Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients

2024/05/2218:27:33 science 1961


Autophagy is one of the hot topics. Now will explain the research ideas of "autophagy in inflammatory-related diseases" in the project design.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

1. Research background

Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients from the time of diagnosis is very short. Because curative treatment is often not feasible due to limited tumor spread, invasion, distant metastasis, and sensitivity to chemotherapy drugs. Therefore, exploring "the mechanism of improving the sensitivity of anti-tumor drugs in non-small cell lung cancer" has become a top priority.

Autophagy plays an important role in cell growth, development, differentiation, aging, drug resistance and cell death. Autophagy can inhibit tumors and also promote tumor survival under conditions of limited nutrition and low oxygen.

YBX1 (Y-box binding protein 1) is a multifunctional protein that regulates transcription by binding to the inverted CCAAT box at the promoter of target genes. It plays a role in tumor invasion and metastasis, drug resistance, cell proliferation, and DNA repair, etc. play an important role in promoting cancer. Recent studies have shown that YBX1 is associated with autophagy in intrahepatic cholestasis of pregnancy. However, the relationship between YBX1 and NSCLC autophagy and its molecular mechanism have not been elucidated.

In this study, the authors found: ① The expression of autophagy-related protein LC3I/II is closely related to the expression of YBX1 in NSCLC tissue samples and cells. According to the hypothesis, LC3I/II is controlled by YBX1; ② Overexpression of YBX1 will cause in vitro and the upregulation of autophagy in vivo; ③ YBX1 knockdown enhances the anti-tumor effect of cisplatin by regulating autophagy; ④ YBX1 can promote autophagy through the p110β/Vps34/beclin1 pathway, which indicates that the YBX1/p110β/Vps34/beclin1 axis Involved in the development of cancer and the progression of NSCLC.

2. Research methods and results

1. Differential expression of YBX1 and autophagy-related protein LC3I/II in NSCLC patients

The author first used western immunoblot to analyze the expression of YBX1 and LC3I/II in NSCLC in NSCLC patient tissues and NSCLC cells. The results showed that both proteins were highly expressed, indicating that YBX1 is related to autophagy.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

2. YBX1 promotes the occurrence of autophagy

The authors constructed YBX1 overexpression A549, Hcc827 cells and knockdown H1299, H460 cells, using western immunoblotting, autophagy double-labeled adenovirus (mRFP-GFP-LC3) and autophagy induction. Or inhibitors to analyze the relationship between YBX1 and autophagy.

First, western immunoblotting analysis found that the reduced expression of YBX1 significantly inhibited the expression of LC3I/II, and the opposite phenomenon was found in overexpressed cells. Next, immunofluorescence and transmission electron microscopy were used to analyze the formation of Autophagosome in the ultrastructure of infected with GFP-LC3, and found that YBX1 promoted the formation of autophagosome. Finally, autophagy induction or inhibitors were used to analyze GFP-LC3 or GFP-LC3II conversion in YBX1 overexpression and knockdown cell lines, and it was found that YBX1 overexpression could promote the formation of GFP-LC3.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

3. Analysis of the mechanism of YBX1-mediated autophagy

The signaling pathway of autophagy includes PI3K/Akt/mTOR and p110 β/Vps34/beclin1, by analyzing the expression of key signaling molecules in YBX1 overexpression and knockdown cell lines , the authors determined that YBX1 controls the occurrence of autophagy by regulating p110 β/Vps34/beclin1.

Considering that YBX1 is a well-known transcription activator, further Jaspar analysis revealed that YBX1 works by binding to the p110β promoter, and the binding relationship between the two was further verified through pulldown assay.In order to further confirm that "YBX1 regulates p110β at the transcription level rather than at the protein level", the authors used actinomycin D to inhibit the expression level of p110β in YBX1 overexpressed tumor cells of RNA polymerase I. The results showed that the high expression of YBX1 did not cause p110β Attenuation of mRNA significance. Through reversion experiments, the authors further verified that YBX1-mediated p110β autophagy of and .

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

4. YBX1 reduces cisplatin-mediated inhibition of NSCLC cell proliferation and promotes cell apoptosis by inducing autophagy

It is known that YBX1 is significantly higher in cisplatin-resistant cell lines than in sensitive cell lines, researchers Further treatment of NSCLC cell lines with cisplatin found that the increase trend of YBX1 and LC3I/II conversion was time-dependent. When YBX1 overexpression and knockdown cell lines were treated with cisplatin, it was found that YBX1 significantly reduced the inhibition of cell activity mediated by cisplatin and inhibited cell apoptosis. In the knockdown cell lines, the authors also found that the expression of C-Caspase 3/9 increased, while the expression of Bcl-2 and Caspase 3 decreased, indicating that apoptosis was activated. This result shows that knocking out YBX1 enhances the anti-proliferative effect of cisplatin in non-small cell lung cancer.

In order to further study the mechanism by which YBX1 regulates the sensitivity of NSCLC to cisplatin, the authors further reduced the expression of Bcl-2 in YBX1 knockdown cells and found that knocking down both YBX1 and Bcl-2 in NSCLC cells could significantly increase the sensitivity to cisplatin. Drug sensitivity.

Literature has shown that Bcl-2, as an anti-apoptotic protein, inhibits autophagy through beclin1. The authors further tested that knockdown of YBX1 can cause a decrease in Bcl-2 and LC3I/II, and a decrease in Bcl-2 can more significantly increase the expression of LC3I/II, indicating that YBX1 regulates the sensitivity of NSCLC cells to cisplatin and autophagy. related.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

5. In vivo experiments

The researchers used subcutaneous injection to construct a YBX1 overexpression mouse model and analyzed the changes in transplanted tumors in the mice. Then cisplatin was added to treat the mice, and it was found that mice overexpressing YBX1 were less sensitive to cisplatin. IHC and WB were used to detect the expression of YBX1 in tumor masses. The results showed that YBX1 overexpression significantly increased the expression of p110β, beclin1, and LC3I/II.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

6. Clinical sample detection

The author used IHC to detect the relationship between the expression of YBX1 and LC3I/II in lung cancer tissues and the prognosis of patients. It was found that high expression of YBX1 has a small survival rate, but LC3I/II has no correlation with the prognosis of patients. In addition, a positive relationship between YBX1 and LC3I/II was also observed.

Autophagy is one of the hot spots. Now let’s explain the research ideas of “autophagy in inflammatory-related diseases” in the project design. 1. Research background Non-small cell lung cancer (NSCLC) is a common malignant tumor. The average survival time of lung cancer patients  - DayDayNews

IV. Summary

This study proves that the expression of YBX1 and LC3I/II in NSCLC cells and tissue specimens is significantly increased. Overexpression of YBX1 significantly promoted autophagy both in vitro and in vivo. YBX1 reduces sensitivity to cisplatin partly by inducing autophagy and not simply by increasing Bcl-2 expression.

Further comprehensive analysis revealed that p110β is a key effector regulated by YBX1 to mediate autophagy. These analyzes illustrate the critical role of p110β/Vps34/beclin1 signaling in autophagy and the integral relationship of p110β in YBX1-mediated regulation of p110β/Vps34/beclin1 transcription. The authors of first confirmed that YBX1 overexpression can promote autophagy through in vivo and in vitro experiments, and then found that YBX1 reduced sensitivity to cisplatin by inducing autophagy. Finally, they studied a further mechanism: YBX1 regulates transcription through p110β/Vps34/beclin1. The whole research idea is very clear and classic, and it is worth learning by everyone.

Source: https://www.nature.com/articles/s41419-020-2555-4#Sec18

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