LncRNA LINC00342 contributes to the growth and metastasis of colorectal cancer via targeting miR-19a-3p/NPEPL1 axis

Abstract

Background: Long intergenic non-protein coding RNA 00342 (LINC00342) has been identified as a novel oncogene. However, the functional role of LINC00342 in colorectal cancer (CRC) remains unclear.

Methods: The expression of LINC00342 is detected by real-time PCR (RT-PCR) analysis. Cell proliferation, migration and invasion and xenograft model are examined to analyze the biological functions of LINC00342 in vitro and in vivo using colony formation, would healing and transwell analyses. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays are used to identify the target interactions between LINC00342, miR-19a-3p and aminopeptidase like 1 (NPEPL1).

Results: LINC00342 was highly expressed in CRC. Down-regulation of LINC00342 inhibited cell proliferation and metastasis of CRC cells. Moreover, knocking down LINC00342 inhibited the tumor growth in vivo. Mechanistic investigation revealed that LINC00342 might sponge miR-19a-3p to regulate NPEPL1 expression. Further investigation indicated that the ontogenesis facilitated by LINC00342 was inhibited due to the depletion of NPEPL1.

Conclusion: LINC00342 promotes CRC progression by competitively binding miR-19a-3p with NPEPL1.

Keywords: Colorectal cancer; Invasion; LINC00342; NPEPL1; Proliferation; miR-19a-3p.

Fig. 1

LINC00342 is significantly upregulated in CRC tissues and cells. a Expression of LINC00342 in non-tumor tissues and CRC tumor tissues determined by real-time PCR; b expression of LINC00342 in colonic epithelial NCM460 cells and four CRC cells (HCT-8, SW480, HT-29 and DLD-1); c subcellular fractionation assay of LINC00342 in SW480 and HT-29 cells, analyzed by RNA-FISH. **P < 0.05 vs. the non-tumor group or NCM460 cells

Fig. 2

LINC00342 requires for the proliferation, migration and invasion of CRC cells. SW480 and HT-29 cells were infected with lentivirus containing sh-NC or sh-LINC00342. The transfection efficiency was confirmed by real-time PCR (a) and green fluorescence microscopy (b); cell proliferation was measured with CCK-8 (c) and colony formation (d) assays; cell migration and invasion was determined by wound healing assay (e) and transwell migration and invasion assays (f); the protein levels of E-cadherin and Vimentin were measured by western blotting (g). *P < 0.05, **P < 0.01 vs. sh-NC group

Fig. 3

MiR-19a-3p is downregulated in CRC and negatively regulated by LINC00342. Expression of miR-19a-3p in CRC tumor tissues (a) and cell lines (b); ^**P < 0.01 vs. the non-tumor group or NCM460 cells. The miRNA target sites of LINC00342 was predicted by using DIANA tools, the miR-19a-3p binding site and the mutational LINC00342 sequence were shown (c); expression of miR-19a-3p in SW480 and HT-29 cells transfected with miR-19a-3p mimic or mimic NC (d); SW480 and HT-29 cells were co-transfected with the wild or mutant type LINC00342 plasmids and miR-19a-3p mimic or mimic NC, and the luciferase activities were measured (e); **P < 0.01 vs. NC mimic group. RIP assays were performed in SW480 and HT-29 cells, and precipitated RNA levels were presented as fold changes in Ago2 relative to IgG immunoprecipitates (f); **P < 0.01 vs. IgG group. Expression of miR-19a-3p in SW480 and HT-29 cells transfected with sh-NC or sh-LINC00342 (g); **P < 0.01 vs. sh-NC group. Expression of LINC00342 in SW480 and HT-29 cells transfected with NC-mimic or miR-19a-3p mimic (h); **P < 0.01 vs. NC mimic group

Fig. 4

MiR-19a-3p inhibits CRC cell proliferation, migration and invasion. The proliferation (a, b), migration and invasion abilities (c, d) and the protein levels of E-cadherin and Vimentin (E) in SW480 and HT-29 cells transfected with mimic NC or miR-19a-3p mimic. **P < 0.01 vs. NC mimic group

Fig. 5

LINC00342 functions as a ceRNA of NPEPL1 by sponging miR-19a-3p. TargetScan was used to predict that NPEPL1 mRNA 3′UTR contains miR-19a-3p binding site, and the mutational 3′UTR of NPEPL1 mRNA were indicated (a); SW480 and HT-29 cells were co-transfected with the wild or mutant type NPEPL1 plasmids and miR-19a-3p mimic or mimic NC, and the luciferase activities were measured (b); ^**P < 0.01 vs. mimic NC group. RIP assay showed that NPEPL1 was a direct target of miR-19a-3p (c); The mRNA and protein levels of NPEPL1 in SW480 and HT-29 cells transfected with mimic NC or miR-19a-3p mimic (d, e); **P < 0.01 vs. mimic NC group. Luciferase reporter assay showed the interaction between LINC00342, miR-19a-3p and NPEPL1 (f); **P < 0.01 vs. NC mimic group. ^##P < 0.05 vs. miR-19a-3p mimic group. The mRNA and protein levels of NPEPL1 in SW480 and HT-29 cells transfected with sh-NC or sh-LINC00342 (g, h); **P < 0.01 vs. sh-NC group

Fig. 6

Knockdown of NPEPL1 rescues the carcinogenesis of LINC00342 on CRC progression. The expression of NPEPL1 and LINC00342 in NPEPL1 and LINC00342-overexpressed SW480 cells (a); **P < 0.01 vs. NC inhibitor group. The proliferation (b, c), migration and invasion abilities (d, e) and the protein levels of E-cadherin and Vimentin (f) in SW480 and HT-29 cells transfected with LINC00342 and/or sh-NPEPL1; **P < 0.01, ***P < 0.001 vs. NC inhibitor group; ^#P < 0.05, ^##P < 0.05 vs. miR-19a-3p inhibitor + sh-NC group

Fig. 7

Silencing LINC00342 inhibits tumorigenesis of CRC cells through in vivo. Representative images of nude mice and tumors (a); the growth curves of tumors derived from HT-29 cells transfected with sh-LINC00342 or si-NC (b); the average weight of tumors (c); the analysis of histological in tumor sections (d); immunohistochemistry assay was conduct to assess the expression levels of Ki67, E-cadherin and vimentin. The photographs were taken at the magnification of × 200 (E, F). *P < 0.05, **P < 0.01, ***P < 0.001 vs. sh-NC group