Downregulation of MiR-31 stimulates expression of LATS2 via the hippo pathway and promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Abstract

Background: Dysregulation of miRNAs is associated with cancer development by coordinately suppressing abundant target genes. Emerging evidence indicates that miR-31 plays a dual role in tumorigenicity. However, whether miR-31 plays as an oncogene in esophageal squamous cell carcinoma (ESCC) and the potential target molecules are still unclear. MiR-31 role in ESCC was investigated and an association of the target molecules with EMT was identified in the progression of ESCC.

Methods: Western blot assays and qRT-PCR was performed to detect the protein and mRNA levels. We investigated the role of miR-31 in the regulation of LATS2 expression in ESCC cell lines via functional assays both in vivo and in vitro. The luciferase reporter assays was conducted to confirm LATS2 is a potential target of miR-31. Immunohistochemistry was used to measure LATS2 and TAZ expression in normal and ESCC tissue.

Results: LATS2 is a component of the Hippo tumor-suppressive signaling pathway. Frequent loss of heterozygosity of LATS2 has been reported in esophageal cancer. We analyzed the reciprocal expression regulation of miR-31 and LATS2 and demonstrated that LATS2 expression was elevated by down-regulation of miR-31 at the post-transcriptional level in ESCC. Moreover, miR-31 significantly suppressed the luciferase activity of mRNA combined with the LATS2 3'-UTR, a key molecule in the Hippo pathway. Then, LATS2 consequently promoted the translocation of TAZ, which was examined using immunohistochemistry. Silencing of miR-31 significantly inhibited the cell proliferation, induced apoptosis and decreased the ability of migration/invasion in vitro. LATS2 impedes ESCC cell proliferation and invasion by suppressing miR-31, as well as mice xenograft model in vivo. Meanwhile, the nuclear localization of LATS2 constrained the phosphorylation of TAZ. Then, the expression level of TAZ was notably heightened with a high risk of recurrence compared to that observed in the low-risk patients, as well as, the higher expression associated with a poor survival.

Conclusions: Our study demonstrated that overexpression of miR-31 undertook an oncogenic role in ESCC by repressing expression of LATS2 via the Hippo Pathway and activating epithelial-mesenchymal transition. LATS2 and TAZ could be potential novel molecular markers for predicting the risk of recurrence and prognosis of ESCC.

Keywords: EMT; Esophageal squamous cell carcinoma; Hippo pathway; LATS2; TAZ; miR-31.

Fig. 1

Expression of miR-31 in ESCC cell lines and tissue samples and the in vitro effects of miR-31 on cell proliferation, migration and invasion in ESCC cells. a The relative expression level of miR-31 in 20 specimens of ESCC (T) and the adjacent nontumor tissues (N) was determined by qRT-PCR. b QRT-PCR analysis miR-31 expression in five human ESCC cell lines and the normal esophageal epithelial cell line (HEEC). c QRT-PCR analysis of the relative expression of miR-31 in each group of ESCC cells transfected with miR-31 mimics and inhibitor. d-e MTT and colony formation assays in ESCC cells overexpressing or underexpressing miR-31. f-g Wound scratch healing assay of ESCC cell showed that change of miR-31 effectively affected cell motility. Photographs were taken immediately (0 h) and at 48 h after wounding, quantification of wound closure was done. h Migration assay and Invasion assay revealed that the upexpression or downexpression of miR-31 promoted or inhibited the invasion ability of ESCC cells. Results are expressed as the mean ± SD of three independent experiments. *: P < 0.05; **: P < 0.01

Fig. 2

MiR-31 negatively regulates the expression of LATS2 by directly targeting the LATS2 3′-UTR. a Schematic representation of miR-31 gene promoter with the putative LATS2-binding sites and the sequences of the point mutations. b Luciferase activity in cells following co-transfection with control or miR-31-encoding plasmids and wild or mutant LATS2 pLUC vectors to predict miR-31 binding site in the 3′-UTR of LATS2. c-d. The mRNA and protein levels of LATS2 were detected in the group of ESCC cell lines than HEEC cell lines via qRT-PCR and western blot assays. e-f. QRT-PCR and western blot analysis showed that miR-31 could negatively regulate LATS2 expression of ESCC cells. g. Immunohistochemical staining of LATS2 protein in primary ESCC tissue samples (Left: ×200; Right: ×400). h-i. Kaplan-Meier survival plots analysis of the association of LATS2 with the OS and PFS of ESCC patients. Values are mean ± SD; *: P < 0.05; **: P < 0.01

Fig. 3

Effects of LATS2 on ESCC cell proliferation, colony formation, migration, and invasion in vitro. a QRT-PCR analysis of the relative expression of LATS2 in two groups of ESCC cells transfected with cDNA/pLATS2, siRNA/LATS2 and their corresponding control. b-c MTT and colony formation assays in ESCC cells after changed expressing of LATS2. d Wound scratch healing assay was performed to evaluate the motility of ESCC cells transfected with cDNA/pLATS2, siRNA/LATS2. Photographs were taken immediately (0 h) and at 48 h after wounding, quantification of wound closure was done. e Migration assay and Invasion assay were conducted to examine the invasion ability of ESCC cells. Results are expressed as the mean ± SD of three independent experiments. *: P < 0.05; **: P < 0.01

Fig. 4

Silencing of LATS2 reverses the effects of anti-miR-31 on phenotypes of ESCC cells. a Quantification of LATS2 expression was achieved by qRT-PCR transfected with anti-miR-31 or co-transfected with siRNA/LATS2. b-c MTT and colony formation assays were conducted to evaluate the proliferative capacity of ESCC cells. d-e. Wound healing assays were used to assess the motility of ESCC cells transfected with anti-miR-31 alone or co-transfected with siRNA/LATS2. f Migration and invasion assay were conducted in ESCC cells transfected with the same type. Results are expressed as the mean ± SD of three independent experiments. *: P < 0.05; **: P < 0.01

Fig. 5

MiR-31 regulates EMT in ESCC cells. a Morphologies of ESCC cell lines after changing the expression of miR-31. b-c QRT-PCR and Western blotting were used to analysis the mRNA and protein levels of EMT markers in ESCC cells after transfection with anti-miR-31. d Immunofluorescence assay was executed the expression of EMT protein markers transfection with anti-miR-31 in ESCC cells. *: P < 0.05; **: P < 0.01

Fig. 6

LATS2 influences EMT in ESCC cells. a-b QRT-PCR and Western blotting were used to analysis the mRNA and protein levels of EMT markers in ESCC cells after transfection with cDNA/pLATS2 and/or siRNA/LATS2. c Immunofluorescence assay was executed the expression of EMT protein markers transfection with cDNA/pLATS2 in ESCC cells. *: P < 0.05; **: P < 0.01

Fig. 7

MiR-31/LATS2 interaction regulates EMT in ESCC cells. a-b QRT-PCR and Western blotting were used to analysis the mRNA and protein levels of EMT markers in ESCC cells after transfection with anti-miR-31 and co-transfection siRNA/LATS2. *: P < 0.05; **: P < 0.01

Fig. 8

LATS2 overexpression leads to the reduction of TAZ, which induces tumor development and correlates with prognosis in ESCC. a-c QRT-PCR and western blot analysis showed that LATS2 could negatively regulate TAZ expression of ESCC cells. b Western blot for LATS2 antibody. d Immunohistochemical staining of TAZ protein in primary ESCC tissue samples (Left: ×200; Right: ×400). e Kaplan-Meier survival plots analysis of the association of TAZ with the OS and PFS of ESCC patients. f Rescue assay confirmed that overexpression of TAZ partially reverse the effects of LATS2 inhibition in ESCC cells via qRT-PCR Values are mean ± SD. g Analysis of correlation between miR-31 and LATS2/TAZ, LATS2 and TAZ expression levels in 164 ESCC tissues specimens. *: P < 0.05; **: P < 0.01

Fig. 9

MiR-31/LATS2/TAZ interaction regulates tumor growth in vivo. a-b Effect of miR-31 on tumor growth in nude minces. Growth curves of tumors resulted from injection of Eca109 cells transfected with anti-miR-31 in nude mice. Immunostaining of LATS2 and TAZ protein stained sections of the transplanted tumors as indicated (original magnification, ×400). c-e Effect of different LATS2 expression on tumor growth in nude mice. Xenograft assay with TE1 stable cells revealed that inhibition of LATS2 improved the volume of the xenograft tumors, while restoration of LATS2 showed a significantly depressed tumor volume. f-g Loss of LATS2 significantly reversed the suppression of tumor growth induced by anti-miR-31. Immunostaining of LATS2 and TAZ protein stained sections was indicated (original magnification, ×400). *: P < 0.05; **: P < 0.01