion The miR-15b-5p/USP9X/PIK3CA/AKT1 signaling pathway may provide potential therapeutic targets for asthma.
Animals ; MicroRNAs/metabolism* ; Asthma/pathology* ; Class I Phosphatidylinositol 3-Kinases/genetics* ; Ubiquitin Thiolesterase/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Humans ; Inflammation/genetics* ; Cell Line ; Female ; Male

OBJECTIVES: To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). METHODS: Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. RESULTS: Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. CONCLUSIONS In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.
Humans ; MicroRNAs/genetics* ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Sjogren's Syndrome/pathology* ; Epithelial Cells/cytology* ; Submandibular Gland/cytology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Apoptosis ; Class Ia Phosphatidylinositol 3-Kinase ; Cells, Cultured

Child ; Humans ; Class I Phosphatidylinositol 3-Kinases/genetics* ; Mutation ; Neoplasms, Germ Cell and Embryonal/genetics*

The present study aimed to explore the underlying mechanism of Wuling Capsules in the treatment of hepatic fibrosis(HF) through network pharmacology, molecular docking, and animal experiments. Firstly, the chemical components and targets of Wuling Capsules against HF were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), Traditional Chinese Medicines Integrated Database(TCMID), GeneCards, and literature retrieval. The protein-protein interaction(PPI) network analysis was carried out on the common targets by STRING database and Cytoscape 3.9.1 software, and the core targets were screened, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. Enrichment analysis was conducted on the core targets and the "drug-core component-target-pathway-disease" network was further constructed. Subsequently, molecular docking between core components and core targets was conducted using AutoDock Vina software to predict the underlying mechanism of action against HF. Finally, an HF model induced by CCl_4 was constructed in rats, and the general signs and liver tissue morphology were observed. HE and Masson staining were used to analyze the liver tissue sections. The effects of Wuling Capsules on the levels of inflammatory factors, hydroxyproline(HYP) levels, and core targets were analyzed by ELISA, RT-PCR, etc. A total of 445 chemical components of Wuling Capsules were screened, corresponding to 3 882 potential targets, intersecting with 1 240 targets of HF, and 47 core targets such as TNF, IL6, INS, and PIK3CA were screened. GO and KEGG enrichment analysis showed that the core targets mainly affected the process of cell stimulation response and metabolic regulation, involving cancer, PI3K-Akt, MAPK, and other signaling pathways. Molecular docking showed that the core components of Wuling Capsules, such as lucidenic acid K, ganoderic acid B, lucidenic acid N, saikosaponin Q2, and neocryptotanshinone, had high affinities with the core targets, such as TNF, IL6 and PIK3CA. Animal experiments showed that Wuling Capsules could reduce fat vacuole, inflammatory infiltration, and collagen deposition in rat liver, decrease the levels of inflammatory cytokines TNF-α, IL-6, and HYP, and downregulated the expressions of PI3K and Akt mRNA. This study suggests that the anti-HF effect of Wuling Capsules may be achieved by regulating the PI3K-Akt signaling pathway, reducing the levels of TNF-α and IL-6 inflammatory factors, and inhibiting the excessive deposition of collagen.
Animals ; Rats ; Interleukin-6 ; Network Pharmacology ; Animal Experimentation ; Tumor Necrosis Factor-alpha ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Liver Cirrhosis/genetics* ; Medicine, Chinese Traditional ; Capsules ; Class I Phosphatidylinositol 3-Kinases ; Collagen ; Drugs, Chinese Herbal/pharmacology*

Objective: To investigate the impact of molecular classification and key oncogenes on the oncologic outcomes in patients with endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) receiving fertility-preserving treatment. Methods: Patients with EC and AEH undergoing progestin-based fertility-preserving treatment and receiving molecular classification as well as key oncogenes test at Obstetrics and Gynecology Hospital, Fudan University from January 2021 to March 2023 were reviewed. Hysteroscopic lesion resection and endometrial biopsy were performed before initiating hormone therapy and every 3 months during the treatment to evaluate the efficacy. The risk factors which had impact on the treatment outcomes in EC and AEH patients were further analyzed. Results: Of the 171 patients analyzed, the median age was 32 years, including 86 patients with EC and 85 patients with AEH. The distribution of molecular classification was as follows: 157 cases (91.8%) were classified as having no specific molecular profile (NSMP); 9 cases (5.3%), mismatch repair deficient (MMR-d); 3 cases (1.8%), POLE-mutated; 2 cases (1.2%), p53 abnormal. No difference was found in the cumulative 40-week complete response (CR) rate between the patients having NSMP or MMR-d (61.6% vs 60.0%; <i>Pi>=0.593), while the patients having MMR-d had increased risk than those having NSMP to have recurrence after CR (50.0% vs 14.4%; <i>Pi>=0.005). Multi-variant analysis showed PTEN gene multi-loci mutation (<i>HRi>=0.413, 95%<i>CIi>: 0.259-0.658; <i>Pi><0.001) and PIK3CA gene mutation (<i>HRi>=0.499, 95%<i>CIi>: 0.310-0.804; <i>Pi>=0.004) were associated with a lower cumulative 40-week CR rate, and progestin-insensitivity (<i>HRi>=3.825, 95%<i>CIi>: 1.570-9.317; <i>Pi>=0.003) and MMR-d (<i>HRi>=9.014, 95%<i>CIi>: 1.734-46.873; <i>Pi>=0.009) were independent risk factors of recurrence in EC and AEH patients. Conclusions: No difference in cumulative 40-week CR rate is found in the patients having NSMP or MMR-d who received progestin-based fertility-preserving treatment, where the use of hysteroscopy during the treatment might be the reason, while those having MMR-d have a higher risk of recurrence after CR. Oncogene mutation of PTEN or PIK3CA gene might be associated with a lower response to progestin treatment. The molecular profiles help predict the fertility-preserving treatment outcomes in EC and AEH patients.
Pregnancy ; Female ; Humans ; Adult ; Hyperplasia ; Progestins ; Fertility Preservation ; Endometrial Neoplasms/pathology* ; Endometrial Hyperplasia/surgery* ; Treatment Outcome ; Precancerous Conditions ; Fertility ; Class I Phosphatidylinositol 3-Kinases ; Retrospective Studies

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all <i>Pi><0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all <i>Pi><0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.
Female ; Humans ; Middle Aged ; Class I Phosphatidylinositol 3-Kinases/metabolism* ; Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis* ; DNA Mismatch Repair/genetics* ; Endometrial Neoplasms/pathology* ; Microsatellite Instability ; Mismatch Repair Endonuclease PMS2/genetics* ; Molecular Typing

Objective: To investigate the spectrum of PIK3CA gene mutations in Chinese women with hormone receptor positive and HER2 negative (HR⁺/HER2^-) breast cancer, to provide the genetic evidence for identifying potential beneficiaries from specific PI3K isoform inhibitors in Chinese women with breast cancer and to develop detection strategies. Methods: A total of 365 breast cancer specimens archived at the Peking Union Medical College Hospital, Beijing, China from January 2017 to October 2017 were screened. Among these patients, 186 HR⁺/HER2^- women with invasive breast cancer were collected. PIK3CA gene mutations were detected using next generation sequencing technology. The gene variant features were then analyzed and compared with reported data. Results: Among the 186 HR⁺/HER2^- breast cancer cases, 40 (21.5%,40/186) cases harbored PIK3CA gene mutations. Exons 9 and 20 of PIK3CA mutations occurred in 92.5%(37/40)of the tumors, which included E545K, E545G, Q546K, E542K, Q546R, P539R, E547D, H1047R, H1047L, H1047Q and N1044Y. Only one case harbored the exon 7 C420R mutation. Additionally, exons 1 (F83C) and 5 (G364R) uncommon mutations were discovered respectively in 2 cases. Based on the finding, 85.0% (34/40) of cases with known mutations could be detected using companion diagnostic methods. Moreover, 25.0% (10/40) of patients had two or three variants, which were composed of E726K/N345K, H1047Q/N345K, H1047R/G364R, H1047R/E453K, E545G/E726K, E542K/E726K, E542K/H1047R, E545K/H1047R/H1047L and E545K/E547D. The lymph node positive rate in these patients with PIK3CA mutation was remarkably higher than those without (i.e., wild type, <i>Pi><0.05). Conclusions: In this group of HR⁺/HER2^- breast cancer patients, common PIK3CA gene mutations account for the vast majority of the mutations. New rare variants in PIK3CA are also identified while their clinical significance needs to be further studied in a large cohort and/or multi-center study.
Humans ; Female ; Breast Neoplasms/genetics* ; East Asian People ; China ; Class I Phosphatidylinositol 3-Kinases/genetics*

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. METHODS: Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of <i>miR-124ai>, phosphatidylinositol-3-kinase catalytic subunit alpha (<i>PIK3CAi>) and its downstream genes <i>Bcl-2i> and <i>Baxi>. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. RESULTS: Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all <i>Pi><0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (<i>Pi><0.05 or <i>Pi><0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (<i>Pi><0.01 or <i>Pi><0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (<i>Pi><0.01 or <i>Pi><0.001), while the expressions of PIK3CA and Bcl-2 were decreased (<i>Pi><0.05 or <i>Pi><0.01 or <i>Pi><0.001), and the ratio of Bcl-2 to Bax was significantly decreased (<i>Pi><0.01 or <i>Pi><0.001). CONCLUSIONS Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.
Animals ; Arthritis, Experimental/metabolism* ; Arthritis, Rheumatoid/genetics* ; Cattle ; Cell Proliferation ; Class I Phosphatidylinositol 3-Kinases/metabolism* ; Mice ; Mice, Inbred DBA ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Synovial Membrane ; bcl-2-Associated X Protein/metabolism*