miR-663a inhibits tumor growth and invasion by regulating TGF-β1 in hepatocellular carcinoma

Abstract

Background: The dysregulation of miR-663a is frequently observed in many human cancers. However, the functional role and precise mechanism of miR-663a have been controversial in hepatocellular carcinoma (HCC) and need to be studied in depth.

Methods: The expression of miR-663a was detected in human cell lines and HCC tissues by quantitative RT-PCR (qRT-PCR), and data from the Cancer Genome Atlas (TCGA). Cell proliferation was investigated using MTS, EdU, colony formation assays, and xenograft animal experiments, and the cell invasion capacity was evaluated using the transwell assay. The target gene of miR-663a was identified by qRT-PCR, Western blot, and dual-luciferase reporter assays. The clinicopathological features of miR-663a and the correlation between miR-663a and TGF-β1 expression were also investigated in the clinical samples of HCC.

Results: miR-663a was significantly downregulated in HCC cells relative to immortal normal liver cells, as indicated using qRT-PCR, and the lower expression of miR-663a was also confirmed in HCC tissue samples and the data from TCGA. The expression of miR-663a in HCC tissue samples was statistically significantly associated with size and the number of tumors. In addition, the upregulation of miR-663a inhibited the proliferation and invasion of HCC cells in vitro. Further study showed that miR-663a directly targeted transforming growth factor beta 1 (TGF-β1) to suppress HCC invasion, and that the inhibitory effect of miR-663a on cell invasion could be regulated by TGF-β1. In vivo studies showed that miR-663a significantly inhibited tumor growth. A negative correlation between miR-663a and TGF-β1 expression was also confirmed from the clinical samples of HCC.

Conclusions: miR-663a acts as a tumor suppressor and exerts a substantial role in inhibiting the proliferation, invasion, and tumorigenesis of HCC by regulating TGF-β1 in vitro and in vivo. These observations indicate that miR-663a may be a suitable diagnostic, therapeutic, and prognostic target for the treatment of HCC.

Keywords: Hepatocellular carcinoma (HCC); Invasion; Proliferation; Transforming growth factor β1 (TGF-β1); miR-663a.

Fig. 1

miR-663a was low-expressed in HCC tissues and cell lines. a qRT- PCR analysis of miR-663a expression in 60 pairs HCC and their adjacent nontumor liver tissues. The relative expression of miR-663a was normalized to U6 snRNA. b Analysis of microarray data from the Cancer Genome Atlas project database (TCGA) database. c qRT- PCR analysis of miR-663a expression in HCC cell lines (Huh-7, HCC-LM3, SK-HEP1, MHCC97H and MHCC97L) and normal immortalized liver cell line (L02). Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001

Fig. 2

miR-663a inhibited HCC cell proliferation abilities. a Level of miR-663a in Huh-7 and SK-HEP1 cells transfected with miR-663a agomiR, antagomiR and respective control as assessed by qRT-PCR. The expression of miR-663a was normalized against U6 snRNA. b The MTS assay analysis was used to evaluate the proliferation of HCC cells transfected with miR-663a agomiR, antagomiR and respective control. c The EdU assay analysis was used to evaluate the proliferation of HCC cells transfected with miR-663a agomiR and control. d The colony formation assay analysis was used to evaluate the proliferation of HCC cells transfected with miR-663a agomiR, antagomiR and respective control. Representative fields are shown (400×). Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001

Fig. 3

Effect of miR-663a on invasion in HCC. Transwell assay analysis was used to evaluate the invasive capacity of HCC cells transfected with miR-663a agomiR, antagomiR and respective control. Representative fields are shown (400×). Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001

Fig. 4

miR-663a directly targeted TGF-β1. a The overlap of TGF-β1 by bioinformatics prediction between miRWalk, TargetScan, PITA, DIANAmT and miRanda programs. b Diagram of the putative binding site of miR-663a on the 3′UTR of TGF-β1 predicted by Targetsan. The mutant sequences of 3′-UTR of TGF-β1 used in luciferase reporter is shown in transverse line. c Relative expression of luciferase reporters with wild type TGF-β1 3′-UTR or mutant TGF-β1 3′-UTR after co-transfection with miR-663a agomir, antagomir or respective control in 293 T. d qRT-PCR analysis of TGF-β1 mRNA expression in the HCC cells transfected by either miR-663a agomir, antagomir or respective control. The expression of TGF-β1 was normalized against GAPDH. e Western blot analysis of TGF-β1 protein expression in the HCC cells transfected by either miR-663a agomir, antagomir or respective control. The expression of TGF-β1 was normalized against Tubulin. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 5

TGF-β1 was a functional target of miR-663a. a Downregulation of TGF-β1 expressions inhibited the invasion of HCC cells, which was similar to those induced by miR-663a agomir. b miR-663a antagomir-induced cell invasion was reversed by the knockdown of TGF-β1. Representative fields are shown (400×). Data are presented as mean ± SD (n = 3). *p < 0.05, ***p < 0.001

Fig. 6

miR-663a suppressed tumorigenesis in vivo. a Tumor formation in nude mice 4 weeks after injection with Huh-7- pLenti-miR-663a and Huh-7- pLenti-NC. b Growth curve of Huh-7- pLenti-miR-663a and Huh-7- pLenti-NC-formed tumors. Volumes of the tumors were measured every 3 days. (Data are presented as mean ± SD (n = 6)). c Volumes of the Huh-7- pLenti-miR-663a and Huh-7- pLenti-NC-formed tumors 4 weeks after the initial injection. d Weights of the Huh-7- pLenti-miR-663a and Huh-7- pLenti-NC-formed tumors 4 weeks after the initial injection. e The expression of miR-663a and TGF-β1 was detected by qRT–PCR in the mouse tumor tissues induced by Huh-7- pLenti-miR-663a and Huh-7- pLenti-NC. (Data are presented as mean ± SD (n = 3)). f The expression of TGF-β1 in tumor tissues was measured by immunohistochemistry and HE staining. Representative fields are shown (400×). **p < 0.01, ***p < 0.001

Fig. 7

Decline of miR-663a related with the up-regulation of TGF-β1 in HCC tissues. (a) Expression of miR-663a and TGF-β1 were detected by the qRT-PCR in 8 pairs of HCC samples. miR-663a was decreased in 6 of 8 HCC samples. N, Patient number. (b) The correlation analysis between miR-663a and TGF-β1 in HCC tissues. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001