ABSTRACT
OBJECTIVE: Epidermal growth factor receptor 2 (C-erbB-2) is one of the most frequently mutated oncogenes in human tumors. We aimed to evaluate the knockout efficiency of clustered regularly interspaced short palindromic repeat (CRISPR) technology using ultrasound microbubble transfection to target C-erbB-2 in human endometrial cancer (HEC)-1A cells. METHODS: Three single guide RNAs (sgRNAs) targeting C-erbB-2 were designed and used to construct CRISPR/CRISPR-associated (Cas)9-C-erbB-2 plasmids. The constructed plasmids were transfected into HEC-1A cells using ultrasound microbubbles. C-erbB-2 knockout cloned cells were identified by green fluorescence. C-erbB-2 mRNA and protein expression was measured by reverse transcription (RT)-PCR and western blotting, respectively. RESULTS: RT-PCR showed that C-erbB-2 mRNA expression was significantly lower in sgRNA1-transfected cells (0.57 ± 0.06) than in blank (1.00 ± 0.09) and negative-control groups (1.02 ± 0.12). Western blotting revealed C-erbB-2 protein expression to be significantly lower in sgRNA1-transfected cells (0.269 ± 0.033) than in blank (0.495 ± 0.059) and negative-control groups (1.243 ± 0.281). However, there was no significant difference in C-erbB-2 protein and mRNA expression in sgRNA2- and sgRNA3-transfected cells compared with controls. CONCLUSION: Ultrasound microbubbles can mediate plasmid transfer into HEC-1A cells to interfere with gene expression and knockout C-erbB-2.
Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Endometrial Neoplasms/genetics , Gene Knockout Techniques , Microbubbles , Receptor, ErbB-2/metabolism , Ultrasonics , Base Sequence , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , RNA, Guide, Kinetoplastida/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Endometrial cancer is one of the chief culprits threatening women's lives. Although numerous epidemiological experiments have been carried out into the aetiology of endometrial cancer, the cause of the disease has been unclear up to now. In recent years, PTPN18, a member of the protein tyrosine phosphatases (PTP) family predicted to be tumour suppressors or oncogenes, has been confirmed to participate in the occurrence and progression of many cancers. Few studies, however, have explained the role in the endometrial cancer. So, it caught our attention to explore if PTPN18 participates in and plays a regulatory role in the proliferation, apoptosis, and metastasis of endometrial cancer. In our results, we found that PTPN18 was overexpressed in endometrial cancer tissue compared to paracancerous tissue by immunohistochemistry. Not only that, silencing of PTPN18 in endometrial cancer cell lines (HEC-1-A and HEC-1-B) can significantly impair proliferation detected by CCK8 assay and flow cytometry (FCM) analyses and inhibit the metastasis of endometrial cancer cells shown by the scratch test and the Transwell experiment. PTPN18 knockdown can promote the apoptosis of endometrial cancer. In addition, nude mice tumour formation assay confirmed the results in vivo. Although the exact function of PTPN18 in endometrial cancer is unclear, the targeted therapy drugs enhancing PTPN18 may be considered in the future treatment of endometrial carcinoma.
Subject(s)
Apoptosis , Down-Regulation , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Endometrial Neoplasms/genetics , Female , Gene Silencing , Humans , Neoplasm Invasiveness , Neoplasm Metastasis , Protein Tyrosine Phosphatases, Non-Receptor/deficiency , Protein Tyrosine Phosphatases, Non-Receptor/geneticsABSTRACT
A highly regioselective cycloaddition procedure of electron-deficient allenes with trifluorodiazoethane (CF3CHN2) is described. In absence of bases, the reaction proceeded smoothly to give 5-(trifluoromethyl)pyrazolines, whereas the utility of Et3N led to the formation of 3-(trifluoromethyl)pyrazoles.
