Experimental Study of EGFR-targeted Adenovirus Vector Mediated tRFs Silencing in Reversing Radioresistance of Cervical Squamous Cell Carcinoma

Abstract

Acquired radioresistance during radiotherapy has been considered as one of the most important reasons for local recurrence or treatment failure of cervical squamous cell carcinoma (CSCC). The tRNA-derived RNA fragments (tRFs) has been reported to involve in various biological processes and may play an important role in radioresistance. The aim of the present study was to analyze the role of tRFs in tumor radiosensitivity, and explore possible mechanisms for targeted intervention of tRFs in reversing CSCC radioresistance. The radioresistant CSCC cell line (SiHa-R) was established. The expression profile of tRFs was used to determine the differentially expressed tRFs in SiHa-R cells. Tumor tissue and serum samples from patients with CSCC were collected, and the relationship between tRFs expression levels and clinical radiotherapy sensitivity was analyzed. Two recombinant replication-deficient adenovirus vectors, Ad.Egr-si-tRFs (promoter Egr-1 with high radiation activation efficiency mediated tRFs silencing sequence) and Ad.CMV-sCAR-EGF (expressed bifunctional fusion protein sCAR-EGF targeting epidermal growth factor receptor), were constructed using gene recombination technique. The dose- and time-effect of radiation-induced tRFs silencing, the expression characteristics of fusion proteins, and their effects on the radiosensitivity of CSCC were observed after targeted infection with adenovirus vectors. Targeted silencing of tRFs to reverse CSCC radioresistance in experimental treatment protocols was explored. Among the detected candidate 2207 tRFs genes, tRF-16-JUO9YED was significantly upregulated in radioresistant CSCC cells. Under the guidance of the fusion protein sCAR-EGF expressed by Ad.CMV-sCAR-EGF, Ad.Egr-si-tRFs could efficiently targeted infect CSCC cells. After tRF-16-JUO9YED silencing mediated by radiation-induced promoter Egr-1, related downstream genes JAG2, SMO, PMM2 and AGK were down-regulated in CSCC cells. It was demonstrated that EGFR-targeted adenovirus vector mediated tRFs silencing might greatly improve radiosensitivity by reducing cell proliferation, inducing more apoptosis, inhibiting migration and invasion, affecting cell cycle and differentiation in radioresistant CSCC cells. This study revealed possible involvement of dysregulated tRFs in the regulation of the process of radioresistance in CSCC cells. EGFR-targeted adenovirus vector mediated tRF-16-JUO9YED silencing may reverse CSCC radioresistance, improve tumor radiotherapy effect, and provide experimental evidence for tumor gene-radiotherapy.