MicroRNA -196b is related to the overall survival of patients with esophageal squamous cell carcinoma and facilitates tumor progression by regulating SOCS2 (Suppressor Of Cytokine Signaling 2)

Figures & data

Table 1. Relationship between miR-196b expression and clinical parameters of patients with ESCC

Variables	Cases (n = 115)	miR-196b expression		P-value
		Low (n = 57)	High (n = 58)
Age				0.509
≤60	61	32	29
>60	54	25	29
Gender				0.768
Male	65	33	32
Female	50	24	26
Smoking				0.780
Absent	58	28	30
Present	57	29	28
Alcohol drinking				0.373
Absent	72	38	34
Present	43	19	24
Pickled veg consumption				0.075
Absent	49	29	20
Present	66	28	38
Location				0.638
Upper	23	12	11
Middle	75	35	40
Lower	17	10	7
Lymph node metastasis				0.010
Negative	80	46	34
Positive	35	11	24
TNM stage				0.004
I–II	83	48	35
III	32	9	23

Table 2. Multivariate Cox analysis of clinical characteristics in relation to overall survival

Characteristics	Multivariate analysis
	HR	95% CI	P
miR-196b	2.099	1.124–3.920	0.020
Age	1.773	0.976–3.221	0.060
Gender	1.500	0.857–2.623	0.155
Smoking	1.022	0.584–1.790	0.938
Alcohold drinking	1.408	0.753–2.634	0.284
Pickled veg consumption	1.496	0.850–2.632	0.163
Location	1.592	0.624–4.059	0.330
Lymph node metastasis	1.956	1.101–3.477	0.022
TNM stage	1.846	1.060–3.215	0.030

Figure 1. miR-196b expression was measured in ESCC tissue and cells. A. miR-196b expression was increased in ESCC tissue samples compared to adjacent normal tissue specimens. the analysis was carried out using paired students’ t-test by graphpad prism software. B. miR-196b expression was raised in four ESCC cell lines than normal esophageal epithelial cell Het-1A. the data were analyzed using one-way ANOVA by graphpad prism software. ***P < 0.001

Figure 2. Kaplan-meier method was conducted to analyze the five-year survival rate of ESCC patients using SPSS software. high expression of the miR-196b group displayed a shorter overall survival rate than the low miR-196b expression group (log-rank test P= 0.005)

Figure 3. Proliferation of TE-1 and Eca-109 cells was facilitated or suppressed after the upregulation or downregulation of miR-196b compared to untreated cells. (a) and (b) expression level of miR-196b was determined in TE-1 and Eca-109 cells by qRT-PCR after transfection with miR-196b mimic, inhibitor, or NCs. (c) and (d) proliferative capacity of TE-1 and Eca-109 cells were measured by CCK-8. (e) and (f) Migratory ability of TE-1 and Eca-109 cells were measured by transwell assay (magnification 200×). (g) and (h) Invasive ability of TE-1 and Eca-109 cells were measured by transwell assay (magnification 200×). The above analysis was performed using one-way ANOVA by graphpad prism software. *P < 0.05, ***P < 0.001

Figure 4. miR-196b interacted with SOCS2. (a) the binding site between SOCS2 and miR-196b is shown. (b) spearman correlation analysis revealed the negative correlation between miR-196b and SOCS2 expression. (c) the cell lysate was incubated with an anti-AGO2 antibody for RIP, and the SOCS2 content was measured by RT-PCR. (d) expression of SOCS2 mRNA was detected using qRT-PCR in TE-1 cells with upregulated or downregulated expression of miR-196b. (e) the expression of SOCS2 protein was measured using a western blot assay. (f) luciferase activity of WT-SOCS2 TE-1 cells group was weakened or enhanced by miR-196b overexpression or knockdown, respectively, but luciferase activity has no change in MUT-SOCS2 TE-1 cells by miR-196b. The above analysis was performed by graphpad prism software.***P < 0.001