A novel long non-coding RNA XLOC_004787, is associated with migration and promotes cancer cell proliferation by downregulating mir-203a-3p in gastric cancer

Abstract

Background: Long noncoding RNAs (lncRNAs) have been identified as important regulatory factors implicated in a wide array of diseases, including various forms of cancer. However, the roles of most lncRNAs in the progression of gastric cancer (GC) remain largely unexplored. This study investigates the biological function and underlying mechanism of a novel lncRNA, XLOC_004787 in GC.

Methods: The location of XLOC_004787 in GES-1 cells and HGC-27 cells were detected by fluorescence in situ hybridization (FISH) assay. The expression levels of XLOC_004787 were assessed using quantitative real-time fluorescence PCR (qRT-PCR) in various cell lines, including GES-1, MGC-803, MKN-45, BGC-823, SGC-7901, and HGC-27 cells. Functional assays such as Transwell migration, cell counting kit-8 (CCK-8), and colony formation experiments were employed to analyze the effects of XLOC_004787 and miR-203a-3p on cell migration and proliferation. Protein levels associated with GC in these cell lines were examined by Western blotting. The intracellular localization of β-catenin and P-Smad2/3 was assessed using immunofluorescence (IF) assay. Additionally, the interaction between XLOC_004787 and miR-203a-3p was investigated using a dual luciferase assay.

Results: XLOC_004787 was localized at both the cytoplasm and nucleus of GES-1 cells and HGC-27 cells. Compared to normal tissues and GES-1 cells, XLOC_004787 expression was significantly upregulated in GC tissues and cells, with the highest and lowest expression observed in SGC-7901 and HGC-27 cells, respectively. Furthermore, a reduced expression of XLOC_004787 was seen to inhibit migration and proliferation in SGC-7901 cells. Western blotting analysis revealed that a decrease in XLOC_004787 expression correspondingly decreased the expression of N-cadherin, mmp2, mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1, and TGF-β, while concurrently increasing E-cadherin expression. This was also associated with diminished expression of P-Smad2/3 in relation to Smad2/3, and reduced P-Gsk3β expression in comparison to Gsk3β. Additionally, the nuclear entry of P-Smad2/3 and β-catenin was reduced by lower XLOC_004787 expression. Amplifying XLOC_004787 expression via pcDNA_XLOC_004787 suggested a potential for cancer promotion. Notably, XLOC_004787 was found to negatively regulate mir-203a-3p expression, with potential binding sites identified between the two. Higher mir-203a-3p expression was observed to decrease migration and proliferation, and enhance E-cadherin expression. Conversely, suppression of mir-203a-3p expression suggested a potential promotion of proliferation and migration in GC cells.

Conclusions: These results suggest that XLOC_004787, found to be upregulated in GC tissues, potentially promotes proliferation and migration in GC cells. This occurs through the activation of TGF-β and Wnt/β-catenin signaling pathways and the expression of EMT-related proteins. Additionally, XLOC_004787 may influence cell migration and proliferation by modulating the signaling pathway via the adsorption and inhibition of mir-203a-3p.

Keywords: Gastric cancer; Migration; Mir-203a-3p; Proliferation; XLOC_004787.

Fig. 1

XLOC_004787 is upregulated both in GC tissues and cells. A and B RNA-FISH assay revealed the cytoplasmic and nuclear location of XLOC_004787 in GES-1 cells and HGC-27 cells (original magnification × 600). C and D XLOC_004787 mRNA expression in 32 paired GC tissues and the adjacent normal tissues by qRT-PCR. E qRT-PCR detected XLOC_004787 levels within GES-1 cells as well as five GC cell lines. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

High expression of XLOC_004787 promoted cell migration and proliferation in GC cells. A qRT-PCR was used to detect the efficiency of knockdown and overexpression of XLOC_004787. B CCK-8 assay was conducted to observe the ability of proliferation in GC cells. C Colony-formation assay was carried out to observe the ability of proliferation in GC cells. D Transwell migration assay was used to detect the efficiency of cell migration (original magnification × 40). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

High expression of XLOC_004787 upregulated the proteins levels related with migration, proliferation and EMT in GC cells. A, C and G By western blotting analysis, the level of E-cadherin increased and the standard of mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1 and TGF-β decreased after XLOC_004787 knockdown in SGC-7901 cells. B, D and H By western blotting detection, the standard of E-cadherin declined and the level of mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1 and TGF-β raised after XLOC_004787 overexpression in HGC-27 cells. E and I Western blotting found that knock down XLOC_004787 in SGC-7901 cells reduced the expression of P-Gsk3β and P-Smad2/3 relative to Gsk3β and Smad2/3. F and J Western blotting showed that upregulated XLOC_004787 in HGC-27 cells enhanced the expression of P-Gsk3β and P-Smad2/3 relative to Gsk3β and Smad2/3. Data were shown as mean ± SEM. The experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

High expression of XLOC_004787 promoted the efficiency of entering the nucleus of P- Smad2/3 and β-catenin. A and C, The amount of P-Smad2/3 and β-catenin entering the nucleus is reduced after XLOC_004787 silencing by IF, scale bar = 25 µm. B and D, The quantity of P-Smad2/3 and β-catenin entering the nucleus is enhanced after XLOC_004787 overexpression by IF (original magnification × 600)

Fig. 5

XLOC_004787 has an inhibitory effect on the expression of mir-203a-3p. A Through the prediction of AnnoLnc, mir-203a-3p had binding site with XLOC_004787. B Fluorescence had been inhibited after transfection of the wild type plasmids, fluorescence inhibition is released after transfection of the mutant plasmid. C mir-203a-3p expression increased after XLOC_004787 knocked down, mir-203a-3p expression decreased after XLOC_004787 overexpressed by qRT-PCR analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

High expression of mir-203a-3p suppressed cell migration and proliferation in GC cells. A qRT-PCR was used to detect the efficiency of knockdown and overexpression of mir-203a-3p. B Colony-formation assay was carried out to observe the ability of proliferation in GC cells. C Transwell migration assay was used to detect the efficiency of cell migration (original magnification × 40). D CCK-8 assay was conducted to observe the ability of proliferation in GC cells. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

High expression of mir-203a-3p suppressed the proteins levels related with migration, proliferation and EMT in GC cells. A, C and G By western blotting analysis, the level of E-cadherin increased and the standard of mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1 and TGF-β decreased after mir-203a-3p overexpression in SGC-7901 cells. B, D and H By western blotting detection, the standard of E-cadherin declined and the level of mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1 and TGF-β raised after mir-203a-3p inhibition in HGC-27 cells. E and I Western blotting found that upregulated mir-203a-3p in SGC-7901 cells reduced the expression of P-Gsk3β and P-Smad2/3 relative to Gsk3β and Smad2/3. F and J Western blotting showed that inhibited XLOC_004787 in HGC-27 cells enhanced the expression of P-Gsk3β and P-Smad2/3 relative to Gsk3β and Smad2/3. Data were shown as mean ± SEM. The experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001