Long non-coding RNA TCL6 enhances preferential toxicity of paclitaxel to renal cell carcinoma cells

Abstract

Background: Recent findings have shown long non-coding RNAs (lncRNAs) are dysregulated in a variety of cancer cells. In this report, we investigate the effect of T-cell leukemia lymphoma 6 (TCL6) on paclitaxel (PTX)-induced apoptosis in Renal cell carcinoma (RCC) cells. Methods: Expression levels of TCL6 in RCC tissues were analyzed via The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Fluorescence in situ hybridization (FISH) was performed to detect the expression of TCL6 in RCC tissues and cells. Two pairs of cell lines were used: TCL6-silenced 786-O cell line and scrambled 786-O cell line, TCL6-overexpressed Caki-1 cell line and Caki-1 scrambled cell line. Cell viability was detected using the MTT assay. Apoptosis was examined by flow cemetery. Dual reporter gene assay was performed to confirm the direct downstream target miRNA of TCL6. Results: Based on RNA sequencing expression data of RCC tissues from TCGA and GEO datasets, the expression deficiency of TCL6 was observed in RCC tissues. Low level of TCL6 was associated with worse overall and disease-free survival of RCC patients. The FISH showed similar results with low expression of TCL6 in RCC tissues and cells. After PTX treatment, a time-dependent decrease in cell viability was observed in TCL6-overexpressed RCC cells and an increase in cell viability was observed in TCL6-silenced cells compared to control cells. Apoptosis induced by PTX was significantly increased in TCL6-overexpressed cells. Inhibition of TCL6 showed a significant decrease in apoptosis. Furthermore, luciferase reporter assay revealed that TCL6 is a direct target gene of miR-221. Conclusions: TCL6 effectively sensitizes RCC to PTX mainly through downregulation of miR-221. Our results suggest that PTX combined with TCL6 might be a potentially more effective chemotherapeutic approach for renal cancer.

Keywords: Apoptosis; Chemotherapy.; Paclitaxel; Renal cell carcinoma.

Figure 1

The downregulation of TCL6 expression was associated with poor prognosis of patients. A. The expression of TCL6 was decreased in RCC tissues (523 cases) compared to the normal tissues (100 cases), which of data was collected from the TCGA database and analyzed by GEPIA platform. B. The TCL6 expression data of RCC (n=448) and normal (n=67) tissues were further analyzed by online TANRIC platform. C. We further analyzed the expression levels of TCL6 in seventy-two RCC and match normal tissues obtained from the GEO database (GSE53757dataset). D. The expression of TCL6 gradually decreased among the different clinical stages of renal cancer (by one-way analysis of variance). Data are shown as means ± SD. Kaplan-Meier curves showed the patient overall survival (E) and disease-free survival (F) of the high TCL6 expression and low TCL6 expression. These graphs were conducted by TANRIC platform. Dotted line: the 95% confidence interval (CI). G. Low level of TCL6 expression was associated with poor overall survival of patients with renal cancer. *P<0.05.

Figure 2

The relative expression levels of TCL6 in RCC tissues cells. A. Confocal FISH images exhibited expression and localization of TCL6 in RCC and adjacent normal renal tissues. TCL6 (red signal) was mainly located in the cytoplasm. Blue DAPI for nucleus staining. B. The levels of TCL6 expression was detected by FISH in HK-2 cells and 5 RCC cell lines. There were less red signals in 5 RCC cell lines compared to HK-2 cells. C. The basal expression of TCL6 in HK-2, 786-O, and Caki-1 cells was evaluated by qRT-PCR. D. 786-O cells were transfected with TCL6 siRNA1, siRNA2, siRNA3, and NC siRNA, respectively. qRT-PCR revealed the expression levels of TCL6 were significantly reduced by treatment with TCL6 siRNA 3. E. The expression levels of TCL6 were elevated in Caki-1 cells transfected with TCL6 overexpression plasmids compared to those cells were treated with the negative control plasmids. Data are shown as means ± SD (n=3). FISH: fluorescence in situ hybridization; *P<0.05.

Figure 3

TCL6 expression is correlated with sensitization of RCC cells to PTX. The indicated concentrations of PTX were treated to two kinds of renal cancer cells for 0, 24, 48, 72, and 96 h. A. The proliferation of TCL6-inhibited 786-O cells were significantly increased as compared to those 786-O cells treated with scramble control siRNAs (si-NC) with different levels of PTX concentration (0, 50, 100, 200 nM) at 96 h. B. After incubation for 96 h, the proliferation of TCL6-increased Caki-1 cells was significantly lower than those cells treated with empty pcDNA3.1 plasmid (oe-NC) with different concentrations of PTX. si-NC: 786-O cells treated with scrambled control siRNAs; si3-TCL6: 786-O cells treated with si3-TCL6 siRNAs; oe-NC: Caki-1 cells treated with empty pcDNA3.1 plasmid. oe-TCL6: Caki-1 cells treated with TCL6 overexpressing plasmids. Data are shown as means ± SD. *P<0.05.

Figure 4

Modulation of PTX cytotoxicity by overexpression (oe-TCL-6) or inhibition (si3-TLC6) of TLC6. A. The apoptosis assays of 786-O cells were performed with (si3-TCL6) or without si3-TCL6 siRNAs (si-NC) under PTX treatments. B. Inversely, up-regulation of TCL6 decreased the apoptosis of Caki-1 cells compared to control cells (oe-NC). Data are shown as means ± SD (n=3). *P<0.05.

Figure 5

TCL6 sensitizes RCC cells to PTX through sponging miR-221. A. The level of miR-221 was negatively correlated with TCL6 expression in renal cancer, which was conducted via TANRIC platform. B. The basal expression of miR-221 in HK-2, 786-O, and Caki-1 cells were evaluated by qRT-PCR. C. The expression levels of miR-221 were detected by qRT-PCR after knockdown of TCL6 in 786-O cells. D. The expression level of miR-221 was further examined with or without upregulation of TCL6 using qRT-PCR. E. TCL6 contains two potential binding sites of miR-221. F. Relative luciferase activity in wild type of TCL6. The decrease in luciferase activity in the wild type indicated specific binding of miR-221 to its target site. Data are shown as means ± SD. *P<0.05.