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. 2019 Jul 5;39(7):BSR20182101.
doi: 10.1042/BSR20182101. Print 2019 Jul 31.

LncRNA ANRIL affects the sensitivity of ovarian cancer to cisplatin via regulation of let-7a/HMGA2 axis

Jin-Tian Miao  1 , Jian-Hua Gao  2 , Yong-Qian Chen  2 , Hong Chen  1 , Hao-Yi Meng  2 , Ge Lou  3
Affiliations

LncRNA ANRIL affects the sensitivity of ovarian cancer to cisplatin via regulation of let-7a/HMGA2 axis

Jin-Tian Miao et al. Biosci Rep. .

Retraction in

Abstract

This paper tried to explore ANRIL expression in ovarian cancer and how it affects cisplatin-sensitivity of ovarian cancer cells via regulation of let-7a/high-mobility group protein A2 (HMGA2) axis. qRT-PCR was used to detect ANRIL and let-7a levels in ovarian cancer tissues and cell lines (SKOV3 and SKOV3/DDP). Then cells were randomly assigned into Blank, negative control siRNA, ANRIL siRNA, let-7a inhibitor, and ANRIL siRNA+let-7a-inhibitor groups. CCK-8 assay was applied for assessing cell viability of cells treated with different concentrations of cisplatin. Flow cytometry was employed to test cell apoptosis rate. qRT-PCR and Western blot were performed for related molecules detection. Nude mice transplanted with SKOV3/DDP cells were used to confirm the effects of ANRIL siRNA on the cisplatin-sensitivity. Ovarian cancer tissues and cisplatin-resistant cells had increased ANRIL expression and decreased let-7a expression, and those patients with higher clinical stage and pathological grade showed higher ANRIL and lower let-7a. Dual-luciferase reporter-gene assay confirmed the targeting relationship between ANRIL and let-7a, and between let-7a and HMGA2. The cell viability and cisplatin IC50 were decreased in ANRIL siRNA group exposed to different concentrations of cisplatin, with enhanced apoptosis, as well as elevated let-7a and declined HMGA2, which would be reversed by let-7a inhibitor. Meanwhile, ANRIL down-regulation enhanced the inhibitory effect of cisplatin on tumor growth of nude mice and reduced tumor weight. Silencing ANRIL expression reduced HMGA2 expression to promote the apoptosis and improve cisplatin-sensitivity of ovarian cancer cells via up-regulating let-7a expression.

Keywords: ANRIL; HMGA2; cisplatin; let-7a; ovarian cancer; sensitivity.

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Conflict of interest statement

The present study obtained the approval of the Ethics Committee of The Affiliated Tumor Hospital of Harbin Medical University and all patients gave written consent to use their tissue samples for research purposes. Besides, all animal experiments in the present study abided by the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health in the U.S.A. [21] and gained the approval of the Ethics Committee for Animal Experiments in our hospital (approval number: KY2016-02).

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Expressions levels of ANRIL and let-7a in ovarian cancer tissues and cisplatin-resistant cell lines
The expression levels of ANRIL(A) and let-7a (B) in ovarian cancer and adjacent tissues. The expression levels of ANRIL (C) and let-7a (D) in HOSEPiCs, SKOV3, and SKOV3/DDP cell lines; *, P<0.05 compared with normal HOSEPiCs; #, P<0.05 compared with SKOV3 cells.
Figure 2
Figure 2. Effect of ANRIL on the cisplatin sensitivity
The effect of ANRIL on the cisplatin sensitivity of SKOV3 (A,B) and SKOV3/DDP (C,D) cells observed after CCK-8 assay. *, P<0.05 compared with the Blank group; #, P<0.05 compared with the ANRIL siRNA group; &, P<0.05 compared with the let-7a-inhibitor group.
Figure 3
Figure 3. The effect of ANRIL on the apoptosis of SKOV3 and SKOV3/DDP cells
(A–D) Cell apoptosis in SKOV3 and SKOV3/DDP cells detected flow cytometry; (E,F) Apoptosis-related proteins (Caspase-3, Bax, and Bcl-2) were detected by western blotting in SKOV3 (E,G) and SKOV3/DDP (F,H) cells; *, P<0.05 compared with the Blank group; #, P<0.05 compared with the ANRIL siRNA group; &, P<0.05 compared with the let-7a-inhibitor group.
Figure 4
Figure 4. LncRNA-ANRIL and let-7a interact with and suppress the expression of each other
(A,B) Schematic representation of the predicted target site for let-7a in lncRNA-ANRIL (A), and for HMGA2 in let-7a; (C,D) Relative luciferase activities of luciferase reporters bearing wild-type or mutant-type lncRNA-ANRIL/HMGA2 following transfection with the indicated let-7a mimic or mimic NC.
Figure 5
Figure 5. The expression levels of ANRIL, let-7a, and HMGA2 in SKOV3 and SKOV3/DDP cells
(A,B) The expression of ANRIL and let-7a in SKOV3 cells detected by qRT-PCR; (C,D) the protein expression level of HMGA2 in SKOV3 cells determined by Western-blot; (E,F) the expression of ANRIL and let-7a in SKOV3/DDP cells detected by qRT-PCR; (G,H) the protein expression level of HMGA2 in SKOV3/DDP cells determined by Western-blot; *, P<0.05 compared with the Blank group; #, P<0.05 compared with the ANRIL siRNA group; &, P<0.05 compared with the let-7a-inhibitor group.
Figure 6
Figure 6. ANRIL siRNA increased cisplatin sensitivity in xenograft nude mice
(A) Representative photographs of tumors in nude mice; (B) images are included to identify and define tumor histology; (C,D) The tumor growth curve (C) and tumor weight (D) of transplantation tumor of nude mice; (E,F) Expression levels of HMGA in each treatment group. Positive cells are stained brown. Quantified protein levels (area %) in representative xenograft tumors are shown; *, P<0.05 compared with the NC siRNA+NS group; #, P<0.05 compared with the ANRIL siRNA+NS group; &, P<0.05 compared with the NC siRNA+cisplatin group.
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