Use of TBRG4 as a drug target in in vitro screening of drugs for treating gallbladder cancer
By inhibiting TBRG4 expression and interfering with the U2AF2/TBRG4/EGFR signaling pathway, the problem of poor treatment effect of gallbladder cancer is solved, the proliferation and invasion of gallbladder cancer cells are effectively inhibited, and a new treatment approach is provided.
Patent Information
- Application Number
- CN202310890259.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-19
- Publication Date
- 2025-09-12
- Estimated Expiration
- 2043-07-19
AI Technical Summary
Existing drugs are not effective in treating gallbladder cancer, and there is a lack of effective molecular targets to improve treatment levels.
By using TBRG4 as a drug target and inhibiting its expression, the U2AF2/TBRG4/EGFR signaling pathway is interfered with, the glycolysis process of gallbladder cancer cells is inhibited, and oxidative phosphorylation is enhanced, thereby inhibiting the proliferation, migration and invasion of cancer cells.
It effectively inhibits the proliferation, migration and invasion of gallbladder cancer cells, providing new molecular mechanisms and clinical application value for the treatment of gallbladder cancer.
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Figure CN116904596B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine and relates to a therapeutic target for gallbladder cancer, specifically the use of TBRG4 as a drug target in in vitro screening of drugs for treating gallbladder cancer. Background Art
[0002] Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, accounting for 80-95% of the overall incidence of biliary malignancies. Due to the insidious onset of gallbladder cancer, low radical resection rate, easy invasion and metastasis, and poor response to chemotherapy and radiotherapy, its overall prognosis is very poor, with a median survival of only 6 months and a 5-year survival rate of only 5-10%. Therefore, gallbladder cancer is also known as the "uncrowned king" of cancer. Compared with the gratifying progress made in the treatment of gastric cancer, colorectal cancer and other cancers in the past decade, the treatment of gallbladder cancer has almost stagnated. Finding the molecules and mechanisms that lead to the progression of gallbladder cancer is an important direction for improving the treatment of gallbladder cancer.
[0003] The FASTK family of proteins is involved in mitochondrial RNA processing required for oxidative phosphorylation. Transforming Growth Factor Beta Regulator 4 (TBRG4) has been found to be involved in mitochondrial energy regulation during cell proliferation and can promote tumor progression. Knockout of TBRG4 can inhibit cell proliferation and promote apoptosis by reducing TGF-β1 expression and inhibiting the PI3K / AKT signaling pathway. However, whether TBRG4 has a similar effect in gallbladder cancer is unknown. Therefore, we need to study the molecular mechanism of TBRG4 in gallbladder cancer and propose a therapeutic target for TBRG4 in gallbladder cancer.
[0004] It is well known that glycolysis is a key factor in the progression of various cancers, including gallbladder cancer. In the hypoxic tumor microenvironment, the shift from oxidative phosphorylation to glycolysis can promote epithelial-mesenchymal transition and tumor metastasis. Therefore, inhibiting glycolysis can inhibit cancer cell proliferation and other biological functions, promoting a metabolic shift from aerobic glycolysis to oxidative phosphorylation. Genes involved in glycolysis and its related pathways have become important targets for therapeutic investigation. Summary of the Invention
[0005] In response to the above technical problems in the prior art, the present invention provides the use of TBRG4 as a drug target in in vitro screening of drugs for treating gallbladder cancer. This use is intended to solve the technical problem that the drugs in the prior art are not effective in treating gallbladder cancer.
[0006] The present invention provides use of TBRG4 as a drug target in in vitro screening of drugs for treating gallbladder cancer, wherein the drug is a drug that inhibits TBRG4 expression.
[0007] The present invention also provides use of a reagent for detecting TBRG4 in preparing a kit for diagnosing gallbladder cancer.
[0008] In vitro and in vivo experiments confirmed that knocking out TBRG4 significantly inhibited the proliferation, invasion, migration, and aerobic glycolysis of gallbladder cancer cells, while overexpressing TBRG4 had the opposite effect. Further experiments confirmed that the U2AF2 / TBRG4 / EGFR signaling pathway activates the PI3K / AKT / mTOR signaling pathway, inhibiting glycolysis in tumor cells.
[0009] The present invention discovered that inhibiting TBRG4 can suppress glycolysis and enhance oxidative phosphorylation in gallbladder cancer, thereby inhibiting the development, invasion, and metastasis of gallbladder cancer. Knocking out TBRG4 inhibited the proliferation, migration, and invasion of gallbladder cancer cells in vitro and in vivo. Knocking out TBRG4 also inhibited glycolysis and enhanced oxidative phosphorylation in gallbladder cancer cells.
[0010] The present invention discovered that TBRG4 mRNA can be directly bound by U2AF2, and that the binding can enhance the stability of TBRG4 and upregulate the expression of TBRG4.
[0011] The present invention found that TBRG4 activates the PI3K / AKT / mTOR signaling pathway by binding to EGFR and upregulating its expression.
[0012] In summary, TBRG4 is bound by U2AF2 upstream, affecting its stability, and binds to EGFR downstream, activating the PI3K / AKT / mTOR signaling pathway to inhibit glycolysis in tumor cells, thereby suppressing the development, invasion, and metastasis of gallbladder cancer. TBRG4 plays a key role in the development and glycolysis of gallbladder cancer, and is an important factor in regulating the development, invasion, and metastasis of gallbladder cancer. It is expected to become a potential target for anti-tumor therapy in the treatment of gallbladder cancer.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] This invention is the first to demonstrate in detail the specific molecular mechanism by which the U2AF2 / TBRG4 / EGFR signaling pathway inhibits glycolysis in gallbladder cancer. TBRG4 may serve as a potential target for the anti-tumor treatment of gallbladder cancer. This invention provides a theoretical basis and clinical application value for subsequent research on therapeutic targets for gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 A shows the expression of TBRG4 in cholangiocarcinoma tissue samples from the TCGA public database.
[0016] Figure 1 B shows that the expression of TBRG4 in gallbladder cancer tissues was significantly higher than that in adjacent adjacent tissues (n=20).
[0017] Figure 1 C shows that TBRG4 is upregulated in gallbladder cancer tissues as shown in representative images of immunohistochemical analysis.
[0018] Figure 1 D shows that TBRG4 mRNA expression levels were upregulated in gallbladder cancer cells compared with normal intrahepatic bile duct epithelial cells (HIBEpiC).
[0019] Figure 1 E shows that TBRG4 protein expression levels are upregulated in gallbladder cancer cells compared with normal intrahepatic bile duct epithelial cells (HIBEpiC).
[0020] Figure 1 F shows the interference and overexpression efficiency of TBRG4 in GBC-SD and SGC-996 cells detected by immunoblotting and qPCR methods.
[0021] Figure 2 A shows a CCK-8 assay confirming that TBRG4 knockdown inhibits the proliferation of gallbladder cancer cells GBC-SD, while overexpression promotes proliferation. This assay was used to examine the migration and invasion abilities of GBC-SD and SGC-996 cells.
[0022] Figure 2 B shows a cell scratch assay confirming that knockdown of TBRG4 inhibits the migration and invasion of gallbladder cancer cells GBC-SD, while overexpression of TBRG4 promotes migration and invasion.
[0023] Figure 2 C shows Transwell assays confirming that knockdown of TBRG4 inhibits the migration and invasion of gallbladder cancer cells GBC-SD, while overexpression of TBRG4 promotes migration and invasion.
[0024] Figure 2 D shows a nude mouse subcutaneous transplant tumor model constructed using a gallbladder cancer cell line. Experiments confirmed that knocking down TBRG4 can inhibit the size and volume of the transplant tumor, while overexpression promotes the size and volume of the transplant tumor.
[0025] Figure 2 E shows that the tumor morphology and TBRG4 expression were confirmed by immunohistochemical analysis of the transplanted tumor tissue.
[0026] Figure 3 A shows the oxygen consumption rates of gallbladder cancer cells GBC-SD with TBRG4 knockdown and the control group, and gallbladder cancer cells SGC-996 with TBRG4 overexpression and the control group.
[0027] Figure 3 B shows the extracellular acidification rate of gallbladder cancer cells GBC-SD with TBRG4 knockdown and control group, and gallbladder cancer cells SGC-996 with TBRG4 overexpression and control group.
[0028] Figure 3 C shows the glucose uptake of gallbladder cancer cells GBC-SD with TBRG4 knockdown and the control group, and gallbladder cancer cells SGC-996 with TBRG4 overexpression and the control group.
[0029] Figure 3 D shows the lactate production in gallbladder cancer cells GBC-SD with TBRG4 knockdown and the control group, and gallbladder cancer cells SGC-996 with TBRG4 overexpression and the control group.
[0030] Figure 3 E shows the expression levels of rate-limiting glycolytic enzymes (PDK1, HK-2, LDHA, GLUT1) in GBC-SD and SGC-996 cells.
[0031] Figure 4 A shows the expression of TBRG4 in the nucleus and cytoplasm of GBC-SD and SGC-996 cells detected by nuclear-cytoplasmic fractionation (U6 was used as a nuclear control, and GAPDH was used as a cytoplasmic control).
[0032] Figure 4 B shows the results of RIP detection, indicating that U2AF2 can bind to TBRG4 mRNA.
[0033] Figure 4 C shows the attenuation of TBRG4 mRNA in gallbladder cancer cells GBC-SD transfected with si-U2AF2. After U2AF2 downregulation, the half-life of TBRG4 mRNA decreased.
[0034] Figure 4 D shows the expression of U2AF2 and its correlation with TBRG4 in cholangiocarcinoma tissues analyzed through the TCGA public database.
[0035] Figure 4 E shows the expression of U2AF2 in GBC-SD and SGC-996 cells detected by immunoblotting and qPCR.
[0036] Figure 4 F shows the knockdown efficiency of U2AF2 in GBC-SD cells detected by immunoblotting and qPCR.
[0037] Figure 4G shows the protein expression of TBRG4 in GBC-SD cells transfected with sh-NC / sh-U2AF2 detected by immunoblotting.
[0038] Figure 4 H shows the mRNA expression of TBRG4 in GBC-SD cells transfected with sh-NC / sh-U2AF2 detected by qPCR.
[0039] Figure 5 A shows the oxygen consumption rate of gallbladder cancer cells GBC-SD with U2AF2 knockdown, TBRG4 overexpression, and both U2AF2 knockdown and TBRG4 overexpression.
[0040] Figure 5 B shows the determination of the extracellular acidification rate of gallbladder cancer cells GBC-SD with U2AF2 knockdown, TBRG4 overexpression, and U2AF2 knockdown and TBRG4 overexpression.
[0041] Figure 5 C shows the glucose uptake in gallbladder cancer cells GBC-SD with U2AF2 knockdown, TBRG4 overexpression, and both U2AF2 knockdown and TBRG4 overexpression.
[0042] Figure 5 D shows the lactate production in gallbladder cancer cells GBC-SD with U2AF2 knockdown, TBRG4 overexpression, and both U2AF2 knockdown and TBRG4 overexpression.
[0043] Figure 5 E shows the expression levels of rate-limiting glycolytic enzymes (PDK1, HK-2, LDHA, GLUT1) in various GBC-SD cells differentially expressing U2AF2 and TBRG4.
[0044] Figure 6 A shows the mRNA expression of EGFR in GBC-SD cells transfected with sh-NC / sh-TBRG4 detected by qPCR.
[0045] Figure 6 B shows the protein expression of EGFR in GBC-SD cells transfected with sh-NC / sh-TBRG4 detected by immunoblotting.
[0046] Figure 6 C shows the correlation between TBRG4 and EGFR detected by co-immunoprecipitation method.
[0047] Figure 6D shows the protein expression of key proteins (PI3K, AKT, mTOR) in the PI3K / AKT pathway in GBC-SD cells transfected with sh-NC / sh-TBRG4, as determined by immunoblotting. DETAILED DESCRIPTION
[0048] Example 1: TBRG4 is highly expressed in gallbladder cancer tissues and cells
[0049] We first evaluated the expression of TBRG4 in cholangiocarcinoma tissue samples from the TGCA public database and found that it was highly expressed in tumor tissues ( Figure 1 A). Therefore, we subsequently detected the mRNA expression level of TBRG4 in 20 pairs of gallbladder cancer and adjacent tissue samples obtained clinically and found that it was highly expressed in gallbladder cancer tissue ( Figure 1 B). Immunohistochemical examinations also revealed that epithelial cells in adjacent tissues were well differentiated, while gallbladder cancer cells were mostly in mitosis and undifferentiated ( Figure 1 C). Compared with human normal intrahepatic bile duct epithelial cells (HIBEpiC), TBRG4 was upregulated in gallbladder cancer cell lines GBC-SD, SGC-996, and NOZ ( Figure 1 D, E), and then we used expression vectors and RNA interference to construct overexpression and knockdown models in gallbladder cancer cells ( Figure 1 F) These results indicate that TBRG4 is not only upregulated in cholangiocarcinoma, but also highly expressed in gallbladder cancer.
[0050] The primers used for quantitative PCR are as follows:
[0051] TBRG4
[0052] Forward: 5′-GTGCTCGCTGCGACATCA-3′.
[0053] Reverse: 5′-GGAGTGGGAAATGGGAG-3′.
[0054] Overexpression and knockdown cell models were constructed using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) to transfect 293T cells with the shRNA sequence or expression vector along with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G. 48 hours after transfection, the virus was retrieved using a 0.45 mm filter. GBC-SD and SGC-966 cells were infected with 1 × 10⁶ lentiviral transfection units in the presence of polyethylene glycol (6 μg / mL; Sigma-Aldrich, St Louis, MO, USA). The shRNA sequences are as follows:
[0055] Human TBRG4 shRNA-1#:
[0056] 5′-ACCGGTCAAGCAGCAATGGTACTTATCTCGAG ATAAGTACCATTGCTGCTTGATTTTT-3′
[0057] Human TBRG4 shRNA-2#:
[0058] 5′-ACCGGGAGCATCTACTCCCTACATAGCTCGAG CTATGTAGGGAGTAGATGCTTTTT-3′
[0059] Example 2: Effects of TBRG4 on the biological functions of gallbladder cancer cells in vivo and in vitro
[0060] We used CCK-8 assay to determine that knockout of TBRG4 could inhibit the proliferation of gallbladder cancer cells GBC-SD, while overexpression of TBRG4 promoted the proliferation of gallbladder cancer cells SGC-996 ( Figure 2 A). TBRG4 is also involved in the migration and invasion of GBC cells. Through scratch and Transwell assays, it was found that knocking down TBRG4 can reduce the migration and invasion levels of gallbladder cancer cells GBC-SD, while overexpression of TBRG4 in gallbladder cancer cells SGC-996 promotes the migration and invasion characteristics of cells ( Figure 2 B, C).
[0061] Subsequently, through an in vivo mouse subcutaneous transplant tumor model, it was found that in the GBC-SD cell mouse model transfected with sh-TBRG4, the subcutaneous transplant tumors were significantly smaller than those in the mouse model derived from gallbladder cancer cell SGC-996 overexpressing TBRG4 ( Figure 2 D). Meanwhile, the tumor morphology and TBRG4 expression were determined by immunohistochemistry ( Figure 2 E).
[0062] The above results indicate that knocking down TBRG4 can inhibit the proliferation, migration and invasion of gallbladder cancer in vitro and inhibit tumor growth in vivo.
[0063] Example 3: Knockdown of TBRG4 inhibits glycolysis in gallbladder cancer cells
[0064] Next, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in gallbladder cancer cells transfected with sh-TBRG4 or pcDNA-TBRG4. We found that knockdown of TBRG4 inhibited aerobic glycolysis in gallbladder cancer cells, while overexpression of TBRG4 promoted glycolysis ( Figure 3 A, B). In GBC-SD gallbladder cancer cells where TBRG4 expression is suppressed, relative glucose uptake and lactate production are relatively low. However, in SGC-996 cells where TBRG4 expression is promoted, glucose uptake and lactate production are significantly increased ( Figure 3 C, D). To confirm the results of the OCR and ECAR assays, we measured the levels of rate-limiting glycolytic enzymes, including PDK1, HK-2, LDHA, and GLUT1. The protein levels of glycolytic enzymes in gallbladder cancer cells with TBRG4 inhibition were significantly lower than those in the control group, while the expression levels of glycolytic enzymes in cells overexpressing TBRG4 were significantly increased ( Figure 3 E) These results suggest that TBRG4 expression can affect pathways regulating glycolysis in gallbladder cancer.
[0065] Example 4: U2AF2 directly binds to TBRG4 mRNA, enhancing its stability and promoting expression
[0066] The relative expression level of TBRG4 in the cytoplasm was higher than that in the nucleus ( Figure 4 A), we found that U2AF2 interacts with TBRG4 mRNA through RIP assay ( Figure 4 B). After knocking down U2AF2 expression in gallbladder cancer cells GBC-SD transfected with si-U2AF2, we found that the half-life of TBRG4 mRNA was reduced ( Figure 4 C), indicating that U2AF2 enhances the stability of TBRG4mRNA. We then evaluated the cholangiocarcinoma tissue samples in the TCGA public database and found that the expression of U2AF2 in cholangiocarcinoma tissue was increased compared with adjacent adjacent tissues ( Figure 4 D), and the expression of U2AF2 and TBRG4 in cancer tissues was significantly correlated (P<0.001, R=0.56). Similarly, the expression of U2AF2 was relatively upregulated in gallbladder cancer cells, and after knocking down the expression of U2AF2, the expression of TBRG4 was also downregulated in gallbladder cancer cells ( Figure 4 EH). These results suggest that U2AF2 can directly bind to TBRG4 mRNA and enhance its stability. U2AF2 expression can affect TBRG4 mRNA expression and protein levels in gallbladder cancer cells.
[0067] The primers used in the quantitative PCR experiment are:
[0068] U2AF2
[0069] Forward:5′-AAGAATGCCACGCTGAGCAC-3′
[0070] Reverse:5′-GTCCTCCAGATCTCCTCATAC-3′
[0071] The shRNA used to construct the knockdown cell model is:
[0072] Human U2AF2 shRNA:
[0073] 5′-ACCGGGGTAGGAACATAGCGTGTTTACTCGAG TAAACACGCTATGTTCCTACCTTTTT-3′
[0074] Example 5: U2AF2 regulates glycolysis through TBRG4 in gallbladder cancer cells
[0075] We went on to measure the OCR and ECAR in gallbladder cancer cells transfected with sh-NC / sh-U2AF2 and / or pcDNA / pcDNA-TBRG4. When U2AF2 was underexpressed, OCR increased. Overexpression of TBRG4 reduced the increase, but the level was still higher than that of control cells. OCR was lowest in cells overexpressing TBRG4. A similar pattern occurred with ECAR. When U2AF2 was inhibited, ECAR decreased, while overexpression of TBRG4 increased ECAR, but its level was still lower than that of control cells. ECAR was highest when TBRG4 was overexpressed ( Figure 5 A, B). Glucose uptake and lactate production were measured in GBC-SD gallbladder cancer cells. It was found that when TBRG4 was overexpressed, glucose uptake and lactate production were the highest. In cells overexpressing TBRG4, interference with U2AF2 reduced glucose uptake and lactate production. In GBC cells where U2AF2 expression was suppressed, glucose uptake and lactate production were the lowest ( Figure 5 C, D). We also measured the levels of rate-limiting glycolytic enzymes, including PDK1, HK-2, LDHA, and GLUT1. When TBRG4 was overexpressed, the levels of glycolytic enzymes were highest, while interference with U2AF2 reduced the levels of glycolytic enzymes ( Figure 5 E) These results indicate that U2AF2 regulates glycolysis through TBRG4 in gallbladder cancer cells.
[0076] Example 6: Knockdown of TBRG4 inhibits EGFR expression and activation of the PI3K / AKT pathway
[0077] To investigate whether TBRG4 regulates the PI3K / AKT pathway in gallbladder cancer cells, we determined the expression of EGFR in gallbladder cancer cells GBC-SD transfected with sh-NC / sh-TBRG4-1 by qPCR and immunoblotting. We found that knockdown of TBRG4 could inhibit the expression levels of EGFR mRNA and protein ( Figure 6 A, B). Co-IP experiments confirmed that TBRG4 directly binds to EGFR ( Figure 6 C). In addition, the expression of key proteins in the PI3K / AKT pathway (including PI3K, AKT, and mTOR) was also reduced in gallbladder cancer cells with TBRG4 knockdown ( Figure 6 D) In summary, TBRG4 directly binds to EGFR, regulating EGFR expression and the PI3K / AKT pathway in gallbladder cancer cells. Knockdown of TBRG4 inhibits the PI3K-AKT pathway. Therefore, upregulation of TBRG4 activates the PI3K-AKT pathway and contributes to the progression of gallbladder cancer.
[0078] The primers used in the quantitative PCR experiment are:
[0079] EGFR Forward:5′-GCATTGATAGAAATGGGCTGCAA-3′
[0080] Reverse: 5′-AGTGGGTCGAGGAGGTTGAG-3′.
[0081] In summary, the research results of this study found that inhibiting TBRG4 can significantly suppress the proliferation, migration, and invasion of gallbladder cancer cells, suggesting that TBRG4 is an important regulatory molecule affecting the progression of gallbladder cancer. Further research showed that the U2AF2 / TBRG4 / EGFR signaling pathway can regulate the glycolysis process of gallbladder cancer cells through the PI3K / AKT pathway. Inhibiting TBRG4 can significantly inhibit glycolysis in gallbladder cancer cells, thus confirming that TBRG4 is an important regulatory molecule for energy metabolism and opening up a new therapeutic approach for the treatment of gallbladder cancer.
Claims
1. Use of TBRG4 as a drug target in in vitro screening of drugs for treating gallbladder cancer, wherein the drug is a drug that inhibits TBRG4 expression.
2. Application of a reagent for detecting TBRG4 in the preparation of a kit for diagnosing gallbladder cancer.