Vitamin D-mediated tsRNA-07804 triggers mitochondrial dysfunction and suppresses non-small cell lung cancer progression by targeting CRKL

Abstract

Objective: tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs with various functions in multiple cancers. Nevertheless, whether vitamin D executes its function in mitochondrial dysfunction and non-small cell lung cancer (NSCLC) progression through tsRNAs remains obscure.

Methods: Differentially expressed tsRNAs between control and vitamin D-treated H1299 cells were acquired by small RNA sequencing. Cell and animal experiments were implemented to elucidate the impacts of vitamin D and tsRNA on mitochondrial dysfunction and NSCLC progression. Dual-luciferase reporter assay, quantitative real-time PCR, western blot and recovery experiments were applied to determine the mechanism of tsRNA in NSCLC.

Results: We discovered that vitamin D receptor resulted in decreased mitochondrial-related functions and vitamin D caused mitochondrial dysfunction of NSCLC cells. tsRNA-07804 was remarkably upregulated in vitamin D-treated H1299 cells. Functional experiments indicated that vitamin D led to mitochondrial dysfunction, repressed the proliferation, migration, invasion, and promoted apoptosis of H1299 cells via regulating tsRNA-07804. Mechanistically, tsRNA-07804 induced mitochondrial dysfunction and inhibited the malignancy of H1299 cells by suppressing CRKL expression. In vivo experiments showed that vitamin D inhibited the tumor growth in NSCLC by increasing tsRNA-07804 expression. Moreover, clinical sample analysis unveiled that tsRNA-07804 had a negative correlation with CRKL.

Conclusions: In conclusion, our study proved that vitamin D induced mitochondrial dysfunction and suppressed the progression of NSCLC through the tsRNA-07804/CRKL axis. Overall, these results unveiled that tsRNA-07804 might act as a potential therapeutic target for NSCLC.

Keywords: CRKL; Mitochondrial dysfunction; Non-small cell lung cancer; Progression; Vitamin D; tsRNA-07804.

Fig. 1

Vitamin D induces mitochondrial dysfunction of NSCLC cells. A Glucose uptake, B lactate production, and C ATP production were detected in H1299 cells treated with vitamin D. D The effect of vitamin D on mitochondrial membrane potential was determined by JC-1 fluorescent probe. E The effect of vitamin D on mitochondrial content was determined by Mito-Tracker Green staining. F The effect of vitamin D on mitochondrial ROS was determined by MitoSOX staining. Scale bar = 50 μm. NC normal control group, VitD Vitamin D treatment group. *P < 0.05, **P < 0.01

Fig. 2

Analysis of tsRNA expression and related function in vitamin D-treated NSCLC cells. A Heatmap displayed differentially expressed tsRNAs in H1299 cells of vitamin D treatment and control groups. GO (B) and KEGG enrichment analysis (C) of vitamin D-regulated differentially expressed tsRNAs

Fig. 3

Vitamin D upregulated tsRNA-07804 to cause mitochondrial dysfunction of NSCLC cells. A qRT-PCR analysis of expression of five candidate tsRNAs in vitamin D-treated H1299 cells. B, C qRT-PCR analysis of overexpression and interference efficiency of tsRNA-07804 in H1299 cells. D Glucose uptake, E lactate production, and F ATP production were detected in in vitamin D-treated H1299 cells transfected with tsRNA-07804 inhibitor. G The effect of knockdown of tsRNA-07804 on mitochondrial membrane potential in vitamin D-treated H1299 cells was determined by JC-1 fluorescent probe. H The effect of knockdown of tsRNA-07804 on mitochondrial content in vitamin D-treated H1299 cells was determined by Mito-Tracker Green staining. I The effect of knockdown of tsRNA-07804 on mitochondrial ROS in vitamin D-treated H1299 cells was determined by MitoSOX staining. Scale bar = 50 μm.*P < 0.05, **P < 0.01

Fig. 4

Vitamin D upregulated tsRNA-07804 to inhibit malignant activities of NSCLC cells. A The impact of vitamin D and tsRNA-07804 overexpression on the cell viability of H1299 cells was assayed by CCK-8. B The effects of vitamin D and tsRNA-07804 interference on the proliferation of H1299 cells were assayed by CCK-8. C The effects of vitamin D and overexpression of tsRNA-07804 on migration and invasion of H1299 cells were measured by transwell assay. Scale bar = 100 μm. D The effects of vitamin D and tsRNA-07804 interference on H1299 cell migration and invasion were assayed via the transwell assay. Scale bar = 50 μm. E The effect of vitamin D and overexpression of tsRNA-07804 on apoptosis of H1299 cells was determined via flow cytometry. F The impact of vitamin D and tsRNA-07804 interference on H1299 cell apoptosis was assayed by flow cytometry. G, H Quantitative analysis of apoptosis regulated by tsRNA-07804 overexpression or knockdown. I, J The protein expression of senescence markers in tsRNA-07804 knockdown H1299 cells treated with vitamin D was detected by western blot. *P < 0.05, **P < 0.01

Fig. 5

tsRNA-07804 negatively regulates CRKL. A Venn diagram showing target genes regulated by tsRNA-07804. B The network of tsRNA-07804-target genes-signaling pathways. C qRT-PCR analysis of the relative expression of five candidate target genes of tsRNA-07804. D Correlation analyses of VDR expression and CRKL expression in the GEPIA database. E The expression of CRKL in LUSC tissues using the TNMplot database. F–H Correlation analyses of CRKL expression and NSCLC patients’ overall survival (OS), first progression (FP), and post progression survival (PPS). The expression of CRKL in H1299 cells after overexpression of tsRNA-07804 was assayed by qRT-PCR (I) and western blot (J). K Dual-luciferase activity detection. *P < 0.05, **P < 0.01

Fig. 6

tsRNA-07804 results in mitochondrial dysfunction of NSCLC cells via downregulating CRKL expression. The effect of CRKL overexpression on A glucose uptake, B lactate production, and C ATP production in tsRNA-07804 overexpressed H1299 cells were assessed by corresponding kits. D The effect of CRKL overexpression on mitochondrial membrane potential in tsRNA-07804 overexpressed H1299 cells was determined by JC-1 fluorescent probe. E Quantitative analysis of JC-1 staining. F The effect of CRKL overexpression on mitochondrial content in tsRNA-07804 overexpressed H1299 cells was determined by Mito-Tracker Green staining. G The effect of CRKL overexpression on ROS in tsRNA-07804 overexpressed H1299 cells was determined by MitoSOX staining. Scale bar = 50 μm. CRKL-OE represents CRKL overexpression. *P < 0.05, **P < 0.01

Fig. 7

tsRNA-07804 suppresses malignant behaviors of NSCLC cells via downregulating CRKL expression. Impacts of CRKL overexpression on proliferation (A), migration and invasion (B), and apoptosis (C) in tsRNA-07804 overexpressed H1299 cells were assayed by CCK-8, transwell, and flow cytometry, respectively. Scale bar = 100 μm. D The protein expression of senescence markers in tsRNA-07804 overexpressed and CRKL overexpressed H1299 cells was detected by western blot. *P < 0.05, **P < 0.01

Fig. 8

Vitamin D inhibits tumor growth via the tsRNA-07804/CRKL axis. A Images of tumors in different treatment groups. B Effects of vitamin D and tsRNA-07804 interference on tumor volume. C Effects of vitamin D and tsRNA-07804 interference on tumor weight. D qRT-PCR was applied to evaluate the effect of vitamin D and tsRNA-07804 interference on the expression of tsRNA-07804. E The impact of vitamin D and tsRNA-07804 interference on CRKL expression was assayed via qRT-PCR. F The effect of vitamin D and tsRNA-07804 interference on CRKL expression was assayed by immunohistochemistry. Scale bar = 100 μm. G, H tsRNA-07804 and CRKL expression in tumor tissues and normal adjacent tissues of NSCLC patients was tested through qRT-PCR (n = 16). I Correlation between tsRNA-07804 and CRKL. *P < 0.05, **P < 0.01