Crocin exerts anti-proliferative and apoptotic effects on cutaneous squamous cell carcinoma via miR-320a/ATG2B

Figures & data

Table 1. Clinicopathological features of cSCC patients enrolled in our study

Variable	n	%
Sex
Male	17	46.9
Female	15	53.1
Tumor thickness
≤2.0 mm	8	25
2.1–5.9 mm	13	40.6
≥ 6.0 mm	11	34.3
Tumor horizontal size
≤20 mm	9	28.1
21–49 mm	11	34.4
≥ 50 mm	12	37.5
Tumor differentiation
Good	19	59.4
Moderate	8	25
Poor	6	18.6
Desmoplastic growth
No	29	90.6
Yes	3	9.4
Tumor site
Ear	9	28.1
Lip	1	3.1
Other	1	3.1
Face, other	12	37.5
Body, other	9	28.1
>1 simultaneous cSCC
Absent	23	71.9
Present	9	28.1

Table 2. Primers in experiments

Gene	Primer 5ʹ→3’
MiR-320a	F: CTCCCCTCCGCCTTCTCTTC
	R: TCCTTTTTCGCCCTCTCAACC
ATG2B	F: AACTGCTGACGAATCCTCAGG
	R: GGGGTTCCAGCTAGGTGAGA
GAPDH	F: GGAGCGAGATCCCTCCAAAAT
	R: GGCTGTTGTCATACTTCTCATGG
U6	F: CTCGCTTCGGCAGCACA
	R: AACGCTTCACGAATTTGCGT

Figure 1. Clinical sample analysis. (a) MiR-320a expression in cSCC tissues and normal adjacent tissues. (b) ATG2B expression in cSCC tissues and normal adjacent tissues. (c) The link between miR-320a and ATG2B expression (Pearson correlation analysis). **P < 0.01, ***P < 0.001 versus the control group

Figure 2. Crocin suppresses the proliferation of cSCC cells and induces their apoptosis. (a) MTT assay to evaluate cell viability. (b) Inhibition rate curve used for the determination of IC50 of crocin on the proliferation of A431 and SCL-1 cells treated with 1, 2 and 4 mM crocin for 48 h. (c) MTT assay to examine cell proliferation. (d) Western blots for cyclin D1 and cleaved caspase-3 expression. NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group

Figure 3. Crocin increases autophagy in cSCC cells. (a) Immunofluorescent staining assay for autophagy. (b) Western blots for BECN1 and LC3B. **P < 0.01 versus the control group

Figure 4. MiR-320a mediates autophagy and the apoptotic effects of crocin on cSCC cells. (a) MiR-320a expression in both cell lines following crocin treatment. (b) MiR-320a expression following transfection. (c) Western blots for BECN1, LC3B and cleaved caspase-3. **P < 0.01, ***P < 0.001 versus the control group

Figure 5. ATG2B expression is upregulated by crocin. (a) The illustration of the complementary sequence between miR-320a and ATG2B. (b) The luciferase activity in wild-type luciferase constructs. (c) The luciferase activity in mutant-type luciferase constructs. (d) ATG2B expression following miR-320a inhibitor transfection. NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group

Figure 6. ATG2B expression is upregulated by crocin. (a) ATG2A mRNA expression. (b) Western blots for ATG2B. (c) ATG2A mRNA expression in both cell lines following si-ATG2B transfection. (d) Western blots for ATG2B and cleaved caspase-3. **P < 0.01, ***P < 0.001 versus the control group

Figure 7. Crocin triggers the apoptosis in cSCC in vivo. (a) The illustration of tumors in animal experiments. (b) Tumor weight. (c) Western blots for cyclin D1, cleaved caspase-3, ATG2B, BECN1 and LC3B. *P < 0.05 versus the control group