Cancer Res Treat.  2018 Jul;50(3):992-1008. 10.4143/crt.2017.226.

Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939-Mediated Transcriptional Repression of Bcl-xL

• Cui C
• Zhai D
• Cai L
• Duan Q
• Xie L
• Yu J

• KMID: 2417888
• DOI: http://doi.org/10.4143/crt.2017.226

Abstract

PURPOSE
Studies have found that long noncoding RNA HEIH (lncRNA-HEIH) is upregulated and facilitates hepatocellular carcinoma tumor growth. However, its clinical significances, roles, and action mechanism in colorectal cancer (CRC) remains unidentified.
MATERIALS AND METHODS
lncRNA-HEIH expression in CRC tissues and cell lines was measured by quantitative real-time polymerase chain reaction. Cell CountingKit-8, ethynyl deoxyuridine incorporation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and nude mice xenografts assays were performed to investigate the roles of lncRNA-HEIH. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays were performed to investigate the action mechanisms of lncRNA-HEIH.
RESULTS
In this study, we found that lncRNA-HEIH is significantly increased in CRC tissues and cell lines. lncRNA-HEIH expression is positively associated with tumor size, invasion depth, and poor prognosis of CRC patients. Enhanced expression of lncRNA-HEIH promotes CRC cell proliferation and decreases apoptosis in vitro, and promotes CRC tumor growth in vivo. Whereas knockdown of lncRNA-HEIH inhibits CRC cell proliferation and induces apoptosis in vitro, and suppresses CRC tumor growth in vivo. Mechanistically, lncRNA-HEIH physically binds to miR-939. The interaction between lncRNA-HEIH and miR-939 damages the binding between miR-939 and nuclear factor κB (NF-κB), increases the binding of NF-κB to Bcl-xL promoter, and promotes the transcription and expression of Bcl-xL. Moreover, Bcl-xL expression is positively associatedwith lncRNA-HEIH in CRC tissues. Blocking the interaction between lncRNA-HEIH and miR-939 abolishes the effects of lncRNA-HEIH on CRC tumorigenesis.
CONCLUSION
This study demonstrated that lncRNA-HEIH promotes CRC tumorigenesis through counteracting miR-939-mediated transcriptional repression of Bcl-xL, and suggested that lncRNA-HEIH may serve as a prognostic biomarker and therapeutic target for CRC.

Keyword

Long noncoding RNA; Colorectal neoplasms; Tumorigenesis; miR-939; NF-κB; Bcl-xL

MeSH Terms

Animals
Apoptosis
Carcinogenesis*
Carcinoma, Hepatocellular
Cell Line
Cell Proliferation
Chromatin Immunoprecipitation
Colorectal Neoplasms*
Deoxyuridine
DNA Nucleotidylexotransferase
Heterografts
Humans
Immunoprecipitation
In Vitro Techniques
Luciferases
Mice
Mice, Nude
Prognosis
Real-Time Polymerase Chain Reaction
Repression, Psychology*
RNA
RNA, Long Noncoding*
DNA Nucleotidylexotransferase
Deoxyuridine
Luciferases
RNA
RNA, Long Noncoding

Figure

• 

  Fig. 1. Long noncoding RNA HEIH (lncRNA-HEIH) is up-regulated in colorectal cancer (CRC) and indicts poor prognosis of CRC patients. (A) The expression of lncRNA-HEIH in 84 paired CRC and adjacent normal mucosa was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ***p < 0.001 by Wilcoxon signed-rank test. (B) The 84 CRC patients were classified into two groups in accordance with the median expression value of lncRNA-HEIH in CRC tissues. Kaplan-Meier survival analysis was performed to detect the association between lncRNA-HEIH expression level and overall survival. p=0.006 by log-rank test. (C) The expression of lncRNA-HEIH in human colon normal epithelial cell line NCM460 and CRC cell lines HT-29, SW480, HCT116, and LoVo was detected by qRT-PCR and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p < 0.01, ***p < 0.001 by Student's t test.

• 

  Fig. 2. Enhanced expression of long noncoding RNA HEIH (lncRNA-HEIH) promotes colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD). from three independent experiments. **p < 0.01, ***p < 0.001 by Student's t test. (E, F) lncRNA-HEIH stably overexpressed and control HT-29 cells were subcutaneously injected into nude mice. Tumor volumes were detected every 7 days (E). Tumor weights were detected at the 28th day after injection (F). (G) Tumors generated from subcutaneous injection with lncRNA-HEIH stably overexpressed and control HT-29 cells were immunohistochemistry stained for Ki-67. Scale bars=50 μm. (H) The same tumors as in panel G were stained for TUNEL. Scale bars=100 μm. For E-H, results are shown as mean±SD from six mice. **p < 0.01 by Mann-Whitney U test.

• 

  Fig. 3. Knockdown of long noncoding RNA HEIH (lncRNA-HEIH) inhibits colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH stably knocked-down and control LoVo cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH stably knocked-down and control LoVo were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH stably knocked-down and control LoVo cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. (E, F) lncRNA-HEIH stably knocked-down and control LoVo cells were subcutaneously injected into nude mice. Tumor volumes were detected every 7 days (E). Tumor weights were detected at the 28th day after injection (F). (G) Tumors generated from subcutaneous injection with lncRNA-HEIH stably knocked-down and control LoVo cells were immunohistochemistry stained for Ki-67. Scale bars=50 μm. (H) The same tumors as in panel G were stained for TUNEL. Scale bars=100 μm. For E-H, results are shown as mean±SD from six mice. **p < 0.01 by Mann-Whitney U test.

• 

  Fig. 4. Long noncoding RNA HEIH (lncRNA-HEIH) physically binds to miR-939 and counteracts the binding between miR-939 and nuclear factor κB (NF-κB). (A) Schematic outlining the predicted miR-939 binding sites on lncRNA-HEIH. The red nucleotides indict the seed sequences of miR-939. (B) HT-29 cell lysates were incubated with biotinylated lncRNA-HEIH or the miR-939 binding sites mutated lncRNA-HEIH (lncRNA-HEIH-mut); after pull-down, RNAs were retrieved and detected by quantitative real-time polymerase chain reaction (qRT-PCR), and normalized to input. (C) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, RNA immunoprecipitation assay (RIP) assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. (D) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, RIP assays with NF-κB specific antibody were performed, and the retrieved RNA was detected by qRT-PCR and normalized to input. Results are shown as mean±standard deviation from three independent experiments. **p < 0.01 by Student's t test.

• 

  Fig. 5. Long noncoding RNA HEIH (lncRNA-HEIH) counteracts miR-939‒mediated transcriptional repression of Bcl-xL. (A) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, chromatin immunoprecipitation (ChIP) assays with nuclear factor κB (NF-κB) specific antibody were performed, and the retrieved DNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to input. (B) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, ChIP assays with NF-κB specific antibody were performed, and the retrieved DNA was detected by qRT-PCR and normalized to input. (C) After co-transfection of lncRNA-HEIH or lncRNA-HEIH-mut and luciferase reporter containing Bcl-xL promoterinto HT-29 cells, the luciferase activities were measured. Results are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. (D) After co-transfection of lncRNA-HEIH knockdown plasmid, miR-939 inhibitors, and luciferase reporter containing Bcl-xL promoter into LoVo cells, the luciferase activities were measured. Results are shown as in panel C. (E) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL mRNA levels were detected by qRT-PCR and normalized to GAPDH. (G) After transient transfection of lncRNA-HEIH or lncRNA-HEIH-mut into HT-29 cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. (H) After co-transfection of lncRNA-HEIH knockdown plasmid and miR-939 inhibitors into LoVo cells, Bcl-xL protein levels were detected by western blot and normalized to GAPDH. Results are shown as mean±standard deviation from three independent experiments. **p < 0.01, ***p < 0.001 by Student's t test.

• 

  Fig. 6. Bcl-xL mRNA level is positively correlated with long noncoding RNA HEIH (lncRNA-HEIH) expression level in colorectal cancer (CRC) tissues. (A) Bcl-xL mRNA levels in 84 paired CRC and adjacent normal mucosa were detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. ***p < 0.001 by Wilcoxon signed-rank test. (B) The correlation between lncRNA-HEIH and Bcl-xL expression levels in 84 CRC tissues was detected by Pearson correlation analysis.

• 

  Fig. 7. The mutation of miR-939 binding sites on long noncoding RNA HEIH (lncRNA-HEIH) abolished the effects of lncRNA-HEIH on colorectal cancer tumorigenesis. (A) The expression of lncRNA-HEIH in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphate dehydrogenase. (B) Cell proliferation rate of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were detected by the Cell Counting Kit-8 assays. (C) Proliferative cells of lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 were labeled with ethynyl deoxyuridine (EdU). Red color indicts EdU-positive cells. Scale bars=100 μm. (D) The level of apoptosis in lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining. Blue color indicts TUNEL-positive cells. Scale bars=100 μm. For A-D, results are shown as mean±standard deviation (SD) from three independent experiments. **p < 0.01, ***p < 0.001 by Student's t test. (E, F) lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were subcutaneously injected into nude mice. Tumor volumes were detected every 7 days (E). Tumor weights were detected at the 28th day after injection (F). (G) Tumors generated from subcutaneous injection with lncRNA-HEIH or lncRNA-HEIH-mut stably overexpressed and control HT-29 cells were immunohistochemistry stained for Ki-67. Scale bars=50 μm. (H) The same tumors as in panel G were stained for TUNEL. Scale bars=100 μm. For E-H, results are shown as mean±standard deviation from six mice. **p < 0.01 by Mann-Whitney U test.

Reference

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