HOXC-AS1-MYC regulatory loop contributes to the growth and metastasis in gastric cancer

Abstract

Background: Gastric cancer (GC) is one of the most prevalent and deadly malignancies worldwide. Accumulating reports have indicated the participation of long non-coding RNAs (lncRNAs) in the onset and progression of GC.

Methods: GSE109476 data was utilized to screen out lncRNAs dysregulated in GC. Gene expressions were determined by qRT-PCR and western blot. Both in vitro and in vivo experiments were carried out to assess the function of HOXC-AS1 in GC. The association between genes was verified via RIP, ChIP, CoIP, RNA pull down and luciferase reporter assays, as appropriate.

Results: HOXC-AS1 was discovered to be upregulated in GC and located both in cytoplasm and in nucleus in GC cells. Functionally, inhibition of HOXC-AS1 restrained GC cell growth and metastasis both in vitro and in vivo. Moreover, HOXC-AS1 was proved to be trans-activated by c-MYC in GC. In return, HOXC-AS1 positively regulated MYC expression in GC through targeting miR-590-3p/MYC axis in cytoplasm and modulating BRG1/β-catenin complex-activated MYC transcription in nucleus. Furthermore, the rescue assays verified that MYC mediated HOXC-AS1-affected GC progression.

Conclusion: Our research illustrated a feedback loop of HOXC-AS1-MYC in aggravating GC cell growth and metastasis, highlighting HOXC-AS1 as a promising target for GC diagnosis and treatment.

Keywords: BRG1; Gastric cancer; HOXC-AS1; MYC; miR-590-3p.

Fig. 1

HOXC-AS1 was overexpressed in GC tissues and cell lines. (a, b) Heat map and volcano plot obtained from analyzing GSE109476. (c) GSE109476 suggested that both HOXC-AS1 and HOXC-AS3 was highly-expressed in GC tissues compared to the adjacent ones. (d) qRT-PCR result of HOXC-AS1 and HOXC-AS3 in 35 pairs of GC tissues collected in our study. (e) The expression of HOXC-AS1 in GC cell lines was examined by qRT-PCR. (f) The potential localization of HOXC-AS1 predicted by lncLocator. (g) FISH analysis of HOXC-AS1 location in GC cells. * P < 0.05, ** P < 0.01

Fig. 2

Knockdown of HOXC-AS1 inhibited cell proliferation, migration, invasion and EMT in GC. (a) qRT-PCR result of HOXC-AS1 in BGC-823 and AGS cells transfected with shCtrl or two shRNAs aiming at HOXC-AS1. (b) The viability of BGC-823 and AGS cells under above transfections was assessed by CCK-8 assays. (c, d) EdU and TUNEL assays were respectively performed to evaluate cell proliferation and apoptosis in BGC-823 and AGS cells with HOXC-AS1 inhibition or not. (e, f) Transwell assays were implemented to estimate the effect of HOXC-AS1 on GC cell migration and invasion. Western blot analysis of E-cadherin, N-cadherin and Vimentin in BGC-823 and AGS under HOXC-AS1 knockdown versus control. ** P < 0.01

Fig. 3

HOXC-AS1 was transcriptionally upregulated by c-MYC in GC. (a) Three online tools including UCSC, JASPAR and PROMO predicted that c-MYC might potentially regulate HOXC-AS1 transcription. (b, e) The expression of MYC and HOXC-AS1 in GC cells with MYC downregulation or overexpression was assayed by qRT-PCR. (f, g) ChIP and luciferase reporter assays revealed that HOXC-AS1 was positively regulated by c-MYC at transcriptional level. (h, i) The predicted binding of c-MYC on HOXC-AS1 promoter region was simulated here. (j) Luciferase reporter assay confirmed the pricise binding of c-MYC to HOXC-AS1 promoter at site from − 1954 to − 1959. * P < 0.05, ** P < 0.01

Fig. 4

HOXC-AS1 triggered MYC expression in GC by absorbing miR-590-3p in cytoplasm. (a) qRT-PCR result of MYC level in GC cells with or without HOXC-AS1 silence. (b) DIANA predicted that there was only two miRNA that interacted with both HOXC-AS1 and MYC. (c) The expression levels of above two miRNAs in GC cell lines were determined by qRT-PCR. (d) qRT-PCR result of the two miRNAs in BGC-823 and AGS cells with HOXC-AS1 inhibition or not. (e) RIP assay proved the co-existence of HOXC-AS1, miR-590-3p and MYC mRNA in RISC. (f, g) Luciferase reporter assay showed the competition binding of HOXC-AS1 and MYC mRNA to miR-590-3p. (H) Relative expression of MYC in indicated BGC-823 and AGS cells was analyzed via qRT-PCR. * P < 0.05, ** P < 0.01

Fig. 5

HOXC-AS1 promoted BRG1 interaction with β-catenin to enhance MYC transcription. (a) qRT-PCR result of the expression of MYC in BGC-823 and AGS cells in response to HOXC-AS1 silence or not, or HOXC-AS1 silence plus miR-590-3p inhibition. (b) The effect of HOXC-AS1 on MYC transcription was assessed by luciferase reporter assay. (c) The binding of HOXC-AS1 to MYC promoter was examined by DNA pull down assay. (d) TOP/FOP flash assay was conducted to estimate the impact of HOXC-AS1 on Wnt/β-catenin activation. (e, f) The influence of HOXC-AS1 on CTNNB1 expression and β-catenin nuclear translocation was determined by qRT-PCR and western blot, as appropriate. (g) The interaction between HOXC-AS1 and BRG1 in GC cells was testified by RIP and RNA pull down assays. (h) The interaction between BRG1 and β-catenin in GC cells with or without HOXC-AS1 knockdown was evaluated by CoIP assay. ** P < 0.01

Fig. 6

Silencing HOXC-AS1 confined GC tumorigenesis and metastasis in vivo. (a) Representative images and tumor volume of AGS cells transfected with shCtrl, shHOXC-AS1, or shHOXC-AS1 + pcDNA3.1/MYC. (b) Mean weight of these tumors. (c) qRT-PCR result of the expression of HOXC-AS1 and MYC in above tumors. (d) The expression of HOXC-AS1, MYC, Ki67, E-cadherin and N-cadherin in those tumors was tested by ISH or IHC staining, as needed. (e) The level of EMT-related proteins was determined by western blot. (f, g) HE staining of livers and lung obtained from in vivo metastatic experiments and quantitation of the metastatic nodules in these livers. ** P < 0.01

Fig. 7

MYC upregulation reversed the suppression of HOXC-AS1 inhibition on the biological processes of GC cells. (a) qRT-PCR and western blot analyses were performed to assay the expression of MYC at both mRNA and protein levels in AGS cells transfected with shCtrl, shHOXC-AS1#1 or shHOXC-AS1#1 together with pcDNA3.1 plasmid containing MYC. (b-e) The viability, proliferation, apoptosis and motility in above AGS cells were assessed respectively by CCK-8, EdU, TUNEL and transwell assays. (f) The level of EMT-associated proteins in indicated AGS cells was determined by western blot. * P < 0.05, ** P < 0.01

Fig. 8

Schematic model of the HOXC-AS1-MYC feedback loop in aggravating GC tumorigenesis and metastasis. C-MYC-activated HOXC-AS1 sponges miR-590-3p in cytoplasm to stabilize MYC mRNA and strengthens BRG1-β-catenin interaction to promote MYC transcription in nucleus in the meantime, leading to prompted MYC expression and resultantly accelerated GC progression