Background Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. Methods CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential.Results CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGFstable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin a3b1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin a3b1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin a3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Conclusions Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin a3b1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin a3b1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.
Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.
Aim: Connective tissue growth factor (CTGF) has been known to play important roles in normal physiological conditions as well as pathological conditions. In gastric cancer, previous study has shown that CTGF expression elevation blocks the adhesion of gastric cancer cells to peritoneum in vitro and in vivo. In this study we aimed to purify CTGF from E. coli using a His‐tag purification system and setup a non‐invasive small animal model to study the effect of recombinant CTGF in gastric cancer progression. Material and methods: Recombinant CTGF‐CT was purified using a His‐tag purification system. Gastric cancer cell was injected with or without recombinant CTGF‐CT into SCID mice intraperitoneally. Growth of gastric cancer tumor was observed using small animal ultrasound and survival was calculated using the Kaplan‐Meier survival probability assay. Results: Recombinant CTGF‐CT was shown to have about 50% inhibition in in vitro adhesion assay (p‐value < 0.001). Ultrasound scans have shown higher average tumor sizes and numbers in non‐treated, and vehicle treated group compared to recombinant CTGF‐CT treated group. 100% mortality of mice in recombinant CTGF‐CT treated group compared to vehicle treated group and non‐treated group were at 2 months and 7 days, 1.5 months, and 1 month respectively. Conclusion: Purified recombinant CTGF‐CT significantly reduced the number and size of gastric cancer tumors in mice determined using non‐invasive ultrasound and is a potential therapeutic drug in gastric cancer therapy. Grant Funding Source: Supported by National Science Councel, Taiwan
Background Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β‐hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. Results We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. Conclusions PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde.
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