Application of SMIM26 protein as a clinical biomarker for renal cancer

Abstract

The present invention discloses the use of the SMIM26 protein as a clinical biomarker for renal cancer. Immunohistochemistry was used to detect SMIM26 protein expression in pan-cancer tissues and adjacent para-cancer samples. The findings revealed significant differential expression of SMIM26 protein between renal tissue and renal cancer tissue, with low expression in clear cell renal cell carcinoma and high expression in adjacent para-cancer tissue. Furthermore, SMIM26 protein expression correlated with survival in renal cancer patients. Therefore, SMIM26 protein can be used as a clinical diagnostic and prognostic biomarker for renal cancer. Furthermore, renal cancer patients with high SMIM26 protein expression have longer survival times, suggesting that SMIM26 may also be used in renal cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of SMIM26 protein as a clinical biomarker for renal cancer. Background Art

[0002] Renal cell carcinoma (RCC) is a malignant tumor that arises from renal tubular epithelial cells and is the most common malignant kidney tumor. Early diagnosis of RCC is difficult, and most RCC patients are already in the mid-to-late stage at the time of diagnosis, severely impacting treatment outcomes. Due to the lack of effective early screening biomarkers, more than 15% of RCC patients have already developed distant metastases at the time of clinical diagnosis, and the five-year survival rate for patients with metastatic RCC is less than 10%. Therefore, exploring new RCC biomarkers and therapeutic targets is the only way to improve the early diagnosis rate and survival rate of RCC.

[0003] SMIM26 (Small Integral Membrane Protein 26) is a novel protein of unknown function located in mitochondria, encoded by the long noncoding RNA LINC00493. There are currently no reports on the expression of SMIM26 in tumors. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a reagent for detecting the expression level of SMIM26 protein for use in preparing a product for predicting the prognosis risk and / or diagnosing renal cancer.

[0005] Another object of the present invention is to provide the use of SMIM26 protein in the preparation of drugs for treating renal cancer.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Use of a reagent for detecting the expression level of SMIM26 protein in the preparation of a product for predicting the prognosis risk and / or diagnosing renal cancer.

[0008] The SMIM26 protein is a protein encoded by long non-coding RNA-LINC00493, and its NCBI accession number is: NM_001348957.2.

[0009] The renal cancer is renal clear cell carcinoma.

[0010] The products include test kits, test strips or chips.

[0011] The present invention combines immunohistochemical experiments to find that SMIM26 protein is lowly expressed in renal cancer and highly expressed in (renal cancer) adjacent cancer tissues. Therefore, the SMIM26 protein provides a new diagnostic and prognostic biomarker for renal cancer.

[0012] Use of SMIM26 protein in preparing a drug for treating renal cancer.

[0013] The present invention has found through experiments that renal cancer patients with high expression of SMIM26 protein have a longer survival time. Therefore, SMIM26 protein can be used in the treatment of renal cancer.

[0014] The renal cancer is renal clear cell carcinoma.

[0015] The present invention has the following advantages and effects compared to the prior art:

[0016] 1. The present invention uses immunohistochemistry to detect the expression of SMIM26 in pan-cancer tissues and adjacent para-cancer samples for the first time. It was found that SMIM26 protein is specifically differentially expressed in renal tissue and renal cancer tissue, with low expression in clear cell renal cell carcinoma (ccRCC) and high expression in adjacent para-cancer tissue. In addition, its expression level is correlated with the survival time of ccRCC patients, making it a highly effective biomarker for the clinical diagnosis of ccRCC.

[0017] 2. The results of large-sample renal cancer and adjacent tissue immunohistochemistry analysis and survival curve analysis in the present invention showed that patients with renal clear cell carcinoma who highly expressed SMIM26 had a relatively long survival time, indicating that SMIM26 is an effective biomarker for the diagnosis and prognosis of renal cell carcinoma, which can accurately predict the occurrence and development of renal clear cell carcinoma and has strong clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 2 is the staining intensity diagram of SMIM26 in pan-cancer and adjacent tumors; A is the staining situation of SMIM26 in pan-cancer and adjacent tumors; B is the staining intensity score result of SMIM26 in pan-cancer and adjacent tumors.

[0019] Figure 2 The expression and statistical graph of SMIM26 in human renal cancer tissue and adjacent tissue (in the figure, T: renal cancer tissue, N: adjacent tissue); among them, A is the expression of SMIM26 in human renal cancer tissue and adjacent tissue; B is the statistical result of SMIM26 in human renal cancer tissue and adjacent tissue.

[0020] Figure 3 It is a scoring reference graph for the staining intensity and positive staining rate of SMIM26; among them, A is a scoring reference graph for the staining intensity of SMIM26; B is a scoring reference graph for the positive staining rate of SMIM26.

[0021] Figure 4 This is a curve chart showing the relationship between SMIM26 expression and survival time of renal cancer patients. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0023] The SMIM26 (Small Integral Membrane Protein 26) described in the present invention is a mitochondrial protein encoded by LINC00493, and its NCBI accession number is NM_001348957.2. The preparation method of the anti-SMIM26 polyclonal antibody involved in the embodiment is as follows:

[0024] (1) The SMIM26 protein coding gene sequence and the pGEX-4T-1 plasmid (commonly available) were digested with restriction endonucleases Xho1 and BamH1, and then ligated using T4 ligase to obtain the recombinant plasmid pGEX-4T1-SMIM26;

[0025] (2) The recombinant plasmid pGEX-4T1-SMIM26 was transformed into Escherichia coli BL21 and induced with 0.5 mM IPTG for 3.5 h in a 37°C constant temperature shaker. The SMIM26 protein was then purified using the GST protein purification system.

[0026] (3) Japanese large-eared white rabbits (Female, 2.1 kg, Wuhan Proteintech Group) were subcutaneously inoculated with 100 mg of SMIM26 protein for immunization, and anti-SMIM26 antibodies were produced in the rabbits;

[0027] (4) Anti-SMIM26 antibodies were purified from Japanese white rabbit serum.

[0028] Example 1 SMIM26 can be used as a biomarker for the diagnosis of renal clear cell carcinoma

[0029] 1. Immunohistochemical detection of SMIM26 protein was performed in 5 cases of various tumor tissues and adjacent tissues (tissue sample chips were purchased from Shanghai Xinchao Biotechnology Co., Ltd.), including renal cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, breast cancer, intestinal cancer, and gastric cancer. Immunohistochemical detection of SMIM26 was performed in 368 cases of renal clear cell carcinoma (tissue samples were obtained from the Sun Yat-sen University Cancer Center) and 281 adjacent tissue samples, and survival correlation analysis was performed. The specific steps were as follows:

[0030] (1) Dyeing

[0031] The tissue sample chips and tissue samples were incubated with anti-SMIM26 polyclonal antibody at a dilution of 1:500 (volume ratio) at 4°C overnight, then washed three times with PBS buffer, incubated with horseradish peroxidase-conjugated secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), and finally developed with diaminobenzidine (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).

[0032] (2) Method for interpretation of staining results:

[0033] Scoring was based on staining intensity: 0 point (0+), 1 point (1+), 2 points (2+).

[0034] Staining positive rate score (X): 0 points (0 ≤ X), 1 point (0 < X ≤ 25%), 2 points (25 < X ≤ 50%), 3 points (51 < X ≤ 75%), 4 points (75 < X ≤ 100%). The positive rate is the proportion of positively stained cells to all cells used.

[0035] Total score: The product of “staining intensity score” and “staining positive rate score” is the total score.

[0036] Figure 3 Shown are the staining intensity of SMIM26 and the scoring reference for the staining positivity rate.

[0037] (3) Survival analysis of patients based on immunohistochemistry results

[0038] According to the total SMIM26 staining score, the patients were divided into two groups: a SMIM26 high expression group (total staining score greater than or equal to 4) and a low expression group (total staining score less than 4). The survival curve was fitted based on the corresponding survival time.

[0039] 2. Results

[0040] Immunohistochemistry test results Figure 1 and Figure 2 As shown: Figure 1 The results showed that among various tumor tissues and adjacent tissues, SMIM26 was specifically lowly expressed in renal clear cell carcinoma compared with adjacent tissues, while there was no obvious differential expression in other tumors (lung cancer, liver cancer, esophageal cancer, pancreatic cancer, breast cancer, intestinal cancer, and gastric cancer). Figure 2 The expression of SMIM26 in 368 renal cancer tissues and 281 adjacent tissue samples was shown. The statistical graph showed that SMIM26 was significantly underexpressed in renal cancer tissues (P<0.001).

[0041] In addition, according to the expression of SMIM26, renal cancer patients were divided into two groups, SMIM26 high expression group and low expression group, and the survival curve was fitted based on the corresponding survival time. Figure 4 As shown, there is a significant positive correlation between the expression level of SMIM26 and the patient's survival time, indicating that SMIM26 is an effective biomarker for the diagnosis of renal cancer and has application value in the clinical diagnosis of renal cancer.

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the expression level of SMIM26 protein in the preparation of a product for predicting the prognosis risk and / or diagnosing renal cancer, characterized in that: The renal cancer is renal clear cell carcinoma.

2. The use according to claim 1, characterized in that: The SMIM26 protein is a protein encoded by long non-coding RNA-LINC00493, and its NCBI accession number is: NM_001348957.

2.

3. The use according to claim 1, characterized in that: The products include test kits, test strips or chips.

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