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Technol Cancer Res Treat. 2024; 23: 15330338241276889.
Published online 2024 Aug 28. doi:10.1177/15330338241276889
PMCID: PMC11363239
PMID: 39194338
Xi-yue Zhang, Bachelor of Medicine,1 Ming Luo, MD,2 Shu Qin, MD,2 Wen-guang Fu, MD,2 and Meng-yu Zhang, MD2
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Abstract
Detect the expression of Farnesoid X Receptor(FXR), Multiple Drug Resistance Associated Protein-1(MRP-1) and Solute Carrier Family 7, Member 5 (SLC7A5) in hepatocellular carcinoma(HCC) of rat model, so as to provide new therapeutic targets for gene therapy of HCC. Sixty male Wistar rats were randomly divided into three groups. The rats in experimental group were given 0.2% diethylnitrosamine (DEN) by gavage with a dose of 10 mg/kg, 3 times a week, and it stopped at 12 weeks. The control group rats were given physiological saline by gavage, while the sham operation group did not receive anything by gavage. At 10 weeks, one rat in the experimental group was euthanized, and the changes of livers were recorded. The procedure was repeated at 12 weeks. After 12 weeks, HCC only occurred in the experimental group. After confirming the formation of the tumor through pathological examination, liver tissues and tumor tissues were taken from the three groups. FXR, MRP-1 and SLC7A5 expression in liver tissues and tumor tissues was detected. After 7 weeks the rats in experimental group ate less, and their weight was significantly reduced. Three months later, HCC was detected in 15 rats in the experimental group. The ratio of FXR/GAPDH mRNA, MRP-1/GAPDH mRNA, SLC7A5/GAPDH mRNA were significantly different among the three groups. Under the light microscope the FXR protein, MRP-1 protein, and SLC7A5 protein react with their respective antibodies, and they showed granular expression. Every pathological section included different numbers of positive cells in each group. FXR expression in HCC of rats was significantly lower than that in normal liver tissues, but MRP-1 and SLC7A5 expression in HCC were significantly higher than that in normal liver tissues, suggesting that drugs targeting FXR, MRP-1 and SLC7A5 may be new strategies for the treatment of HCC.
Keywords: FXR, MRP-1, SLC7A5, HCC, DEN
Introduction
Primary liver cancer is one of the widely occurring malignant tumors in the world. HCC is the main histological subtype of liver cancer, accounting for an extremely high proportion in primary cancer. It ranks among the top five causes of cancer-related deaths in various countries around the world. Hepatitis B and C virus infection, nitrite intake, moldy food, smoking and diabetes are the main risk factors for liver cancer. Due to the lack of obvious symptoms and signs in HCC patients in the early subclinical and subclinical stages, some patients may experience mild upper abdominal tenderness, some patients may present with gastrointestinal symptoms, or the earliest manifestation may be caused by metastatic lesions in other areas. However, patients usually do not notice this and do not have the awareness to undergo examination and treatment. But when symptoms such as jaundice, vomiting, diarrhea, ascites, and cachexia appear, it is already in the late stage, and the medical treatment methods available are very limited, with poor results.1–3 For HCC patients, surgical resection is the first choice, but only a portion of early and mid stage patients can receive surgical treatment. According to Barcelona's classification, only HCC patients in the first two stages can meet the requirements for R0 resection(No cancer cells at the cutting edge, complete resection), but patients in the later three stages often cannot be completely removed. Although some patients underwent palliative surgery and received postoperative use of anti-tumor drugs to improve treatment outcomes, overall, there was no significant improvement in the 5-year survival rate of patients. Some patients are unable to undergo surgery due to multiple metastases of the tumor within and outside the liver, invasion of surrounding tissues by the tumor, severe heart and lung disease, or severe renal dysfunction. Alternative therapies include radiofrequency ablation, cryotherapy, ultrasound focusing, and radioactive therapy. However, clinical validation has found that although these methods can partially prolong survival, their effectiveness in improving patient prognosis is not significant. Therefore, constantly exploring new treatment methods is a long-term and sustained process.
Gene therapy has emerged and gradually developed in the treatment of tumors in recent years, and has been applied to the treatment of some tumors, but its applicability is limited. We can see from the results of multiple medical centers and research bases around the world that the effectiveness of gene therapy alone is very limited. However, when combined with methods such as interventional therapy and immunotherapy, it has better effects on some patients with unresectable tumors. However, currently discovered genes and developed targeted drugs are mainly used to inhibit tumor angiogenesis, and are rarely directly related to the division and proliferation of tumor cells. Therefore, we are committed to searching for genes that can cause tumor tissue necrosis and inhibit tumor cell proliferation as therapeutic targets. Farnesylate X receptor (FXR) is related to bile acid metabolism, its gene and protein expression plays an important role in the occurrence and development of colon, breast and liver tumors. According to statistics from the TCGA database and GTEX database, there is a decrease in FXR in 24 known tumors. MRP-1 and SLC7A5 are the target gene of FXR,4–6 the MRP-1 is an ATP binding transporter cassette and it is resistant to many anticancer drugs, SLC7A5 is an important member of the L-type amino acid transporter, it mainly responsible for transporting large, branched, aromatic amino acids, including some essential amino acids, providing a carrier channel for the entry and exit of amino acids in tumor cells. Due to the discovery of reduced FXR in various tumor tissues and the discovery of FXR agonists, as well as the clear regulation of MRP-1 and SLC7A5 by FXR, we investigated the expression of FXR, MRP-1 and SLC7A5 in HCC, regardless of whether their role in HCC is enhanced or decreased, in an attempt to shed the light on new targets for HCC treatment.
Materials and Methods
Statement
The reporting of this study conforms to ARRIVE 2.0 guidelines.7 Animal care guidelines abided by the ‘Guide for the Care and Use of Laboratory Animals, eighth Edition’.8
Animals
The Animal experimental center of Southwest Medical University provided the Wistar rats(male, 180 ± 5 g, 6 weeks old). They were randomly divided into experimental group, control group and sham operation group (n = 20 for each group).
Experimental Methods
Materials: DEN(Sichuan Vicki Biotechnology Co., Ltd), Pentobarbital sodium(Hubei Hongyunlong Biotechnology Co., Ltd), Hematoxylin eosin staining solution(Jinhua Lisheng Equipment Co., Ltd), anti-FXR antibody (Chemicon USA), anti-MRP-1 antibody (Chemicon USA), anti-SLC7A5 antibody (Chemicon USA), BIO-RAD T100 real-time PCR detection system(USA). Prepare paraffin tissue sections using a paraffin slicer, formalin, paraffin, and alcohol. The sense and antisense primers used to detect FXR mRNA levels were as follows: 5′-CCTCATTGTCTCCCCGACTTA-3′ and 5′-ACTTGTGACG AAAGATCTCCG-3′. The sense and antisense primers used to detect MRP-1 mRNA levels were as follows: 5′-GCAGGGCTACTTCTACACCG-3′ and 5′-TCATCGCCATCACAGCATTG-3′.The sense and antisense primers used to detect SLC7A5 mRNA levels were as follows: 5′-CCGTGAACTGCTACAGCGT-3′ and 5′-CTTCCCGATCTGGACGAAGC-3′.The sense and antisense primers used to detect GAPDH mRNA were as follows: 5′-GATGGTGGGTATGGGTCAGAA-3′ and 5′- CTAGGAGCCAGGGCA GTAATC-3′. The 2-ΔΔCt method was used to express the data.
Establish animal model and test All the Wistar rats were kept in an environment with a temperature of 25°C and a relative humidity of 60%. Provide the day and night light variation cycles with 12 h of light and 12 h of darkness. After a week of stable feeding, the rats in experimental group were given 0.2% diethylnitrosamine (DEN) by gavage with a dose of 10 mg/kg, 3 times a week, and it stopped at 12 weeks. The control group rats were given physiological saline by gavage, while the sham operation group did not receive anything by gavage. A modified tail suspension test (TST) was used to evaluate the vitality of the rats. At 10 weeks, one rat in the experimental group was euthanized(The rat is placed in an IVC cage, and a CO2 gas pipe is connected at the inlet of the water bottle. The valve of the gas bottle is opened, and CO2 is injected into the euthanasia box at a rate of 10% to 30% of the volume per minute, allowing CO2 to fill the cage. After the rats fainted and lost their exercise ability, increase the gas flow, and the maximum flow shall not exceed 0.5Kpa. Confirm that the breathing of the rats disappears, close CO2 after Mydriasis, and observe for another two minutes to determine the death of the animals) and the changes of livers were recorded. The procedure was repeated at 12 weeks. After 12 weeks, confirming the formation of the tumor through pathological examination, blood is extracted from the tail of rats for liver function testing, liver tissues and tumor tissues were taken from the three groups. HE staining showed the tissues of HCC, liver tissues of control group and sham operation group. Real-time polymerase chain reaction (RT-PCR) was used to detect the expression of FXR mRNA, MRP-1 mRNA and SLC7A5 mRNA (RNA was extracted from HCC and normal liver tissues), GAPDH served as internal control. Immunohistochemical SP method was used to analyze the expression of FXR protein, MRP-1 protein and SLC7A5 protein. Under the light microscope, FXR protein reacted with anti-FXR antibody, MRP-1 protein reacted with anti-MRP-1 antibody and SLC7A5 protein reacted with anti-SLC7A5 antibody, they showed granular expression. Every pathological section was randomly divided into 6 regions, and 200 cells were observed in each region.
Statistical Analysis
The experimental data is expressed as mean ± standard deviation. The data analysis was conducted using SPSS26.0 statistical software. The t test was used to evaluate the difference between two groups, the chi-square test was used to evaluate the data obtained from immunohistochemistry, and P < .05 indicates a statistically significant difference. Linear regression is used to determine the relationship among three genes.
Results
Changes in Rats
Three rats in the experimental group died three months later, while the other two groups had no dead rats. After 7 weeks, the food intake of the experimental group rats gradually decreased, and their weight also decreased significantly (Table 1, Table 2). After blood drawn exa mination, it was found that there were differences in liver function among the three groups of rats (Table 3). After 12 weeks, pathological examination confirmed the presence of HCC in 15 rats in the experimental group (Figure 1).
Figure 1.
Pathological examination.
(A) HCC in experimental group.
(B) normal liver tissues in control group.
(C) normal liver tissues in sham operation group.
(D)The dividing cells of HCC are represented by green arrows (magnification 100×).
(E) normal liver tissues in control group. HE stain (magnification 100×).
(F) normal liver tissues in sham operation group. HE stain (magnification 100×).
After 12 weeks, HCC was found in 15 rats in the experimental group, all of which were infiltrating masses. The tumor cells were multinucleated, and the endoplasmic reticulum and mitochondria were swollen significantly. However, the liver tissues of the control group and the sham operation group only showed mild inflammation and edema, and a small amount of inflammatory cells were infiltrated.
Table 1.
Daily Food-Intake (g).
week | control group (n = 20) | sham operation group(n = 20) | experimental group (n = 20) |
---|---|---|---|
7 | 25.17 ± 0.18 | 24.36 ± 0.14 | 19.75 ± 0.08 |
8 | 25.91 ± 0.21 | 25.28 ± 0.19 | 20.36 ± 0.12 |
9 | 26.74 ± 0.26* | 26.53 ± 0.23^ | 20.91 ± 0.13 |
10 | 27.15 ± 0.29# | 27.03 ± 0.27& | 19.86 ± 0.11 |
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Note: *P < .05 compared with the experimental group, ^P < .05 compared with the experimental group, #P < .05 compared with the experimental group, &P < .05 compared with the experimental group. After 7 weeks the rats in experimental group ate less, due to the formation and progression of HCC, their food intake is significantly reduced compared with the other two groups.
Table 2.
Body Mass (g).
week | control group (n = 20) | sham operation group(n = 20) | experimental group (n = 20) |
---|---|---|---|
7 | 295 ± 5.4 | 293 ± 4.9 | 286 ± 3.7 |
8 | 323 ± 6.1 | 320 ± 5.8 | 292 ± 5.2 |
9 | 361 ± 6.5* | 357 ± 6.3^ | 294 ± 5.3 |
10 | 378 ± 7.2# | 371 ± 6.8& | 283 ± 3.5 |
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Note: *P < .05 compared with the experimental group, ^P < .05 compared with the experimental group, #P < .05 compared with the experimental group, &P < .05 compared with the experimental group. After 7 weeks the rats in experimental group ate less, due to the formation and progression of HCC, their weight was significantly reduced compared with the other two groups.
Table 3.
Changes of Liver Function.
Related indicators | control group (n = 20) | sham operation group(n = 20) | experimental group (n = 20) |
---|---|---|---|
(ALT)/(U/L) | 50.27 ± 2.01$ | 52.36 ± 2.05$$ | 87.46 ± 2.73 |
(AST)/(U/L) | 48.73 ± 1.90& | 47.65 ± 1.68&& | 90.32 ± 3.12 |
(TC)/(mmol/L) | 2.97 ± 0.06 | 3.05 ± 0.08 | 3.03 ± 0.07 |
(TBA)/(μmol/L) | 1.71 ± 0.03# | 1.78 ± 0.05## | 2.37 ± 0.11 |
(TBIL)/(μmol/L) | 3.73 ± 0.17 | 3.82 ± 0.21 | 3.79 ± 0.18 |
(DBIL)/(μmol/L) | 0.81 ± 0.03* | 0.85 ± 0.04** | 1.97 ± 0.08 |
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Note: $P < .05 compared with the experimental group, &P < .05 compared with the experimental group, #P < .05 compared with the experimental group,*P <.05 compared with the experimental group, $$P < .05 compared with the experimental group, &&P < .05 compared with the experimental group,##P < .05 compared with the experimental group, **P < .05 compared with the experimental group. The levels of direct bilirubin, total bile acids, alanine aminotransfease and aspartate transaminase, in experimental group were higher compared with the other two groups.
ALT: alanine aminotransfease, AST: aspartate transaminase, TC:total cholesterol, TBA: total bile acids, TBIL: Total bilirubin, DBIL: direct bilirubin
Analysis of FXR, MRP-1 and SLC7A5 Expression
Through RT-PCR, we found that there were changes in the expression of FXR, MRP-1 and SLC7A5 in tumor cells compared to normal liver cells. In the experimental group, control group, and sham surgery group, after 15 cycles, the FXR/GAPDH ratios were 19 ± 1.2, 39 ± 2.1 and 40 ± 2.3, respectively (t = 3.117, P < .05, between the experimental group and the control group; t = 3.128, P < .05, between the experimental group and the sham surgery group), and the MRP-1/GAPDH ratios were 47 ± 2.8, 21 ± 1.6 and 20 ± 1.5, respectively (t = 3.327, P < .05, between the experimental group and the control group; t = 3.254, P < .05, between the experimental group and the sham surgery group), The SLC7A5/GAPDH ratios were 43 ± 2.2, 19 ± 0.7 and 20 ± 1.0, respectively (t = 3.271, P < .05, between the experimental group and the control group; t = 3.183, P < .05, between the experimental group and the sham surgery group). The experimental group showed statistical differences compared to the control group and the sham surgery group(Figure 2).
Figure 2.
Analysis of FXR mRNA, MRP-1 mRNA and SLC7A5 mRNA expression by RT-PCR.
Through RT-PCR we found that in HCC, control group and sham operation group the FXR/GAPDH ratios were 19 ± 1.2, 39 ± 2.1 and 40 ± 2.3, respectively. The MRP-1/GAPDH ratios were 47 ± 2.8, 21 ± 1.6 and 20 ± 1.5, respectively. The SLC7A5/GAPDH ratios were 43 ± 2.2, 19 ± 0.7 and 20 ± 1.0, respectively. After fifteen cycles, there was significant statistical difference among the three groups. Using IMAGE J software for quantitative analysis of immunohistochemistry images, the average optical density of FXR, MRP-1 and SLC7A5 were obviously different among the three groups.
The FXR protein, MRP-1 protein, and SLC7A5 protein react with their respective antibodies, as shown in Figure 3. Every pathological section included 1200 cells. FXR protein is expressed as follows: A total of 224 positive cells (18.7%) were in the experimental group, 1006 positive cells (83.8%) were in the control group, 1032 positive cells (86.0%) were in the sham operation group, and there was significant difference among the three groups (χ2 = 34.89, P < .05, between experimental group and control group; χ2 = 36.17, P < .05, between experimental group and sham operation group.). MRP-1 protein is expressed as follows: A total of 972 positive cells (81.0%) were in the experimental group, 206 positive cells (17.2%) were in the control group, 190 positive cells (15.8%) were in the sham operation group, and there was significant difference among the three groups (χ2 = 32.76, P < .05, between experimental group and control group; χ2 = 33.01, P < .05, between experimental group and sham operation group.). SLC7A5 protein is expressed as follows: A total of 994 positive cells (82.8%) were in the experimental group, 242 positive cells (20.2%) were in the control group, 226 positive cells (18.8%) were in the sham operation group, and there was significant difference among the three groups (χ2 = 34.39, P < .05, between experimental group and control group; χ2 = 35.22, P < .05, between experimental group and sham operation group.). Using IMAGE J software for quantitative analysis of immunohistochemistry images, the average optical density of FXR, MRP-1 and SLC7A5 were obtained as follows:In the experimental group, control group and sham surgery group, the average optical density of FXR were 0.11 ± 0.02, 0.29 ± 0.03 and 0.30 ± 0.02 (t = 2.995, P < .05, between the experimental group and the control group; t = 3.062, P < .05, between the experimental group and the sham surgery group), the average optical density of MRP-1 were 0.33 ± 0.04, 0.15 ± 0.02 and 0.17 ± 0.03 (t = 3.187, P < .05, between the experimental group and the control group; t = 3.139, P < .05, between the experimental group and the sham surgery group), the average optical density of SLC7A5 were 0.32 ± 0.03, 0.12 ± 0.01 and 0.14 ± 0.02 (t = 3.207, P < .05, between the experimental group and the control group; t = 3.198, P < .05, between the experimental group and the sham surgery group).
Figure 3.
Analysis of FXR protein, MRP-1 protein and SLC7A5 protein expression by immunohistochemical assay.
From photo a to photo c: FXR expression in experimental group, control group and sham operation group(magnification 200×).
From photo d to photo f: MRP-1 expression in experimental group, control group and sham operation group(magnification 200×).
From photo g to photo i: SLC7A5 expression in experimental group, control group and sham operation group(magnification 200×).
FXR protein reacted with the anti-FXR antibody, MRP-1 protein reacted with anti-MRP-1 antibody and SLC7A5 protein reacted with anti-SLC7A5 antibody. Every pathological section included 1200 cells. FXR protein expression, MRP-1 protein expression and SLC7A5 protein expression were significant difference among the three groups.
The results of linear regression analysis are presented in Table 4. Using MRP-1 and SLC7A5 as independent variables and FXR as the dependent variable for linear regression analysis, the regression coefficient value of MRP-1 in the experimental group was 0.590 (t = 2.702, P = .019 < .05), indicating that MRP-1 will have a significant impact on FXR. The regression coefficient value of SLC7A5 is 0.165 (t = 0.814, P = .431 > .05), indicating that SLC7A5 does not have an impact on FXR. In the control group, there was no significant correlation between changes in MRP-1, SLC7A5, and FXR. In the sham surgery group, the regression coefficient of MRP-1 was 0.660 (t = 2.995, P = .008 < .01), indicating that MRP-1 has a significant impact on FXR. The regression coefficient of SLC7A5 was 0.491 (t = 2.105, P = .051 > .05), indicating that SLC7A5 does not have an impact on FXR. Summary analysis shows that there is a significant correlation between the changes in MRP-1 and FXR, but the correlation between the changes in SLC7A5 and FXR is not strong.
Table 4.
The Results of Linear Regression Analysis.
Non standardized coefficient | Standardization coefficient | t | P | Collinearity diagnosis | |||
---|---|---|---|---|---|---|---|
B | Standard error | Beta | VIF | Tolerance | |||
constant | −15.490 | 12.688 | - | −1.221 | .246 | - | - |
MRP-1 | 0.590 | 0.218 | 0.600 | 2.702 | .019* | 1.010 | 0.990 |
SLC7A5 | 0.165 | 0.203 | 0.181 | 0.814 | .431 | 1.010 | 0.990 |
R2 | 0.414 | ||||||
Adjustment R2 | 0.317 | ||||||
F | F (2,12) = 4.245,p = 0.040 | ||||||
D-Wvalue | 1.912 |
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A | |||||||
---|---|---|---|---|---|---|---|
Non standardized coefficient | Standardization coefficient | t | P | Collinearity diagnosis | |||
B | Standard error | Beta | VIF | Tolerance | |||
constant | 26.749 | 5.272 | - | 5.074 | .000** | - | - |
MRP-1 | 0.025 | 0.350 | 0.021 | 0.071 | .944 | 2.130 | 0.469 |
SLC7A5 | 0.626 | 0.364 | 0.516 | 1.722 | .103 | 2.130 | 0.469 |
R2 | 0.283 | ||||||
Adjustment R2 | 0.199 | ||||||
F | F (2,17) = 3.355,P = .059 | ||||||
D-Wvalue | 1.733 |
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B | |||||||
---|---|---|---|---|---|---|---|
Non standardized coefficient | Standardization coefficient | t | P | Collinearity diagnosis | |||
B | Standard error | Beta | VIF | Tolerance | |||
constant | 17.065 | 4.024 | - | 4.241 | .001** | - | - |
MRP-1 | 0.660 | 0.220 | 0.531 | 2.995 | .008** | 1.568 | 0.638 |
SLC7A5 | 0.491 | 0.233 | 0.373 | 2.105 | .051 | 1.568 | 0.638 |
R2 | 0.659 | ||||||
Adjustment R2 | 0.619 | ||||||
F | F (2,17) = 16.448,P = .000 | ||||||
D-Wvalue | 1.508 |
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C
A) The results of linear regression analysis in experimental group.
B) The results of linear regression analysis in control group.
C) The results of linear regression analysis in sham operation group.
Dependent variable:FXR, * P < .05 ** P < .01
The results of linear regression analysis shows that there is a significant correlation between the changes in MRP-1 and FXR, but the correlation between the changes in SLC7A5 and FXR is not strong.
Discussion
Primary liver cancer includes HCC and cholangiocellular carcinoma, HCC accounts for the main part. The incidence rate of HCC is high in China and even in Southeast Asia. In the preclinical and subclinical stages of HCC, there are usually no obvious symptoms, while various symptoms and signs are more common in the middle and late stages. Some of the main clinical symptoms include jaundice, vomiting, diarrhea, decreased appetite, weight loss, or liver mass. Some patients have low fever, cachexia, gastrointestinal bleeding, etc After long-term basic research and clinical validation, a feasible treatment method has been established under existing technological conditions. But we know that there are many factors related to HCC, mainly including hepatitis, alcoholism, aflatoxin, immune factors, obesity, etc However, the changes in tumor genes, various cellular and humoral factors in the tumor microenvironment, the mechanisms of tumor invasion and metastasis, and the reasons for tumor escape from drug treatment are still not very clear.9,10 For example, most liver cancer occurs on the basis of cirrhosis. Whether it is hepatitis or alcohol that causes damage to the liver, the basic structure of the liver will undergo changes during the continuous damage and repair of liver cells. The composition of normal liver lobules will continue to disappear, new fibrous tissue will appear, ultimately leading to cirrhosis. After the onset of cirrhosis, as the disease continues to progress, the structural destruction and regeneration of the liver are in a continuous alternating process. In this case, normal liver cells will gradually lose their ability to differentiate and regenerate, requiring stem cells in the liver to differentiate and form new liver cells. During differentiation, stem cells can become normal liver cells under normal circumstances. However, after the normal structure of the liver is disrupted, the normal differentiation of stem cells will be affected. If mutations occur in the early stages of differentiation, it may lead to the appearance of poorly differentiated tumors. If the differentiation level develops to a higher level, it may lead to the appearance of moderately differentiated tumors. However, it is not entirely clear what factors lead to differentiation and variation, as well as their principles and key points of action. This can explain that although current comprehensive treatment methods have shown certain effects, the improvement in patient prognosis is not significant. Usually, surgery is preferred for resectable tumors. If the tumor is limited to a portion of the liver lobe or segment, radical tissue resection can be performed, and the patient's prognosis is good. However, if the tumor invades large blood vessels and perihepatic tissue, accompanied by metastasis to other organs, surgery may not be able to completely remove it, and even surgery may not be considered. However, the effectiveness of chemotherapy, microwave ablation, and ultrasound focusing methods is limited, and the 5-year survival rate has not been significantly improved. Therefore, it is necessary to continuously explore more effective treatment methods. Gene therapy is a new method that has emerged in recent years and has good application prospects. However, currently developed drugs mainly target genes involved in tumor angiogenesis, and there are few directly related to tumor cell growth and proliferation. Therefore, we strive to search for genes related to the pathogenesis of HCC as therapeutic targets in order to achieve more significant therapeutic effects.11–13
FXR is usually expressed on the surface of hepatocytes and bile ducts. It has many functions, especially important in several aspects. For example, it affects tumor growth through specific signal transduction pathways. When FXR is highly expressed, tumor growth is inhibited. On the contrary, when FXR is reduced, tumor growth may continue. Therefore, we found that the expression of FXR in tumor cells of HCC was significantly lower than that in normal liver tissues.14,15 MRP-1 is a member of the ATP-binding cassette transporter superfamily. The MRP gene is located on the p13.1 band of chromosome 16 and consists of 6500 bp. It encodes a transmembrane glycoprotein of 1531 amino acids with a molecular weight of 190 kD. The occurrence of drug resistance is due to the increased activity of the ATP dependent glutathione S conjugate carrier. The output carrier of glutathione S binding, also known as the “GS-X pump,” can excrete anionic conjugates from the cell and play a role in clearing exogenous toxins. MRP-1 can mediate the excretion of glutathione sulfur conjugates, glucuronide glycoside conjugates, and sulfate conjugates from cells in drugs. The steps of MRP-1 transport may be: GSH synthesis → GSH coupling with drugs → MRP-1 pumping drugs out of the cell.16–18 MRP-1 is not only located in plasma cell membrane, but also in endoplasmic reticulum and Golgi follicles, suggesting that MRP-1 can also isolate drugs in cells, so that drugs cannot bind to target sites, which indirectly leads to drug resistance. Previous studies have found that MRP-1 expression increased in tumor tissues (lung cancer, colon cancer, gastric cancer), and the expression of MRP-1 becomed more pronounced as the degree of tumor differentiation decreases. A similar phenomenon was also found in our experiment, where the expression of MRP-1 in liver cancer tissues was significantly higher than that in normal liver tissues. This is also an important reason for the alternating resistance of various chemotherapy drugs and targeted drugs in the treatment of liver cancer. Amino acids are the fundamental components that make up proteins and participate in many important metabolic pathways within cells. Abnormal transport of amino acids can lead to severe amino acid absorption and metabolic disorders. Therefore, the transport of amino acids has important pathological significance. In mammals, as amino acids belong to small polar substances, they cannot freely pass through the cell membrane and must be assisted by corresponding amino acid transporters on the cell membrane. SLC7A5 is an important member of L-type amino acid transporter 1 (LAT1), responsible for transporting large, branched, aromatic amino acids, including some essential amino acids. During the proliferation, migration and invasion of tumor cells, a large amount of amino acids are required to provide nutrients, and the amino acid transporter SLC7A5 provides a carrier channel for the entry and exit of amino acids in tumor cells.19–21 Therefore, amino acid transporters play a crucial role in tumor occurrence and progression. Therefore, in our experimental results, we observed the expression level of SLC7A5 in tumor tissues increased obviously.That is to say, SLC7A5 plays an important role in the growth of liver cancer tissues. As the tumor changes, SLC7A5 will also undergo corresponding changes.
In the experimental results, we found several significant changes. According to existing database information, among HCC with differential expression of FXR,MRP-1 and SLC7A5, the patients have differential prognosis and survival time. Patients with low FXR expression and high expression of MRP-1 and SLC7A5 both showed significant 5-year survival reductions. The risk factors for other functional impairments are increased in patients with low FXR expression and those with high SLC7A5 expression. Therefore, the expression of FXR, MRP-1 and SLC7A5 is closely related to the prognosis of patients. We established animal models to observe the expression of the FXR, MRP-1 and SLC7A5, in preparation for subsequent drug targeted therapy. As the tumor tissues grew, the food intake and weight of rats in experimental group gradually decreased compared to normal rats, while liver function was gradually affected; As FXR in tumor tissues decreases, MRP-1 and SLC7A5 show a significant increase. Previous studies have shown that hepatocytes have membrane polarity and are composed of a basal membrane (blood sinus surface) and a capillary bile duct membrane (intrahepatic bile duct surface) with different biochemical structures and biological functions. Bile salt carriers distributed on the capillary bile duct membrane include bile salt export pump (BSEP) and MRP-1,22–24 which discharge bile acid from hepatocytes into bile ducts. On the contrary the bile salt carriers distributed on the basement membrane include multiple drug resistance associated protein-2 (MRP-2) and histocompatibility transporter α-β(OSTα- OSTβ), which can discharge the newly synthesized and reabsorbed bile acid into venous blood. When FXR decreases, bile acids in liver cells increase, MRP-1 expression is upregulated, and MRP-2 expression decreases. In our experiment, similar changes were also found. The bile acid content of the experimental group rats increased, and the changes in FXR and MRP-1 in tumor cells were opposite. FXR was lower than that of normal liver cells, while MRP-1 was significantly higher. Therefore, there are several issues that have sparked our thinking. Firstly, FXR regulates the expression of the SHP gene to form the FXR-SHP pathway. When regulating the transcription of the SHP gene, FXR can bind to the upstream and downstream IR1 sites of the gene to form a end-to-end transcriptional circular structure. Moreover, the binding strength between FXR and the newly discovered downstream IR1 site is significantly higher than that of the upstream IR1 site. This leads to changes in FXR expression, changes in bile acid content, and gradual tumor formation. MRP-1 is regulated by the MAPK signaling pathway through changes in GSH(Glutathione) status, When GSH and GSSG undergo back and forth changes, the expression of MRP-1 will be significantly affected. FXR can affect the MAPK signaling pathway, leading to changes in MRP-1. Does MRP-1 have a reverse effect on FXR? Secondly, after the increase of MRP-1, in addition to changing the drug resistance of tumors, will it participate in the metabolism of bile acids, affect the degeneration of liver cells, and even the production of new tumors? Thirdly, which drugs may interfere with changes in FXR and MRP-1 expression, further affecting tumor cells? The existing research results on SLC7A5 indicate a phenomenon. When FXR is upregulated by agonists, the content of TGF-β decreases, on the contrary when FXR decreases, the content of TGF-β increases.It may be a regulatory mechanism. Due to TGF-β is the upstream regulator gene of SLC7A5, TGF-β has a similar trend in changes to SLC7A5, so changes in FXR may directly affect the expression of SLC7A5,25,26 which exhibit opposite trends. In our experiment, we observed that with the decrease of FXR in tumor tissues, the expression of SLC7A5 significantly increased compared to normal liver tissues. That is to say, when tumors grow actively, the demand for amino acids increases significantly. Based on the changes in MRP1 and SLC7A5, we have also drawn some conclusions worth considering from the experiment. Firstly, the absence of FXR is an important cause of liver tumor formation, and the changes in corresponding target genes are worth further research. Secondly, with the formation and development of tumors, the components required for tumor cell generation increase, and the demand for amino acids significantly increases. Resistance to various drugs will continue to increase. And, due to the discovery that there is a significant correlation between the changes in MRP-1 and FXR, but the correlation between the changes in SLC7A5 and FXR is not strong.This indicates that the amino acid demand of tumor cells is constantly increasing, but their drug resistance can be regulated. Thirdly, different drugs targeting FXR, MRP-1 and SLC7A5 are currently being tested in animals or have been used in clinical settings. Therefore, further observation and experimentation are needed to determine whether these drugs can overcome tumor resistance and inhibit tumor progression, and whether their use alone or in combination will produce different effects.27,28
Meanwhile, our research also has limitations. Firstly, in our study, we evaluated the changes in FXR, MRP-1 and SLC7A5 in HCC. Although the signaling pathways that cause these changes have been partially understood, their detailed mechanisms need to be further explored. Secondly, our sample size is not large and further observation is needed to determine if there are any biases. Once again, we have discovered changes in FXR, MRP-1 and SLC7A5 in HCC, but whether these changes can be transformed into new therapeutic targets and whether they can be applied in clinical tumor treatment still needs to be verified through more basic research and clinical trials. At present, although some targeted drugs have been used in the treatment of HCC, their main goal is not the growth and proliferation of tumor cells, but the inhibition and destruction of tumor blood vessels. However, long-term use also has various side effects. Some patients may experience gastrointestinal bleeding, rash in various parts of the body, and renal dysfunction. Moreover, current targeted drugs have specific therapeutic sites and may not be widely applicable. More importantly, once the patient cannot tolerate stopping medication, the tumor may rapidly grow and metastasize. The existing research results facilitate our understanding of gene expression changes and related treatment methods in HCC, and these findings can be gradually applied in clinical practice.29,30 But many issues require deeper observation and research. In this study, we achieved several objectives. First, we successfully established a rat model of HCC. Secondly, we observed the expression changes of FXR, MRP-1, and SLC7A5 in normal liver tissues and tumor tissues. This also suggests that we can explore new treatment pathways by improving the therapeutic effects of existing drugs and preventing tumor cell proliferation. Finally, these changes have preliminarily laid the foundation for us to conduct targeted drug experiments. So, this study may help us explore new therapeutic targets for HCC according to the changes in FXR, MRP-1 and SLC7A5. In future experiments, the drugs that can act on FXR, MRP-1 and SLC7A5, as well as their mechanisms of action, will be the focus of our research.
Footnotes
Consent for Publish: All authors have approved the manuscript and agree with publication in Technology in Cancer Research & Treatment
Data Availability: All data generated or analysed during this study are included in this published article.
The authors declared no potential conflicts of interest with the research, authorship, and publication of this article.
Ethics Approval and Informed Consent: The study protocol was approved by the Ethics Committee of Southwest Medical University, Luzhou, Sichuan Province, China. Number:20240108-002. Animal welfare guidelines abided by China Laboratory Animal Welfare Law and Animal management regulations, Number: GB/T 35892-20181.
Funding: This research is supported by Metabolic Hepatobiliary Pancreatic Diseases Key Laboratory of Luzhou City and the Luzhou Municipal Bureau of Science, Technology and Talent Work (grant number 2023JYJ026).
ORCID iD: Meng-yu Zhang https://orcid.org/0000-0001-6409-8543
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