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. 2016 Sep 20;7(38):62474-62489.
doi: 10.18632/oncotarget.11518.

"VSports在线直播" Long noncoding RNA CCAT1 acts as an oncogene and promotes chemoresistance in docetaxel-resistant lung adenocarcinoma cells

Jing Chen  1 , Kai Zhang  1 , Haizhu Song  1 , Rui Wang  1 , Xiaoyuan Chu  1 , Longbang Chen  1
Affiliations

Long noncoding RNA CCAT1 acts as an oncogene and promotes chemoresistance in docetaxel-resistant lung adenocarcinoma cells

VSports注册入口 - Jing Chen et al. Oncotarget. .

V体育官网入口 - Abstract

Chemoresistance remains one of the major obstacles in clinical treatment of lung adenocarcinoma (LAD). Indeed, docetaxel-resistant LAD cells present chemoresistance and epithelial-to-mesenchymal transition phenotypes. Long non-coding RNAs (lncRNAs) are known to promote tumorigenesis in many cancer types VSports手机版. Here, we showed that the lncRNA colon cancer-associated transcript-1 (CCAT1) was upregulated in docetaxel-resistant LAD cells. Furthermore, downregulation of CCAT1 decreased chemoresistance, inhibited proliferation, enhanced apoptosis and reversed the epithelial-to-mesenchymal transition phenotype of docetaxel-resistant LAD cells. We also found that the oncogenic function of CCAT1 in docetaxel-resistant LAD cells depended on the sponging of let-7c. In turn, the sponging of let-7c by CCAT1 released Bcl-xl (a let-7c target), thereby promoting the acquisition of chemoresistance and epithelial-to-mesenchymal transition phenotypes in docetaxel-resistant LAD cells. Our data reveal a novel pathway underlying chemoresistance and the epithelial-to-mesenchymal transition in docetaxel-resistant LAD cells. .

Keywords: chemoresistance; epithelial-to-mesenchymal transition; let-7c; lncRNA CCAT1; lung adenocarcinoma (LAD) V体育安卓版. .

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"VSports app下载" Conflict of interest statement

No conflicts of interest to disclose.

Figures

Figure 1
Figure 1. CCAT1 expression in LAD and its association with patient prognosis
A. Differences in CCAT1 expression levels between LAD tissues and pair-matched noncancerous tissues. The expression of CCAT1 was normalized to that of GAPDH. Statistical differences between samples were analyzed with paired samples t-test (n = 36, p<0.01). B. Expression level of CCAT1 in parental and docetaxel-resistant LAD cell lines. Data are presented as mean ± standard error based on at least three independent experiments. *p<0.05, **p<0.01.
Figure 2
Figure 2. Role of CCAT1 in chemosensitivity of parental or docetaxel-resistant LAD cells
A. qRT-PCR assay was performed to examine the expression of CCAT1 48 h after transfection of SPC-A1 or H1299 cells with CCAT1 (or control) and of SPC-A1/DTX or H1299/DTX cells with si-CCAT1 (or siRNA control). Cells transfected with null were regarded as Mock. B. IC50 values for docetaxel in SPC-A1 cells transfected with CCAT1 and SPC-A1/DTX cells transfected with si-CCAT1. C.-D. MTT and colony formation assays on SPC-A1 cells transfected with CCAT1, and SPC-A1/DTX cells transfected with siCCAT1. E.-F. Flow cytometry of SPC-A1 cells transfected with CCAT1, and SPC-A1/DTX cells transfected with siCCAT1. G. Western blot of apoptosis related proteins (activated caspase-3, total caspas-3, activated caspase-9, total caspase-9, activated PARP and total PARP). Error bars represent the mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01 vs. control group.
Figure 3
Figure 3. Forced expression of CCAT1 facilitates acquisition of an EMT phenotype in LAD cells
A. The phenotype of docetaxel-resistant LAD cells and the corresponding parental cells. The docetaxel-resistant LAD cells present a fibroblast-like morphology (typical of mesenchymal phenotype) while the corresponding parental cells present a round-like morphology (typical of epithelial phenotype). B. Western blot of epithelial markers (E-cadherin, β-catenin) and mesenchymal markers (N-cadherin, vimentin), C. transwell assay to measure metastasis capacity, and D. immunofluorescence analysis of EMT markers in docetaxel-resistant LAD cells and parental LAD cells. E. Western blot and F. Immunofluorescence analysis of epithelial markers (E-cadherin, β-catenin) and mesenchymal markers (N-cadherin, vimentin), and G. transwell assay to measure metastasis capacity in SPC-A1 cells transfected with CCAT1, and in SPC-A1/DTX cells transfected with si-CCAT1. Error bars represent the mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01 vs. control group.
Figure 4
Figure 4. Effect of CCAT1 on chemoresistance and EMT of docetaxel-resistant LAD cells in vivo
SPC-A1/DTX cells were transfected with shRNA-CCAT1 or shRNA-control and injected subcutaneously into nude mice. When the average tumor size reached approximately 50 mm3, docetaxel was administered through intraperitoneal injection at a dose of 1 mg/kg every other day for three doses in total. A. Growth curve of tumor volumes and representative photographs of tumor-bearing mice and tumors formed 28 days after the first administration of docetaxel. B.-C. Immunostaining of Ki-67, and PCNA proteins and TUNEL-stained sections of the transplanted tumors (original magnification, ×400). D. Western blot of epithelial markers (E-cadherin, β-catenin) and mesenchymal markers (N-cadherin, vimentin). *p<0.05, **p<0.01 vs. control group.
Figure 5
Figure 5. Let-7c is a target of CCAT1
A. qRT-PCR analysis of let-7c in parental and docetaxel-resistant LAD cells. B.-C. Effect of dysregulated CCAT1 expression on the level of let-7c measured by qRT-PCR. D. CCAT1 levels measured by qRT-PCR after ectopic expression or knockdown of let-7c. E. Top: Bioinformatics predicted let-7c binding sites at two distinct positions in CCAT1. Bottom: RIP assay to detect the association between CCAT1 and let-7c in SPC-A1 and SPC-A1/DTX cells. The positive and negative control refer to U1 and IgG, respectively. F. Luciferase activity in SPC-A1 cells co-transfected with let-7 and luciferase reporters containing no insert, CCAT1, or mutant CCAT1 (containing two mutations, let-7c-3p and let-7c-5p). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. N.S. means “not significant”, *p<0.05, **p<0.01 vs. control group.
Figure 6
Figure 6. Role of let-7c in chemoresistance of docetaxel-resistant LAD cells
A. MTT and colony formation assays, B. flow cytometry, C. western blot of epithelial markers (E-cadherin, β-catenin) and mesenchymal markers (N-cadherin, vimentin), D. immunofluorescence analysis of changes in epithelial and mesenchymal markers, and E. transwell assay to test for metastasis capacity of let-7c inhibitor-transfected SPC-A1 cells and let-7c mimics-transfected SPC-A1/DTX cells. Error bars represent the mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01 vs. control group.
Figure 7
Figure 7. The function of CCAT1 in chemoresistance of LAD cells was partially reversed by let-7c
LAD cells were stably transfected with pLent/CCAT1 or control vector and docetaxel-resistant LAD cells were stably transfected with sh-CCAT1 or shRNA-control. A. IC50 values of SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 was partially reversed by let-7c mimics and let-7c inhibitor, respectively. B.-C. MTT and colony formation assays were performed to determine the proliferation capacity of SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 in the presence of let-7c mimics and let-7c inhibitor, respectively. D. The apoptosis rate of SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 was partially reversed by let-7c mimics and let-7c inhibitor, respectively. Error bars represent the mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01 vs. control group. E. The expression of epithelial markers (E-cadherin, β-catenin) and mesenchymal markers in SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 detected by western blotting was partially reversed by let-7c mimics and let-7c inhibitor, respectively. F. Immunofluorescence analysis of changes in epithelial markers and mesenchymal markers in SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 after treatment with let-7c mimic or let-7c inhibitor, respectively. G. The metastasis capacity of SPC-A1 cells stably transfected with pLent/CCAT1 and SPC-A1/DTX cells stably transfected with sh-CCAT1 was partially reversed by let-7c mimics and let-7c inhibitor, respectively. Error bars represent the mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01 vs. control group.
Figure 8
Figure 8. CCAT1 positively regulates the let-7c target gene Bcl-xl in LAD tissues
A. The ectopic expression of CCAT1 upregulated Bcl-xl at both transcript and protein levels in two parental LAD cell lines. B. In docetaxel-resistant cells, si-CCAT1 decreased Bcl-xl at both transcript and protein levels. C. The expression of Bcl-xl in 36 paired samples of primary LAD and NT cells (p<0.01), showing positive correlation between Bcl-xl expression and CCAT1 expression in 36 LAD tissues (2-tailed Spearman's correlation, r=0.779, p<0.01). D. qRT-PCR detection of let-7c expression in 36 paired LAD and NT samples (p<0.01), showing negative correlation between CCAT1 and let-7c expression levels in 36 LAD tissues (Spearman's correlation analysis, r = −0.696; p<0.01). E. Schematic overview of CCAT1 regulatory signaling. Data are presented as mean ± SD of at least three independent experiments.
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