Blocking Modification of Eukaryotic Initiation 5A2 Antagonizes Cervical Carcinoma via Inhibition of RhoA/ROCK Signal Transduction Pathway

Abstract

Cervical carcinoma is one of the leading causes of cancer-related death for female worldwide. Eukaryotic initiation factor 5A2 belongs to the eukaryotic initiation factor 5A family and is proposed to be a key factor involved in the development of diverse cancers. In the current study, a series of in vivo and in vitro investigations were performed to characterize the role of eukaryotic initiation factor 5A2 in oncogenesis and metastasis of cervical carcinoma. The expression status of eukaryotic initiation factor 5A2 in 15 cervical carcinoma patients was quantified. Then, the effect of eukaryotic initiation factor 5A2 knockdown on in vivo tumorigenicity ability, cell proliferation, cell cycle distribution, and cell mobility of HeLa cells was measured. To uncover the mechanism driving the function of eukaryotic initiation factor 5A2 in cervical carcinoma, expression of members within RhoA/ROCK pathway was detected, and the results were further verified with an RhoA overexpression modification. The level of eukaryotic initiation factor 5A2 in cervical carcinoma samples was significantly higher than that in paired paratumor tissues ( P < .05). And the in vivo tumorigenic ability of HeLa cells was reduced by inhibition of eukaryotic initiation factor 5A2. Knockdown of eukaryotic initiation factor 5A2 in HeLa cells decreased the cell viability compared with normal cells and induced G1 phase cell cycle arrest ( P < .05). Moreover, the cell migration ability of eukaryotic initiation factor 5A2 knockdown cells was dramatically inhibited. Associated with alterations in phenotypes, RhoA, ROCK I, and ROCK II were downregulated. The above-mentioned changes in eukaryotic initiation factor 5A2 knockdown cells were alleviated by the overexpression of RhoA. The major findings outlined in the current study confirmed the potential of eukaryotic initiation factor 5A2 as a promising prognosis predictor and therapeutic target for cervical carcinoma treatment. Also, our data inferred that eukaryotic initiation factor 5A2 might function in carcinogenesis of cervical carcinoma through an RhoA/ROCK-dependent manner.

Keywords: ROCK I; ROCK II; RhoA; cervical carcinoma; eukaryotic initiation 5A2.

Figure 1.

The expression of eIF5A2 is upregulated in clinical cervical carcinoma (CC) samples. A, Quantitative analysis result of real-time quantitative polymerase chain reaction (qPCR) detection of clinical samples. B, Quantitative analysis result and representative images of Western blotting assay of clinical samples.

Figure 2.

Knockdown of eIF5A2 inhibited the growth of cervical carcinoma in vivo. A, Quantitative analysis results of growth curve of solid tumors in different groups; tumor volumes at the last 4 recording time points were significantly inhibited by knockdown of eIF5A2. B, Representative images of the morphology of solid tumors in different groups. *Significantly different from the control group, P < .05. #Significantly different from the NC group, P < .05. NC, negative control.

Figure 3.

Knockdown of eIF5A2-attenuated cell viability and anchorage-independent growth capability in HeLa cells. A, The results of MTT assay; cell viabilities at the last 4 time points were significantly inhibited by the inhibition of eIF5A2. B, The results of colony formation assay; cell numbers in sheIF5A2 cells were significantly reduced compared with the control or NC group. *Significantly different from the control group, P < .05. ^#Significantly different from the NC group, P < .05. NC, negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.

Figure 4.

Knockdown of eIF5A2-induced G1 phase arrest in cell cycle distribution. *Significantly different from the control group, P < .05. ^#Significantly different from the NC group, P < .05. NC, negative control.

Figure 5.

Knockdown of eIF5A2 decreased the cell migration ability in HeLa cells. A, Representative images and quantitative analysis results of scratch assay; wound healing rate of the sheIF5A2 group was significantly lower than those in the control and NC groups. B, Representative images and quantitative analysis results of transwell experiment; cell numbers moving through the porous member of the sheIF5A2 group were significantly lower than those in the control and NC groups. *Significantly different from the control group, P < .05. ^#Significantly different from the NC group, P < .05. NC, negative control.

Figure 6.

Eukaryotic initiation factor 5A2 determined the proliferation and mobility of cervical carcinoma (CC) cells through an RhoA/Rho-associated kinase (ROCK)-dependent manner. A, Quantitative analysis results and representative images of Western blotting assay; the expression of RhoA, ROCK I, and ROCK II was downregulated by knockdown of eIF5A2. B, Results of MTT assay; overexpression of RhoA attenuated the negative effect of eIF5A2 knockdown. C, Results of scratch assay; overexpression of RhoA attenuated the negative effect of eIF5A2 knockdown. *Significantly different from the control group, P < .05. ^#Significantly different from the NC group, P < .05. ^aSignificantly different from the NC group, P < .05. ^bSignificantly different from the sheIF5A2 group, P < .05. ^cSignificantly different from the vector group, P < .05. NC, negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.