Long non-coding RNA TUSC7 acts a molecular sponge for miR-10a and suppresses EMT in hepatocellular carcinoma - PubMed (original) (raw)

. 2016 Aug;37(8):11429-41.

doi: 10.1007/s13277-016-4892-6. Epub 2016 Mar 22.

Zhikui Liu 1, Bowen Yao 1, Changwei Dou 1, Meng Xu 1, Yumo Xue 1, Linglong Ding 1, Yuli Jia 1, Hongyong Zhang 1, Qing Li 1, Kangsheng Tu 1, Yang Jiao 2 3, Qingguang Liu 4, Cheng Guo 5

Affiliations

Long non-coding RNA TUSC7 acts a molecular sponge for miR-10a and suppresses EMT in hepatocellular carcinoma

Yufeng Wang et al. Tumour Biol. 2016 Aug.

Abstract

Despite advances in the roles of long non-coding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) in cancer biology, which has been identified as a tumor suppressor by regulating cell proliferation, apoptosis, migration, invasion, cell cycle, and tumor growth, the function of TUSC7 in hepatocellular carcinoma (HCC) remains unknown. In this study, we observed that the expression of TUSC7 was immensely decreased in HCC. Clinically, the lower expression of TUSC7 predicted poorer survival and may be an independent risk factor for HCC patients. Moreover, TUSC7 inhibited cell metastasis, invasion, and epithelial-to-mesenchymal transformation (EMT) through competitively binding miR-10a. Furthermore, we found that TUSC7 could decrease the expression of Eph tyrosine kinase receptor A4 (EphA4), a downstream target of miR-10a as well as an EMT suppressor, through TUSC7-miR-10a-EphA4 axis. Taken together, we demonstrate that TUSC7 suppresses EMT through the TUSC7-miR-10a-EphA4 axis, which may be a potential target for therapeutic intervention in HCC.

Keywords: EMT; EphA4; HCC; TUSC7; miR-10a.

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Conflict of interest statement

Compliance with ethical standards This study was reviewed and approved by the Research Ethics Committee of the Xi’an Jiaotong University. All patients provided informed consent before surgery. All samples were handled according to the ethical and legal standards. Conflicts of interest None

Figures

Fig. 1

Fig. 1

The expression levels of TUSC7 in HCC. Comparing differences in the expression levels of TUSC7 between a HCC and matched non-tumor tissues, b aggressive and non-aggressive tumor tissues, c HCC tissues arising from recurrent and non-recurrent groups, and d HCC cell lines and the immortalized hepatic cell line LO2. Values are depicted as mean ± SD; *p < 0.05, by t test

Fig. 2

Fig. 2

Prognostic significance of TUSC7 in HCC cases. Kaplan-Meier 3-year a overall and b disease-free survival curves of HCC patients according to the level of TUSC7 expression. The low TUSC7 group (≤0.33, n = 53); the high TUSC7 group (>0.33, n = 22). The mean expression value (0.33) obtained for TUSC7 of the 75 HCC samples detected by qRT-PCR was chosen as the cutoff value. *p < 0.05, by log-rank test

Fig. 3

Fig. 3

Wound healing assays to assess the effect of TUSC7 on cell mobility. a qRT-PCR analysis revealed that TUSC7 expression in Hep3B cells was reduced most obviously by _TUSC7_-siRNA3 and pcDNA/TUSC7 largely increased the TUSC7 expression in MHCC97H cells. b Wound healing assays to assess the effect of TUSC7 on cell mobility in Hep3B cells. c Wound healing assays to assess the effect of TUSC7 on cell mobility in MHCC97H cells.*P < 0.05, by t test

Fig. 4

Fig. 4

Transwell assays to assess the effect of TUSC7 on migration and invasion in HCC cells. a The effect of TUSC7 on migration and invasion ability in Hep3B cells. b The effect of TUSC7 on migration and invasion ability in MHCC97H cells. *P < 0.05, by t test

Fig. 5

Fig. 5

TUSC7 inhibited EMT progression in HCC. a_–_d Immunohistochemistry staining of E-cadherin and vimentin in HCC tissues. In cases of high TUSC7 expression tissue group (a, b), there was strong E-cadherin and no detectable vimentin protein expression in the same tissue section. In contrast, in the cases of low TUSC7 expression tissue group (c, d), there was no detectable E-cadherin and strong vimentin protein expression. Values are depicted as mean ± SD; **p < 0.001, by _t_ test. _Scale bar_ = 100 μm. _e_, _f_ Expression of EMT mRNA markers was assessed by qRT-PCR in the low _TUSC7_ expression tissue group (≤0.33, _n_ = 38) and high _TUSC7_ expression tissues group (>0.33, n = 37), both groups from HCC samples. g Hep3B and MHCC97H cells with different TUSC7 levels were subjected to western blot for E-cadherin and vimentin. Representative western blot showed that downregulation of TUSC7 obviously increased protein expression of vimentin and reduced E-cadherin expression in HCC cells. *P < 0.05, by t test

Fig. 6

Fig. 6

TUSC7 targets miR-10a. a TUSC7 binding sequence in miR-10a-WT and sequence of miR-10a-Mut. b TUSC7 overexpression significantly suppressed the luciferase activity that carried wild-type but not mutant-type miR-10a. And TUSC7 overexpression almost had no effect on the luciferase activity that carried neither wild-type nor mutant-type miR-10a. n = three repeats with similar results; **p < 0.01, by t test. c, d qRT-PCR revealed that TUSC7 could negatively regulate miR-10a expression; e, f qRT-PCR revealed that TUSC7 could positively regulate the mRNA expression level of EphA4. *P < 0.05, by t test

Fig. 7

Fig. 7

miR-10a reverses the inhibitory effects of TUSC7 on HCC cells. a–d Wound healing assays showed that miR-10a largely reversed the inhibitory effect of TUSC7 on cell mobility. e Western blot revealed that miR-10a largely reversed the inhibitory effect of TUSC7 on EMT. f–i Transwell assays showed that miR-10a could largely reverse the inhibitory effect of TUSC7 on cell migration and invasion.* P <0.05, ** P <0.01, *** P <0.001, by t test

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