Yonsei Med J.  2019 Aug;60(8):727-734. 10.3349/ymj.2019.60.8.727.

Long Noncoding RNA MALAT1 Regulates Hepatocellular Carcinoma Growth Under Hypoxia via Sponging MicroRNA-200a

• Zhao ZB
• Chen F
• Bai XF

• KMID: 2452953
• DOI: http://doi.org/10.3349/ymj.2019.60.8.727

Abstract

PURPOSE
Hepatocellular carcinoma (HCC) is a common cancer worldwide. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA (lncRNA), has been reported to be aberrantly expressed in hypoxic cancer cells. MALAT1 plays a significant role in many malignancies, including HCC. The aim of this study was to explore the role of MALAT1 in hypoxic HCC cells and its underlying regulatory mechanism.
MATERIALS AND METHODS
Quantitative reverse transcription PCR (qRT-PCR) assay was performed to detect the mRNA levels of MALAT1 and microRNA-200a (miR-200a) in HCC cells. Cell invasion and migration ability were evaluated by Transwell assay. Starbase v2.0 and luciferase reporter assay were employed to identify the association between MALAT1 and miR-200a. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively.
RESULTS
MALAT1 levels were significantly upregulated in HCC cells under hypoxia. Hypoxia promoted proliferation, migration, and invasion, and blocked apoptosis in Hep3B cells, which were weakened by knockdown of MALAT1. Starbase v2.0 showed that MALAT1 and miR-200a have a complementarity region, and luciferase reporter assay verified that MALAT1 interacted with miR-200a in Hep3B cells. Moreover, MALAT1 negatively regulated the expression of miR-200a. miR-200a levels were dramatically downregulated in HCC cells under hypoxia. Upregulation of miR-200a inhibited proliferation, migration, and invasion, and induced apoptosis in Hep3B cells under hypoxia. Interestingly, downregulation of miR-200a partially reversed the tumor-suppressive effect of knockdown of MALAT1 on Hep3B cells in hypoxic condition.
CONCLUSION
LncRNA MALAT1 was involved in proliferation, migration, invasion, and apoptosis by interacting with miR-200a in hypoxic Hep3B cells, revealing a new mechanism of MALAT1 involved in hypoxic HCC progression.

Keyword

MALAT1; miR-200a; HCC; proliferation; hypoxic

MeSH Terms

Adenocarcinoma
Anoxia*
Apoptosis
Carcinoma, Hepatocellular*
Cell Proliferation
Down-Regulation
Flow Cytometry
Luciferases
Lung
Polymerase Chain Reaction
Reverse Transcription
RNA, Long Noncoding*
RNA, Messenger
Up-Regulation
Luciferases
RNA, Long Noncoding
RNA, Messenger

Figure

• 

  Fig. 1 MALAT1 was upregulated in hepatocellular carcinoma cells by hypoxia. qRT-PCR assay was performed to measure the expression of MALAT1 in Huh7, SNU-423, PLC, and Hep3B cells cultured under normoxic or hypoxic condition. *p<0.05. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

• 

  Fig. 2 Knockdown of MALAT1 suppressed growth and induced apoptosis of Hep3B cells after hypoxia challenge. Hep3B cells were transfected with si-NC or si-MALAT1. For N-control group, cells were cultured under normoxia condition for 72 hours; for H-control group, cells were first challenged with hypoxia (1% oxygen) for 24 hours, followed by culturing under normoxia condition for 48 hours. (A) MALAT1 expression level in each treatment group was measured by qRT-PCR. (B) Hep3B cell proliferation in each treatment group was evaluated by MTT assay. (C and D) Hep3B cell apoptotic rate in each treatment group was detected by flow cytometry. *p<0.05: when compared with N-control group, †p<0.05: when compared with H-si-NC group. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

• 

  Fig. 3 Knockdown of MALAT1 suppressed migration and invasion of Hep3B cells promoted by hypoxia. Hep3B cells were transfected with si-NC or si-MALAT1, and normoxia and hypoxia treatment were performed as described in Fig. 2A. Cell migration (A) and invasion (B) were evaluated by Transwell assay. *p<0.05: when compared with N-control group, †p<0.05: when compared with H-si-NC group. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

• 

  Fig. 4 MALAT1 interacted with miR-200a in hypoxic Hep3B cells. (A) The predicted binding sites between miR-200a and MALAT1 mRNA through Starbase v2.0. Luciferase reporter plasmids containing wild-type (WT) or mutated (MUT) MALAT1 binding sites of miR-200a were established. (B and C) Hep3B cells were co-transfected with MALAT1-WT or MALAT1-MUT luciferase reporter and miR-200a, miR-NC, anti-NC, or anti-miR-200a, followed by the determination of luciferase activity at 48 h after transfection. (D) Hep3B cells were transfected with si-NC, si-MALAT1, pcDNA3.1 empty vector, and pcDNA-MALAT1 overexpression plasmid, and miR-200a expression level was measured by qRT-PCR. *p<0.05: when compared with N-control group, †p<0.05: when compared with H-miR-NC group. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

• 

  Fig. 5 Upregulation of miR-200a inhibited the proliferation, invasion, migration, and induced apoptosis in Hep3B cells under hypoxia. Hep3B cells in (B–F) were treated as described in Fig. 2. (A) qRT-PCR analysis of the expression of miR-200a in Huh7, SNU-423, PLC, and Hep3B cells with or without hypoxia challenge. (B) miR-200a expression level in Hep3B cells was measured by qRT-PCR assay. (C) Cell proliferation was evaluated by MTT assay. (D) Cell apoptotic rate was detected by flow cytometry. (E and F) Cell migration and invasion were evaluated by Transwell assay. *p<0.05: when compared with N-control group, †p<0.05: when compared with H-miR-NC group. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

• 

  Fig. 6 Downregulation of miR-200a partially reversed the effect of MALAT1 knockdown on hypoxia-challenged Hep3B cells. Hep3B cells were transfected with si-MALAT1 or si-MALAT1+anti-miR-200a and were challenged with normoxia or hypoxia as described in Fig. 2. (A) miR-200a expression in each group was evaluated by qRT-PCR. (B) Cell proliferation was detected by MTT assay. (C) Cell apoptotic rate was detected by flow cytometry. (D and E) Cell migration and invasion were measured by Transwell assay. *p<0.05: when compared with H-control group, †p<0.05: when compared with H-si-MALAT1 group. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

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