Korean J Physiol Pharmacol.  2019 Nov;23(6):449-458. 10.4196/kjpp.2019.23.6.449.

Long non-coding RNA T-cell leukemia/lymphoma 6 serves as a sponge for miR-21 modulating the cell proliferation of retinoblastoma through PTEN

• Tao S
• Wang W
• Liu P
• Wang H
• Chen W

• KMID: 2461037
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.449

Abstract

Retinoblastoma (Rb) is one of the most common eye malignancies occur in childhood. The crucial roles of non-coding RNAs, particularly long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been widely reported in Rb progression. In the present study, we found the expression of lncRNA T-cell leukemia/lymphoma 6 (TCL6) was significantly downregulated in Rb tissues and cell lines. Knockdown of lncRNA TCL6 promoted cell proliferation while reduced cell apoptosis in Rb cells. Moreover, lncRNA TCL6 serves as a sponge for miR-21, a previously-reported oncogenic miRNA in Rb, by direct targeting to negatively regulated miR-21 expression, therefore modulating Rb proliferation through miR-21. TCL6 overexpression inhibited Rb cell proliferation while miR-21 overexpression exerted an opposing effect; the effect of TCL6 overexpression was partially attenuated by miR-21 overexpression. PTEN/PI3K/AKT signaling pathway was involved in lncRNA TCL6/miR-21 axis modulating Rb cell proliferation. Taken together, lncRNA TCL6 serves as a tumor suppressor by acting as a sponge for miR-21 to counteract miR-21-mediated PTEN repression.

Keyword

Cell proliferation; miRNA 21; Phosphate and tension homology deleted on chromsome ten (PTEN); Retinoblastoma

MeSH Terms

Apoptosis
Cell Line
Cell Proliferation*
MicroRNAs
Porifera*
Repression, Psychology
Retinoblastoma*
RNA, Long Noncoding*
RNA, Untranslated
T-Lymphocytes*
MicroRNAs
RNA, Long Noncoding
RNA, Untranslated

Figure

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  Fig. 1 Expression of long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6) in retinoblastoma (Rb) tissues and cell lines. (A) Expression of lncRNA TCL6 in normal retina tissues (n = 8) and Rb tissue specimens (n = 22) were detected using real-time PCR. (B) Expression of lncRNA TCL6 in a normal cell line, ARPE-19, and two Rb cell lines, Y79 and WERI-Rb-1, were detected using real-time PCR. The data are presented as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01.

• 

  Fig. 2 Effect of long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6) knockdown on retinoblastoma (Rb) cell proliferation and apoptosis. (A) LncRNA TCL6 knockdown was achieved in Y79 and WERI-Rb-1 cells by transfection of si-TCL6, compared to si-NC (negative control), as confirmed by real-time PCR. Y79 and WERI-Rb-1 cells were transfected with si-TCL6 and examined for cell viability using MTT assay (B), cell apoptosis using Flow cytometry (C), and DNA synthesis capacity using 5-ethynyl 2-deoxyuridine (EDU) assay (D). Apollo staining (red) and DAPI staining (blue) were performed to detect the EDU positive cells; The arrow indicated positive cells (×400). The data are presented as mean ± standard deviation of three independent experiments. **p < 0.01.

• 

  Fig. 3 miR-21 is a direct target of long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6). (A, B) TCL6 overexpression (OE) vectors or miR-21 mimics were transfected into in Y79 and WERI-Rb-1 cells. The levels of TCL6 and miR-21 were determined using real-time PCR. (C) miR-21 expression in Y79 and WERI-Rb-1 cells examined using realtime PCR. (D, E) miR-21 expression in response to TCL6 knockdown or overexpression was detected in Y79 and WERIRb-1 cells. (F) The schematic diagram showing predicted miR-21 binding site in lncRNA TCL6. Two kinds of TCL6 luciferase reporter gene vectors containing wild or mutated miR-21 binding site were constructed. HEK293 cells were cotransfected with the above vectors and miR-21 mimics and examined for luciferase activity. NC, negative control; Wt, wild-type; mut, mutant-type. The data are presented as mean ± standard deviation of three independent experiments. **p < 0.01.

• 

  Fig. 4 The dynamic effect of long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6) and miR-21 on retinoblastoma (Rb) cell proliferation and apoptosis. Y79 and WERI-Rb-1 cells were co-transfected with lncRNA TCL6 overexpressing vector (lncTCL6 OE) and miR-21 mimics, and examined for cell viability using MTT assay (A) and DNA synthesis capacity using 5-ethynyl 2-deoxyuridine (EDU) assay (B). The arrow showed the EdU positive cells. OD, optical density. The data are presented as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01 compared to vector + negative control (NC) mimics; #p < 0.05, ##p < 0.01, compared to TCL6 OE + NC mimics group.

• 

  Fig. 5 PTEN/PI3K/AKT signaling pathway is involved in long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6)/miR-21 axis modulating retinoblastoma (Rb) cell proliferation. (A) Y79 and WERI-Rb-1 cells were co-transfected with lncRNA TCL6 overexpressing (OE) vector and miR-21 mimics, and examined for protein levels of p-PTEN, PTEN, p-AKT, and AKT. (B) Y79 and WERI-Rb-1 cells were transfected with si-TCL6 and examined for protein level of p-PTEN, PTEN, p-AKT, and AKT. (C, D) The activity of PI3K was determined by ELISA. **p < 0.01 compared to vector + negative control (NC) mimics or NC group; #p < 0.05, ##p < 0.01, compared to TCL6 OE + NC mimics group.

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  Fig. 6 Expression and correlation of miR-21 and PTEN in tissue specimens. (A, B) Expression of miR-21 and PTEN mRNA expression in retinoblastoma (Rb) and non-cancerous tissue specimens was detected using real-time PCR. (C, D) Correlation between long non-coding RNA T-cell leukemia/lymphoma 6 (lncRNA TCL6) and miR-21, between lncRNA TCL6 and PTEN in Rb tissues was analyzed using Pearson's correlation analysis. The data are presented as mean ± standard deviation of three independent experiments. **p < 0.01, compared to the normal group.

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