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. 2017 Apr 25;8(17):27929-27942.
doi: 10.18632/oncotarget.15846.

Down-regulation of long non-coding RNA RP11-708H21.4 is associated with poor prognosis for colorectal cancer and promotes tumorigenesis through regulating AKT/mTOR pathway

Longci Sun  1 , Chunhui Jiang  1 , Chunjie Xu  1 , Hanbing Xue  2 , Hong Zhou  1 , Lei Gu  1 , Ye Liu  1 , Qing Xu  1
Affiliations

Down-regulation of long non-coding RNA RP11-708H21.4 is associated with poor prognosis for colorectal cancer and promotes tumorigenesis through regulating AKT/mTOR pathway

Longci Sun et al. Oncotarget. .

Abstract

Long non-coding RNAs (lncRNAs) serve critical roles in cancer development and progression. Herein, through next generation RNA sequencing and experimental validations, we determined the expression status of RP11-708H21.4 in colorectal cancer (CRC) and explored its clinical significance and biological functions in CRC. Differentially expressed lncRNAs from CRC samples and corresponding normal mucosa tissues was screened through RNA sequencing, and RP11-708H21.4 was selected for further experimental validation. The expression levels of RP11-708H21.4 in CRC tissues and cell lines were determined using qRT-PCR. Also, the relationship between the clinicopathological features and RP11-708H21.4 expression was analyzed. Cell viability was examined by CCK-8 and colony assays; cell migration and invasion were detected by transwell assays; cell cycle and cell apoptosis were analyzed by flow cytometry. The chemosensitivity of CRC cells to 5-Fluorouracil (5-FU) was also determined using CCK-8 assay. CRC xenograft tumor models were established to determine the biological functions of RP11-708H21.4 in vivo. Levels of cell cycle-related proteins and AKT/mTOR pathway-related proteins were detected by western blot assay. RP11-708H21.4 expression was aberrantly decreased in CRC, and its expression was closely associated with aggressive clinicopathologic features and unfavorable prognosis of CRC patients. Overexpressed RP11-708H21.4 suppresses CRC cell proliferation through inducing G1 arrest. Moreover, up-regulation of RP11-708H21.4 inhibits cell migration and invasion, causes cell apoptosis, and enhances 5-FU sensitivity of CRC cells. Finally, increased RP11-708H21.4 expression blocked AKT/mTOR pathway, and repressed in vivo CRC xenograft tumor growth. The results indicated that RP11-708H21.4 might have potential roles as a biomarker and a therapeutic target for CRC.

Keywords: RNA sequencing; RP11-708H21.4; colorectal cancer; long noncoding RNA; tumorigenesis.

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Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1
(A) Volcano plot of P values as a function of the weighted fold change for lncRNAs in CRC tissues. (B) Heatmap of expression profiles for the 117 lncRNAs that showed significant expression changes (39 up-regulated and 78 down-regulated).
Figure 2
Figure 2. RP11-708H21.4 was significantly down-regulated in CRC
(A) Levels of RP11-708H21.4 were determined in 149 CRC tissues and their corresponding normal mucosa by qRT-PCR. (B) RP11-708H21.4 was evidently down-regulated in 72% of CRC tissues compared with their corresponding normal mucosa. (C) Levels of RP11-708H21.4 were determined in six CRC cell lines (DLD-1, SW-480, HT-29, SW-620, HCT-116 and LOVO) and normal intestinal epithelial cell line (HIEC). Data are presented as means ± SDs based on at least three independent experiments. Asterisk indicates statistically significant changes: ***P < 0.001; **P < 0.01 compared with NC; Student's t-test. (D) Kaplan-Meier survival curve analysis showed that lower expression of RP11-708H21.4 is correlated with shorter overall survival of CRC patients. P value was assessed by log-rank test.
Figure 3
Figure 3. RP11-708H21.4 suppresses CRC cell proliferation in vitro through induction of cell cycle arrest
(A) CCK-8 assay was performed to measure the proliferation of HT-29 and HCT-116 cell lines transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4. (B) The representative pictures of colony formation assay for crystal violet-stained HT-29 and HCT-116 cells were shown. (C) Cell cycle analysis showed that significant G1 phase arrest and S phase decrease were observed in HT-29 and HCT-116 cell lines transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4. (D) Western blotting showed that Cyclin D1 expression was decreased, whereas p27 expression was increased at nuclear protein levels after the up-regulation of RP11-708H21.4 in HT-29 and HCT-116 cells. Data are presented as means ± SDs based on at least three independent experiments. Asterisk indicates statistically significant changes: **P < 0.01; *P < 0.05 compared with NC; Student's t-test.
Figure 4
Figure 4. RP11-708H21.4 facilitates CRC cell apoptosis and represses cell invasion in vitro
(A) Cell apoptosis analysis of HT-29 and HCT-116 cell lines transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4. (B) Transwell assay was performed to measure the invasion and migration of HT-29 and HCT-116 cell lines transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4. The cells were counted under a microscope in five randomly selected fields. Data are presented as means ± SDs based on at least three independent experiments. Asterisk indicates statistically significant changes: ***P < 0.001; **P < 0.01 compared with NC; Student's t-test.
Figure 5
Figure 5. RP11-708H21.4 enhances 5-FU sensitivity in CRC cells
CCK-8 assay was conducted to detect the growth of (A) HT-29 and (B) HCT-116 cell lines transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4 at different concentrations of 5-FU (2, 4, 8, 16 and 32 μM). Data are presented as means ± SDs based on at least three independent experiments. Asterisk indicates statistically significant changes: ***P < 0.001; **P < 0.01; *P < 0.05 compared with NC; Student's t-test.
Figure 6
Figure 6. RP11-708H21.4 blocks AKT/mTOR pathway in CRC cells
Western blotting showed that phosphorylation of AKT, mTOR and S6K1 was significantly repressed in RP11-708H21.4 overexpressed HT-29 and HCT-116 cells.
Figure 7
Figure 7. RP11-708H21.4 represses CRC xenograft tumor growth in vivo
(A) CRC xenograft tumor models were developed in nude mice by HT-29 cells transfected with empty vector (control) or pcDNA3.1-RP11-708H21.4. Tumor volume was calculated every 3 days. (B) The representative pictures of tumors from respective groups of nude mice were shown. (C) Tumor weight of each tumor sample from two groups was represented. Data are presented as means ± SDs based on at least three independent experiments. Asterisk indicates statistically significant changes: ***P < 0.001; **P < 0.01 compared with NC; Student's t-test.
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