Application of mitochondrial autophagy proteins NIX and PINK1 in preparation of kit for evaluating curative effect of sorafenib on liver cancer

Abstract

The invention discloses application of mitochondrial autophagy proteins NIX and PINK1 in preparation of a kit for evaluating the curative effect of liver cancer on sorafenib, and belongs to the technical field of biology. The expression quantity of the nematode autophagy protein NIX and the nematode autophagy protein PINK1 in liver cancer tissue or primary focus puncture tissue is detected through a reagent or a kit, and it is proved that the nematode autophagy protein NIX and the nematode autophagy protein PINK1 affect the liver cancer treatment effect of sorafenib by regulating mitochondrial autophagy; the clinical significance and value of the mitochondrial autophagy proteins NIX and PINK1 in the sorafenib treatment effect of the liver cancer are defined.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the use of mitochondrial autophagy proteins NIX and PINK1 in preparing a kit for evaluating the efficacy of sorafenib on liver cancer. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor. There are about 800,000 new cases of liver cancer every year in the world. According to the latest cancer report in my country, the mortality rate of liver cancer has risen from the third place to the second place, and about 336,000 people die of liver cancer every year. Most liver cancer patients are diagnosed in the middle and late stages, and have lost the opportunity for surgical treatment. They can only choose non-surgical treatment. In drug treatment, sorafenib is currently the first molecular targeted drug in the world that has been proven to be effective for liver cancer.

[0003] However, evidence from multicenter, randomized, double-blind Phase III clinical trials around the world shows that although sorafenib can indeed benefit patients with advanced liver cancer and prolong their survival, the extension time is extremely limited, with an average of only 2.8 months. Therefore, how to improve its anti-cancer efficacy is a prominent issue facing clinicians and pharmacologists. Studies have shown that liver cancer cells are generally less sensitive to the in vitro effects of sorafenib, with a killing rate of only 20%-30%. This may be related to drug-induced autophagy of liver cancer cells, and similar cell autophagy phenomena have also been found in other traditional anti-tumor drugs, and even restrict the development of a variety of new anti-liver cancer drugs.

[0004] Autophagy in a general sense is usually non-selective. When cell autophagy clears damaged organelles in a targeted manner, it manifests as corresponding selective autophagy, such as mitochondrial autophagy (mitophagy), peroxisomal autophagy (pexophagy), endoplasmic reticulum autophagy (reticulophagy), etc. Reports have found that various types of selective autophagy are closely related to the occurrence and development of tumors. Considering that mitochondria are the control center of cell apoptosis and cell life activities, they are more important for clearing tumor cells, suggesting that the exploration should start from the study of mitochondrial autophagy.

[0005] In mammalian cells, the most common pathways mediating mitophagy are the Pink1 / Parkin pathway and the BNIP3 / NIX pathway. When the mitochondrial membrane is depolarized, Pink1 accumulates on the outer membrane of mitochondria, activates and recruits Parkin to ubiquitinate damaged mitochondria, which are connected to LC3 protein through autophagy receptors to form mitophagosomes. The aggregation of damaged mitochondria caused by insufficient or absent Pink1 / Parkin activity is an important pathogenic mechanism of Parkinson's disease. In contrast, Nix is ​​located in the outer membrane of mitochondria and can directly interact with Atg8 protein or ATG8 homolog LC3 to cause mitophagy and mediate mitochondrial clearance during reticulocyte maturation. However, certain interactions have also been found between the two pathways. For example, Parkin can ubiquitinate NIX and interfere with the formation of NIX-dependent autophagy.

[0006] In recent years, the relationship between mitochondrial autophagy and tumors has begun to emerge. It has been reported that Parkin is a "molecular switch" of cells, which determines the fate of cells in response to different stress factors. The PAPK2 gene encoding Parkin is located at the fragile site of chromosome 6, and its gene mutation is associated with the onset of human colon cancer, glioma, lung cancer, hepatocellular carcinoma, etc. Fujiwara et al. found that the proliferation ability of liver cells in Parkin gene knockout mice was enhanced and hepatocellular carcinoma could occur, suggesting that the PAPK2 gene may be a tumor suppressor gene. In addition, studies have found that sorafenib inhibits the mitochondrial electron transport chain and promotes the recruitment of Parkin to damaged mitochondria, thereby inducing mitochondrial autophagy and causing tumor cell death. It is speculated that Pink1 / Parkin-mediated mitochondrial autophagy may have an inhibitory effect on tumor growth. BNIP3 and NIX (also known as BNIP3L) promote mitochondrial autophagy under hypoxic conditions. Studies have found that under the action of doxorubicin, the expression of mitochondrial autophagy protein NIX in colorectal cancer stem cells increased, and inhibition of NIX significantly enhanced the sensitivity of cancer cells to doxorubicin, suggesting that NIX expression may have a protective effect on tumor cells.

[0007] At present, the relationship between the clinical outcome of liver cancer and the occurrence and development of mitophagy under different sorafenib sensitivity states is still unclear, and the interaction points of the NIX and Pink1 signaling pathways in the process are still unclear. There are no reports on the application of mitophagy proteins NIX and PINK1 in the preparation of kits for evaluating the efficacy of sorafenib in liver cancer. Summary of the invention

[0008] The purpose of the present invention is to solve the above problems existing in the prior art and provide the use of mitochondrial autophagy proteins NIX and PINK1 in the preparation of a kit for evaluating the efficacy of sorafenib in liver cancer. The present invention uses a new perspective of mitochondrial autophagy and the mechanism of action of NIX and PINK1-dependent mitochondrial autophagy in sorafenib treatment of liver cancer to verify the influence of NIX and PINK1 on sorafenib-based liver cancer drug treatment regimens; thereby seeking new ideas and new targets for the clinical treatment and efficacy evaluation of liver cancer with sorafenib.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows:

[0010] In a first aspect, the present invention provides the use of mitochondrial autophagy proteins NIX and PINK1 in the preparation of a reagent or kit for evaluating the efficacy of sorafenib in liver cancer.

[0011] Furthermore, the reagent or kit detects the expression levels of mitochondrial autophagy proteins NIX and PINK1 in liver cancer tissue or primary lesion puncture tissue.

[0012] Furthermore, the reagent or kit uses an immunohistochemical method to detect the expression levels of mitochondrial autophagy proteins NIX and PINK1 in liver cancer tissue or primary lesion puncture tissue.

[0013] In a second aspect, the present invention provides the use of mitochondrial autophagy proteins NIX and PINK1 in the preparation of a reagent or a kit for determining the sensitivity of liver cancer patients to sorafenib.

[0014] Furthermore, the reagent or kit detects the expression levels of mitochondrial autophagy proteins NIX and PINK1 in liver cancer tissue or primary lesion puncture tissue.

[0015] In a third aspect, the present invention provides a method for determining the prognosis of a liver cancer patient taking sorafenib, the method comprising detecting the expression levels of mitochondrial autophagy proteins NIX and PINK1 in liver cancer tissue or primary lesion puncture tissue.

[0016] The beneficial effects of the present invention are as follows:

[0017] On the basis of existing research on the relationship between mitochondrial autophagy and tumors, the present invention further studies and shows that the autophagosomes with mitochondria as substrates in liver cancer Hep3B cells stimulated by sorafenib increase; in immunoblotting detection, sorafenib can induce a significant increase in the expression of NIX, a marker of cell mitochondrial autophagy, in a concentration-dependent manner, and the latter is negatively correlated with the shear changes of Caspase3, a drug-induced apoptosis signal, suggesting that high expression of NIX induced in liver cancer may be unfavorable for drug treatment. Unlike the high expression of NIX, the basal level of Pink1 in liver cancer Hep3B cells is low, and sorafenib alone has little effect on the expression of Pink1 and Parkin in cells. The latter uses melatonin to stimulate the mitochondrial autophagy activity of the Pink1 / Parkin pathway, and it is found that it can improve the efficacy of sorafenib in the treatment of liver cancer, providing new evidence for the anti-cancer effect of mitochondrial autophagy in the Pink1 / Parkin pathway.

[0018] The present invention explores new targets for evaluating the clinical efficacy of sorafenib, which can achieve individualized targeted treatment and reduce the pain of liver cancer patients. In addition, the present invention interprets the efficacy and prognosis of sorafenib for liver cancer from a new perspective of mitochondrial autophagy-related pathways, which has great clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Immunohistochemical detection of the expression of mitochondrial proteins NIX and PINK1 (×400); A1-A3 in the figure are the expression of NIX in liver cancer tissues, paracancerous tissues and normal control group, and B1-B3 are the expression of PINK1 in liver cancer tissues, paracancerous tissues and normal control tissues;

[0020] Figure 2 The expression of mitochondrial NIX protein and mRNA under the action of different concentrations of sorafenib;

[0021] Figure 3 The expression of mitochondrial PINK1 protein and mRNA under different concentrations of sorafenib;

[0022] Figure 4 The expression of mitochondrial NIX protein and mRNA at different time of sorafenib treatment;

[0023] Figure 5 The expression of mitochondrial PINK1 protein and mRNA at different time of sorafenib treatment;

[0024] Figure 6 Western blot was used to detect the expression of NIX protein, apoptosis protein and autophagy protein in starved HepG2 cells treated with different concentrations of sorafenib;

[0025] Figure 7 To investigate the effect of NIX overexpression on the mortality of HepG2 liver cancer cells induced by sorafenib.

[0026] Figure 8 To investigate the effect of NIX overexpression on autophagy and apoptosis of HepG2 cells induced by sorafenib.

[0027] Fig. 9 To investigate the effect of overexpression of PINK1 on autophagy and apoptosis in HepG2 cells;

[0028] Fig.10 To investigate the effects of overexpression of PINK1 on HepG2 cell clones, proliferation ability, death and apoptosis;

[0029] Fig.11 The response of liver cancer cells stably overexpressing PINK1 to sorafenib;

[0030] Fig.12 This is a nude mouse liver cancer model and the tumor image after intervention;

[0031] Fig.13 Figure 2 is the tumor growth curve (without drug intervention and after sorafenib intervention). DETAILED DESCRIPTION

[0032] The various liver cancer cell lines (including HepG2, Hep3B, Huh7) involved in the specific embodiments of the present invention were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0033] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased on the market.

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0035] Example 1

[0036] Thirty-nine cases of hepatocellular carcinoma tissues and their adjacent tissues were collected, and 36 cases of normal liver tissues adjacent to hepatic hemangioma during the same period were taken as controls. Immunohistochemistry was used to detect the expression levels of mitochondrial autophagy proteins NIX and PINK1. The results showed that there were significant differences in the expression levels of mitochondrial proteins NIX and PINK1 between cancer tissues, adjacent tissues and normal control groups (P < 0.05). Among them, the expression level of NIX in liver cancer tissues was significantly higher than that in adjacent tissues, and the expression level of NIX in adjacent tissues was significantly higher than that in the normal control group; the expression level of PINK1 in adjacent tissues was significantly higher than that in liver cancer tissues, and the expression level of PINK1 in liver cancer tissues was significantly higher than that in the normal control group; Figure 1 .

[0037] Example 2

[0038] The liver cancer cell line HepG2 was taken and revived, passaged and cultured according to conventional methods. The liver cancer cells in the logarithmic growth phase were divided into 5 groups and inoculated into 6-well culture plates. When the cells were completely attached to the wall and grew, gradient final concentrations of sorafenib (2.5, 5, 10, 20 μM) or equal volumes of solvent DMSO were added, and the cells were incubated in a 5% CO2, 37°C incubator for 24 hours, and the cells were collected. The total cell protein was extracted, and the expression of mitochondrial proteins NIX and Pink1 was detected by western blotting. The total cell RNA was extracted, and the expression of NIX and Pink1 mRNA was detected by qRT-PCR. Research results: The results of HepG2 cells under the action of different concentrations of sorafenib are as follows: As the concentration of sorafenib increases, the expression of NIX protein in HepG2 cells shows a downward trend. Figure 2 (A, B), the mRNA expression of NIX also showed a downward trend, e.g. Figure 2 (C); PINK1 protein expression in HepG2 cells showed an increasing trend. Figure 3 (A, B), the mRNA expression of PINK1 also showed an increasing trend, e.g. Figure 3 (C).

[0039] Example 3

[0040] The liver cancer cell line HepG2 was taken and revived, passaged and cultured according to conventional methods. The liver cancer cells in the logarithmic growth phase were divided into 5 groups and inoculated into 6-well culture plates. When the cells were completely attached to the wall and grew, the final concentration of sorafenib (10μM) or an equal volume of solvent DMSO was added, and the cells were incubated in a 5% CO2, 37°C incubator for 6, 12, 24, and 48 hours, and the cells were collected respectively. The total cell protein was extracted, and the expression of mitochondrial proteins NIX and Pink1 was detected by western blotting. The total cell RNA was extracted, and the expression of NIX and Pink1 mRNA was detected by qRT-PCR. Research results: The results of HepG2 cells under the same concentration of sorafenib for different time periods are as follows: With the increase of time, the expression of NIX protein in HepG2 cells showed a downward trend. Figure 4 (A, B), the mRNA expression of NIX also showed a downward trend, e.g. Figure 4 (C); PINK1 protein expression in HepG2 cells showed an increasing trend. Figure 5 (A, B), the mRNA expression of PINK1 also showed an increasing trend, e.g. Figure 5 (C).

[0041] Example 4

[0042] The liver cancer cell line HepG2 was taken and revived, passaged and cultured according to the conventional method. The liver cancer cells in the logarithmic growth phase were divided into 5 groups and inoculated into 6-well culture plates until the cells were completely attached to the wall and grew. The conventional culture medium with 1% FBS was replaced, but gradient final concentrations of sorafenib (2.5, 5, 10, 20 μM) or an equal volume of solvent DMSO were added, and the stimulation was continued for 24 hours in a 5% CO2, 37°C incubator. Cell proteins were collected, and western blotting was used to detect the expression of mitochondrial autophagy protein NIX, non-selective autophagy signal P62 and apoptosis protein caspase3. Research results: Starvation treatment can promote the induction of autophagy in HepG2 cells. As the concentration of sorafenib increases, the expression of NIX protein shows a downward trend, the apoptosis protein Caspase3 protein decreases, and the autophagy protein P62 decreases. Figure 6 , indicating that as the concentration of sorafenib increases, cells undergo autophagic apoptosis, in which the mitochondrial protein NIX is involved in the occurrence of cell autophagy and apoptosis.

[0043] Example 5

[0044] The liver cancer cell lines NIX-cDNA-HepG2 cells and NC-HepG2 cells were taken and revived, passaged and cultured according to conventional methods. The stably transfected cells in the logarithmic growth phase were grouped and inoculated in 100mm culture dishes and cultured in a 37°C, 5% CO2 incubator. 10μM sorafenib and an equal volume of solvent DMSO were added respectively, and the stimulation intervention was carried out in a 5% CO2, 37°C incubator for 24 hours. Cell proteins were collected, and western blotting was used to detect the expression of mitochondrial protein NIX, non-selective autophagy signal P62 and apoptosis protein caspase3. In addition, trypan blue staining was performed to observe the cell mortality rate. Research results: Trypan blue results showed that there was no statistical difference in mortality between the NIX overexpression group and the NC group (P>0.05). After the addition of sorafenib, it was found that the mortality rate of the NIX overexpression group was lower than that of the NC group, which was statistically significant. Figure 7 This suggests that overexpression of NIX can reduce the killing effect of sorafenib on liver cancer cells. Further WB detection found that overexpression of NIX can inhibit sorafenib-induced autophagy in liver cancer HepG2 cells, thereby reducing the expression of apoptosis protein Casepase3. Figure 8 .

[0045] Example 6

[0046] The liver cancer cell line NC-HepG2 / PINK1-cDNA-HepG2 was taken and revived, passaged and cultured according to conventional methods. The stably transfected PINK1-cDNA-HepG2 cells and NC-HepG2 cells in the logarithmic growth phase were grouped and inoculated in 100mm culture dishes and cultured in a 37°C, 5% CO2 incubator for 24 hours. Cell proteins were collected, and western blotting was used to detect the expression of mitochondrial protein PINK1, non-selective autophagy signals P62, LC3, and apoptosis protein caspase3; Research results: Compared with the NC group, after overexpression of PINK1, the expression level of autophagy protein P62 decreased, and the expression of LC3B increased, indicating that overexpression of PINK1 can induce autophagy in liver cancer cells; the expression of apoptosis protein caspase3 increased, indicating that overexpression of PINK1 promotes the occurrence of cell apoptosis, such as Fig. 9 .

[0047] On this basis, we further performed plate monoclonal experiments, CCK8 assay, trypan blue staining, and flow cytometry to verify the effects of overexpression of PINK1 on HepG2 cell cloning, proliferation ability, mortality, and apoptosis. Results: Compared with the NC group and the normal group, overexpression of PINK1 promoted the mortality rate and apoptosis rate of HepG2 liver cancer cells; it was found that overexpression of PINK1 could inhibit the proliferation and cloning ability of liver cancer cells, such as Fig.10 .

[0048] Example 7

[0049] The liver cancer cell line NC-HepG2 / PINK1-cDNA-HepG2 was taken and revived, passaged and cultured according to conventional methods. Stably transfected PINK1-cDNA-HepG2 cells and NC-HepG2 cells in the logarithmic growth phase were grouped and inoculated in 100mm culture dishes. When the cells were completely attached to the wall, the conventional culture medium with 10% FBS was replaced and sorafenib (10μM) was added according to the experimental requirements. The cells were cultured in a 5% CO2, 37℃ incubator for 24h, and the cell proteins were collected. The expression of mitochondrial protein PINK1, non-selective autophagy signals P62, LC3 and apoptosis protein caspase3 was detected by western blotting; the cell death rate was detected by trypan blue staining. Results: Compared with the control group, the expression of autophagy protein P62 decreased, LC3B expression increased, and apoptosis protein caspase3 expression increased in HepG2 cells overexpressing PINK1 under the action of sorafenib, indicating that overexpression of PINK1 can induce the occurrence of cell autophagy. Under the action of sorafenib, cell autophagy is enhanced, promoting the apoptosis of liver cancer cells. Fig.11Trypan blue test results: Compared with the control group, the cell death rate in the PINK1 overexpression group (32.76% ± 2.05% VS 52.36% ± 1.14%) increased by 19.60%, and compared with the PINK1 overexpression group, the cell death rate in the PINK1 overexpression group + sorafenib group increased by 21.37% (30.99% ± 2.59% VS 52.36% ± 1.14%). Fig.11 .

[0050] Example 8

[0051] Huh7, NC-Huh7, and NIX-cDNA-Huh7 cells were cultured at a concentration of 1×10 7 Each 0.2 ml cell suspension was implanted on the right lower edge of the rib cage of 90 nude mice. When the tumor volume of all mice grew to ≥100 mm 3 After that, simultaneous drug intervention was started, and the mice were randomly divided into a control group, a vehicle group, a sorafenib group (30 mg / Kg), a melatonin group (10 mg / kg), and a sorafenib combined with melatonin group, with 6 mice in each group. Sorafenib was administered orally + melatonin was injected intraperitoneally for 14 consecutive days. The longest diameter (L) of the tumor and the perpendicular diameter (D) were measured every 3 days. The tumor volume (V) was calculated according to the following formula, and the weight of the nude mice was measured. After the last administration of the drug, the nude mice were fasted for 12 hours. The nude mice were anesthetized with 10% chloral hydrate at 0.01 mL / g body weight, and then killed by cervical dislocation, and the tumor tissue was peeled off. The tumor weight was recorded and photographed. Fig.12 . Research results: (1) Fig.13 Without drug intervention, the tumor volume of each group of nude mice increased over time, and the growth of NIX-cDNA-Huh7 tumor was more obvious (P<0.05). Under the intervention of sorafenib, the tumor volume of NC-Huh7 group was significantly smaller than that of NIX-cDNA-Huh7 on days 18-21 (P<0.05). (2) After sorafenib intervention, the tumor inhibition rates of NC-Huh7 group and NIX-cDNA-Huh7 group were 63.7% and 45.9%, respectively (P<0.05). This suggests that high expression of NIX protein can promote tumor growth and increase insensitivity to sorafenib.

[0052] In summary, based on the results of the above embodiments, the present invention illustrates that NIX and PINK1 affect the efficacy of sorafenib in treating liver cancer by regulating mitochondrial autophagy; and proves the clinical significance and value of mitochondrial autophagy proteins NIX and PINK1 in the efficacy of sorafenib in treating liver cancer.

[0053] The research methods and results described above express several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians and medical workers in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. Use of mitochondrial autophagy protein in the preparation of a reagent or kit for evaluating the efficacy of sorafenib in liver cancer, characterized in that: The mitochondrial autophagy protein is NIX or PINK1.

2. The use of the mitophagy protein according to claim 1 in the preparation of a reagent or kit for evaluating the efficacy of sorafenib in liver cancer, characterized in that: The reagent or kit detects the expression level of the mitochondrial autophagosome protein in liver cancer tissue or primary lesion puncture tissue.

3. The use of the mitophagy protein as described in claim 2 in the preparation of a reagent or kit for evaluating the efficacy of sorafenib in liver cancer, characterized in that: The reagent or kit uses an immunohistochemical method to detect the expression level of the mitochondrial autophagy protein in liver cancer tissue or primary lesion puncture tissue.

4. Use of mitochondrial autophagy protein in the preparation of a reagent or kit for determining the sensitivity of liver cancer patients to sorafenib, characterized in that: The mitochondrial autophagy protein is NIX or PINK1.

5. Use of the mitophagy protein according to claim 4 in the preparation of a reagent or a kit for determining the sensitivity of liver cancer patients to sorafenib, characterized in that: The reagent or kit detects the expression level of the mitochondrial autophagy protein in liver cancer tissue or primary lesion puncture tissue.

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