ABSTRACT
OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Subject(s)
Animals , Humans , Annexin A5 , Apoptosis , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Cells, Cultured , Flow Cytometry , Genotype , In Situ Hybridization , Larva/physiology , Phenotype , RNA/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Rho Guanine Nucleotide Exchange Factors/metabolism , Sincalide/analysis , Spectrophotometry/methods , Zebrafish/physiologyABSTRACT
ABSTRACT Objective: To study the expression patterns of long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) and the changes in cell proliferation, apoptosis, migration and invasion induced by silencing CCAT1 in bladder cancer cells. Materials and Methods: The expression levels of CCAT1 were determined using realtime quantitative polymerase chain reaction in cancerous tissues and paired normal tissues from 34 patients with bladder cancer. The relationship between clinical characteristics and CCAT1 expression was analyzed. And then we conducted cell experiments. Bladder urothelial carcinoma cell lines T24 and 5637 cells were transfected with CCAT1 small interfering RNA (siRNA) or scramble siRNA. Cell proliferation and apoptosis changes were determined using a Cell Counting Kit-8 (CCK-8) assay and a flow cytometry assay. Migration and invasion changes were measured using a wound healing assay and a trans-well assay. microRNAs (miRNAs) were predicted by Starbase 2.0, and their differential expression levels were studied. Results: CCAT1 was significantly upregulated in bladder cancer (P < 0.05). CCAT1 upregulation was positively related to tumor stage (P = 0.004), tumor grade (P = 0.001) and tumor size (P = 0.042). Cell proliferation, migration and invasion were promoted by abnormally expressed CCAT1. miRNAs miR-181b-5p, miR-152-3p, miR-24-3p, miR-148a-3p and miR-490-3p were potentially related to the aforementioned functions of CCAT1. Conclusion: CCAT1 plays an oncogenic role in urothelial carcinoma of the bladder. In addition, CCAT1 may be a potential therapeutic target in this cancer.
Subject(s)
Humans , Male , Female , Aged , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , RNA, Long Noncoding/analysis , Sincalide/analysis , Time Factors , Wound Healing/genetics , Down-Regulation , Gene Expression , Gene Expression Regulation, Neoplastic , Up-Regulation , Cell Movement/genetics , MicroRNAs/genetics , RNA, Small Interfering , Cell Line, Tumor , Cell Proliferation/genetics , Real-Time Polymerase Chain Reaction , Flow CytometryABSTRACT
An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.