ARHGAP1 overexpression inhibits proliferation, migration and invasion of C-33A and SiHa cell lines

Figures & data

Table 1 Primers used in qRT-PCR analysis

Gene	Primer sequence	Species	Amplicon size (bp)
ARHGAP1	Forward: 5′-CAGCGTGTGATGGAATCAG-3′ Reverse: 5′-TGGAGGAAGGAGCCATAAG-3′	Human	179
MMP2	Forward: 5′-TTGACGGTAAGGACGGACTC-3′ Reverse: 5′-GGCGTTCCCATACTTCACAC-3′	Human	134
ZEB1	Forward: 5′-GCTGTAAGTGCCATTTCTC-3′ Reverse: 5′-GTCCCAGTGTTTCAGTTTC-3′	Human	159
CCNB1	Forward: 5′-CTCCGGTGTTCTGCTTCTC-3′ Reverse: 5′-GCTGTTCTTGGCCTCAGTC-3′	Human	183
Twist	Forward: 5′-AGTCCGCAGTCTTACGAG-3′ Reverse: 5′-GCTTGCCATCTTGGAGTC-3′	Human	228
PCNA	Forward: 5′-GCCTGACAAATGCTTGCTGAC-3′ Reverse: 5′-TTGAGTGCCTCCAACACCTTC-3′	Human	223
GAPDH	Forward: 5′-CACCCACTCCTCCACCTTTG-3′ Reverse: 5′-CCACCACCCTGTTGCTGTAG-3′	Human	110

Abbreviations: CCNB1, Cyclin B1; MMP2, matrix metallopeptidase 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; qRT-PCR, quantitative real-time polymerase chain reaction; Twist, twist family bHLH transcription factor 1; ZEB1, zinc finger E-box binding homeobox 1.

Figure 1 ARHGAP1 expression in cervical cancer tissues and their adjacent normal tissues.

Notes: (A) The mRNA expression data of ARHGAP1 obtained from TCGA were much lower in tumor tissues than in normal tissues. (B) The expression levels of ARHGAP1 were lower in cervical carcinoma tissues than in paracancerous tissues. n=3, mean ± standard deviation. ***P<0.001 vs normal or paracancerous tissues.
Abbreviation: TCGA, The Cancer Genome Atlas.

Table 2 Correlation between the expression of ARHGAP1 and clinicopathologic features

Clinicopathological parameter	ARHGAP1		P-value
	Low-ARHGAP1 group (number of patients)	High-ARHGAP1 group (number of patients)
Age (years)			0.551
<54	13	9
>54	6	7
Size (cm^2)			0.025
<5	12	5
>5	13	5
Lymphatic invasion			0.001
Yes	20	8
No	5	2
Pathologic stages			0.387
I	6	4
II	15	10
Distant metastasis			0.272
Yes	6	7
No	14	8
Depth of tumor invasion			0.185
T1 + T2	11	8
T3 + T4	6	10

Figure 2 ARHGAP1 expression in cervical carcinoma cell lines.

Notes: (A) ARHGAP1 mRNA expression was determined by qRT-PCR in HeLa, CaSKi, C-33A, SiHa and C4-1 cell lines. (B) Protein levels of ARHGAP1 were determined by Western blot in HeLa, CaSKi, C-33A, SiHa and C4-1 cell lines. (C) Expression of ARHGAP1 protein in C-33A cell lines following transduction with lenti-ARHGAP1. (D) Expression of ARHGAP1 protein in SiHa cell lines following transduction with lenti-ARHGAP1. (E) Expression of ARHGAP1 protein in HeLa cell lines following transduction with lenti-shRNA-ARHGAP1. n=3, mean ± SD. ***P<0.001 vs HeLa cells.
Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; lenti-ARHGAP1, lentivirus targeting ARHGAP1; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; shNC, shRNA-NC.

Figure 3 Cell survival was assessed by CCK-8 assay.

Notes: (A) The growth of C-33A cells was markedly obstructed by ARHGAP1 overexpression in a time-dependent manner. (B) The mean cell proliferation rates of C-33A cells that were transfected with lenti-ARHGAP1 for 72 h were significantly weakened. (C) The growth of SiHa cells was markedly obstructed by ARHGAP1 overexpression in a time-dependent manner. (D) The mean cell proliferation rates of SiHa cells that were transfected with lenti-ARHGAP1 for 72 h were significantly weakened. (E) The growth of HeLa cells was markedly increased by ARHGAP1 knockdown in a time-dependent manner. (F) The mean cell proliferation rates of HeLa cells that were transfected with lenti-shRNA-ARHGAP1 for 72 h were significantly increased. n=3, mean ± SD. ***P<0.001 vs control groups.
Abbreviations: CCK-8, Cell Counting Kit-8; OD, optical density; lenti-ARHGAP1, lentivirus targeting ARHGAP1; NC, negative control; shNC, shRNA-NC.

Figure 4 Effects of ARHGAP1 on migration of both C-33A and SiHa cells.

Notes: (A) Migration ability of C-33A cells was identified by Transwell assay in different groups. (B) The number of migrating C-33A cells was markedly declined by ARHGAP1 overexpression. (C) Migration ability of SiHa cells was identified by Transwell assay in different groups. (D) The number of migrating SiHa cells was markedly declined by ARHGAP1 overexpression. (E) Migration ability of HeLa cells was identified by Transwell assay in different groups. (F) The number of migrating HeLa cells was markedly increased by ARHGAP1 knockdown. n=3, mean ± SD. **P<0.01, ***P<0.001 vs control groups. (A, C, E) magnification ×200.
Abbreviations: NC, negative control; shNC, shRNA-NC.

Figure 5 Effects of ARHGAP1 on invasion of C-33A and SiHa cells.

Notes: (A, B) After transduction with lenti-ARHGAP1, invasive ability and the number of invasive C-33A cells were identified by Transwell invasion assay. (C, D) Effects of ARHGAP1 overexpression on invasion of SiHa cells. (E, F) After transduction with lenti-shRNA-ARHGAP1, invasive ability and the number of invasive HeLa cells were identified by Transwell invasion assay. n=3, mean ± SD. ***P<0.001 vs control groups. (A, C, E) magnification ×200.
Abbreviations: lenti-ARHGAP1, lentivirus targeting ARHGAP1; NC, negative control; shNC, shRNA-NC.

Figure 6 Effects of ARHGAP1 on the protein expressions of MMP2, ZEB1, CCNB1, Twist and PCNA in C-33A and SiHa cells.

Notes: (A) ARHGAP1 overexpression evidently decreased the mRNA expression of all the genes in C-33A cells. (B) The protein levels of all the proteins were declined in C-33A cells. (C) ARHGAP1 overexpression evidently decreased the mRNA expression of all the genes in SiHa cells. (D) The protein levels of all the proteins were declined in SiHa cells. (E) ARHGAP1 knockdown evidently increased the mRNA expression of all the genes in HeLa cells. (F) The protein levels of all the proteins were increased in HeLa cells. n=3, mean ± SD. **P<0.01, ***P<0.001 vs control groups.
Abbreviations: CCNB1, Cyclin B1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP2, matrix metallopeptidase 2; PCNA, proliferating cell nuclear antigen; NC, negative control; shNC, shRNA-NC; Twist, twist family bHLH transcription factor 1; ZEB1, zinc finger E-box binding homeobox 1.