A biomarker for gastric cancer metastasis and application thereof

Abstract

The application belongs to the technical field of molecular biology diagnosis, and provides a biomarker for gastric cancer metastasis and application thereof, wherein the biomarker for gastric cancer metastasis is SLC16A12. The applicant finds that the expression of SLC16A12 protein is down-regulated in gastric cancer tissues, and the expression of SLC16A12 protein is reduced in tissues of patients with gastric cancer metastasis. Overexpression of SLC16A12 can inhibit the scratch healing, migration and invasion ability of gastric cancer cells; and knockdown of SLC16A12 can promote the wound healing, migration and invasion of gastric cancer cells. Based on the above finding, the application proposes the application of SLC16A12 as a gastric cancer metastasis diagnosis and prevention target, and provides a new strategy and idea for developing an anti-gastric cancer metastasis drug.

Description

Technical Field

[0001] This application belongs to the field of molecular biology diagnostic technology. Specifically, this application provides a biomarker for gastric cancer metastasis and its application. Background Technology

[0002] Gastric cancer is a common malignant tumor of the digestive system, frequently occurring in the antrum and angle of the stomach. Its incidence and mortality rate rank fifth among malignant tumors. Gastric cancer progresses rapidly and is prone to metastasis, posing a major challenge to clinical treatment. Therefore, inducing tumor cell death and inhibiting its metastatic ability has become an important strategy in the treatment of gastric cancer. Currently, the complex pathogenesis of gastric cancer is not fully understood, leading to difficulties in early diagnosis and treatment. Coupled with the combined influence of genetic and environmental factors, most patients are diagnosed at an advanced stage. The generally accepted mechanism of gastric cancer involves the joint participation of multiple factors, multiple targets, and multiple signaling pathways. Despite the current clinical use of multidisciplinary comprehensive therapies such as surgery, radiotherapy, and immunotherapy, the 5-year survival rate remains below 50%, often accompanied by adverse reactions and the risk of recurrence and metastasis. Therefore, fully elucidating the molecular mechanisms of gastric cancer development and progression is crucial for achieving early and accurate diagnosis and effective treatment, and is of great significance for improving patient prognosis and quality of life.

[0003] Molecular biomarkers, due to their ability to reveal the essence of disease development at the molecular level, have become a research hotspot in the field of oncology diagnosis and treatment. Precision medicine based on molecular levels is gradually changing clinical practice in gastric cancer. However, despite the promising prospects of molecular biomarkers in gastric cancer diagnosis and treatment, there is still a lack of specific molecular targets and related products that can accurately predict metastasis risk and provide effective prognostic assessment. SLC16A12 protein belongs to the solute carrier (SLC) superfamily, the second largest class of membrane proteins after G protein-coupled receptors (GPCRs), primarily responsible for the transmembrane transport of substances such as creatine, amino acids, ions, and drugs. Studies have shown that SLC16A12 expression is reduced in colorectal cancer tissues, and its changes precede mucosal morphological abnormalities, suggesting it may serve as a potential biomarker for early diagnosis and prevention. In gastric cancer, the high methylation level of SLC16A12 is negatively correlated with telomere shortening, suggesting its potential involvement in the malignant transformation process. However, the specific mechanisms by which SLC16A12 participates in regulating gastric cancer metastasis remain lacking in systematic and in-depth research. Summary of the Invention

[0004] On the one hand, this application provides a biomarker for gastric cancer metastasis, wherein the biomarker for gastric cancer metastasis is SLC16A12.

[0005] On the other hand, this application provides the application of a reagent for the specific detection of SLC16A12 in the preparation of a kit for diagnosing gastric cancer metastasis.

[0006] Furthermore, the reagent for specifically detecting SLC16A12 is a reagent for detecting the level of SLC16A12 mRNA in gastric cancer tissue.

[0007] Furthermore, the reagent used to detect the SLC16A12 mRNA level in gastric cancer tissue is a PCR detection reagent.

[0008] Furthermore, the PCR detection reagent includes upstream and downstream primers with sequences SEQ ID NO.1 and SEQ ID NO.2.

[0009] Furthermore, the reagent for specifically detecting SLC16A12 is a reagent for detecting the SLC16A12 protein level in gastric cancer tissue.

[0010] Furthermore, the reagent used to detect the SLC16A12 protein level in gastric cancer tissue was a Western blot reagent.

[0011] Furthermore, the gastric cancer metastasis refers to gastric cancer lymph node metastasis.

[0012] Furthermore, the kit also includes cell lysis reagents and protein or nucleic acid extraction reagents.

[0013] On the other hand, this application provides the use of SLC16A12 or reagents that overexpress SLC16A12 in the preparation of drugs that inhibit gastric cancer metastasis.

[0014] The "reagent for specific detection of SLC16A12" mentioned in this application refers to a reagent that can specifically detect the level of SLC16A12 protein or mRNA, such as one containing primers / probes, antibodies, etc., but does not contain the general detection components of specific detection reagents, such as polymerases and buffers, and does not belong to "reagent for specific detection of SLC16A12".

[0015] Given the known gene and protein sequences of SLC16A12 (including but not limited to various versions obtained from databases such as Genbank and EMBL), those skilled in the art can routinely design PCR detection reagents, such as probes and primers, to detect mRNA levels, and perform various PCR detection methods, such as qPCR, to quantify mRNA levels. Those skilled in the art can also routinely design methods and reagents for detecting protein levels, such as antigen-antibody reaction-based detection methods and reagents, such as Western blot and ELISA methods and reagents. Those skilled in the art can also directly use existing or commercially available SLC16A12 mRNA or protein detection reagents. Attached Figure Description

[0016] Figure 1The analysis of SLC16A12 and gastric cancer tissue in the TCGA database shows the correlation: Part A is adjacent normal tissue and cancerous tissue; Part B is different stages of gastric cancer; Part C is different metastatic stages; and Part D is the prognostic survival of gastric cancer patients.

[0017] Figure 2 The expression levels of SLC16A12 in normal cells and gastric cancer cells are shown.

[0018] Figure 3 This shows the expression level of SLC16A12 in adjacent and gastric cancer tissues of patients with metastatic gastric cancer.

[0019] Figure 4A Displaying an AGS fluorescence image.

[0020] Figure 4B The image shows the fluorescence of HGC-27.

[0021] Figure 4C The results of qRT-PCR verification of SLC16A12 mRNA expression level in AGS are shown.

[0022] Figure 4D The results of qRT-PCR verification of SLC16A12 mRNA expression level in HGC-27 are shown.

[0023] Figure 4E The Western blot in AGS was used to verify the protein expression level of SLC16A12.

[0024] Figure 4F The Western blot analysis of HGC-27 shows the protein expression level of SLC16A12.

[0025] Figure 5 The effect of knockdown / overexpression of SLC16A12 on the scratch healing ability of gastric cancer cells is shown: Part A is overexpression of SLC16A12; Part B is knockdown of SLC16A12.

[0026] Figure 6A This study demonstrates the effect of SLC16A12 overexpression on the invasive ability of gastric cancer cells.

[0027] Figure 6B This demonstrates the effect of knocking down SLC16A12 on the invasive ability of gastric cancer cells.

[0028] Figure 6C This study demonstrates the effect of SLC16A12 overexpression on the migration ability of gastric cancer cells.

[0029] Figure 6D This demonstrates the effect of knocking down SLC16A12 on the migration ability of gastric cancer cells. Detailed Implementation

[0030] Example 1: TCGA database analysis of SLC16A12 expression in gastric cancer patient tissues Experimental methods: Gastric cancer sample data and corresponding clinical information were downloaded from the TCGA database (https: / / portal.gdc.cancer.gov). Expression levels in TPM format were extracted and normalized using log2(TPM+1). A total of 449 samples were included, comprising 34 adjacent normal tissues and 415 gastric cancer tissues. The t-test was used to compare expression differences between the two groups, one-way ANOVA was used to compare expression levels at different time stages, and the log-rank test was used to analyze survival differences.

[0031] Experimental results: like Figure 1 As shown in the TCGA data analysis, the expression level of SLC16A12 was significantly reduced in 415 gastric cancer tissues compared to 34 normal tissues. Figure 1 As shown in Part B, SLC16A12 expression was lower in gastric cancer tissues at different clinical stages than in normal tissues, with the lowest expression level observed in Stage I, and significantly lower than in Stages II–IV. Figure 1 As shown in Part C, SLC16A12 expression was further decreased in metastatic gastric cancer tissues compared to non-metastatic tissues. Figure 1 As shown in Part D, the survival analysis results showed that patients in the SLC16A12 high expression group had significantly longer overall survival.

[0032] In summary, SLC16A12 is downregulated in gastric cancer tissues, and its low expression is closely related to tumor size, lymph node metastasis, and patient prognosis.

[0033] Example 2: Western blot detection of expression levels of SLC16A12 in normal gastric mucosal cells and gastric cancer cells. Experimental methods: To investigate the differential expression of SLC16A12 at the protein level, Western blot was used to detect the expression of SLC16A12 in normal gastric mucosal cells GES-1 and four gastric cancer cell lines (AGS, HGC-27, MKN-45, and MGC-803). The five cell lines were seeded in 100 mm culture dishes and cultured for 24 hours. After removing the supernatant, the cells were washed once with 5 mL of PBS buffer. 1 mL of trypsin was added for digestion, and after digestion, complete culture medium was added to terminate the reaction. The cell suspension was collected and centrifuged at 1000 rpm for 5 minutes to collect the cell pellet.

[0034] Preparation of protein lysis buffer: Add 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of 100 mM PMSF to each 1 mL of pre-chilled Lysis Buffer, and mix well. Add the lysis buffer to the cell pellet, place on a shaker at 4°C and shake vigorously for 30 seconds, then incubate on ice for 4 minutes. Repeat this process 5 times. Then centrifuge at 12,000 g for 5 minutes at 4°C, collect the supernatant as the whole protein extract, and quantify the protein concentration.

[0035] Protein samples were separated by SDS-PAGE gel electrophoresis (Epizyme Biomedical Technology, China), then transferred to a PVDF membrane and blocked with 5% skim milk. The membrane was then incubated with primary antibody (overnight at 4°C) and secondary antibody (incubated at room temperature for 1.5 hours). Finally, ECL chemiluminescence solution (ThermoFisher Scientific, USA) was added for chemiluminescence detection, and the grayscale values ​​of the protein bands were statistically analyzed using ImageJ software.

[0036] Experimental results: Western blot results showed that ( Figure 2 Compared with normal gastric mucosal cells GES-1, the expression of SLC16A12 protein was significantly reduced in all four gastric cancer cell lines, with the most significant downregulation observed in the AGS and HGC-27 cell lines. This result further confirms that SLC16A12 is underexpressed in gastric cancer cells.

[0037] Example 3: Western blot detection of SLC16A12 expression levels in adjacent normal tissues and gastric cancer tissues of metastatic gastric cancer. Experimental methods: To investigate the correlation between SLC16A12 protein expression and gastric cancer metastasis, this study collected surgical tissue specimens from 8 patients pathologically diagnosed with gastric cancer and accompanied by lymph node / distant metastasis. Two types of tissue were collected from each patient: adjacent normal tissue (>5 cm from the tumor margin and pathologically confirmed to be cancer-free) and primary gastric cancer tissue. Immediately after excision, the tissues were repeatedly rinsed with pre-cooled PBS buffer to remove surface blood and contaminants.

[0038] The cleaned tissue was cut into small fragments and placed in a homogenizer. RIPA lysis buffer containing a mixture of protease inhibitors was added, and the mixture was thoroughly homogenized in an ice bath. The homogenate was transferred to a 1.5 mL centrifuge tube and incubated on ice for 30 min to allow for complete lysis. Subsequently, the mixture was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant (i.e., the total protein solution) was carefully aspirated into a new pre-chilled centrifuge tube, the precipitate was discarded, and protein concentration was quantified.

[0039] Protein samples were separated by SDS-PAGE gel electrophoresis (Epizyme Biomedical Technology, China), then transferred to a PVDF membrane and blocked with 5% skim milk. The membrane was then incubated with primary antibody (overnight at 4°C) and secondary antibody (incubated at room temperature for 1.5 hours). Finally, ECL chemiluminescence solution (ThermoFisher Scientific, USA) was added for chemiluminescence detection, and the grayscale values ​​of the protein bands were statistically analyzed using ImageJ software.

[0040] Experimental results: like Figure 3 As shown in the results, Western blot analysis revealed that the expression of SLC16A12 protein was significantly reduced in metastatic gastric cancer tissues (M) compared to adjacent normal tissues (N), suggesting that SLC16A12 is expressed at low levels in metastatic gastric cancer tissues.

[0041] Example 4: Construction of a stable cell line with knockdown / overexpression of SLC16A12 Experimental methods: Cell line construction and viral transfection: AGS and HGC-27 gastric cancer cell lines were selected and seeded in 60 mm culture dishes. Lentiviral transfection was performed when cell confluence reached 30%. 5 μL of lentivirus suspension and 40 μL of viral infection enhancement medium were added to each well of a 6-well plate, and the plates were incubated. Ten hours after transfection, the medium was replaced with serum-containing complete medium, and the cells were cultured for another 48 hours. Puromycin was then added to a final concentration of 1.0 μg / mL for selection, and this process was continued for 72 hours. Positive cells were then passaged and cultured on a larger scale. Transfection efficiency verification: Green fluorescent protein expression was observed using fluorescence microscopy, and SLC16A12 expression was detected at the mRNA and protein levels using qRT-PCR and Western blot, respectively, to evaluate the transfection effect. The primer sequences for SLC16A12 are shown in Table 1.

[0042] Table 1 Primer sequences for SLC16A12

[0043] Experimental results: like Figure 4A and Figure 4B As shown, the green fluorescence signal in the cells gradually increases with prolonged transfection time, indicating successful viral transfection. Figures 4C-4FqRT-PCR and Western blot results showed that, compared with the control group transfected with empty vector virus, the mRNA and protein expression levels of SLC16A12 were significantly increased in the overexpression group, while both were significantly decreased in the knockdown group. These results indicate that stable cell lines with SLC16A12 knockdown and overexpression were successfully constructed.

[0044] Example 5: In vitro experiment to detect the effect of SLC16A12 overexpression / knockdown on gastric cancer scratching ability. Experimental methods: SLC16A12 cells transfected with AGS knockdown or overexpression and HGC-27 stably transfected cells were seeded in 60 mm culture dishes. When cell confluence reached 90%, a uniform scratch was made on the cell surface using a sterile pipette tip. Cells were gently washed with PBS buffer to remove detached cells, and the dishes were then incubated for 24 hours. The scratched area was photographed at the same location at 0 and 24 hours post-scratching. ImageJ software was used to analyze changes in scratch width and calculate cell migration rate. Cell migration rate = ((0h scratch distance - 24h scratch distance) / 0h scratch distance) × 100%.

[0045] Experimental results: like Figure 5 As shown in Part A, compared with the corresponding control group, overexpression of SLC16A12 inhibited the scratch healing rate of AGS and HGC-27 cells; Figure 5 As shown in Part B, knocking down SLC16A12 promoted the speed of scratch healing in gastric cancer cells. The results indicate that SLC16A12 overexpression can inhibit the migration ability of gastric cancer cells.

[0046] Example 6: Effect of SLC16A12 knockdown / overexpression on the number of invasive and migrating gastric cancer cells. Experimental methods: Cell migration assay: SLC16A12 cells with knockdown or overexpression of AGS and HGC-27 were transfected and stable cell lines were selected. Single-cell suspensions were prepared and cell counts were performed. An appropriate amount of cell suspension (2 × 10⁴ cells) was seeded into the upper chamber of a Transwell chamber using serum-free medium. Complete medium containing 30% serum was added to the lower chamber as a chemokine. The culture plate was incubated at 37°C in a 5% CO₂ incubator for 24 hours. After incubation, the chamber was removed, the medium was discarded, and the cells were washed with PBS. Cells were fixed with 4% paraformaldehyde for 30 minutes, washed again with PBS, and stained with 0.1% crystal violet solution for 30 minutes. After staining, the cells were washed, air-dried, and photographed under a microscope at random fields of view. Cells that migrated to the lower surface were counted using ImageJ software.

[0047] Cell invasion assay: Based on the migration assay, matrix gel was pre-coated onto the polycarbonate membrane of the Transwell chamber to simulate the extracellular matrix environment. Preparation of the reagent gel: C10 buffer solution (10×) was diluted with serum-free F12 medium at a volume ratio of 1:19 to obtain a 0.5× working solution. 0.5 mL of gel A (2×) was mixed with 0.5 mL of serum-free F12 medium to prepare gel A (1×) stock solution. The gel A (1×) stock solution was further diluted 15-fold using C10 buffer solution (0.5×) pre-equilibrated at 37℃, and thoroughly mixed to obtain the matrix gel working solution. 0.1 mL of the working solution was uniformly added to the upper chamber of the Transwell, placed in a 24-well plate, and incubated at 4℃ for 3 hours to allow the matrix gel to polymerize and form a stable coating layer. Subsequent cell seeding, culture, fixation, staining, and counting procedures were the same as in the migration assay.

[0048] Experimental results: like Figure 6A As shown, overexpression of SLC16A12 can reduce the number of invasive cells in gastric cancer cells; Figure 6B As shown, knocking down SLC16A12 significantly increases the number of cells that successfully invade; Figure 6C As shown, overexpression of SLC16A12 can reduce the number of migrating cells in gastric cancer cells; Figure 6D As shown, knocking down SLC16A12 significantly increased the number of cells that successfully migrated. These results confirm that high expression of SLC16A12 is associated with the inhibition of gastric cancer invasion and migration.

Claims

1. A biomarker for gastric cancer metastasis, wherein the biomarker for gastric cancer metastasis is SLC16A12.

2. Application of reagents for specific detection of SLC16A12 in the preparation of kits for diagnosing gastric cancer metastasis.

3. The application according to claim 2, wherein the reagent for specifically detecting SLC16A12 is a reagent for detecting the level of SLC16A12 mRNA in gastric cancer tissue.

4. According to the application described in claim 3, the reagent for detecting the SLC16A12 mRNA level in gastric cancer tissue is a PCR detection reagent.

5. The application according to claim 4, wherein the PCR detection reagent comprises upstream and downstream primers with sequences SEQ ID NO.1 and SEQ ID NO.

2.

6. In the application according to claim 2, the reagent for specifically detecting SLC16A12 is a reagent for detecting the SLC16A12 protein level in gastric cancer tissue.

7. According to claim 6, the reagent for detecting the SLC16A12 protein level in gastric cancer tissue is a Western blot reagent.

8. The application according to any one of claims 2-7, wherein the gastric cancer metastasis is gastric cancer lymph node metastasis.

9. The application according to any one of claims 2-8, wherein the kit further comprises a cell lysis reagent and a protein or nucleic acid extraction reagent.

10. Application of SLC16A12 or reagents that overexpress SLC16A12 in the preparation of drugs that inhibit gastric cancer metastasis.

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