Effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA.

Lijie He,Heqiang Feng,Dandan Lv,Haina Li,Jing Wang,Dandan Chang

doi:10.1515/med-2020-0147

Lijie He, Heqiang Feng + Show 4 more

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https://doi.org/10.1515/med-2020-0147

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Abstract

ObjectiveThis article aims to investigate the effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA.MethodsHeLa cells of cervical cancer were divided into five groups: blank control group, negative control group (miRNA-200b mimic NC), miRNA-200b mimic group, RhoA-negative control group, and RhoA overexpression group. Cells were collected 48 h after transfection. The expression levels of miRNA-200b were detected by RT-PCR. Target relationship between miRNA-200b and RhoA was verified by the dual-luciferase reporter assay. RhoA mRNA and protein expression were detected by western blot and RT-PCR methods. Flow cytometry was used to detect the apoptosis of cells in each group, and the CCK8 method was used to detect the proliferation of cells in each group. The mRNA and protein expression of Bax and cyclin D1 were detected by RT-PCR and western blot.ResultsThe results of the dual luciferase reporter assay showed that RhoA was the target gene of microRNA 200b. Compared with the blank control group and the miRNA-200b mimic-NC group, the proportion of apoptotic cells increased significantly in the miRNA-200b mimic group, and the proliferation of cells was inhibited (P < 0.05). After overexpression of RhoA, the percentage of apoptotic cells decreased and the ability of cell proliferation increased significantly (P < 0.05).ConclusionmiRNA-200b can inhibit the proliferation and promote the apoptosis of cervical cancer cells by targeting the RhoA gene.

Highlights

Cervical cancer has become the second most prevalent malignancy among young and middle-aged women in China, accounting for more than 28% of cases worldwide
HeLa cells of human cervical cancer were purchased from the cell bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; fetal calf serum was purchased from Gibco, USA; Lipofectamine2000 was purchased from Invitrogen of the United States; RIPA lysate, BCA protein quantitation kits, ECL chemiluminescent solution, and CCK8 kits were purchased from Beyotime Biotechnology Co., Ltd, China; rabbit anti-human RhoA monoclonal antibody (No EPR18134), rabbit anti-human Cyclin D1 monoclonal antibody (No EPR2241), rabbit anti-human Bax monoclonal antibody (No EPR18283), rabbit anti-human VEGF monoclonal antibody (No Y103), and mouse antihuman TGF-β1 monoclonal antibody (No Y103) TB21 were purchased from Abcam, USA; horseradish peroxidaselabeled goat anti-rabbit IgG and goat anti-mouse IgG were purchased from ZSBIO, China
Compared with the blank control group and mimic-NC group, the miRNA-200b mimic group showed a significant increase in the proportion of cell apoptosis and inhibited cell proliferation, and the P values were 0.001 and 0.001, respectively; in the RhoA overexpression group, the proportion of cell apoptosis decreased and the cell proliferation ability was significantly enhanced and the P values were 0.001 and 0.001, respectively, and there was a significant difference (P < 0.05); the apoptosis and the proliferation ability in the blank control group were compared with those in the mimicNC group and the RhoA NC group, and the results showed that the P values were 0.987 and 0.965, respectively, and there was no significant difference (P > 0.05)

Summary

Introduction

Cervical cancer has become the second most prevalent malignancy among young and middle-aged women in China, accounting for more than 28% of cases worldwide. The incidence and mortality of cervical cancer have been increasing significantly in China [1]. MicroRNAs (miRNAs) are endogenous singlestranded small RNAs that can play an important role in the development and progression of human tumors through posttranslational modifications [2]. A previous study has reported that miRNAs are closely related to the proliferation, apoptosis, and invasion of cervical cancer cells and are considered as new targets for the diagnosis, treatment, and prognostic evaluation of cervical cancer [3]. Studies have found that RhoA is overexpressed in a variety of cancer tissues and cancer cells and is closely related to tumor invasion and metastasis [6,7]. Bax is a member of the Bcl-2 family and plays a role in promoting apoptosis.

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