Downregulation of hsa_circTLK1 represses non-small cell lung cancer progression by regulating miR-876-3p/SRSF7 axis

Abstract

Background: This study clarified the expression of cicrTLK1 in non-small cell lung cancer (NSCLC) and explored its role in cancer growth, metastasis and immune escape, providing a potential molecular target and theoretical basis for NSCLC treatment.

Methods: The expression levels of circTLK1, miR-876-3p and SRSF7 were determined by RT-qPCR assay. The localization of circTLK1 in NSCLC cells was determined by FISH assay. EdU and cell plate clone formation assay were applied to explore cell proliferation. Wound healing test and Transwell assay were applied to measure the migration and invasion ability. Cell apoptosis rate was detected by FCM assay. Western blotting assay was adopted to measure the protein expression of SRSF7. Dual-luciferase reporter gene assay was applied to assess the interaction between miR-876-3p and circTLK1, and between miR-876-3p and SRSF7. The ability of cirTLK1 to regulate tumor formation in vivo was examined by tumor transplantation experiments in nude mice.

Results: The relative expression of circTLK1 was increased in NSCLC cell lines. Knockdown of circTLK1 prohibited the proliferation, migration, and invasion, and promoted apoptosis rate, but miR-876-3p inhibitor reversed the effects of circTLK1 knockdown. In addition, silencing of circTLK1 overtly restrained the growth of transplanted tumors in vivo, and inhibited immune escape. In addition, circTLK1 interacted with miR-876-3p, and SRSF7 was concluded to be the target gene of miR-876-3p.

Conclusion: In this study, we researched the inhibitory effect of circTLK1knockdown on NSCLC progression and immune escape, and further elucidated the potential regulatory mechanism of circTLK1/miR876-3p/SRSF7 axis.

Keywords: SRSF7; hsa_circTLK1; miR-876-3p; non-small cell lung cancer.

Fig. 1

Increased expression of circTLK1 and reduced expression of miR-876-3p were discovered in NSCLC cells (A) Schematic illustrating of the generation of circTLK1. (B, C) RT-qPCR assay for the relative expression of circTLK1and miR-876-3p in NSCLC cells (NCI–H23, A549, NCI–H1734, SK-MES-1) and HBE cells. (D) The expression of circTLK1 and TLK1 was measured by a RT-qPCR assay upon RNase R treatment (The original image is provided in the Supplementary file S-Fig. 1D). (E) The cellular distribution of circTLK1 and miR-876-3p was analyzed by FISH. (F) The subcellular location of circTLK1 was examined by RT-qPCR. **p < 0.01.

Fig. 2

MiR-876-3p was targeted by circTLK1 (A) The binding sites between miR-876-3p and circTLK1 were predicted by Circular RNA Interactome. (B) Dual-luciferase reporter assay for the luciferase activity of WT-circTLK1 and MUT-circTLK1. (C) CircTLK1 enrichment was detected by RNA immunoprecipitation assay in cells transfected with miR-NC or miR-876-3p. (D) The expression of miR-876-3p was measured by RT-qPCR. **p < 0.01.

Fig. 3

Reduced of circTLK1 hindered the malignant progression of NSCLC cells by regulating miR-876-3p (A) The expression of circTLK1. (B) The expression of miR-876-3p. (C) DNA synthesis was determined by EdU assay. (D) Cell plate clone formation assay for the clonogenicity of transfected cells. (E) The cell apoptosis of NCI–H1734 and A549 cells was evaluated by FCM. (F) The migrated ability of NCI–H1734 and A549 cells was assessed through Wound healing test. (G) The cell invasion of NCI–H1734 and A549 cells was evaluated by Transwell assay. **p < 0.01.

Fig. 4

SRSF7 was a target of miR-876-3p (A) The potential binding sites between SRSF7 3’UTR and miR-876-3p were predicted by TargetScan. (B) Dual-luciferase reporter assay for the luciferase activity of WT- SRSF7 and MUT-SRSF7. (C) Western blotting assay for the protein expression of SRSF7 in NCI–H1734 and A549 cells (The original image is provided in the Supplementary file S-Fig. 4C). **p < 0.01.

Fig. 5

SRSF7 silencing suppressed the deterioration of NSCLC cells (A) The protein expression of SRSF7 (The original image is provided in the Supplementary file S-Fig. 5A). (B) EdU assay. (C) The effect of si-SRSF7 on the clonogenicity was evaluated by Cell plate clone formation assay. (D) The effect of si-SRSF7 on cell apoptosis was evaluated by FCM. (E) The effect of si-SRSF7 on cell migration ability was assessed through Wound healing test. (F) The effect of si-SRSF7 on cell invasion was evaluated by Transwell assay. **p < 0.01.

Fig. 6

CircTLK1 regulated the oncogenic activities via the miR-876-3p/SRSF7 axis(A) Western blotting assay for the protein expression of SRSF7 in NCI–H1734 and A549 cells (The original image is provided in the Supplementary file S-Fig. 6A). (B) EdU assay. (C) Cell plate clone formation assay for the clonogenicity of transfected cells. (D) The cell apoptosis of NCI–H1734 and A549 cells was evaluated by FCM. (E) The migrated ability of NCI–H1734 and A549 cells was assessed through Wound healing test. (F) The cell invasion of NCI–H1734 and A549 cells was evaluated by Transwell assay. **p < 0.01.

Fig. 7

CircTLK1 activated CD8⁺ T cells in NSCLC cells (A) CCK-8 assay. (B) FCM assay. (C–E) The expression levels of IFN-γ, IL-2 and TNF-α were detected by ELISA. **p < 0.01.

Fig. 8

CircTKL1 knockdown suppressed the xenograft growth in vivo (A) The expression of circTLK1 in xenograft tumors was detected by RT-qPCR. (B) The expression of miR-876-3p in xenograft tumors was detected by RT-qPCR. (C) Tumor volume of nude mice was recorded every 5 days. (D, E) Tumor weight was measured after injection for 30 days. (F) IHC analysis was used to detect the expression of SRSF7 and Ki67 in tumor tissues. **p < 0.01.