western blot analyses of fam83a knocked down human cervical cancer cells

자궁경부암의 표적 유전자로서 FAM83A 의 검증

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.

저자 스포트라이트: 자궁경부암에서 FAM83A의 역할 탐구 

자궁경부암 세포 성장 및 시스플라틴 감수성에서의 역할을 검증하기 위한 FAM83A 의 녹다운

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  

FAM83A가 인간 자궁경부암 세포를 쓰러뜨린 웨스턴 블롯 분석

위의 처리된 세포를 얼음처럼 차가운 PBS로 세척하여 잔류 배지를 제거하고 PMSF가 포함된 RIPA 용해 완충액 50마이크로리터를 추가하여 세포를 용해합니다. 튜브에 세포를 수집합니다. 완전한 용해를 지키기 위하여 몇 분 동안 얼음에 세포를 잠복하고, 그 후에 12에 4 섭씨 온도에 분리기, 15 분 동안 000 g.그런 다음 세포 용해물을 로딩 버퍼와 혼합하고 혼합물을 섭씨 95도에서 100도에서 수조에서 5-10분 동안 가열하여 단백질을 변성시킵니다. 각 샘플에서 10마이크로리터의 총 단백질을 SDS0-PAGE 겔에 로드합니다. 일정한 볼륨에서 젤을 실행tag80볼트의 e.브로모페놀 블루가 분리 겔의 바닥에 도달하면 전기영동을 중지합니다. 그런 다음 얼음 수조에서 습식 전달 시스템을 사용하여 겔에서 분리된 단백질을 폴리비닐리덴 디플루오라이드 멤브레인으로 옮깁니다. 전사 완료 후, 멤브레인을 제거하고 차단 완충액에서 실온에서 약 1시간 동안 배양한다.HRP에 접합된 2차 항체로 멤브레인을 배양합니다. 화학발광 기질을 사용하여 멤브레인의 단백질 밴드를 시각화하고 화학발광 이미징 시스템을 사용하여 신호를 캡처합니다. 웨스턴 블롯 분석은 FAM83A의 억제된 발현이 Bax 및 cleaved-caspase-3 단백질을 증가시키고, BCL-2의 발현을 감소시키며, 시토크롬 c 방출을 증가시킨다는 것을 보여주었다.

western-blot-analyses-fam83a-knocked-down-human-cervical-cancer

Research

JoVE Journal

Cancer Research

Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells

203 조회수.  Yangtze University. 위의 처리된 세포를 얼음처럼 차가운 PBS로 세척하여 잔류 배지를 제거하고 PMSF가 포함된 RIPA 용해 완충액 50마이크로리터를 추가하여 세포를 용해합니다. 튜브에 세포를 수집합니다. 완전한 용해를 지키기 위하여 몇 분 동안 얼음에 세포를 잠복하고, 그 후에 12에 4 섭씨 온도에 분리기, 15 분 동안 000 g.그런 다음 세포 용해물을 로딩 버퍼와 혼합하고 혼합물을 섭씨 95도에?...

비디오: 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 ...