Name: Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR
Uploaded: 2023-06-09T12:20:00
Description: Watch this Scientific Journal Video about Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR at JoVE.com

determining gene expression in salidroside treated mcf-7 cells using qrt-pcr

Het mechanisme van salidroside bij het onderdrukken van MCF-7-celmigratie en -proliferatie

As one of the most commonly diagnosed cancers and most common malignancies, the latest statistics indicate that 2.3 million cases of breast cancer emerged around the world by 2020, accounting for 11.7% of all cancer cases1. Common symptoms of breast cancer include breast tenderness and tingling, breast lumps and pain, nipple discharge, erosion or sunken skin, and enlarged axillary lymph nodes1,2. Even more alarming, the number of new cases and the overall incidence of breast cancer continues to increase at an overwhelming rate each year, accounting for 6.9% of cancer-related deaths1. At present, breast cancer intervention still mainly involves chemotherapy, surgery, radiotherapy, and comprehensive treatment. Although treatment can effectively reduce the recurrence rate and mortality rate of patients, long-term treatment application often causes produce multidrug resistance, large-area hair loss, nausea and vomiting, and serious mental and psychological burden2,3. Notably, the potential risk of multiple organ metastases from breast cancer also forces people to seek novel herbal sources of drug therapy4,5.
Phosphoinositide 3 kinase (PI3K)-mediated signaling is implicated in the growth, proliferation, and survival of breast cancer through splicing that affects the expression of multiple genes6. As a downstream signal-sensing protein of PI3K, numerous evidence suggests that protein kinase B (AKT) could couple with the mammalian target of rapamycin (mTOR) protein to further increase breast cancer7,8,9. Moreover, the deactivation of PI3K/AKT/mTOR signaling has also been claimed to be a key component in drugs inhibiting malignant proliferation and stimulating apoptosis in breast cancer10,11,12. It is well known that extreme hypoxia in the tumor microenvironment forces a massive surge in hypoxia-inducible factor 1 alpha (HIF-1&#945;), which further worsens the progression of breast cancer13,14,15. In parallel, AKT stimulation also leads to excessive accumulation of HIF-1&#945;, limiting apoptosis in breast cancer samples16,17. Interestingly, the activation of PI3K-AKT-HIF-1&#945; signaling has been confirmed to be involved in pathologic progression and metastasis in a variety of cancers, including lung cancer18, colorectal cancer19, ovarian cancer20, and prostate cancer21. In addition to being orchestrated by HIF-1&#945;, forked head transcription factor 1 (FoxO1) overexpression is also triggered by AKT signaling stimulation, which promotes cycle arrest and the inhibition of apoptosis in breast cancer cells22,23. Together, the above solid evidence suggests that the inhibition of the cascade of PI3K-AKT-HIF-1&#945;-FoxO1 signaling may be a potential novel target for drug therapy in breast cancer.
Salidroside (Sal) has been widely demonstrated to exert anti-cancer24,25, anti-hypoxia26,27,28,29, and immune-enhancing pharmacological activities30. It is a light brown or brown powder that is easily soluble in water, is a type of phenylethanoid glycoside, and has a chemical structure formula of C14H20O7 and a molecular weight of 300.331,32. Modern pharmacological investigations have demonstrated that Sal can promote the apoptosis of gastric cancer cells by restraining PI3K-AKT-mTOR signaling24. Further evidence also suggests that the suppression of PI3K-AKT-HIF-1&#945; signaling by Sal treatment may contribute to the apoptosis of cancer cells by enhancing their sensitivity to chemotherapeutic agents25. Evidence also suggests that Sal restricts cell migration and invasion and causes cycle arrest by promoting apoptosis in the human breast cancer MCF-7 cells33,34. However, it remains to be seen whether Sal can regulate PI3K-AKT-HIF-1&#945;-FoxO1 signaling and inhibit the malignant proliferation of MCF-7 cells. Therefore, this protocol aimed to explore the effects of Sal on MCF-7 cell migration, invasion, cell cycle, and apoptosis via the PI3K-AKT-HIF-1&#945;-FoxO1 pathway. The integrated research strategies comprising conventional, low-cost, and independent experiments, such as cell migration and invasion assessments, apoptosis and cell cycle detection by flow cytometry, reactive oxygen species (ROS) and Ca2+ fluorescence determination, etc., can provide a reference for the overall design of experiments for anti-cancer research with traditional herbal medicine. The experimental process of this study is shown in Figure 1.

Auteur in de schijnwerpers: onderzoek naar de moleculaire mechanismen van salidroside bij de behandeling van borstkanker 

Onderzoek naar de farmacologische werking en het moleculaire mechanisme van salidroside bij het remmen van MCF-7-celproliferatie en -migratie

We focus on exploring active compounds derived from natural products and their potential in treating breast cancer. So specifically we aim to elucidate the pharmacologic effects of salidroside and its molecular mechanisms in combining breast cancer. Currently, reliable experimental techniques are lacking to validate salidroside's molecular targets in treating breast cancer.One significant finding in the field is a potential molecular mechanism of salidroside in enhancing the proliferation and migration of MCF7 cells through the regulation of PI3K/AKT/HIF-1 alpha/FoxO1 signaling. The laboratory's future research directly is to explore the pathophysiological mechanisms underlying breast cancer onset, development, prognosis and outcome. Additionally, we screen for active small molecular drugs derived from natural sources that may add in breast cancer prevention and treatment.

Watch this Scientific Journal Video about Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR at JoVE.com

Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR  

Bepaling van genexpressie in met speekdroside behandelde MCF-7-cellen met behulp van qRT-PCR

Oogst de MCF-7-cellen door centrifugalisatie bij 560-G gedurende drie minuten, bij vier graden Celsius. Voeg 500 microliter gebufferd RL-1 tot vijf keer 10 toe aan de zes cellen en meng grondig tot er geen celmassa's meer zichtbaar zijn. Breng vervolgens de celhomogenaten over naar een DNA-reinigingskolom die is ingebed in de verzamelbuis.Centrifugeer de monsters gedurende twee minuten bij 85-50-G bij vier graden Celsius. Verwijder na het centrifugeren de DNA-reinigingskolom en bewaar het supernatans in de opvangbuis. Voeg 800 microliter buffer RL-2 en 500 microliter van het supernatant toe en meng voorzichtig.Breng vervolgens 700 microliter van het mengsel over in een kolom met alleen RNA die is ingebed in de verzamelbuis. Centrifugeer de buis bij 8, 550-G gedurende één minuut, bij vier graden Celsius. Gooi vervolgens de doorstroming weg in de opvangbuis.Was de RNA-kolom eerst met 500 milliliter buffer RW-1 en vervolgens met 700 milliliter buffer RW-2, door één minuut te centrifugeren bij 8, 550-G en vier graden Celsius bij elke wasbeurt. Na het verwijderen van de resterende RW-2 door opnieuw te centrifugeren, brengt u de kolom met alleen RNA over naar een nieuwe verzamelbuis. Voeg 100 microliter RNAs-vrij gedeïoniseerd water, voorverwarmd op 65 graden Celsius, toe aan het midden van het membraan in de kolom met alleen RNA en wacht twee minuten.Centrifugeer vervolgens gedurende één minuut bij 8, 550-G bij vier graden Celsius om de RNA-oplossing op te vangen. Stel het reactiemengsel in voor PCR en stel vervolgens de PCR-reactiecondities van het systeem in. Voer de QRT PCR-procedure uit.QRT PCR toonde aan dat behandeling met cilindriciteit de genexpressie van de pro-apoptotische factoren CC-9, CC-7, CC-3, vim en vax bevorderde, terwijl het de genexpressie van anti-apoptotische BCL-2 remde. Verder verlaagde toediening van cilindricide de genexpressieniveaus van PI-3K, AKT, M-tor, HIF1-Alpha en Fox-01.

Research

JoVE Journal

Medicine

Determining Gene Expression in Salidroside Treated MCF-7 Cells Using qRT-PCR

Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration

Salidroside (Sal) contains anti-carcinogenic, anti-hypoxic, and anti-inflammatory pharmacological activities. However, its underlying anti-breast cancer ...

Evaluating the Migration and Invasion of Salidroside Treated MCF-7 Cells in a Wound Scratch Assay

Evaluatie van de migratie en invasie van met speeksel behandelde MCF-7-cellen in een wondkrastest

Determining Na+-K+-ATPase and Ca2+-Mg2+-ATPase Activity in Salidroside Treated MCF-7 Cells

Bepaling van de activiteit van Na+-K+-ATPase en Ca 2+-mg2+-ATPase in met speeksel behandelde MCF-7-cellen

Flow Cytometry Analysis of Apoptosis and the Cell Cycle in Salidroside Treated MCF-7 Cells

Flowcytometrie-analyse van apoptose en de celcyclus in met salidroside behandelde MCF-7-cellen

DCFH-DA and Fluo-4 AM Fluorescence Staining of Salidroside Treated MCF-7 Cells

DCFH-DA en Fluo-4 AM fluorescentiekleuring van met speeksel behandelde MCF-7-cellen

Analyzing Protein Expression in Salidroside Treated MCF-7 Cells by Western Blotting

Analyse van eiwitexpressie in met speeksel behandelde MCF-7-cellen door Western Blotting