LncRNA gas5 regulates granulosa cell apoptosis and viability following radiation by X-ray through sponging miR-205-5p and Wnt/β-catenin signaling pathway ingranulosa cell tumor of ovary

Yan Li,Wei Xia,Ying Ye,Juhua Zhuang,Guoyu Wang,Saifei He,Xing Ma,Jun Li

doi:10.4314/tjpr.v19i12.2

Yan Li, Wei Xia + Show 6 more

Open Access

https://doi.org/10.4314/tjpr.v19i12.2

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Abstract

Purpose: The study explored the role of lncRNA gas5 in ovarian granulosa cells exposed to X-ray in granulosa cell tumor of ovary(GCTO). Methods:Exposed the KGN cell line (KALANG, Beijing, China) to X-ray to mimic the radiotherapy for GCSO patients in vitro, cell viability was checked by CCK8 assays. RT-qPCR detected the RNA expression of apoptosis-related genes while Western Blot for biomarkers in wnt/β-catenin signaling. Differential expressions of lncRNA gas5 were examined after cells exposed to X ray for 0,24,48hs. We over expressed gas5 and assessed resultant cell viabilities, apoptosis and signaling. The sponging between gas5 and miR-205-5p was verified through Luciferase Assay. CCK8, RT-qPCR and Western Blot were applied for investigations into the correlation between miR-205-5p and cell viability and apoptosis after miR-205-5p augmentation. Similarly, the interactions between the gas5 and miR-205-5p were assessed after co-transfection of miR-205-5p mimics and oe-gas5. Last, wnt inhibitor was used to study the role of signaling pathway in KGN cells. Results: Exposure of KGN toX-ray reduced cell viabilities and increased apoptosis. Gas5 had reduced expression in cells while miR-205-5p increased. Gas5 upregulation could protect the cells from apoptosis and add to the cell viability and activation of wnt//β-catenin signaling. lncRNA gas5 targeted miR-205-5p and miR-205-5p mimics could counteract functions of up-regulated lncRNA gas5, regulating Wnt/β-catenin signaling pathway. Inactivation in Wnt/β-catenin could suppress cell viability. Conclusions: lncRNA gas5 regulated the cell apoptosis and viability after cellular radiation, which might be a potential therapeutic target to combine into radiotherapy for GCTO patients in clinical stage. Keywords: Ovary, proliferation, apoptosis, lncRNA gas5, x-ray

Highlights

Conclusions: lncRNA gas5 regulated the cell apoptosis and viability after cellular radiation, which might be a potential therapeutic target to combine into radiotherapy for granulosa cell tumor of ovary (GCTO) patients in clinical stage
Starbase v2.0 predicted that miR-205-5p targeted lncRNA gas5 (Figure 3A)
Luciferase reporter assay was applied to make sure of the binding sites between lncRNA gas5 and miR-205-5p, showing that the luciferase activity only decreased significantly in the group that was co-transfected with wild-type of gas5 and miR-205-5p mimics, which indicated that wild type of miR-205-5p could directly bind lncRNA gas5 (Figure 3B). miR-205-5p expression was higher in the group with X-ray exposure than untreated cells (Figure 3C)

Summary

INTRODUCTION

Proliferation, differentiation and apoptosis in granulosa cell tumor of ovary (GCTO). When exposed to X-ray, expression of lncRNA-p21 increased, inhibiting β-catenin signaling and inducing apoptosis, leading to increase in sensitivity of CRC to radiation [12]. KGN cells were cotransfected with 25ng lncRNA gas wt or lncRNA gas mut with 20ul mimics NC or miR-205-5p mimics through LipofectamineTM 3000 Transfection Reagent (Thermo Fisher, USA). The X-ray radiated cells had lower expressions of antiapoptosis genes (Bcl-2 and Bcl-xL) and higher level of caspase-3, suggesting that irradiation promoted apoptosis (Figure 1B-D). Expressions of lncRNA gas were detected in cells exposed to X-ray respectively for 0h,24h and 48 hr. Compared to normal cells(0h), lncRNA gas was expressed lower in X-ray treated cells (Figure 2A). The expression of lncRNA gas decreased significantly as the exposure time increased (Figure 2B, C).

RESULTS

DISCUSSION

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