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. 2020 Aug;22(2):1449-1457.
doi: 10.3892/mmr.2020.11196. Epub 2020 May 29.

lncRNA MALAT1 modulates cancer stem cell properties of liver cancer cells by regulating YAP1 expression via miR‑375 sponging

Liying Zhao #  1 , Guohua Lou #  2 , Aichun Li  2 , Yanning Liu  2
Affiliations

lncRNA MALAT1 modulates cancer stem cell properties of liver cancer cells by regulating YAP1 expression via miR‑375 sponging

Liying Zhao et al. Mol Med Rep. 2020 Aug.

Abstract

Liver cancer stem cells (CSCs) are functionally defined by their ability to undergo self‑renewal, and may contribute to metastasis, recurrence and drug resistance in liver cancer. The long non‑coding RNA metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) has been implicated in tumor formation and metastasis of liver cancer. However, the exact mechanism by which MALAT1 modulates liver CSC features remains largely unknown. In the present study, the expression level of MALAT1 was elevated in cancer spheroids compared with the corresponding levels noted in parental liver cancer cells, whereas the suppression of MALAT1 resulted in markedly reduced sphere formation and decreased expression of stemness factors in liver cancer cells. Dual‑luciferase assay and RNA pull‑down assays further indicated an interaction between MALAT1 and microRNA (miR)‑375, and identified Yes‑associated protein 1 (YAP1) as a direct target of miR‑375 in liver cancer cells. In addition, YAP1 expression was correlated with MALAT1 in liver cancer. The reduced expression of YAP1 caused by knockdown of MALAT1 with MALAT1 small interfering RNA (si‑MALAT1) could be partially abolished by miR‑375 inhibition, suggesting that MALAT1 may regulate YAP1 expression by sponging miR‑375. Furthermore, YAP1 overexpression rescued the decrease in CSC features of liver cancer cells caused by si‑MALAT1, further supporting that MALAT1‑mediated YAP1 signaling was required for the stem‑like characteristics of liver CSCs. The present study revealed that MALAT1 may promote CSC properties of liver cancer cells by upregulating YAP1 expression via sponging miR‑375. The MALAT1/miR‑375/YAP1 axis may serve as a novel target for liver cancer therapy, particularly for the eradication of liver CSCs.

Keywords: liver cancer; cancer stem cell; metastasis-associated lung adenocarcinoma transcript 1; microrna-375; Yes-associated protein 1.

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Figures

Figure 1.
Figure 1.
MALAT1 is overexpressed in liver cancer. (A) RT-qPCR analysis of MALAT1 expression in liver tumor tissues and paired non-cancerous hepatic tissues. (B) Relative expression of MALAT1 in three liver cancer cell lines was detected by RT-qPCR. Data are presented as the mean ± SD. **P<0.01, n=3. MALAT1, metastasis-associated lung adenocarcinoma transcript 1; RT-qPCR, reverse transcription-quantitative PCR.
Figure 2.
Figure 2.
MALAT1 expression is associated with CSC features of liver cancer cells. (A) Reverse transcription-quantitative PCR detection of MALAT1 expression levels in spheroids and their parental cells. (B) Sphere formation assay on self-renewal capacities of si-MALAT1- and si-Ctrl-transfected liver cancer cells. Scale bar, 100 µm. (C) Western blot analysis and gray value assay of the expression levels of stemness factors in HepG2 cells transfected with si-MALAT1 or si-Ctrl. Data are presented as the mean ± SD. *P<0.05, **P<0.01, n=3. MALAT1, metastasis-associated lung adenocarcinoma transcript 1; si-, small interfering RNA; CSC, cancer stem cells; UT, untreated control cells.
Figure 3.
Figure 3.
MALAT1 is a molecular sponge for miR-375. (A) Base-pairing between MALAT1 and miR-375. (B) Dual-luciferase activity of the WT and MUT MALAT1 reporters in the presence of miR-375 or miR-NC mimics in 293T cells. (C) RNA pull-down assay for detection of the interaction between MALAT1 and miR-375 in Huh7 cells. Data are presented as the mean ± SD. **P<0.01, n=3. MALAT1, metastasis-associated lung adenocarcinoma transcript 1; MUT, mutant; WT, wild-type; miR, microRNA; NC, negative control; ns, not significant; bio, 3′-biotin.
Figure 4.
Figure 4.
miR-375 directly targets YAP1. (A) Target site of miR-375 in YAP1 3′-UTR. (B) Dual-luciferase activity of the WT and MUT YAP1 3′-UTR reporters in the presence of miR-375 or miR-NC mimics in 293T cells. (C) Western blot analysis and gray value assay of YAP1 expression in Huh7 cells transfected with miR-375 mimics, miR-375 inhibitors, and their corresponding controls (miR-NC). Data are presented as the mean ± SD. **P<0.01, n=3. UT, untreated control cells; ns, not significant; YAP1, Yes-associated protein 1; WT, wild-type; MUT, mutant; miR, microRNA; UTR, untranslated region; NC, negative control; ORF, open reading frame.
Figure 5.
Figure 5.
MALAT1 can regulate YAP1 expression by sponging miR-375. Western blot analysis of (A) YAP1 expression in the Huh7 cells transfected with si-MALAT1 or si-Ctrl, and (B) in the Huh7 cells co-transfected with si-MALAT1 and miR-375 inhibitor or si-MALAT1 and control inhibitor (miR-NC). (C) Western blot analysis of YAP1 expression in three liver cancer cell lines. (D) Spearman correlation analysis of MALAT1 and miR-375 expression levels in tumor samples. YAP1 expression levels in the T and paired N samples were detected by (E) Western blot analysis and subsequently (F) semi-quantified, relative to GAPDH. Data are presented as the mean ± SD. **P<0.01, n=3. MALAT1, metastasis-associated lung adenocarcinoma transcript 1; YAP1, Yes-associated protein 1; miR, microRNA; si-, small interfering RNA; NC, negative control; T, hepatic tumor tissues; N, non-cancerous hepatic tissues; UT, untreated control cells.
Figure 6.
Figure 6.
YAP1 mediates the regulation of MALAT1 on CSC features of liver cancer cells. (A) YAP1 overexpression in non-spherical Huh7 cells (box) or YAP1 suppression in spherical Huh7 cells (circle), followed by sphere formation assay (arrows). (B) At 2 weeks after sphere formation, UT, oeYAP1 and oeCtrl spheres of Huh7 cells were collected for Western blot analysis of expression of the stemness markers. (C) UT spheres, verteportin-treated spheres and vehicle-treated spheres from Huh7 cells were collected for Western blot analysis at 2 weeks after sphere formation. (D) Sphere formation assay and (E) Western blot analysis of expression of stemness factors on Huh7 cells transfected with si-MALAT1, or co-transfected with si-MALAT1 and oeYAP1 or oeCtrl. Scale bar, 100 µm. UT, untreated control cells. YAP1, Yes-associated protein 1; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; CSC, cancer stem cell; oe, overexpression plasmid; Ctrl, control; si-, small interfering RNA; p-, phosphorylated.
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