OBJECTIVE: To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect. METHODS: The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments. RESULTS: The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05). CONCLUSION Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.
Humans ; Bone Neoplasms/genetics* ; Cell Proliferation ; MicroRNAs/genetics* ; Osteosarcoma/genetics* ; rab3 GTP-Binding Proteins/genetics* ; RNA, Messenger

OBJECTIVE: To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. METHODS: Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. RESULTS: Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. CONCLUSION RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.
Humans ; rab GTP-Binding Proteins/metabolism* ; Triple Negative Breast Neoplasms ; Cell Line, Tumor ; rab27 GTP-Binding Proteins/metabolism* ; RNA, Small Interfering/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVE: To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. METHODS: Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. RESULTS: Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05). CONCLUSIONS TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*

OBJECTIVE: To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients. METHODS: High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated. RESULTS: A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons. CONCLUSION The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.
Core Binding Factor Alpha 2 Subunit/genetics* ; GTP Phosphohydrolases/genetics* ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Membrane Proteins/genetics* ; Mutation ; Nucleophosmin ; Prognosis ; Proto-Oncogene Proteins p21(ras)/genetics* ; fms-Like Tyrosine Kinase 3

Child ; Epilepsy, Generalized ; Family ; GTP-Binding Proteins/genetics* ; Humans ; Infant ; Spasms, Infantile/genetics* ; Tumor Suppressor Proteins/genetics*

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.
Arachis/genetics* ; Fabaceae/genetics* ; GTP-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Salt Stress ; Stress, Physiological/genetics* ; Tobacco/genetics*

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gα_q/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gα_q/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gα_q/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.
Humans ; Mice ; Animals ; Lipoylation ; NIH 3T3 Cells ; Uveal Neoplasms/genetics* ; Melanoma/genetics* ; Cell Proliferation ; Proto-Oncogene Proteins c-bcl-2 ; GTP-Binding Protein alpha Subunits, Gq-G11/genetics*

Hypertension is a clinical syndrome characterized by elevated systemic arterial blood pressure, which may be accompanied by functional or organic damage of heart, brain, kidney and other organs. The pathogenesis and development of hypertension are affected by genetic, environmental, epigenetic, intestinal microbiota and other factors. They are the result of multiple factors that promote the change of blood pressure level and vascular resistance. G protein coupled receptors(GPCRs) are the largest and most diverse superfamily of transmembrane receptors that transmit signals across cell membranes and mediate a large number of cellular responses required by human physiology. A variety of GPCRs are involved in the control of blood pressure and the maintenance of normal function of cardiovascular system. Hypertension contributes to the damages of heart, brain, kidney, intestine and other organs. Many GPCRs are expressed in various organs to regulate blood pressure. Although many GPCRs have been used as therapeutic targets for hypertension, their efficacy has not been fully studied. The purpose of this paper is to elucidate the role of GPCRs in blood pressure regulation and its distribution in target organs. The relationship between GPCRs related to intestinal microorganisms and blood pressure is emphasized. It is proposed that traditional Chinese medicine may be a new way to treat hypertension by regulating the related GPCRs via intestinal microbial metabolites.
Blood Pressure ; GTP-Binding Proteins ; Gastrointestinal Microbiome ; Humans ; Hypertension/genetics* ; Receptors, G-Protein-Coupled/metabolism*

OBJECTIVES: This study aims to investigate the effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma (TSCC). METHODS: Forty-eight TSCC cases were selected from the Maxillofacial Surgery Center of Qingdao Municipal Hospital from 2017 to 2019. The expression of RhoE in the specimens (TSCC and adjacent tissues) was detected by immunohistochemistry, and RhoE mRNA and protein were extracted to further detect the expression of RhoE. SCC-4 and CAL-27 cells were selected for RESULTS: The expression level of RhoE in TSCC was significantly lower than that in adjacent tissues ( CONCLUSIONS RhoE expression is low in TSCC. Over expression RhoE in TSCC can significantly decrease its migration and invasion abilities. Hence, RhoE may play an important role in regulating the metastasis and invasion of TSCC and provide a new target for gene therapy.
Carcinoma, Squamous Cell ; Cell Line, Tumor ; Humans ; Matrix Metalloproteinase 2 ; Neoplasm Invasiveness ; Tongue ; Tongue Neoplasms ; rho GTP-Binding Proteins/genetics* ; rho-Associated Kinases

OBJECTIVE: To explore the clinical and genetic characteristics of a child featuring developmental delay. METHODS: The child was subjected to whole exome sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: Whole genome sequencing revealed that the child has carried compound heterozygous variants c.2607-1G>C and c.899 + 2dupT of the RAB3GAP1 gene, which were respectively derived from her mother and father. CONCLUSION A rare case of Warburg micro syndrome type 1 was diagnosed. The phenotype of the child was consistent with the literature, in addition with dysplasia of palatine arch, prominent high palatal arch and tooth dysplasia. Above finding has provided a basis for genetic counseling and prenatal diagnosis for the family.
Abnormalities, Multiple/genetics* ; Adult ; Cataract/genetics* ; Child ; Cornea/abnormalities* ; Female ; Humans ; Hypogonadism/genetics* ; Intellectual Disability/genetics* ; Male ; Microcephaly/genetics* ; Mutation ; Optic Atrophy/genetics* ; Whole Exome Sequencing ; rab3 GTP-Binding Proteins/genetics*