Abstract
Yes-associated protein 1 (YAP1) is an important signaling pathway activator molecule. Studies have shown that it is involved in the occurrence of malignant tumors. This study identified a microRNA (miR/miRNA) targeting the 3′ untranslated region (3″ utr) of the YAP1 gene and evaluated its biological impact on human cervical cancer cells and related molecular mechanisms. qPCR and western blotting were used to detect the levels of miR-375 and YAP1 in HeLa cells. TargetScan software was used to identify the binding sites of YAP1 and miR-375. The MTT method was used to determine the viability of HeLa cells transfected with miR-375 mimic and YAP1 interference vector, the Transwell chamber experiment was used to detect the invasion of HeLa cells after transfection, the apoptosis of HeLa cells after transfection was detected by flow cytometry, and the western blotting was used to detect the epithelial mesenchymal transition (EMT) of HeLa cells after transfection. The expression of miR-375 in HeLa cells was significantly lower than that of normal control cervical cells, and the expression of YAP1 in HeLa cells was significantly higher than that of normal control cervical cells. TargetScan analysis showed that miR-375 was bound to the 3′ UTR of YAP1. qPCR and western blot analysis showed that transfection of miR-375 mimics inhibited YAP1 expression in HeLa cells. Transfection of miR-375 mimic and YAP1 interference vector inhibited HeLa cell invasion and EMT and promoted HeLa cell apoptosis. These findings indicate that miR-375 inhibits the malignant development of human cervical cancer cells by regulating the expression of YAP1.
Highlights
Cervical cancer is one of the most common malignant tumors in gynecology
Normal cervical epithelial cells H8 and cervical cancer cells HeLa were purchased from the Shanghai Cell Bank; the RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from GIBCO, USA; the primers of miR-NC, miR-375 mimic, si-NC, si-Yes-associated protein 1 (YAP1), miR-375, and U6 were all purchased from Shanghai Gima Co., Ltd.; wild-type (WT) and mutant (MUT) YAP1 plasmids were synthesized by Shanghai Shenggong Biological Company; the RT-PCR kit, YAP1, E-cadherin, β-catenin, vimentin primary antibody and the corresponding secondary antibody, and Transwell chamber were all purchased from Sigma in the United States; RNA extraction kits, LipofectamineTM 2000, MTT, and apoptosis kits were all purchased from Wuhan Boster Biotech; both Trizol reagent and the luciferase detection kit were purchased from BioVision, USA
Untreated H8, HeLa cells, and transfected cells of each group were collected and centrifuged at 4°C and 8 cm 1000 r/min for 10 min, and the supernatant was discarded. ey were operated on ice, added with 50 μL of RIPA lysate, and centrifuged at 4°C and 12000 g for 15 min, and after 30 min, the supernatant was taken for BCA protein quantification. 40 μg of sample was added to the buffer solution, boiled at 100°C for 5 minutes, and separated by 12% polyacrylamide gel electrophoresis (SDS-PAGE), polyvinylidene fluoride (PVDF) was transferred to the membrane, and TBST solution was blocked for 2 hours
Summary
Cervical cancer is one of the most common malignant tumors in gynecology. It has a high morbidity and mortality rate, and it is showing a young trend. E occurrence of cervical cancer is a process of multifactor influence and multistage development, and its regulatory mechanism is complex. E abnormal increase of YAP1 expression leads to the continuous proliferation of downstream tumor cells, which participate in the early occurrence of malignant tumors [6]. Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells transform into mesenchymal cells. It is a common physiological phenomenon in mammalian development.
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