LincROR promotes tumor growth of colorectal cancer through the miR-145/WNT2B/WNT10A/Wnt/β-catenin regulatory axis

Abstract

Colorectal cancer (CRC) is a prevalent form of malignant tumor, and the current clinical treatments are far from satisfactory. Identifying new therapeutic targets is therefore essential for clinical practices. The long intergenic non-protein coding RNA lincROR has been shown to play a significant role in the tumorigenesis of various cancers. However, the molecular mechanism underlying lincROR-mediated CRC tumorigenesis remains unclear. In the present study, we found that knockdown of lincROR significantly inhibited cell viability in vitro, while its overexpression promoted tumor growth in vivo. Mechanistically, lincROR acted as a miRNA sponge for miR-145, thereby elevating the expression of the target genes WNT2B and WNT10A. The overexpression of WNT2B and WNT10A definitely activated the Wnt/β-catenin pathway, thus led to promoting tumorigenesis in CRC. In summary, our findings identified lincROR as a novel activator of the Wnt/β-catenin pathway by serving as a miRNA sponge for miR-145 and facilitating tumorigenesis, which suggests that lincROR may be a potential therapeutic target for CRC patients.

Fig 1. Knockdown of lincROR suppressed the cell viability and induced the inactivation of Wnt/β-catenin signalling in CRC cells.

(A), The lincROR expression was examined in a panel of CRC cells. (B), LincROR expression was suppressed in SW62 cells infected with shROR. (C-E), Cell viability (C) and colony formation (D, E) were assessed in the lincROR knockdown cells. (F), The TOP flash luciferase activity was examined in the lincROR knockdown cells using dual luciferase assays. (G-H), β-catenin expression was determined in the lincROR silenced SW620 cells by Western blotting and qRT-PCR analysis. (I), The expression of several downstream targets of Wnt/β-catenin pathway was analysed in lincROR knockdown cells through qRT-PCR examination. Data were shown as mean ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; versus the corresponding control group.

Fig 2. LincROR-targeted MiR-145 acted as a negative regulator of CRC cells growth via suppressing Wnt/β-catenin signalling.

(A), The predicted binding sites of miR-145 in lincROR were shown in red, and the binding sequence was inserted into the pmirGLO vector. (B), The CRC cells were co-transfected with miR-145 mimics and WT or MUT luciferase reporter, and the luciferase activity was measured by dual luciferase reporter assay. (C), The miR-145 expression was measured by qRT-PCR examination with lincROR overexpression or knockdown. (D-E), The cell viability was evaluated by MTT assay in miR-145 mimics or anti-miR-145 transfected SW620 cells. (F-I), the colony formation was examined in miR-145 mimics or anti-miR-145 transfected CRC cells. (J-M), The mRNA and protein levels of β-catenin were examined in CRC cells with miR-145 or anti-miR-145 transfection. (N-O), The relative expression of several downstream target genes of the Wnt/β-catenin pathway were examined by qRT-PCR in the miR-145 or anti-miR-145 transfected SW620 cells. Data were shown as mean ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; versus the corresponding control group.

Fig 3. WNT2B and WNT10A were real targets of miR-145 in CRC cells.

(A), The binding sequence of miR-145 in WNT2B/WNT10A 3’UTR was inserted into the dual-luciferase reporter vector to generate the WT plasmids. In contrast, this binding sequence was deleted to generate MUT plasmids. (B-C), Following transfection with miR-145, the luciferase activities of WT and MUT reporters were assessed. (D-F), The mRNA and protein expression levels of WNT2B and WNT10A were detected in CRC cells with miR-145 or anti-miR-145 transfection into SW620 cells. (G-I) The mRNA and protein expression levels of WNT2B and WNT10A were examined by qRT-PCR and western blot examination in lincROR silencing or overexpressing SW620 cells. Data were shown as mean ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; versus the corresponding control group.

Fig 4. LincROR activated the Wnt/β-Catenin signalling pathway by targeting miR-145.

(A-B), the mRNA expression levels of WNT2B and WNT10A were examined in the lincROR overexpressing SW620 cells with miR-145 or anti-miR-145 transfection. (C), The protein expression levels of β-catenin, WNT2B, and WNT10A were examined in the lincROR overexpressing SW620 cells with miR-145 or anti-miR-145 transfection. (D-E), The mRNA expression level of β-catenin was assessed in the lincROR overexpressing SW620 cells with miR-145 or anti-miR-145 transfection. F, The mRNA expression levels of several downstream targets of Wnt/β-catenin signalling were examined in the lincROR overexpressing SW620 cells with miR-145 or anti-miR-145 transfection. Data were shown as mean ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; versus the corresponding control group.

Fig 5. LincROR overexpression promoted the tumor growth of CRC in vivo.

(A), Representative images of burdened tumors in nude mice (n = 5). (B-C), Tumor weights and volumes were analyzed in the lincROR overexpression group and the control group. (D), The immunofluorescence analysis of Ki-67 was performed on tumor tissues (Scale bar, 20 μm.). (E), The immunohistochemical analysis of β-catenin, WNT2B, and WNT10A was conducted on tumor tissues (Scale bar, 100 μm.). Data were shown as mean ± SD (n = 5). *, P < 0.05; ***, P < 0.001; vs NC; versus the corresponding control group.