Figures & data
Figure 1. circFCHO2 expression was elevated in GC, associating with poor outcome.
(A) The expression of circFCHO2 in 30 pairs of tumor tissues and normal tissues adjacent to tumors was assessed using qRT-PCR. (B) The relationship between circFCHO2 and lymph node metastasis (and lung metastasis) of GC patients was researched via using qRT-PCR. (C) Kaplan–Meier curve was made to detect the 60-month survival and metastasis-free survival of GC patients. (D) circFCHO2 expression in cell lines was evaluated by qRT-PCR. (E) Actinomycin D treatment of GC cells was performed to detect the stability of circFCHO2 structure. (F) RNase R was applied for circFCHO2 structure stability detection. (G) The structure of circFCHO2 was monitored by the agarose gel analysis of the PCR production. (H) RNAFISH experiment was used to research the expression and location of circFCHO2 in GC cells. (I) Nuclear and cytoplasmic separation and qRT-PCR were used to detect the expression of circFCHO2 in the cytoplasm and nucleus of GC cells. *** P < 0.001.
Figure 2. circFCHO2 silencing weakened the proliferation, invasion, angiogenesis and stem cell characteristics of GC cells.
(A) The transfection efficiency of cells was determined by qRT-PCR. (B) The expression of FCHO2 mRNA in GC cells was determined by qRT-PCR. (C) CCK-8 assay was applied to assay the proliferation ability of GC cells. (D) EDU staining was used to assess the proliferation ability of GC cells. (E) Transwell experiment was conducted to explore the invasion ability of GC cells. (F) Tube formation experiment was carried out to analyze the angiogenesis ability of GC cells. (G) Spheroidization experiment was used to research the stem cell characteristic detection of GC cells. ** P < 0.01 or *** P < 0.001.
Figure 3. circFCHO2 exerted as a sponge of miR-194-5p.
(A) miR-194-5p, miR-151a-3p and miR-526b-5p were predicted as potential targets of circFCHO2. (B) RNA pull-down was applied to detect the binding relationship between circFCHO2 and the three mRNAs (miR-194-5p, miR-151a-3p and miR-526b-5p). (C) The expression of miR-194-5p in GC patients was investigated using qRT-PCR. (D) Pearson’s correlation analysis researched the correlation between miR-194-5p and circFCHO2 in tumor tissues of GC patients. (E) The expression of miR-194-5p in cell lines was detected by qRT-PCR. (F) The binding site of circFCHO2 and miR-194-5p. (G) Dual-luciferase reporter gene assay. (H) RIP experiment was employed to detect the binding of circFCHO2 and miR-194-5p. (I) miR-194-5p expression in the transfected GC cells was determined by qRT-PCR. *** P < 0.001.
Figure 4. miR-194-5p knockdown partially reversed the inhibition of circFCHO2 silencing on the malignant phenotype of GC cells.
(A) Transfection efficiency of GC cells was explored by qRT-PCR. *** P < 0.001. (B) The proliferation ability of GC cells was researched by CCK-8 assay. (C) EDU staining was carried out to evaluate the proliferation ability of GC cells. (D) The invasion ability of GC cells was detected by transwell experiment. (E) The angiogenesis ability of GC cells was evaluated by tube formation experiment. (F) The cancer stem cell characteristics of GC cells was assessed by spheroidization experiment. ** P < 0.01 or *** P < 0.001 vs. sh-NC group. # P < 0.05 or ## P < 0.01 or ### P < 0.001 vs. sh-circFCHO2 group.
Figure 5. circFCHO2 promoted the activity of the JAK1/STAT3 pathway by sponging miR-194-5p.
(A) JAK1 contained mutual binding site of circFCHO2. (B) Dual-luciferase reporter gene assay. *** P < 0.001. (C) JAK1 expression in the 30 GC patients was explored by qRT-PCR. *** P < 0.001. (D and E) Pearson’s correlation analysis was carried out to research the correlation between JAK1 and miR-194-5p or circFCHO2. (F) The expression of JAK1 protein in cells was detected by Western blot. *** P < 0.001. (G and H) The expression of JAK1 protein in the transfected GC cells was researched through Western blot. *** P < 0.001. (I) The expression of JAK1 and p-STAT3/STAT3 proteins in the transfected GC cells was investigated by Western blot. *** P < 0.001 vs. sh-NC group. ## P < 0.01 or ### P < 0.001 vs. sh-circFCHO2 group.
Figure 6. JAK1 overexpression partially reversed the inhibition of miR-194-5p on the malignant phenotype of GC cells.
(A) The expression of JAK1 protein in the transfected GC cells was monitored by Western blot. *** P < 0.001. (B) The proliferation ability of GC cells was assessed by CCK-8 assay. (C) EDU staining was used to reflect the proliferation ability of GC cells. (D) The invasion ability of GC cells was detected by transwell experiment. (E) The angiogenesis ability of GC cells was investigated by tube formation experiment. (F) The cancer stem cell characteristics of GC cells was evaluated by spheroidization experiment. *** P < 0.001 vs. miR-NC group. # P < 0.05 or ## P < 0.01 or ### P < 0.001 vs. miR-194-5p group.
Figure 7. circFCHO2 silencing weakened the in vivo growth and lung metastasis of GC.
(A and B) The volume and weight of xenograft tumor in nude mice was monitored. (C) IHC was applied to monitor the expression of Ki67 protein in xenograft tumor. (D) The expression of circFCHO2, miR-194-5p and JAK1 mRNA in xenograft tumor was explored by qRT-PCR. (E) Western blot was used to research the expression of JAK1, p-STAT3 and STAT3 proteins in xenograft tumor. (F) HE staining was applied to detect the nodules in lung tissues of mice. (G) The original images of lung nodules. (H) The number of mice with or without lymphatic metastasis in each group. *** P < 0.001.
Figure 8. circFCHO2 in serum exosomes was a sensitive and effective biomarker for the diagnosis of GC.
(A) Serum exosomes of GC patients and healthy participants were isolated. TEM was used for the observation of serum exosomes. (B) Markers of exosomes were researched by Western blot. (C) circFCHO2 expression in the serum exosomes of GC patients and healthy participants was researched by using qRT-PCR. (D) ROC curve was used to analyze the potential of circFCHO2 as a diagnostic marker for GC patients. *** P < 0.001.
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All the data used to support the findings of this study are included within the article.