western blot analyses of fam83a knocked down human cervical cancer cells

אימות FAM83A כגן מטרה בסרטן צוואר הרחם

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.

מחבר זרקור: חקר התפקיד של FAM83A בסרטן צוואר הרחם 

הפלת FAM83A כדי לאמת את תפקידו בצמיחת תאי סרטן צוואר הרחם וברגישות לציספלטין

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  

ניתוחי כתמים מערביים של FAM83A הפילו תאי סרטן צוואר הרחם האנושיים

שטפו את התאים המטופלים לעיל עם PBS קר כקרח כדי להסיר כל מדיה שיורית, והוסיפו 50 מיקרוליטר של חיץ RIPA lysis המכיל PMSF כדי ליזה את התאים. לאסוף את התאים בצינור. לדגור על התאים על קרח במשך כמה דקות כדי להבטיח ליזה מלאה, ולאחר מכן צנטריפוגה בארבע מעלות צלזיוס ב 12, 000 גרם במשך 15 דקות.לאחר מכן מערבבים את הליזט התא עם חיץ העמסה ומחממים את התערובת ב 95 עד 100 מעלות צלזיוס במשך חמש עד 10 דקות באמבט מים כדי לנטרל את החלבונים. יש להעמיס 10 מיקרוליטר של חלבון כולל מכל דגימה על ג'ל SDS0-PAGE. הפעל את הג'ל תחת מתח קבוע של 80 וולט.עצור את האלקטרופורזה כאשר כחול הברומופנול מגיע לתחתית ג'ל ההפרדה. לאחר מכן השתמש במערכת העברה רטובה באמבט קרח כדי להעביר את החלבונים המופרדים מהג'ל אל קרום פוליווינילידן דיפלואוריד. לאחר השלמת ההעברה, מוציאים את הממברנה ודגרים על הממברנה בחיץ חוסם בטמפרטורת החדר למשך כשעה.לדגור על הממברנה עם נוגדן משני מצומד HRP. דמיינו את רצועות החלבונים על הממברנה באמצעות מצע כימילומינסנטי וללכוד את האות באמצעות מערכת הדמיה כימילומינסנטית. ניתוח כתמים מערביים הראה כי הביטוי המודחק של FAM83A הגדיל את החלבונים Bax ו-cleaved-caspase-3, הפחית את הביטוי של BCL-2 והגביר את שחרור ציטוכרום c.

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Research

JoVE Journal

Cancer Research

Western Blot Analyses of FAM83A Knocked Down Human Cervical Cancer Cells

203 Views.  Yangtze University. שטפו את התאים המטופלים לעיל עם PBS קר כקרח כדי להסיר כל מדיה שיורית, והוסיפו 50 מיקרוליטר של חיץ RIPA lysis המכיל PMSF כדי ליזה את התאים. לאסוף את התאים בצינור. לדגור על התאים על קרח במשך כמה דקות כדי להבטיח ליזה מלאה, ולאחר מכן צנטריפוגה בארבע מעלות צלזיוס ב 12, 000 גרם במשך 15 דקות.לאח...

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 ...