tsRNA-GlyGCC promotes colorectal cancer progression and 5-FU resistance by regulating SPIB

Abstract

Background: tRNA-derived small RNAs (tsRNAs) are newly discovered non-coding RNA, which are generated from tRNAs and are reported to participate in several biological processes in diseases, especially cancer; however, the mechanism of tsRNA involvement in colorectal cancer (CRC) and 5-fluorouracil (5-FU) is still unclear.

Methods: RNA sequencing was performed to identify differential expression of tsRNAs in CRC tissues. CCK8, colony formation, transwell assays, and tumor sphere assays were used to investigate the role of tsRNA-GlyGCC in 5-FU resistance in CRC. TargetScan and miRanda were used to identify the target genes of tsRNA-GlyGCC. Biotin pull-down, RNA pull-down, luciferase assay, ChIP, and western blotting were used to explore the underlying molecular mechanisms of action of tsRNA-GlyGCC. The MeRIP assay was used to investigate the N(7)-methylguanosine RNA modification of tsRNA-GlyGCC.

Results: In this study, we uncovered the feature of tsRNAs in human CRC tissues and confirmed a specific 5' half tRNA, 5'tiRNA-Gly-GCC (tsRNA-GlyGCC), which is upregulated in CRC tissues and modulated by METTL1-mediated N(7)-methylguanosine tRNA modification. In vitro and in vivo experiments revealed the oncogenic role of tsRNA-GlyGCC in 5-FU drug resistance in CRC. Remarkably, our results showed that tsRNA-GlyGCC modulated the JAK1/STAT6 signaling pathway by targeting SPIB. Poly (β-amino esters) were synthesized to assist the delivery of 5-FU and tsRNA-GlyGCC inhibitor, which effectively inhibited tumor growth and enhanced CRC sensitive to 5-FU without obvious adverse effects in subcutaneous tumor.

Conclusions: Our study revealed a specific tsRNA-GlyGCC-engaged pathway in CRC progression. Targeting tsRNA-GlyGCC in combination with 5-FU may provide a promising nanotherapeutic strategy for the treatment of 5-FU-resistance CRC.

Keywords: 5-FU resistance; CRC; JAK1/STAT6; METTL1; SPIB; m7G; tsRNA.

Fig. 1

tsRNA-GlyGCC is overexpressed in CRC tissues. A-C. The difference of tsRNA profiles, volcano plot and heat map displaying differentially expressed tsRNA between five pairs of CRC and corresponding normal tissues; D. tsRNA-GlyGCC was significantly upregulation in CRC tissues; E. The structure of tRNA; F. tsRNA-GlyGCC was originated from tRNA-Gly-GCC; G. tsRNA-GlyGCC was mainly located in cell cytoplasm; H. The product of tsRNA-GlyGCC was confirmed by Sanger Sequencing; I. tsRNA-GlyGCC was located in cell cytoplasm by using IF assay; J. FISH assay was used for tsRNA-GlyGCC detection; K. qPCR was conducted to examine the expression of tsRNA-GlyGCC in normal, response CRC, and non-response CRC tissues, *p < 0.05, **p < 0.01, ***p < 0.01. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 2

tsRNA-GlyGCC promote cell proliferation and reduce cell apoptosis in CRC. A.The tsRNA-GlyGCC inhibitor significantly decrease cell proliferation, while tsRNA-GlyGCC mimics promote cell reproductive capacity; B-D. tsRNA-GlyGCC inhibitor reduced anchorage-dependent growth in 5-FU resistance CRC cells; E-G. tsRNA-GlyGCC inhibitor reduced cell migration using transwell assays; H-J. tsRNA-GlyGCC inhibitor induced apoptosis using FACS analysis. K-M. tsRNA-GlyGCC inhibitor decreased the growth of cell spheroids. *p < 0.05, **p < 0.01, ***p < 0.01. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 3

tsRNA-GlyGCC plays an oncogenic role in vivo. A. Flow chart of animal experiment; B. Body weight of mice in different groups; C. Representative photos of tumours; D: Tumor growth measured using a line chart; E. Tumor weights in different groups; F. Tunnel assay was used to detect cell apoptosis in different groups; G. Protein level of Ki67 in tumor tissues in different groups. *p < 0.05, **p < 0.01. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 4

tsRNA-GlyGCC directly targets SPIB. A. Identification of potential target genes of tsRNA-GlyGCC; B-C. The mRNA expression in cells transfected with tsRNA-GlyGCC mimics; D. The mRNA level of SPIB in CRC and adjacent normal tissues; E. Correlation analysis between SPIB and tsRNA-GlyGCC; F. The binding site between SPIB and tsRNA-GlyGCC; G. Luciferase assay was used to check the luciferase activity of wide type and mut type groups; H-I. WB assay was used to detect the protein level of SPIB when transfected with tsRNA-GlyGCC inhibitor. *p < 0.05, **p < 0.01. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 5

SPIB is a transcriptional repressor of STAT6. A-B. WB assay was used to detect the protein level of JAK1/STAT6; C. The binding sites of SPIB in STAT6 promoter region by using JASPAR database; D. Schematic diagrams of promoters of STAT6; E-F. ChIP assay was used to verify that SPIB could bind to STAT6 promoter region; G. Construction dual-luciferase reporter plasmids based on the binding sites of SPIB; H. Luciferase assay was used to detect luciferase activity in HCT116 cells overexpressing SPIB after transfection of pGL enhancer plasmids containing wide type or mut type STAT6 promoter. *p < 0.05, **p < 0.01, ***p < 0.001. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 6

m⁷G modification on tsRNA-GlyGCC. A.The schematic diagram of m7G-MeRIP assay; B. m7G-MeRIP assay was performed to verification of m7G modification sites; C-D. The expression of METTL1 in CRC tissues; E. Interference efficiency of METTL1 by using sh-plasmid; F. tsRNA-GlyGCC was downexpressed in CRC cells transfected with sh-METTL1-1 and sh-METTL1-2; G. Interference with METTL1 expression could reduce the sequencing which containing site 29 enrichment. **p < 0.01, ***p < 0.001. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 7

Characterization of PAE@^5−FUts-inhibitor complex. A.TEM images of PAE@^5−FUts-inhibitor complex; B. Particle size of the PAE@^5−FUts-inhibitor complex; C. Zeta potential of PAE and PAE@^5−FUts-inhibitor; D. Biodistribution of PAE@^5−FUts-inhibitor; E. Stability analysis of PAE@^5−FUts-inhibitor; F. The expression of tsRNA-GlyGCC.**p < 0.01, ***p < 0.001. All data are representative of at least three independent experiments and are presented as the means ± SD

Fig. 8

The antitumor effects of PAE@^5−FUts-inhibitor in vivo. A.The schematic diagram of mice treatment; B. Representative photos of tumours; C-D. The tumor volume and weight in different groups; E. HE staining of tumour cells in different groups; F. FISH assay was conducted to detect the expression of tsRNA-GlyGCC in different groups; G. Immunofluorescence staining was used to detect Ki67 expression in different groups; H-I. IHC was used to detected the expression of SPIB (H) and STAT6 (I) in tumor tissues; J-K.Tunnel assay was used to apoptosis in different groups; **p < 0.01, ***p < 0.001. All data are representative of at least three independent experiments and are presented as the means ± SD