Silencing CDR1as inhibits colorectal cancer progression through regulating microRNA-7

Figures & data

Figure 1 CDR1as is highly expressed in CRC.

Notes: (A) qRT-PCR analysis of CDR1as expression in 40 paired CRC tissues and adjacent normal tissues. (B) qRT-PCR analysis of CDR1as expression in four colon cancer cell lines and two normal epithelial cell lines. (C) Kaplan–Meier curve of OS with CDR1as in CRC patients. *P<0.05, **P<0.01, and ***P<0.001.
Abbreviations: CRC, colorectal cancer; qRT-PCR, quantitative reverse transcription polymerase chain reaction; OS, overall survival; HR, hazard ratio; CI, confidence interval.

Table 1 Correlations between CDRlas expression and clinico-pathological parameters in CRC

Characteristic	n	CDRlas expression, median (range)	P-value
Sex			0.003*
Female	83	2.18 (0.53–4.98)
Male	99	3.87 (1.46–8.93)
Age (years)			0.584
<60	68	2.68 (0.55–7.22)
≥60	114	2.83 (1.00–7.80)
Location			0.561
Right colon	40	2.55 (0.81–5.54)
Left colon	57	2.85 (0.75–10.43)
Rectum	85	2.85 (1.02–7.18)
Tumor size (cm)			0.032*
<5	111	2.57 (0.52–7.16)
≥5	71	3.87 (1.46–8.13)
Histology			0.166
Adenocarcinoma	149	2.95 (1.13–7.54)
Mucinous adenocarcinoma	33	1.88 (0.38–8.25)
Primary pT stage			0.003*
1/2	37	1.09 (0.24–4.70)
3/4	145	2.95 (1.40–8.08)
Primary pN stage			0.002*
0	95	1.57 (0.77–5.79)
1/2	87	2.65 (1.09–7.26)
Synchronous distant metastasis			0.221
No	158	2.74 (0.75–7.24)
Yes	24	3.36 (2.33–8.31)
Stage			0.433
I, II	88	2.59 (0.76–7.64)
III, IV	94	2.83 (1.24–7.48)
Preoperative CEA level			0.331
<5	99	2.95 (0.77–7.81)
5–19	49	2.47 (0.89–4.21)
≥20	34	2.96 (1.38–9.87)

Note:

* represents P<0.05.

Abbreviations: CRC, colorectal cancer; CEA, carcinoembryonic antigen.

Figure 2 CDR1as downregulation inhibits tumor cell proliferation.

Notes: (A) qRT-PCR analysis of CDR1as expression and cell viability analysis in shCON and shCDR1as lentivirus-infected HCT-116 and DLD-1 cells. (B) Cell-cycle distributions of HCT-116 and DLD-1 cells infected with shCON or shCDR1as lentivirus for 72 h. (C) CDR1as knockdown did not significantly increase apoptosis rate in colon cancer HCT-116 cells. (D) Colony images and counts of shCON and shCDR1as lentivirus-infected HCT-116 and DLD-1 cells. *P<0.05, **P<0.01 and ***P<0.001.
Abbreviations: qRT-PCR, quantitative reverse transcription polymerase chain reaction; shCON, control-scrambled shRNA; shCDR1as, shRNA targeting CDR1as; OD, optical density.

Figure 3 CDR1as downregulation suppresses tumor cell invasion.

Notes: (A) Invasion images and counts of colon cancer cells infected with control or shCDR1as lentivirus for 72 h (magnification 100×). (B) Western blot analysis of MMP-9 in HCT-116 and DLD-1 cells infected with control or shCDR1as lentivirus for 72 h. **P<0.01 and ***P<0.001.
Abbreviations: shCON, control-scrambled shRNA; shCDR1as, shRNA targeting CDR1as.

Figure 4 CDR1as promotes cell growth and invasion through partially blocking miR-7.

Notes: (A) qRT-PCR analysis of miR-7 expression in shCON and shCDR1as lentivirus-infected HCT-116 and DLD-1 cells for 72 h. (B) Growth curves of miRNA-Ctrl and miR-7 mimics in HCT-116 and DLD-1 cells for 5 days. (C) Cell-cycle distributions of HCT-116 and DLD-1 cells transfected with miRNA-Ctrl or miR-7 mimics for 48 h. (D) miR-7 mimics did not significantly increase apoptosis rate in colon cancer HCT-116 cells. (E) Growth curves of shCON, shCDR1as, and shCDR1as plus the miR-7 inhibitor in HCT-116 and DLD-1 cells. (F) Invasion images and counts of HCT-116 and DLD-1 cells treated by shCON, shCDR1as, and shCDR1as plus the miR-7 inhibitor. *P<0.05 and **P<0.01.
Abbreviations: miR-7, microRNA-7; qRT-PCR, quantitative reverse transcription polymerase chain reaction; shCON, control-scrambled shRNA; shCDR1as, shRNA targeting CDR1as; miRNA, microRNA; Ctrl, control; OD, optical density.

Figure 5 CDR1as positively regulates EGFR and IGF-1R in CRC.

Notes: (A) qRT-PCR analysis of miR-7-targeted genes in HCT-116 cells infected with shCON and shCDR1as lentivirus for 72 h. (B) Western blot analysis of EGFR and IGF-1R following shCON and shCDR1as lentivirus infection for 72 h in HCT-116 cells. (C) Western blot analysis of EGFR and IGF-1R in HCT-116 cells treated by shCON, shCDR1as, and shCDR1as plus the miR-7 inhibitor for 72 h. (D) EGFR and IGF-1R mRNA levels were positively correlated with CDR1as expression in CRC tumor samples. **P<0.01.
Abbreviations: CRC, colorectal cancer; qRT-PCR, quantitative reverse transcription polymerase chain reaction; miR-7, microRNA-7; shCON, control-scrambled shRNA; shCDR1as, shRNA targeting CDR1as.

Figure S1 CDRlas expression is not correlated with miR-7.\

Note: Pearson correlation analysis of CDRlas and miR-7 expression in 182 CRC tumor samples.

Abbreviations: miR-7, microRNA-7; CRC, colorectal cancer.

Table S1 Specific real-time qPCR primers

Genes	Forward and reverse primers
PAK1	F 5′-CAGCCCCTCCGATGAGAAATA-3′
	R 5′-CAAAACCGACATGAATTGTGTGT-3′
EGFR	F 5′-AGGCACGAGTAACAAGCTCAC-3′
	R 5′-ATGAGGACATAACCAGCCACC-3′
IGF-1R	F 5′-TCGACATCCGCAACGACTATC-3′
	R 5′-CCAGGGCGTAGTTGTAGAAGAG-3′
PTK2	F 5′-TGGTGCAATGGAGCGAGTATT-3′
	R 5′-CCAGGTGGTTGGCTCACTA-3′
YY1	F 5′-GGCAGAATTTGCTAGAATGAAG-3′
	R 5′-AGTTGGTGTCGTTTTAGTTTTG-3′
XRCC2	F 5′-TGCTTTATCACCTAACAGCACG-3′
	R 5′-TGCTCAAGAATTGTAACTAGCCG-3′
PAX6	F 5′-TGGGCAGGTATTACGAGACTG-3′
	R 5′-ACTCCCGCTTATACTGGGCTA-3′
β Actin	F 5′-CAACCGCGAGAAGATGACCC-3′
	R 5′-GAGGCGTACAGGGATAGCAC-3′

Abbreviation: qPCR, quantitative polymerase chain reaction.

Table S2 Correlations between miR-7 expression and clinicopathological parameters in CRC

Characteristic	n	miR-7 expression, median (range)	P-value
Sex			0.357
Female	83	2.78 (0.76–6.72)
Male	99	3.34 (1.18–7.12)
Age (years)			0.335
<60	68	2.54 (0.48–6.13)
≥60	114	2.92 (1.04–7.02)
Location			0.713
Right colon	40	2.71 (0.84–5.63)
Left colon	57	2.85 (0.78–8.41)
Rectum	85	2.80 (1.00–5.78)
Tumor size (cm)			0.092
<5	111	3.54 (1.22–7.98)
≥5	71	2.73 (0.63–6.82)
Histology			0.553
Adenocarcinoma	149	2.64 (1.01–7.38)
Mucinous adenocarcinoma	33	2.43 (0.75–6.43)
Primary pT stage			0.148
1/2	37	2.65 (0.97–5.68)
3/4	145	2.14 (1.05–7.76)
Primary pN stage			0.658
0	95	2.08 (1.30–7.39)
1/2	87	2.15 (0.58–7.45)
Synchronous distant metastasis			0.493
No	158	3.10 (2.13–7.19)
Yes	24	2.95 (0.82–6.29)
Stage			0.348
I, II	88	2.96 (1.26–7.38)
III, IV	94	2.32 (0.64–6.90)
Preoperative CEA level			0.632
<5	99	2.88 (0.67–7.31)
5–19	49	2.35 (0.80–5.03)
≥20	34	3.27 (1.43–7.97)

Abbreviations: CRC, colorectal cancer; CEA, carcinoembryonic antigen.