Fig 2: Overexpression of SPOCK2 or SPRED1 restrains LUAD cell growth in vivo. Nude mice were injected subcutaneously with 2 × 106 of stable transfected A549 cells to construct a xenograft tumor model. A Representative pictures and the tumor volume curves of A549 xenograft tumors. *P < 0.05, **P < 0.01 vs the pcDNA3.1 group; two-way ANOVA; n = 5. B The qRT-PCR and western blot analysis of SPOCK2 and SPRED1 in A549 tumors. **P < 0.01 vs the pcDNA3.1 group; t-test; n = 5, repeated three times. C Typical images of PCNA immunohistochemistry staining. n = 5; magnification, 400×. D Graphical summary of the effects of EZH2, SPOCK2, and SPRED1 in LUAD: EZH2-mediated H3K27me3 transcription repressed SPOCK2 and SPRED1, and suppressed the expression of SPOCK2 and SPRED1 in tumor cells of LUAD, thereby facilitating the proliferation, migration, and invasion of LUAD cells. All data are represented as mean ± SD

Fig 3: Screening of downstream targets of EZH2. EZH2 overexpression vector and its negative control vector were constructed and transfected into A549 cells. A Representative western blot showing EZH2 and H3K27me3 expression levels in A549 cells. n = 3. B The relative mRNA values of DLC1, GATA6, CCL2, SLFN11, RUNX3, SPRED1, and SPOCK2 were evaluated by qRT-PCR. *P < 0.05, **P < 0.01 vs the pcDNA3.1 group; t test; n = 3, repeated three times. C The western blot analysis of SPRED1 and SPOCK2. n = 3. A549 cells were treated with different concentrations (0, 0.25, 0.5, 1, 2 μM) of tazemetostat (EZH2 inhibitor) for 72 h, and then preformed D qRT-PCR analysis for SPOCK2 and SPRED1. **P < 0.01 vs the 0 μM tazemetostat group; one-way ANOVA; n = 3, repeated three times. E Western blot to evaluate the expression of SPRED1, SPOCK2, EZH2 and H3K27me3 upon treatment with tazemetostat. n = 3. F A549 cells were transfected with pGL3-basic or the PGL3-SPOCK2-promoter or PGL3-SPRED1-promoter, and EZH2 overexpression plasmid, then treated with or without 2 μM of tazemetostat. After 48 h, the cells were collected to measure the luciferase activity of SPOCK2 and SPRED1. **P < 0.01 vs the PGL3-Basic group, PGL3-SPOCK2-promoter group, or PGL3-SPRED1-promoter group; one-way ANOVA; n = 3. All data are represented as mean ± SD

Fig 4: MiR-665 directly binds to mRNA of SPRED1. (A) The targets of miR-665 were predicted on the basis of well-known public miRNA databases (RNA22-HSA, miRDB, and TARGETSCAN). Venn diagram shows the predicted targets. (B) The wild-type and corresponding mutant sequences of three predicted binding sites in the 3′-UTR of SPRED1 are highlighted. (C, D) Relative luciferase activity was analyzed after the reporter plasmids or mock reporter plasmids were co-transfected into HepG2 cells and SMMC7721 cells infected with or without anti-miR-665 lentivirus. NC: negative control. **P < 0.01.

Fig 5: Schematic representation of the role and molecular mechanism of sevoflurane in HCC. Sevoflurane is one of the commonly used anesthesia drugs which can assist surgeons in completing the removal of the primary tumor. For tiny HCC lesions or metastatic liver cancer cells, sevoflurane can eliminate the inhibition of tumor suppressor protein SPRED1 by decreasing the expression of miR-665 in liver cancer cells, reducing the phosphorylation level of ERK, and ultimately decreasing MMP-9 expression, thus inhibiting the metastasis ability of tumor cells. Sevoflurane can not only meet the anesthesia needs for surgery, but also have a certain antitumor effect on HCC. HCC: hepatocellular carcinoma; ERK: extracellular signal–regulated kinase; MMP-9: matrix metalloproteinase-9.