Application of GMFB protein as a biological marker for renal clear cell carcinoma

Abstract

The present invention relates to the use of GMFB protein as a biological marker for renal clear cell carcinoma. The present invention analyzed the expression level of GMFB in the KIRC patient population in the TCGA database and clarified that GMFB is associated with the pathogenesis of KIRC. Next, the prognosis of KIRC patients with different GMFB expression levels was analyzed, and it was found that the expression level of GMFB was significantly correlated with the prognosis of KIRC patients. Finally, CCK8 experiments confirmed that knocking down GMFB can significantly increase the viability of renal clear cell carcinoma cells, while overexpressing GMFB can significantly inhibit the viability, proliferation, migration and invasion ability of renal clear cell carcinoma cells, and significantly inhibit the mitochondrial membrane potential of renal clear cell carcinoma cells. The present invention provides a new therapeutic target for renal clear cell carcinoma.

Description

Technical Field

[0001] The present invention relates to a use of protein GMFB, and in particular to an application of GMFB protein as a biological marker of renal clear cell carcinoma. Background Art

[0002] Kidney cancer is one of the ten most common cancers worldwide. An estimated 300,000 people worldwide develop kidney cancer each year, with approximately half of these cases dying from the disease. Renal clear cell carcinoma (KIRC), originating from the epithelial cells of the proximal convoluted tubules of the kidney, has become one of the fastest-growing areas of oncology, accounting for approximately 75% of kidney cancer cases. KIRC is the most common subtype, characterized by its high invasiveness and metastatic rate, the highest mortality rate, and the highest incidence of immune and vascular invasion. Due to its rich vascularity, KIRC can metastasize to the lungs and bones at an early stage, spreading directly or via lymph nodes. Clinically, surgical treatment is the primary approach, but treatment varies significantly depending on the tumor's TNM stage. KIRC patients can be identified using abdominal imaging, but these tumors are insensitive to chemoradiation and chemotherapy, and the five-year survival rate for advanced patients is less than 10%. Some studies have shown that 20%-40% of patients experience local or distant recurrence after nephrectomy. The molecular mechanisms involved in the pathogenesis of KIRC are diverse, including direct changes in chromatin conformation, angiogenesis, and glucose metabolism. In the past few years, many studies have focused on oncogenes in renal cell carcinoma, leading to the discovery of recurrent alterations in KIRC. Therefore, it is necessary to explore the underlying molecular mechanisms of KIRC and gain a deeper understanding of KIRC.

[0003] GMFB (glia maturation factor beta) is a glial maturation factor with an actin depolymerizing factor homology (ADF-H) domain. It is highly conserved throughout evolution from yeast to mammals. This protein is ubiquitously expressed but primarily enriched in brain tissue, where it participates in various biological activities, including chemotaxis, endocytosis, and cell adhesion. Currently, no studies have linked GMFB to renal cancer. Summary of the Invention

[0004] In order to find markers closely related to renal clear cell carcinoma and provide clinical reference for early diagnosis, grading and staging prediction, and prognosis judgment of renal clear cell carcinoma, the present invention provides an application of GMFB gene and protein as biological markers of renal clear cell carcinoma.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The present invention provides application of the GMFB gene as a biological marker for renal clear cell carcinoma.

[0007] The present invention also provides the use of GMFB protein as a biological marker for renal clear cell carcinoma.

[0008] The present invention also provides the use of the GMFB gene as a biomarker in preparing a reagent / kit for diagnosing or progressing renal clear cell carcinoma.

[0009] The present invention also provides the use of GMFB protein as a biomarker in preparing a reagent / kit for diagnosing renal clear cell carcinoma or for disease progression.

[0010] The present invention also provides the use of the GMFB gene as a biomarker in the preparation of a drug for preventing or treating renal clear cell carcinoma.

[0011] The present invention also provides the use of GMFB protein as a biomarker in the preparation of drugs for preventing or treating renal clear cell carcinoma.

[0012] The present invention also provides use of a substance that promotes GMFB gene expression in preparing a drug for preventing and / or treating renal clear cell carcinoma.

[0013] The present invention also provides use of a substance that increases the content or activity of GMFB in preparing a drug for preventing and / or treating renal clear cell carcinoma.

[0014] The present invention also provides use of a reagent for detecting the expression level of GMFB protein in preparing a reagent / kit for diagnosing renal clear cell carcinoma or for the progression of the disease.

[0015] In order to study the application of GMFB as a biological marker in KIRC, the present invention analyzed the expression level of GMFB in the KIRC patient population in the TCGA database and clarified that GMFB is associated with the pathogenesis of KIRC. Next, the prognosis of KIRC patients with different GMFB expression levels was analyzed. It was found that the expression level of GMFB was significantly correlated with the prognosis of KIRC patients. Finally, CCK8, Transwell migration and proliferation, and scratch experiments confirmed that knocking down GMFB can significantly increase the viability of renal clear cell carcinoma cells, while overexpressing GMFB significantly inhibited the migration, invasion and proliferation ability of renal clear cell carcinoma cells, and significantly reduced mitochondrial membrane potential. The present invention provides a new therapeutic target for renal clear cell carcinoma.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] 1) The present study found that GMFB is a differentially expressed gene in renal clear cell carcinoma, and its expression level can be used as an evaluation indicator for renal clear cell carcinoma.

[0018] 2) The present study found that the expression level of GMFB can be used as an indicator of patient prognosis.

[0019] 3 The present study found that knocking down GMFB can significantly increase the proliferation ability of human renal clear cell carcinoma cell Caki-2 cells. Overexpression of GMFB in Caki-2 inhibits the proliferation, migration and invasion of Caki-2 and reduces mitochondrial membrane potential, suggesting that GMFB can become a therapeutic target for renal clear cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1. GMFB mRNA and protein levels are lowly expressed in renal clear cell carcinoma;

[0021] FIG1 includes FIG1(A) and FIG1(B);

[0022] Figure 2 Using human embryonic kidney 293T cells as a normal control, the protein and mRNA levels of GMFB in renal clear cell carcinoma epithelial cells Caki-2 were significantly decreased;

[0023] Figure 3. The overall survival (OS) and disease-specific survival (DFS) of patients with clear cell renal cell carcinoma with high GMFB expression were significantly higher than those with low GMFB expression (OS: P = 0.00018; DFS: P = 0.00013). Therefore, GMFB is significantly correlated with the prognosis of patients with clear cell renal cell carcinoma and can be used as a prognostic marker for clear cell renal cell carcinoma.

[0024] FIG3 includes FIG3(A) and FIG3(B);

[0025] Figure 4. Detection of overexpression and knockdown efficiency in Caki-2 cells. GMFB knockdown and overexpression were confirmed at the protein and mRNA levels.

[0026] FIG4 includes FIG4(A) and FIG4(B);

[0027] Figure 5 .In Caki-2 cells, knockdown of GMFB significantly enhanced cell viability, while overexpression of GMFB significantly reduced cell viability, confirming that GMFB is a potential target for the treatment of renal clear cell carcinoma.

[0028] Figure 6 .Transwell experiments confirmed that the migration and invasion abilities of Caki-2 cells overexpressing GMFB were significantly reduced.

[0029] Figure 7 .Scratch experiments confirmed that the proliferation ability of Caki-2 cells overexpressing GMFB was significantly reduced.

[0030] Figure 8 .JC-1 staining results showed that the membrane potential of Caki-2 cells overexpressing GMFB was significantly reduced. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1

[0033] Analysis of GMFB protein expression in the TCGA database revealed low GMFB expression in clear cell renal cell carcinoma. Statistical analysis of clear cell renal cell carcinoma patient cohorts revealed statistically significant differences, suggesting that GMFB mRNA expression levels can be used as a diagnostic marker for clear cell renal cell carcinoma.

[0034] In this example, human embryonic kidney 293T cells were used as a normal control to measure GMFB mRNA and protein levels in 293T cells and Caki-2 cells, a clear renal cell carcinoma. 293T cells and Caki-2 cells were cultured in DMEM / HG and DMEM / F12 supplemented with 10% serum and 1% penicillin / streptomycin, respectively. They were cultured at 37°C, 5% CO2, and 95% air. GMFB protein levels were assessed by Western blot: cells were washed 1-2 times with PBS buffer, then 200 μl of RIPA lysis buffer was added and lysed on ice for 30 hours, vortexing for a few seconds every ten minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C. Protein was quantified using BCA. SDS-PAGE was performed with 30 μg / well sample loaded. Electrophoresis was performed at 90 V for 30 minutes, followed by 120 V to the bottom. Protein was transferred to a PVDF membrane at 300 mA for 2 hours. Rabbit GMFB antibody and anti-rabbit HRP-conjugated secondary antibody were used to detect the corresponding protein expression, with actin as an internal control. GMFB mRNA levels were detected by RT-qPCR: Wash 1-2 times with PBS buffer, add 1 ml of Trizol lysis buffer, and add 200 μl of chloroform (trichloromethane) so that the lower layer is transparent and the upper layer is red. Mix by inverting and incubating at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes, so that the upper layer is transparent and the lower layer is red. Transfer 400 μl of the upper aqueous phase to a new EP tube, add an equal amount of isopropanol, and add 1 μl of glycogen to aid RNA precipitation. Mix well and incubate at room temperature for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Discard the supernatant, blot with absorbent paper to remove excess liquid, and add 75% DEPC ethanol to the same amount as the Trizol. Centrifuge at 12000rpm for 5 minutes at 4℃, discard the supernatant, absorb the excess liquid with absorbent paper, and air dry for 5 minutes. Add 20μl ddH2O and measure the RNA concentration by Nandrop. 10 μl of cDNA was reverse transcribed using the RT Master Mix. The reverse transcription system consisted of 2 μl of 5× RT Master Mix, 1 μg of RNA, and ddH₂O to 10 μl. Reverse PCR conditions were: 37°C for 15 min; 85°C for 5 s; and storage at 4°C. The cDNA sample was diluted tenfold before reverse transcription. The Q-PCR reaction kit used Tiangen's SuperRealPreMix Plus (SYBR Green) and was detected using the Bio-Rad CFX Manager 2.1 Detection System. GAPDH was used as an internal control, and data were processed using the 2^(-ΔΔCT) method. The Q-PCR reaction system consisted of 2 μl of DNA sample, 0.5 μl of each 10 μM forward and reverse primer, 10 μl of 2× SuperRealPreMix, and ddH₂O to 20 μl. The reaction conditions were: 95°C for 5 min, 95°C for 30 s, and 60°C for 60 s, for 40 cycles.

[0035] Refer to Figure 1 and Figure 2 The results showed that compared with 293T cells, the mRNA and protein levels of GMFB in Caki-2 cells were significantly reduced, and this experimental result was consistent with the results of bioinformatics analysis.

[0036] The prognosis of patients with clear cell renal cell carcinoma at different GMFB expression levels in the TCGA database was then analyzed. Referring to Figure 3, the overall survival (OS) and disease survival (DFS) of patients with clear cell renal cell carcinoma with high GMFB expression were significantly higher than those with low GMFB expression, and the results were statistically significant (OS: P = 0.00018; DFS: P = 0.00013). Therefore, GMFB is significantly correlated with the prognosis of patients with clear cell renal cell carcinoma and can be used as a prognostic marker for clear cell renal cell carcinoma. It was found that the expression level of GMFB is significantly correlated with the prognosis of patients with clear cell renal cell carcinoma.

[0037] Western Blot and RT-qPCR were used to detect the efficiency of overexpression and knockdown of GMFB in Caki-2 cells, using the same experimental methods as above.

[0038] The CCK8 kit was used to detect the proliferation of Caki-2 cells with GMFB knockdown and overexpression: 2000 cells were added to each well of a 96-well plate in 100 μL format, with triplicate wells per group. At 12, 24, and 48 hours after transfection, 10 μL of CCK-8 solution was added to each well. The cells were incubated in a cell culture incubator for another hour, and the absorbance was measured at 450 nm. Figure 4 shows the efficiency of detecting overexpression and knockdown in Caki-2 cells, confirming GMFB knockdown and overexpression at both protein and mRNA levels. Figure 5The study showed that knocking down GMFB significantly enhanced cell proliferation in Caki-2 cells, while overexpressing GMFB significantly reduced cell proliferation, confirming that GMFB is a potential target for the treatment of clear cell renal cell carcinoma. The results showed that 24 and 48 hours after transfection, the viability of Caki-2 cells with GMFB knockdown was significantly enhanced, while the viability of Caki-2 cells with GMFB overexpression was significantly decreased.

[0039] A Transwell assay was used to examine the migration and proliferation of GMFB-overexpressing Caki-2 cells. Cells were digested with 0.25% trypsin / EDTA, the pellet collected after centrifugation, and resuspended in DMEM / F12 medium supplemented with 0.5% FBS. The cell suspension concentration was adjusted to 5 x 10 cells / ml. Transwell inserts with 8 μm pores were placed in 24-well plates. 100 μL of the cell suspension was inoculated into the upper chamber of the Transwell, and 500 μL of complete culture medium was added to the lower chamber. Three replicates were set up for each well. The 24-well plates were then incubated in a 37°C, 5% CO2 incubator for 24 hours. The inserts were then removed and the upper chamber liquid discarded. Any cells that had not crossed the membrane were wiped clean from the upper chamber membrane surface with a cotton swab. The cells were fixed with methanol for 10 minutes at room temperature, washed three times with PBS, and stained with crystal violet for 30 minutes at room temperature. Under a 200x optical microscope, cells were counted in five random fields and the average value was used to represent the migration ability of RPE cells. For cell migration assays, Matrigel was not required in the Transwell chamber; for cell invasion assays, Matrigel was required in the Transwell chamber.

[0040] Experimental results reference Figure 6 , showing that the migration and invasion of Caki-2 cells overexpressing GMFB were significantly reduced.

[0041] Migration of GMFB-overexpressing Caki-2 cells was assessed using a wound wound assay. 48 hours after cell transfection, a 200-μl pipette tip was used to create a scratch in the center of each cell monolayer with uniform force. The bottom of the culture plate was marked with a marker. The plates were washed three times with PBS and replaced with serum-free basal medium. After 24 hours, the wound healing was observed under a microscope.

[0042] Experimental results reference Figure 7 , showing that the wound healing rate of Caki-2 cells overexpressing GMFB is reduced.

[0043] The JC-1 staining kit was used to detect changes in mitochondrial membrane potential in Caki-2 cells overexpressing GMFB. 48 hours after cell transfection, aspirate the culture medium, wash the cells once with PBS, add 1 ml of cell culture medium (optionally containing serum), add 1 ml of JC-1 staining buffer, and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes. After the 37°C incubation, aspirate the supernatant, wash twice with JC-1 staining buffer, and add 2 ml of cell culture medium (optionally containing serum). Observe under a fluorescence microscope.

[0044] Experimental results reference Figure 8 , showing that the mitochondrial membrane potential of Caki-2 cells overexpressing GMFB was reduced.

[0045] The present invention found that GMFB is expressed at low levels at both RNA and protein levels in patients with clear cell renal cell carcinoma. Furthermore, patients with clear cell renal cell carcinoma who had high GMFB expression had significantly better prognosis than those with low GMFB expression. Finally, we found that reducing GMFB gene expression in clear cell renal cell carcinoma cells significantly enhanced the proliferation of the cancer cells, while overexpressing GMFB significantly inhibited the migration, invasion, and proliferation of clear cell renal cell carcinoma cells and significantly reduced mitochondrial membrane potential. This suggests that increasing GMFB expression is a potential therapeutic target for clear cell renal cell carcinoma.

[0046] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. Use of the GMFB gene in the preparation of drugs for preventing or treating renal clear cell carcinoma.

2. Application of GMFB protein in the preparation of drugs for preventing or treating renal clear cell carcinoma.

3. Use of a reagent for detecting GMFB mRNA or protein expression in the preparation of a reagent / kit for the diagnosis or prognosis of renal clear cell carcinoma.

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