Involvement of Bmi-1 gene in the development of gastrointestinal stromal tumor by regulating p16Ink4A/p14ARF gene expressions: An in vivo and in vitro study

Abstract

Objective: This study was conducted in order to explore the role that Bmi-1 plays during the development of a gastrointestinal stromal tumor (GIST) by regulation of the p16^Ink4A and p14^ARF expressions.

Methods: Eighty-six patients diagnosed with GIST were selected to take part in this experiment. The Bmi-1 protein expressions in GIST and adjacent normal tissues were detected using immunohistochemistry and further analyzed by using photodensitometry. To monitor and track the progression of the GIST, a 3-year follow-up was conducted for all affected patients. After cell transfection, the GIST cells were assigned into the control group (without transfection), the negative control (NC) group (transfected with Bmi-1-Scramble plasmid), and the Bmi-1 shRNA group (transfected with the pcDNA3.1-Bmi-1 shRNA plasmid). Protein and mRNA expressions collected from Bmi-1, p16^lnk4A, P14^ARF, cyclin D1, and CDK4 were measured using both the RT-qPCR and western blotting methods Cell senescence was assessed and obtained by using the β-Galactosidase (β-Gal) activity assay. The use of a Soft agar colony formation assay and CCK-8 assay were performed in order to detect the cell growth and subsequent proliferation. Cell invasion and migration were analyzed using the Transwell assay and scratch test.

Results: Bmi-1 in the GIST tissues was found to be significantly higher and the p16^lnk4A and P14^ARF expressions were lower than those in the adjacent normal tissues. Bmi-1 was negatively correlated with p16^lnk4A and P14^ARF expressions according to the correlation analysis. Bmi-1 expression was associated with the TNM stage, postoperative recurrence, metastasis, tumor size, and the 5-year survival rate. Area under ROC curve was calculated at 0.884, and sensitivity, specificity, and accuracy of Bmi-1 predicting the GIST were 67.44%, 97.67%, and 65.12%, respectively. Patients exhibiting a high Bmi-1 expression in the GIST tissues had lower survival rates than those with low Bmi-1 expression. In comparison with the control group, P14^ARF, and p16^lnk4A were up-regulated, while cyclinD 1 and CDK4 were down-regulated, cell senescence was promoted, and cell proliferation, invasion, and migration also showed some regression in the Bmi-1 shRNA group.

Conclusions: These collection of data indicated that the down-regulated Bmi-1 might inhibit the proliferation, invasion, and migration of GIST cells and can be subsequently linked to the incidence and developing a prognosis of GIST.

Keywords: Bmi-1; Gastrointestinal stromal tumor; In vivo and in vitro; p14(ARF) gene; p16(Ink4A) gene.