western blot analyses of fam83a knocked down human cervical cancer cells

FAM83A'nın Rahim Ağzı Kanserinde Hedef Gen Olarak Doğrulanması

Cervical cancer is a global concern as it is one of the leading types of gynecological malignancy worldwide and is the major cause of cancer-related mortality in women1. Radical surgery and chemoradiotherapy are associated with high cure rates at the primary stage. However, treatment outcomes for patients at the advanced stage of cervical cancer who develop metastatic disease are very unfavorable2. Therefore, it is crucial to further understand the biological mechanisms underlying the migration and invasion of cervical cancer cells and identify potential therapeutic targets for the prevention and treatment of this disease.
Identifying target genes involved in cancer progression and finding ways to inhibit their expression or action present promising treatment options. In this study, we identified FAM83 as a cancer-causing gene and further investigated its inhibitory effects on C33a and HeLa cells. FAM83 family oncogenes (FAM83A-H) are widely reported in human cancers3,4. Recently, FAM83A was reported to be upregulated in lung5, breast6, ovarian7, and pancreatic8 cancers,indicating that FAM83A plays an important role in cancer progression via promoting the proliferation, invasion, stem-cell-like traits, and drug resistance in the tumor cells. Importantly, FAM83A was identified as one of the novel candidate genes associated with cervical lesion progression and carcinogenesis9. Despite the confirmation of elevated FAM83A expression in human cervical cancer cells, the specific impact and underlying mechanisms of FAM83A in cervical cancer remain unclear.
In this study, we outline the protocols involved in the identification of FAM83A as a tumor target gene in cervical cancer and use a small interfering RNA (siRNA) for the knockdown of the FAM83A gene in HeLa and C33a cells. 5-Ethynyl-2&#39;-deoxyuridine (EdU) staining was performed to determine the effects on tumor cell proliferation, while wound healing and porous membrane insert assays helped evaluate tumor cell migration and invasion abilities.
Western blotting was performed to determine the levels of apoptosis-related proteins, and JC-1 staining was employed to evaluate mitochondrial function alterations. Thus, we reported that FAM83A plays a critical role in cell proliferation, metastasis, and invasion in cervical cancer. Through PI3K/AKT-pathway-associated mitochondrial dysfunction and apoptosis, FAM83A knockdown sensitized cervical cancer cells to cisplatin (diaminedichloroplatinum, DDP). This study provides a new target for cervical cancer and possibly other cancers and a reference for the development of strategies to overcome the resistance of cancer cells to certain chemotherapeutic drugs.

Yazar Spotlight: Rahim Ağzı Kanserinde FAM83A'nın Rolünü Keşfetmek 

Rahim Ağzı Kanseri Hücre Büyümesi ve Sisplatin Duyarlılığındaki Rolünü Doğrulamak için FAM83A'nın Yıkılması

The scope of our research as focus are investigating the role of FAM83A in cervical cancer growths and cisplatin sensitivity. By addressing this question, we hope to contribute to the scientific understanding of the cervical cancer biology and potentially identify real therapeutic targets for this disease. We found LA sentencing FAM83A expirations as a scientist, cervical cancer cells uses platin treatment.Enhancing the drug satisfies a factor, and inhibiting the cell viability and migration. Our laboratory will focus and foreign research question in the future illustrating the medical mechanisms of FAM83A of FAM83A in cervical cancer. FAM83A in cervical cancer.Developing FAM83A inhibitors or technicalities. Developing FAM83A inhibitors or technicalities. The therapies starting the correlation between FAM83A and the patient prognosis between FAM83A and the patient prognosis and investigating the role of FAM83A in and investigating the role of FAM83A and investigating the orle of FAM83A in other types of cancer.

Watch this Scientific Journal Video about Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells at JoVE.com

Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells  

FAM83A'nın Western Blot Analizleri İnsan Rahim Ağzı Kanseri Hücrelerini Yıktı

Kalıntı ortamı çıkarmak için yukarıdaki işlem görmüş hücreleri buz gibi soğuk PBS ile yıkayın ve hücreleri parçalamak için PMSF içeren 50 mikrolitre RIPA lizis tamponu ekleyin. Hücreleri bir tüpte toplayın. Tam lizizi sağlamak için hücreleri buz üzerinde birkaç dakika inkübe edin, ardından 15 dakika boyunca 12.000 g'da dört santigrat derecede santrifüjleyin.Daha sonra hücre lizatını bir yükleme tamponu ile karıştırın ve proteinleri denatüre etmek için karışımı bir su banyosunda beş ila 10 dakika boyunca 95 ila 100 santigrat derecede ısıtın. Her numuneden 10 mikrolitre toplam proteini bir SDS0-PAGE jeli üzerine yükleyin. Jeli 80 voltluk sabit bir voltaj altında çalıştırın.Bromofenol mavisi ayırma jelinin dibine ulaştığında elektroforezi durdurun. Daha sonra, jelden ayrılan proteinleri bir poliviniliden diflorür membranına aktarmak için bir buz banyosunda ıslak bir transfer sistemi kullanın. Transferin tamamlanmasından sonra, zarı çıkarın ve zarı oda sıcaklığında yaklaşık bir saat boyunca bir bloke edici tampon içinde inkübe edin.Membranı, HRP'ye konjuge edilmiş ikincil bir antikor ile inkübe edin. Bir kemilüminesan substrat kullanarak membran üzerindeki protein bantlarını görselleştirin ve bir kemilüminesans görüntüleme sistemi kullanarak sinyali yakalayın. Western blot analizi, FAM83A'nın baskılanmış ekspresyonunun Bax ve bölünmüş-kaspaz-3 proteinlerini arttırdığını, BCL-2'nin ekspresyonunu azalttığını ve sitokrom c salınımını arttırdığını gösterdi.

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Research

JoVE Journal

Cancer Research

Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells

203 Görüntüleme.  Yangtze University. Kalıntı ortamı çıkarmak için yukarıdaki işlem görmüş hücreleri buz gibi soğuk PBS ile yıkayın ve hücreleri parçalamak için PMSF içeren 50 mikrolitre RIPA lizis tamponu ekleyin. Hücreleri bir tüpte toplayın. Tam lizizi sağlamak için hücreleri buz üzerinde birkaç dakika inkübe edin, ardından 15 dakika boyunca 12.000 g'da dört santigrat derecede santrifüjleyin.Daha sonra hücre lizatını bir yükleme tamponu ile karıştırın ve proteinleri denatüre etmek...

Video: Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells  

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps involved in the identification of a tumor target gene FAM83A in cervical cancer. First, the Cancer Genome Atlas dataset was employed to validate the expression and prognostic significance of FAM83A in women. A small interfering RNA (siRNA) was used for knockdown of the FAM83A gene in HeLa and C33a cells. Next, 5-ethynyl-2&#39;-deoxyuridine (EdU) staining was conducted to determine the effects on the proliferation capabilities of the tumor cells. Wound healing and porous membrane insert assays were performed to evaluate tumor cell migration and invasion abilities.
Western blotting was used to quantify apoptosis-related protein levels. JC-1 staining was employed to evaluate mitochondrial function alterations. Furthermore, cisplatin (diaminedichloroplatinum, DDP) intervention was used to assess the therapeutic potential of the target gene. Flow cytometry and colony formation assays were conducted to further validate the anticancer characteristics of the gene. As a result, FAM83A knockdown was shown to inhibit the proliferation, migration, and invasion of cervical cancer cells and sensitize these cells to cisplatin. These comprehensive methodologies collectively validate FAM83A as a tumor-associated target gene, holding promise as a potential therapeutic target in the prevention and treatment of cervical cancer.

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps ...