Long non-coding RNA PCED1B antisense RNA 1 promotes gastric cancer progression via modulating microRNA-215-3p / C-X-C motif chemokine receptor 1 axis

Figures & data

Table 1. The correlations of PCED1B-AS1 with clinicopathological features of patients with gastric cancer

Characteristics	Patients(n)	PCED1B-AS1expression		P values
		Low expression (n = 21)	High expression (n = 27)
Age (years)
≤ 60	32	12	20	0.217
> 60	16	9	7
Tumor size (cm)
≤ 5	16	11	5	0.014*
> 5	32	10	22
Gender
Male	31	12	19	0.342
Female	17	9	8
TNM stage
I – II	27	16	11	0.014*
III–IV	21	5	16
Tumor differentiation
Well + moderate	29	14	15	0.435
Poor	19	7	12
Lymph node metastasis
Absent	28	17	11	0.005**
Present	20	4	16
Distant metastasis
Absent	21	8	13	0.486
Present	27	13	14

*P < 0.05.

Table 2. The primer sequence used in this study

gene	primer sequence
PCED1B-AS1	Forward: 5ʹ-TCAAGCCAATCAGCTGACAC-3’
	Reverse: 5ʹ- AAACAAATGCCCTGCTTGAC-3’
CXCR1	Forward: 5ʹ-CTGACCCAGAAGCGTCACTTG −3’
	Reverse: 5ʹ-CCAGGACCTCATAGCAAACTG-3’
miR-215-3p	Forward: 5ʹ-TGGATTTGGACGCATTGGTC-3’
	Reverse: 5ʹ-TTTGCACTGGTACGTGTTGATA-3’
GAPDH	Forward: 5ʹ-ACCCAGAAGACTGTGGATGG-3’
	Reverse: 5ʹ- TTCAGCTCAGGGATGACCTT-3’
U6	Forward: 5ʹ-TGCGGGTGCTCGCTTCGGCAGC-3’
	Reverse: 5ʹ-CCAGTGCAGGGTCCGAGGT-3’

Figure 1. PCED1B-AS1 is highly expressed in GC tissues and cell lines

A. The expression of PCED1B-AS1 in the tissues of GC patients was analyzed by GEPIA database.B. The relationship between the expression of PCED1B-AS1 and the the overall survival time of GC patients was analyzed by Kaplan-Meier Plotter database.C. The expression of PCED1B-AS1 was detected by qRT-PCR in GC and normal tissues.D. The expression of PCED1B-AS1 in GES-1 cells and GC cell lines (HGC-27, KATO III, NCI-N87 and AGS) was detected by qRT-PCR.*P < 0.05, ** P < 0.01, and *** P < 0.001

Figure 2. The effect of PCED1B-AS1 on GC cells’ proliferation, migration, invasion and EMT

A. The expression of PCED1B-AS1 in GC cells transfected with si-PCED1B-AS1 and pc-PCED1B-AS1 was detected by qRT-PCR.B-C. CCK-8 and EdU methods were used to detect the regulatory effect of PCED1B-AS1 on the proliferation of GC cells.D-E. Transwell assay was used to detect the regulatory effect of PCED1B-AS1 on migration and invasion of GC cells.F. Western blot assay was used to detect the regulatory effect of PCED1B-AS1 on protein levels of EMT markers (E-cadherin, N-cadherin and Vimentin) in GC cells.* P < 0.05, ** P < 0.01, and *** P < 0.001.

Table 3. The correlations of miR-215-3p with clinicopathological features of patients with gastric cancer

Characteristics	Patients(n)	miR-215-3p expression		P values
		Low expression (n = 17)	High expression (n = 31)
Age (years)
≤60	32	11	21	0.831
>60	16	6	10
Tumor size (cm)
≤5	16	4	12	0.037*
>5	32	13	9
Gender
Male	31	13	18	0.202
Female	17	4	13
TNM stage
I – II	27	6	21	0.030*
III–IV	21	11	10
Tumor differentiation
Well + moderate	29	4	25	<0.001***
Poor	19	13	6
Lymph node metastasis
Absent	28	7	21	0.074
Present	20	10	10
Distant metastasis
Absent	21	6	15	0.382
Present	27	11	16

*P < 0.05, and *** P < 0.001.

Figure 3. PCED1B-AS1 serves as a ceRNA by sponging miR-215-3p in GC

A. The expression of PCED1B-AS1 in the nuclear fraction and cytoplasmic fraction of GC cells was detected by qRT-PCR. U6 and GAPDH were detected in nuclear and cytoplasmic fractions.B. Potential binding sites between PCED1B-AS1 and miR-215-3p were analyzed through the LncBase Predict v.2 database.C. RIP assay was used to validate the interaction between PCED1B-AS1 and miR-215-3p.D. The targeting relationship between PCED1B-AS1 and miR-215-3p was confirmed by dual-luciferase reporter gene assay.E. qRT-PCR was used to detect the expression of miR-215-3p in 48 cases of GC tissues and normal adjacent tissues.F. The expression of miR-215-3p in GES-1 cells and GC cell lines (HGC-27, KATO III, NCI-N87 and AGS) was detected by qRT-PCR.G. Spearman’s correlation analysis was used to analyze the correlation between PCED1B-AS1 expression and miR-215-3p expression in tissues.H. qRT-PCR was used to detect the expression of miR-215-3p in GC cells, after PCED1B-AS1 was overexpressed or knocked down.*P < 0.05, ** P < 0.01, and *** P < 0.001.

Table 4. The correlations of CXCR1 with clinicopathological features of patients with gastric cancer

Characteristics	Patients(n)	CXCR1 expression		P values
		Low expression (n = 20)	High expression (n = 28)
Age (years)
≤60	32	14	18	0.679
>60	16	6	10
Tumor size (cm)
≤5	16	8	8	0.408
>5	32	12	20
Gender
Male	31	13	18	0.959
Female	17	7	10
TNM stage
I – II	27	11	16	0.883
III–IV	21	9	12
Tumor differentiation
Well + moderate	29	14	15	0.251
Poor	19	6	13
Lymph node metastasis
Absent	28	12	16	0.843
Present	20	8	12
Distant metastasis
Absent	21	13	8	0.012*
Present	27	7	20

*P < 0.05 Statistically significant.

Figure 4. CXCR1 is a target mRNA of miR-215-3p

A. The potential binding site between miR-215-3p and CXCR1 was predicted by TargetScan database.B-C. The expression of CXCR1 was detected by qRT-PCR and Western blot assays. N, normal tissues; T, tumor tissues.D. The correlation between CXCR1 and the expression of miR-215-3p and PCED1B-AS1 was analyzed by Spearman’s correlation analysis.E. The targeting relationship between CXCR1 and miR-215-3p was confirmed by dual-luciferase reporter assay.F. Western blot was used to detect the expression of CXCR1 protein in HGC-27 and NCI-N87 cells transfected with miR-215-3p mimics or inhibitors. **P < 0.01, and ***P < 0.001

Figure 5. PCED1B-AS1 promotes the proliferation, migration, invasion and EMT of GC cells by regulating the miR-491-5p/CXCR1 axis

A-B. MiR-215-3p mimic or si-CXCR1 was co-transfected with pc-PCED1B-AS1 into NCI-N87 cells, and the expression of miR-215-3p and CXCR1 detected by qRT-PCR.C-D. CCK-8 and EdU assays were used to detect the proliferation of GC cells after transfection.E-F. Transwell assay was used to detect the migration and invasion of GC cells after transfection.G. Western blot assay was used to detect the protein levels of E-cadherin, N-cadherin, and Vimentin in GC cells after transfection.*P < 0.05, ** P < 0.01, and *** P < 0.001.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.