Application of CHAF1B in preparation of medicine for regulating and controlling autophagy activity of breast cancer cells

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of targeted CHAF1B in preparation of a medicine for regulating and controlling autophagy activity of breast cancer cells. The invention reveals that targeted CHAF1B can activate autophagic death of breast cancer cells for the first time, provides a new strategy for molecular targeted therapy of breast cancer, and shows a good anti-tumor effect; the CHAF1B is proved to be remarkably and highly expressed in breast cancer tissues and is related to prognosis of patients, so that the CHAF1B can be used as a potential diagnosis, treatment and prognosis monitoring target of the breast cancer; the invention discusses that CHAF1B targeted MAPK signal channel induces autophagic death, provides theoretical support for application of CHAF1B as a new therapeutic target, enriches cognition of CHAF1B for regulating breast cancer cell proliferation molecular mechanism, provides a new thought and direction for searching for a new target for breast cancer treatment, and provides a new foundation for application of CHAF1B targeted MAPK signal channel induced autophagic death and application of CHAF1B targeted MAPK signal channel induced autophagic death. And a theoretical basis and an experimental basis are provided for clinically developing a new treatment target.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of CHAF1B in preparing a drug for regulating the autophagy activity of breast cancer cells. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women and the most common cancer worldwide. In recent years, although the application of molecular targeted drugs has significantly improved the prognosis of breast cancer patients, some patients still face the risk of recurrence and metastasis, resulting in poor prognosis. Studies have found that the progression of breast cancer is often accompanied by a variety of epigenetic regulatory abnormalities, mainly including DNA methylation, histone modification, non-coding RNA expression and chromatin structure remodeling. Targeting these epigenetic modification abnormalities, DNA methylation inhibitors-Azacitidine, histone modification inhibitors-Vorinostat and BRD4 inhibitors (JQ1) have achieved good therapeutic effects in the treatment of breast cancer, showing the great potential of targeted epigenetic modification in the treatment of breast cancer.

[0003] Autophagy is a process proposed by Christian de Duve in 1963, in which lysosomes engulf and degrade unused proteins and damaged organelles in cells to maintain the homeostasis of the intracellular environment. In the anti-tumor process, autophagy has a dual regulatory role: on the one hand, it can induce tumor cell death by activating autophagy, and on the other hand, it can maintain the body's energy supply by activating autophagy when the body is in a state of starvation or stress. During the treatment process, most tumor cells will undergo necrosis, apoptosis or autophagic death and be eliminated, while a small number of tumor cells can improve their own survival ability under metabolic stress stimulation through autophagy and become residual cells to continue to proliferate, leading to tumor recurrence and metastasis. For example, studies have found that the rapid proliferation of breast cancer cells often leads to a hypoxic state in the local tumor microenvironment, and hypoxia inhibits the autophagic death of breast cancer cells, inducing drug resistance and lung metastasis; in addition, autophagy can also affect the invasion and migration ability of breast cancer cells by regulating the release of exosomes.

[0004] Chromatin assembly factor-1 (CAF-1) is a heterotrimeric complex that participates in chromatin assembly during DNA replication and repair, while chromatin assembly factor-1B (CHAF1B) is the p60 subunit of the CAF-1 family. Existing studies have mainly focused on the role of CHAF1B in the nucleosome assembly process involved in DNA replication. However, no studies have reported its anti-tumor effect in breast cancer cells and its mechanism related to cell autophagy. In addition, the role of autophagy in breast cancer progression, drug resistance and metastasis has not been thoroughly studied. Therefore, the application of CHAF1B in the preparation of drugs that regulate the autophagy activity of breast cancer cells is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide the use of CHAF1B in the preparation of a drug for regulating the autophagy activity of breast cancer cells, aiming to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions: Application of CHAF1B in the preparation of drugs that regulate the autophagic activity of breast cancer cells. The expression of CHAF1B in breast cancer cells is knocked down by siCHAF1B-6 and siCHAF1B-8 to inhibit the proliferation and cell viability of breast cancer cells and inhibit tumor growth; autophagic death of breast cancer cells is activated by inhibiting CHAF1B; The sequence information of siCHAF1B-8 is as follows: Sense strand: 5′-CCAGCUGGAUGUGUGGAAUTT-3′; Antisense strand: 5′-AUUCCACAUCCAGCUGGTT-3′; The sequence information of siCHAF1B-6 is as follows: Sense strand: 5′-ACGGAAAGUCUGGACCCUUTT-3′; Antisense strand: 5′-AAGGGUCCAGACUUUCCGUTT-3′.

[0007] Furthermore, inhibition of CHAF1B can target and inhibit the MAPK signaling pathway through an epigenetic mechanism to activate autophagic death of breast cancer cells.

[0008] Compared with the prior art, the present invention has the following beneficial effects: The present invention reveals for the first time that targeting CHAF1B can activate the autophagic death of breast cancer cells, which provides a new strategy for molecular targeted therapy of breast cancer and shows good anti-tumor effects. At the same time, the present invention confirms that CHAF1B is significantly highly expressed in breast cancer tissue and is related to the prognosis of patients. This result shows that CHAF1B can be used as a potential target for diagnosis, treatment and prognosis monitoring of breast cancer. The present invention not only clarifies the expression characteristics and prognostic value of CHAF1B in breast cancer, but also deeply explores its molecular mechanism of activating autophagic death of breast cancer cells by targeting the MAPK signaling pathway. These findings provide solid theoretical support and practical significance for the clinical application of CHAF1B as a new molecular therapeutic target, and at the same time enrich the cognition of the molecular mechanism of CHAF1B regulating breast cancer cell proliferation, and provide new ideas and research directions for finding new targets for breast cancer treatment. In addition, this also provides a theoretical basis and experimental basis for the clinical development of new therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The molecular mechanism diagram of the present invention.

[0010] Figure 2 Figure 3 is the expression of CHAF1B in breast cancer tissue and its relationship with prognosis; A is the expression of CHAF1B in tumor tissue and normal tissue of breast cancer patients detected by TCGA database; B is the expression of CHAF1B detected by TCGA database through pairing (i.e. tumor and normal tissue from the same person); C is the relationship between CHAF1B expression and overall survival of breast cancer patients; D is Western Blot analysis of CHAF1B expression in cancer tissue and adjacent tissue of 8 clinical breast cancer patients (the upper and lower figures are from different patients, respectively).

[0011] Figure 3 To inhibit the effect of CHAF1B expression on breast cancer cell viability and proliferation; A is the Western Blot method to detect the expression level of CHAF1B in cells; B is the growth curve experiment to detect the cell proliferation ability; C is the CCK-8 method to detect cell viability; D is the clone formation experiment to detect the cell proliferation ability.

[0012] Figure 4 PI staining was used to detect the number of cell death.

[0013] Figure 5 To inhibit CHAF1B from inducing autophagy in breast cancer cells by inhibiting the MAPK signaling pathway; A is the CCK-8 method to detect the effect of pretreatment with different death inhibitors on cell viability; B is the Western Blot experiment to detect the expression of autophagy-related proteins; C is the KEGG enrichment analysis result diagram.

[0014] Figure 6 To knock down CHAF1B to inhibit the proliferation of breast cancer cells in vivo; A is a schematic diagram of the tumor volume of each group; B is the HE staining and Ki67 expression of the tumors of each group; C is the weight trend of mice during treatment; D is the HE toxicity detection of the internal organs (heart, liver, spleen, lung, and kidney) of each group of mice. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] Figure 1 This is a molecular mechanism diagram of the present invention. CHAF1B targets and regulates the MAPK signaling pathway through an epigenetic mechanism, thereby regulating the autophagic activity of breast cancer cells.

[0017] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0018] Example 1: CHAF1B is highly expressed in breast cancer tissues and is associated with prognosis; First, the TCGA database was used for analysis, and the results showed that compared with normal breast tissue, the expression of CHAF1B in breast cancer tissue was significantly upregulated ( Figure 2 A and B), and was significantly negatively correlated with the prognosis of breast cancer patients ( Figure 2 C in the middle), indicating that patients with high CHAF1B expression (High) have a poor prognosis. To further verify, we collected tissue samples from breast cancer patients for Western blot detection. The results showed that compared with adjacent tissues (Peritumor, P), CHAF1B was significantly more highly expressed in breast cancer tissues (Tumor, T) ( Figure 2 The above results suggest that CHAF1B may be a potential target for diagnosis, treatment and prognosis monitoring of breast cancer.

[0019] Example 2: Inhibition of CHAF1B can inhibit breast cancer cell proliferation and cell viability; First, two effective siRNAs were designed to knock down CHAF1B expression in breast cancer cells, denoted as siCHAF1B-6 and siCHAF1B-8, respectively. The specific sequence information is as follows: siCHAF1B-8: Sense strand: 5'-CCAGCUGGAUGUGUGGAAUTT-3' (as shown in SEQ ID NO. 1); Antisense strand: 5'-AUUCCACAUCCAGCUGGTT-3' (as shown in SEQ ID NO. 2); siCHAF1B-6: Sense strand: 5'-ACGGAAAGUCUGGACCCUUTT-3' (as shown in SEQ ID NO. 3); Antisense strand: 5'-AAGGGUCCAGACUUUCCGUTT-3' (as shown in SEQ ID NO. 4).

[0020] like Figure 3 As shown in A, compared with the control group (siNC, sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3', as shown in SEQ ID NO.5; antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3', as shown in SEQ ID NO.6), the knockdown groups (siCHAF1B-6 and siCHAF1B-8) significantly inhibited the expression of CHAF1B, indicating that the knockdown efficiency was good. The growth curve experiment results showed ( Figure 3 (B) Compared with the control group, the number of cells in the knockdown group was significantly reduced. CCK-8 results showed ( Figure 3 C), compared with the control group, the cell viability of the knockdown group was significantly inhibited. The results of the clone formation experiment showed that ( Figure 3 (D) Compared with the control group, the number of cells in the knockdown group was significantly reduced. The above results suggest that inhibition of CHAF1B can significantly inhibit the proliferation ability and cell viability of breast cancer cells.

[0021] Example 3: Inhibition of CHAF1B can induce autophagic death of breast cancer cells by inhibiting the MAPK signaling pathway; PI staining results showed that ( Figure 4 Compared with the control group (siNC), knocking down the expression of CHAF1B can induce breast cancer cell death. In order to explore the way in which knocking down CHAF1B induces breast cancer cell death, we added different death inhibitors for treatment. The results showed that ( Figure 5 In the figure (A), autophagy inhibitors (CQ, chloroquine) can significantly reverse the number of cell deaths caused by CHAF1B knockdown, suggesting that CHAF1B may regulate the autophagic death of breast cancer cells. KEGG enrichment analysis results showed ( Figure 5 Middle C), knockdown of CHAF1B significantly inhibited the MAPK signaling pathway. Western Blot results showed ( Figure 5Middle B), compared with the control group, knockdown of CHAF1B significantly downregulated the expression of autophagy-related proteins (mTOR, P62, LC3II). The above results indicate that inhibition of CHAF1B can activate autophagic death of breast cancer cells by inhibiting the MAPK signaling pathway.

[0022] Example 4, the regulatory effect of CHAF1B on breast cancer cells in vivo; In order to detect the regulatory effect of CHAF1B on breast cancer cell proliferation in vivo, a breast cancer cell line 4T1 was used to establish a nude mouse breast cancer subcutaneous transplant tumor model, and the drug was treated for 21 days to observe the therapeutic effect of knocking down CHAF1B on breast cancer in nude mice. Figure 6 As shown in A, compared with the control group (siNA), the CHAF1B knockdown group (siCHAF1B) significantly inhibited the tumor volume and the expression of the proliferation marker Ki67 ( Figure 6 The above results suggest that knocking down CHAF1B can inhibit the proliferation of breast cancer cells in vivo. In addition, there was no significant change in the body weight of mice in each group during the treatment period ( Figure 6 C), and no obvious toxicity was observed in the internal organs (heart, liver, spleen, lung, and kidney) ( Figure 6 (D) shows that siCHAF1B treatment has no obvious toxic side effects on nude mice. In summary, CHAF1B can regulate the proliferation of breast cancer cells in vivo.

[0023] Conclusion: This invention reveals for the first time that targeting CHAF1B can activate the autophagic death of breast cancer cells, which provides a new strategy for molecular targeted therapy of breast cancer and shows good anti-tumor effects. At the same time, this invention confirms that CHAF1B is significantly highly expressed in breast cancer tissues and is associated with the prognosis of patients. This result indicates that CHAF1B can be used as a potential target for diagnosis, treatment and prognosis monitoring of breast cancer. This invention not only clarifies the expression characteristics and prognostic value of CHAF1B in breast cancer, but also deeply explores its molecular mechanism of activating the autophagic death of breast cancer cells by targeting the MAPK signaling pathway. These findings provide solid theoretical support and practical significance for the clinical application of CHAF1B as a new molecular therapeutic target, and at the same time enrich the understanding of the molecular mechanism of CHAF1B regulating breast cancer cell proliferation, and provide new ideas and research directions for finding new targets for breast cancer treatment. In addition, this also provides a theoretical basis and experimental basis for the clinical development of new therapeutic targets.

[0024] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. The use of CHAF1B in the preparation of a drug for regulating the autophagy activity of breast cancer cells, characterized in that: Knocking down the expression of CHAF1B in breast cancer cells by siCHAF1B-6 and siCHAF1B-8 inhibited the proliferation and cell viability of breast cancer cells and suppressed tumor growth; Activated the autophagic death of breast cancer cells by inhibiting CHAF1B; The sequence information of siCHAF1B-8 is as follows: Sense strand: 5′-CCAGCUGGAUGUGUGGAAUTT-3′; Antisense strand: 5′-AUUCCACAUCCAGCUGGTT-3′; The sequence information of siCHAF1B-6 is as follows: Sense strand: 5′-ACGGAAAGUCUGGACCCUUTT-3′; Antisense strand: 5′-AAGGGUCCAGACUUUCCGUTT-3′.

2. The use according to claim 1, characterized in that: Inhibition of CHAF1B can target and inhibit the MAPK signaling pathway through an epigenetic mechanism to activate autophagic death of breast cancer cells.

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