Lnc-LRRTM4 promotes proliferation, metastasis and EMT of colorectal cancer through activating LRRTM4 transcription

Abstract

Numerous mechanisms have shown that long noncoding RNAs (lncRNAs) promote the development of colorectal cancer (CRC), but the role of lnc-LRRTM4 in the progression of CRC remains unclear. In this article, we found that lnc-LRRTM4 was highly expressed in CRC tissues and cell lines and that lnc-LRRTM4 could promote the proliferation and metastasis of CRC cells. These consequences were achieved by lnc-LRRTM4 directly binding to the promoter of LRRTM4 to induce its transcription. Moreover, lnc-LRRTM4 enhanced the growth of CRC cells in vivo by promoting cell cycle progression and reducing apoptosis. Taken together, our results revealed that lnc-LRRTM4 promotes the proliferation and metastasis of CRC cells, suggesting that it may be a potential diagnostic and therapeutic target for CRC.

Keywords: CRC; EMT; LRRTM4; Long noncoding RNA; lnc-LRRTM4.

Fig. 1

Lnc-LRRTM4 is highly expressed in CRC and predicts poor prognosis. (A) Hierarchical clustering showing significantly differentially expressed lncRNAs of CRC tissues and para-tumor tissues from the GEO database (GSE156732) (FC ≥ 4 or ≤ -4, p < 0.05). (B) The relative expression of lnc-LRRTM4 in CRC tissues and normal tissues from TCGA database. (C) The relative expression of lnc-LRRTM4 in 50 pairs of CRC tissues and corresponding para-tumor tissues. (D) The expression of lnc-LRRTM4 in normal intestinal epithelial cell line FHC and CRC cell lines (HCT116, SW480, DLD1, LoVo, HCT8, SW620 and HT29). (E) Kaplan–Meier analysis of the OS rate in CRC patients in the TCGA database with high or low expression of lnc-LRRTM4. (F) Kaplan–Meier analysis of the DFS rate in CRC patients in the TCGA database with high or low expression of lnc-LRRTM4. Data are the means ± SD (n = 3 independent experiments), ** p < 0.01, *** p < 0.001

Fig. 2

Lnc-LRRTM4 promotes the proliferation and metastasis of CRC cells. (A) The expression of lnc-LRRTM4 after knockdown in HCT116 and SW480 cells. (B) The expression of lnc-LRRTM4 after overexpression in LoVo cells. (C) The effects of lnc-LRRTM4 knockdown on proliferation in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on proliferation in LoVo cells. (D) The effects of lnc-LRRTM4 knockdown on migration in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on migration in LoVo cells. (E) Statistical analysis of cell numbers from (D). (F) The effects of lnc-LRRTM4 knockdown on invasion in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on invasion in LoVo cells. (G) Statistical analysis of cell numbers from (F). Data are the means ± SD (n = 3 independent experiments), ** p < 0.01, *** p < 0.001

Fig. 3

Lnc-LRRTM4 activates EMT to accelerate the progression of CRC. (A) The effects of lnc-LRRTM4 knockdown on cell cycle in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on cell cycle in LoVo cells. (B) The effects of lnc-LRRTM4 knockdown on apoptosis in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on apoptosis in LoVo cells. (C) The effects of lnc-LRRTM4 knockdown on cell cycle proteins and apoptosis proteins in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on cell cycle proteins and apoptosis proteins in LoVo cells. (D) The effects of lnc-LRRTM4 knockdown on EMT proteins in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on EMT proteins in LoVo cells. Data are the means ± SD (n = 3 independent experiments), * p < 0.05, ** p < 0.01

Fig. 4

Lnc-LRRTM4 enhances LRRTM4 expression to accelerate CRC progression. (A) Image of RNA-FISH staining, lnc-LRRTM4 probes are red, nucleus is blue (1000×). (B) Image showed the nearby gene of lnc-LRRTM4 in the NCBI database. (C) The effects of lnc-LRRTM4 knockdown on LRRTM4 mRNA in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on LRRTM4 mRNA in LoVo cells. (D) The effects of lnc-LRRTM4 knockdown on LRRTM4 protein in HCT116 and SW480 cells and the effects of lnc-LRRTM4 overexpression on LRRTM4 protein in LoVo cells. (E) The effects of LRRTM4 knockdown on proliferation in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on proliferation in LoVo cells. (F) The effects of LRRTM4 knockdown on migration in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on migration in LoVo cells. (G) Statistical analysis of cell numbers from (F). (H) The effects of LRRTM4 knockdown on invasion in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on invasion in LoVo cells. (I) Statistical analysis of cell numbers from (H). Data are the means ± SD (n = 3 independent experiments), ** p < 0.01, *** p < 0.001

Fig. 5

Lnc-LRRTM4 enhances LRRTM4 transcription to promote CRC progression. (A) The effects of LRRTM4 knockdown on cell cycle in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on cell cycle in LoVo cells. (B) The effects of LRRTM4 knockdown on apoptosis in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on apoptosis in LoVo cells. (C) The effects of LRRTM4 knockdown on cell cycle proteins and apoptosis proteins in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on cell cycle proteins and apoptosis proteins in LoVo cells. (D) The effects of LRRTM4 knockdown on EMT proteins in HCT116 and SW480 cells and the effects of LRRTM4 overexpression on EMT proteins in LoVo cells. (E) Probe of lnc-LRRTM4 and negative control LacZ were used for ChIRP, followed by qPCR of LRRTM4 promoter in HCT116 and SW480 cells. (F) Luciferase plasmid of LRRTM4 promoter was transfected into HCT116 and SW480 cells with or without lnc-LRRTM4 siRNA, followed by detection of luciferase activity. Data are the means ± SD (n = 3 independent experiments), * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 6

Lnc-LRRTM4-mediated cell proliferation and metastasis require LRRTM4. (A) The effects of LRRTM4 overexpression on proliferation in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on proliferation in lnc-LRRTM4 overexpression LoVo cells. (B) The effects of LRRTM4 overexpression on migration in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on migration in lnc-LRRTM4 overexpression LoVo cells. (C) Statistical analysis of cell numbers from (B). (D) The effects of LRRTM4 overexpression on invasion in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on invasion in lnc-LRRTM4 overexpression LoVo cells. (E) Statistical analysis of cell numbers from (D). Data are the means ± SD (n = 3 independent experiments), * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 7

LRRTM4 is essential for Lnc-LRRTM4-mediated cell proliferation and metastasis. (A) The effects of LRRTM4 overexpression on cell cycle in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on cell cycle in lnc-LRRTM4 overexpression LoVo cells. (B) The effects of LRRTM4 overexpression on apoptosis in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on apoptosis in lnc-LRRTM4 overexpression LoVo cells. (C) The effects of LRRTM4 overexpression on cell cycle proteins and apoptosis proteins in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on cell cycle proteins and apoptosis proteins in lnc-LRRTM4 overexpression LoVo cells. (D) The effects of LRRTM4 overexpression on EMT proteins in lnc-LRRTM4 stable knockdown HCT116 and SW480 cells and the effects of LRRTM4 inhibition on EMT proteins in lnc-LRRTM4 overexpression LoVo cells. Data are the means ± SD (n = 3 independent experiments), * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 8

Lnc-LRRTM4 promotes cell proliferation in vivo. (A) Image of xenografts formed by lnc-LRRTM4 stable knockdown HCT116 cells and control cells. (B) Volume of xenografts formed by lnc-LRRTM4 stable knockdown HCT116 cells and control cells. (C) Weight of xenografts formed by lnc-LRRTM4 stable knockdown HCT116 cells and control cells. (D) The expression of lnc-LRRTM4 in xenografts formed by lnc-LRRTM4 stable knockdown HCT116 cells and control cells. (E) IHC staining of Ki67, LRRTM4, caspase 3, Cyclin D1 and snail expression in xenografts formed by lnc-LRRTM4 stable knockdown HCT116 cells and control cells. Data are the means ± SD (n = 6 independent experiments), ** p < 0.01