Yonsei Med J.  2019 Sep;60(9):842-853. 10.3349/ymj.2019.60.9.842.

Long Intergenic Non-Protein Coding RNA 665 Regulates Viability, Apoptosis, and Autophagy via the MiR-186-5p/MAP4K3 Axis in Hepatocellular Carcinoma

• Shan Y
• Li P

• KMID: 2457480
• DOI: http://doi.org/10.3349/ymj.2019.60.9.842

Abstract

PURPOSE
Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown.
MATERIALS AND METHODS
The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model.
RESULTS
Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth.
CONCLUSION
LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.

Keyword

HCC; LINC00665; miR-186-5p; MAP4K3; autophagy

MeSH Terms

Apoptosis*
Autophagy*
Binding Sites
Blotting, Western
Carcinoma, Hepatocellular*
Cell Survival
Computational Biology
Flow Cytometry
Heterografts
Humans
Immunoprecipitation
In Vitro Techniques
Luciferases
Microscopy, Fluorescence
RNA
RNA, Long Noncoding*
Up-Regulation
Luciferases
RNA
RNA, Long Noncoding

Figure

• 

  Fig. 1 LINC00665 expression upregulated in HCC. qRT-PCR assay was performed to measure the expression of LINC00665 in HCC tissues and cells. (A) The expression of LINC00665 in HCC tissues and matched normal adjacent tissues. (B) The expression of LINC00665 in HCC cell lines (Huh-7, HepG2, HCCLM6, MHCC-97H, and Hep3B) and human liver cell line HL-7702. (C) The correlation between the LINC00665 gene expression and overall survival in 24 patients with HCC. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665; HCC, hepatocellular carcinoma.

• 

  Fig. 2 Downregulation of LINC00665 inhibits hepatocellular carcinoma proliferation and induces apoptosis and autophagy in vitro. HepG2 and Huh-7 cells were transfected with siLINC00665 or siNC. (A) LINC00665 expression was detected in HepG2 and Huh-7 cells by qRT-PCR. (B) MTT assay was performed to evaluate cell proliferation. (C) Cell apoptotic rate was detected by flow cytometry. (D) Autophagy formation was analyzed by transfection of GFP-LC3 into HepG2 and Huh-7 cells. (E) Western blot was performed to detect the protein expression of Beclin-1 and LC3. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665.

• 

  Fig. 3 LINC00665 interacts with miR-186-5p. (A) The binding sites between miR-186-5p and LINC00665 were predicted by starBase v2.0, and the luciferase reporter plasmids containing the wild-type (wt) or mutated (mut) LINC00665 binding sites of miR-186-5p were established. (B) The luciferase activity was measured in Huh-7 cells co-transfected with LINC00665-wt or LINC00665-mut luciferase reporter and miR-186-5p mimic or miR-NC. (C) The RIP assay was performed, and expression of LINC00665 was detected in samples bound to the Ago2 antibody or IgG in Huh-7 cells. (D) Detection of LINC00665 expression levels using qRT-PCR in samples pulled down by biotinylated miR-186-5p or negative control. (E) Expression levels of miR-186-5p in Huh-7 cells transfected with siLINC00665, siNC, pcDNA LINC00665, or pcDNA3.0 vector. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665.

• 

  Fig. 4 LINC00665 attenuates the tumor-suppressive effect of miR-186-5p in HCC cells in vitro. (A and B) The expression levels of miR-186-5p were measured in HCC tissues and cell lines by qRT-PCR. (C–G) HepG2 and Huh-7 cells were transfected with miR-NC, miR-186-5p mimic, pcDNA3.0 vector+miR-186-5p mimic, and pcDNA LINC00665+miR-186-5p mimic. (C) miR-186-5p expression was detected in HepG2 and Huh-7 cells by qRT-PCR. (D) Cell proliferation was detected by MTT assay. (E) Cell apoptotic rate was analyzed by flow cytometry. (F) Autophagy formation was analyzed by transfection of GFP-LC3 into HepG2 and Huh-7 cells. (G) Western blot was performed to detect the protein expression of Beclin-1 and LC3. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665; HCC, hepatocellular carcinoma.

• 

  Fig. 5 MAP4K3 a target gene of miR-186-5p. (A) The binding sites between miR-186-5p and MAP4K3 were predicted by TargetScan, and the luciferase reporter plasmids containing the wild-type (wt) or mutated (mut) MAP4K3 binding sites of miR-186-5p were established. (B) The luciferase activity was measured in Huh-7 cells co-transfected with MAP4K3-wt or MAP4K3-mut luciferase reporter and miR-186-5p mimic or miR-NC. (C) The RIP assay was performed, and expression of MAP4K3 was detected in the samples bound to the Ago2 antibody or IgG in Huh-7 cells transfected with miR-186-5p mimic or miR-NC. (D) Detection of MAP4K3 expression level using qRT-PCR in Huh-7 cells transfected with NC mimic, miR-186-5p mimic, NC inhibitor, or miR-186-5p inhibitor. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665.

• 

  Fig. 6 Downregulation of miR-186-5p attenuates the tumor-suppressive effect of MAP4K3 knockout in HCC cells in vitro. (A and B) The expression levels of MAP4K3 were measured in HCC tissues and cell lines by qRT-PCR. (C–G) HepG2 and Huh-7 cells were transfected with si-NC, siMAP4K3, inhibitor negative control+siMAP4K3, and miR-186-5p inhibitor+siMAP4K3. (C) MAP4K3 expression was detected in HepG2 and Huh-7 cells by qRT-PCR. (D) Cell proliferation was detected by MTT assay. (E) Cell apoptotic rate was analyzed by flow cytometry. (F) Autophagy formation was analyzed by transfection of GFP-LC3 into HepG2 and Huh-7 cells. (G) Western blot was performed to detect the protein expression of Beclin-1 and LC3. *p<0.05. HCC, hepatocellular carcinoma.

• 

  Fig. 7 LINC00665 regulates the expression of MAP4K3 by interacting with miR-186-5p. (A) The correlation between LINC00665 and miR-186-5p levels was analyzed in HCC tissues. (B) The correlation between MAP4K3 and miR-186-5p levels was analyzed in HCC tissues. (C) The correlation between LINC00665 and MAP4K3 levels was analyzed in HCC tissues. (D) Expression levels of MAP4K3 in HepG2 and Huh-7 cells transfected with siNC, siLINC00665, inhibitor negative control+ siLINC00665, and miR-186-5p inhibitor+siLINC00665. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665; HCC, hepatocellular carcinoma.

• 

  Fig. 8 Knockdown of LINC00665 inhibits tumor growth in vivo. HepG2 and Huh-7 cells transfected with siLINC00665 or siNC were subcutaneously injected into nude mice. (A and B) Tumor volume was measured at 5, 10, 15, 20, and 25 days after implantation of HepG2 or Huh-7 cells, and tumor weight was measured at 25 days after implantation of HepG2 or Huh-7 cells. (C and D) Mice were euthanized for the analysis of LINC00665 and MAP4K3 by qRT-PCR and Western blot at 25 days after implantation of HepG2 or Huh-7 cells. (E) Western blot was performed to examine the expression of Beclin-1 and LC3. *p<0.05. LINC00665, long intergenic non-protein coding RNA 665.

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