Fig 2: SIRT6 inhibits the viability and migration of CRC cells through regulating Lin28b/NRP-1 axis(A) Western blot assay of the protein expression of SIRT6, NRP-1, and Lin28b in the SW480 and SW620 cells transfected with OE-SIRT6. *p < 0.05 versus OE-NC-transfected cells. (B) qRT-PCR detection of the expression of miR-25 in the SW480 and SW620 cells co-cultured with NCM460-EVs/HCT116-EVs. *p < 0.05 versus NCM460-EVs-treated cells. (C) Western blot assay of the protein expression of SIRT6, NRP-1, Lin28b, in the SW480 and SW620 cells co-cultured with NCM460-EVs/HCT116-EVs. *p < 0.05 versus NCM460-EVs-treated cells. (D) qRT-PCR detection of Lin28b expression in the SW480 and SW620 cells transfected with OE-Lin28b. *p < 0.05 versus OE-NC-transfected cells. (E) Western blot assay of the protein expression of Lin28b in the SW480 and SW620 cells transfected with OE-Lin28b. *p < 0.05 versus OE-NC-transfected cells. (F) qRT-PCR detection of the expression of NRP-1 in the SW480 and SW620 cells transfected with OE-NRP-1. *p < 0.05 versus OE-NC-transfected cells. (G) Western blot assay detection of the expression of NRP-1. *p < 0.05 versus OE-NC-transfected cells. (H) OE-Lin28b or OE-NRP-1 and OE-SIRT6 were used to treat SW480 and SW620 cells; western blot assay detection of the expression of SIRT6, NRP-1, Lin28b. *p < 0.05 versus OE-NC-transfected cells, #p < 0.05 versus OE-SIRT6-transfected cells. (I) ChIP experiment to identify the effect of SIRT6 on the acetylation of Lin28b promoter region. *p < 0.05 versus OE-NC-transfected cells. (J) RIP test of the binding of Lin28b to NRP-1. *p < 0.05 versus OE-NC-transfected cells. (K) Cell viability detected by CCK-8 assay. *p < 0.05 versus OE-NC-treated cells, #p < 0.05 versus OE-SIRT6-treated cells. (L) Cell migration detected by Transwell assay. *p < 0.05 versus OE-NC treated cells, #p < 0.05 versus OE-SIRT6 treated cells. (M) Cell invasion detected by Transwell assay. In (K)–(M), SW480 and SW620 cells were transfected with OE-Lin28b or OE-NRP-1 and OE-SIRT6 alone, or co-transfected with OE-SIRT6 and OE-Lin28b, OE-NRP-1, and OE-SIRT6 in combination. *p < 0.05 versus OE-NC-transfected cells, #p < 0.05 versus OE-SIRT6-transfected cells. The data were measurement data, expressed as mean ± SD. The unpaired t test was used for analysis between the two groups, one-way analysis of variance was used for comparison among multiple groups, and two-way analysis of variance was used for comparison between different groups at different time points.

Fig 3: Rate of positive nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-6 (SIRT6) expression, detected by immunohistochemistry, in lung tissue from 86 Chinese Han patients with non-small cell lung cancer (NSCLC): (a) Representative photomicrographs showing sirtuin 6 (SIRT6) protein signal intensity (graded as zero, weak, moderate and strong). Strong staining was found in normal lung specimens, moderate staining was found in TNM I–II specimens and weak or zero staining was found in TNM III–IV specimens. Magnification × 200; (b) Rate of positive SIRT6 expression was significantly lower in cancer specimens versus normal lung specimens, and the rate of positive SIRT6 expression decreased with tumour stage. Data presented as mean ± SD (*P < 0.05 versus normal lung specimens; #P < 0.05 versus TNM I–II specimens; One-way analysis of variance)

Fig 4: miR-25 in CRC cell-derived EVs promotes the tumorigenic and metastatic potentials of CRC cells in vivo through inhibiting SIRT6SW620 cells were infected with lentivirus expressing OE-SIRT6 and treated with HCT116-EVs/HCT116-EVs-miR-25 agomir, while non-infected SW620 cells were treated with NCM460-EVs/HCT116-EVs/HCT116-EVs-miR-25 agomir. The SW620 cell suspension was subcutaneously injected into nude mice. (A) Tumor growth curves showing tumor volume. (B) Weight of the tumors formed in nude mice. (C) SIRT6, Lin28b, and NRP-1 expression in tumor tissues measured by western blot assay. (D) qRT-PCR detection of the expression of miR-25 in the tumor tissues. (E) H&E staining of the mouse lung tissues to observe the metastasis. (F) H&E staining of the mouse liver tissues to observe the metastasis. ∗p < 0.05 versus NCM460-EVs group, #p < 0.05 versus HCT116-EVs group, &p < 0.05 versus HCT116-EVs-miR-25 agomir group. The data were measurement data, expressed as mean ± SD. The data among multiple groups were analyzed by one-way analysis of variance, and comparison of tumor volume at different time points was performed using repeated-measures analysis of variance; n = 6.

Fig 5: The mechanism graph of the regulatory network and function of miR-25 shuttled by CRC cell-derived EVs in CRCIn CRC cell-derived EVs, miR-25 targets and inhibits the expression of SIRT6, while SIRT6 inhibits the expression of Lin28b/NRP-1, leading to suppressed viability, migration, and invasion of CRC cells. Thus, CRC cell-derived EVs loaded with high miR-25 expression promote the tumorigenesis and metastasis of CRC cells.