Application of slc6a12 in diagnosis marker and / or therapeutic target of liver cancer metastasis

Abstract

The application provides application of SLC6A12 in a diagnostic marker and / or a therapeutic target of liver cancer metastasis, and belongs to the technical field of disease diagnosis or biological medicine. The application provides application of SLC6A12 gene and / or SLC6A12 protein in a diagnostic marker and / or a therapeutic target of liver cancer metastasis. The application detects that SLC6A12 gene or SLC6A12 protein is differentially expressed in situ liver cancer and liver cancer metastatic tumor from the clinical liver cancer patient and animal level respectively, and the expression amount is significantly increased in the liver cancer metastatic tumor. The application can obviously reduce the invasion and metastasis ability of liver cancer cells in a primary liver cancer mouse model by knocking down the expression of SLC6A12 gene and / or SLC6A12 protein. Therefore, the application of SLC6A12 gene or protein as a marker and a therapeutic target in liver cancer metastasis provides a new idea for the diagnosis and treatment of liver cancer metastasis.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis or biopharmaceutical technology, specifically relating to the application of SLC6A12 as a diagnostic biomarker and / or therapeutic target for liver cancer metastasis. Background Technology

[0002] Liver cancer is the fourth leading cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers. Metastasis is the leading cause of death for most cancer patients. Metastasis is the process by which primary cancer cells spread to secondary sites. A growing body of research has found that metastatic cancer cells selectively and dynamically modulate their metabolism at each of the continuous multi-step processes involved in metastasis. Furthermore, many metastatic tumors have metabolic characteristics that differ from those of the primary tumor, enabling them to survive and grow in the new environment. Currently, metabolic restriction is considered a potential barrier to cancer cell metastasis. Many studies have shown that metabolites and reactive oxygen species (ROS) support a shift in cell state to a metastatic phenotype with invasive and migratory properties. In addition to ROS, several metabolites have been found to act as signaling molecules that promote epithelial-mesenchymal transition (EMT), which is associated with the metastatic phenotype. For example, fumarate is an epigenetic modifier that can lead to EMT transition. Furthermore, some studies have provided evidence that lactate and pyruvate can promote tumor cell migration and invasion by inducing various signaling pathways. Therefore, discovering the metabolic characteristics of metastatic tumors and identifying potential metastases that can induce EMT and promote metastasis may provide a therapeutic window for preventing or reducing metastasis.

[0003] Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in microorganisms, plants, and animals. GABA is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS), but it is also present in a wide range of non-neural tissues, including the peripheral nervous system and endocrine system. GABA binds to its specific transmembrane receptors and acts as an inhibitory neurotransmitter in the brain. Interestingly, GABA and its receptors have been found to be associated with tumor progression in several solid tumors, including breast cancer, colon cancer, pancreatic cancer, and gastric cancer. Previous studies have focused on GABA receptors, which can activate downstream signaling pathways such as the ERK, EGFR, or Wnt pathways, promoting cancer cell growth. For example, GAD1-mediated GABA both promotes tumor cell proliferation by activating its receptor and inhibits T cell infiltration. However, GABA has also been shown to retain androgen receptors (AR) in the cell nucleus. Therefore, GABA may regulate cancer progression in a manner independent of its receptors, and the underlying mechanisms remain to be explored. Furthermore, environmental GABA levels are tightly controlled by high-affinity sodium-dependent GABA transporters. SLC6A1, SLC6A11, SLC6A12, and SLC6A13, members of solute transporter family 6 (a family of neurotransmitters and sodium cotransporters), are key members for GABA transport, and their expression is tissue-specific. SLC6A12 mediates cellular uptake of betaine and GABA in a sodium- and chloride-dependent process; in fact, SLC6A12 has a higher affinity for GABA than betaine. However, the biological role of GABA transporters in tumors remains unknown. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an application of SLC6A12 as a diagnostic biomarker and / or therapeutic target for liver cancer metastasis. It has been verified that the expression level of SLC6A12 is differentially expressed in in situ liver cancer and metastatic cancer, and that overexpression of SLC6A12 promotes the invasion and metastasis of liver cancer, while knockdown of SLC6A12 can significantly reduce the invasion and metastasis of primary liver cancer in mouse models.

[0005] This invention provides the application of the SLC6A12 gene and / or SLC6A12 protein as a diagnostic biomarker and / or therapeutic target for liver cancer metastasis.

[0006] This invention provides the application of a reagent for detecting the expression levels of the SLC6A12 gene and / or SLC6A12 protein in the preparation of diagnostic reagents or diagnostic kits for liver cancer metastasis.

[0007] Preferably, the reagent for detecting the expression level of the SLC6A12 gene includes qPCR detection primers for the SLC6A12 gene;

[0008] The qPCR detection primers for the SLC6A12 gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0009] Preferably, the reagent for detecting the expression level of SLC6A12 protein includes an antibody against SLC6A12 protein.

[0010] This invention provides the use of reagents for knocking out or reducing the expression of the SLC6A12 gene and / or SLC6A12 protein, reagents for inhibiting the biological function of SLC6A12 protein, or reagents for reducing γ-aminobutyric acid (GABA) cellular uptake in the preparation of drugs for the prevention and / or treatment of liver cancer metastasis.

[0011] Preferably, the reagents for knocking out or reducing the expression level of the SLC6A12 gene include at least one of the following: small interfering RNA, sgRNA, gene editing vector containing said sgRNA, shRNA, and recombinant lentiviral vector or recombinant lentivirus containing said shRNA.

[0012] Preferably, the small interfering RNA includes hSLC6A12#1-siRNA with a nucleotide sequence as shown in SEQ ID NO:3 and / or hSLC6A12#2-siRNA with a nucleotide sequence as shown in SEQ ID NO:4.

[0013] Preferably, the shRNA comprises the nucleotide sequence shSLC6A12 as shown in SEQ ID NO:5.

[0014] This invention provides the application of the SLC6A12 gene and / or SLC6A12 protein as a diagnostic biomarker and / or therapeutic target for hepatocellular carcinoma (HCC) metastasis. This invention detected differential expression of the SLC6A12 gene or SLC6A12 protein in primary HCC and HCC metastases at both clinical HCC patient and animal levels, with a significantly increased expression level in HCC metastases. This invention significantly reduced the invasion and metastasis of primary HCC in a mouse model by knocking down the expression of the SLC6A12 gene and / or SLC6A12 protein. Therefore, the application of the SLC6A12 gene or its protein as a biomarker and therapeutic target for HCC metastasis provides new insights for the diagnosis and treatment of HCC metastasis. Attached Figure Description

[0015] Figure 1 The image shows the expression results of SLC6A12 in the orthotopic (P) and metastatic (M) sites of hepatocellular carcinoma (HCC) patients. A. Real-time qPCR detection results; B. Representative immunohistochemical images of SLC6A12 in tissue sections; C. Western blotting results; n = 10; scale bar = 50 μm.

[0016] Figure 2 Representative immunohistochemical images (left) and quantification (right) of SLC6A12, divided into three cohorts (high, medium, or low) based on tissue scores, including 27 pairs (A and B) and 17 pairs (C and D) of primary HCC tumors (P) and metastatic tumors (M) from patients; SI: stage I, SII: stage II; SIII: stage III; scale bar = 50 μm;

[0017] Figure 3 The results show the expression of SLC6A12 in the primary and metastatic sites of a mouse model of spontaneous metastasis of hepatocellular carcinoma in situ. A. Western blotting results; B. Representative immunohistochemical images of SLC6A12 in tissue sections; n=3, scale bar=50μm;

[0018] Figure 4 To investigate the effects of SLC6A12 knockdown on HCC cell line metastasis and invasion, the following analysis was conducted: A. Real-time qPCR was used to detect the expression profiles of GABA transporters in Huh7 and MHCC97H cell lines. B and C. Effects of SLC6A12 knockdown followed by a supplementary control and the SLC6A12 transporter metabolite GABA on HCC cell line Huh7 (B) and invasion (C); D and E. Effects of SLC6A12 knockdown followed by a supplementary control and the SLC6A12 transporter metabolite GABA on HCC cell line MHCC97H (D) and invasion (E); F. Real-time qPCR analysis was used to demonstrate the knockdown efficiency of SLC6A12 in Huh7 and MHCC97H.

[0019] Figure 5 To illustrate the effect of SLC6A12 knockdown on a mouse model of spontaneous metastasis of hepatocellular carcinoma in situ, the results are shown in the following images: A. Bioluminescence imaging monitoring of tumors in the whole animal; B. Representative bright-field images (left) and quantitative results (right) of HCC lung metastases, with red arrows indicating lung metastases; C. H&E (top), Ki67 (middle), and GABA (bottom) staining of lung metastases tissue sections, n=3. Detailed Implementation

[0020] This invention provides the application of the SLC6A12 gene and / or SLC6A12 protein as a diagnostic biomarker and / or therapeutic target for liver cancer metastasis.

[0021]

[0022] In this invention, the liver cancer preferably includes hepatocellular carcinoma. The hepatocellular carcinoma is preferably represented by Huh7 and MHCC97H, illustrating the relationship between the expression levels of the SLC6A12 gene and / or SLC6A12 protein and liver cancer cell metastasis.

[0023] In the embodiments of the present invention, the expression levels of SLC6A12 gene and / or SLC6A12 protein were significantly increased in both clinical liver cancer patients (in situ liver cancer and liver cancer metastasis) and animals, compared with in situ tumors.

[0024] In another embodiment of the present invention, two HCC cell lines, Huh7 and MHCC97H, were used as experimental subjects. Knocking down the expression of the SLC6A12 gene or its protein can effectively inhibit the metastasis and invasion of HCC cell lines. At the same time, after knocking down SLC6A12, supplementing with the SLC6A12 transport metabolite GABA can restore SLC6A12 and reduce its impact on metastasis and invasion.

[0025] In another embodiment of the present invention, using an orthotopic liver cancer mouse model as the experimental subject, knocking down the expression level of the SLC6A12 gene and its protein in mice can effectively inhibit HCC metastasis. At the same time, supplementing SLC6A12 transport metabolite GABA after knocking down SLC6A12 can restore SLC6A12 and reduce its impact on metastasis and invasion.

[0026] This invention provides the application of a reagent for detecting the expression levels of the SLC6A12 gene and / or SLC6A12 protein in the preparation of diagnostic reagents or diagnostic kits for liver cancer metastasis.

[0027] In this invention, the reagent for detecting the expression level of the SLC6A12 gene preferably includes qPCR detection primers for the SLC6A12 gene. In an embodiment of this invention, the qPCR detection primers for the SLC6A12 gene preferably include a forward primer with the nucleotide sequence shown in SEQ ID NO:1 and a reverse primer with the nucleotide sequence shown in SEQ ID NO:2. The diagnostic kit preferably also includes a qPCR detection buffer. In an embodiment of this invention, the qPCR detection buffer is 2×SYBR GreenMix.

[0028] In this invention, the method of using the diagnostic kit preferably includes the following steps:

[0029] The relative expression level of the SLC6A12 gene in the test sample was detected using qPCR primers. The relative expression level was used to determine whether the sample had hepatocellular carcinoma (HCC) metastasis: a significantly higher relative expression level compared to the control indicated a higher risk of HCC metastasis, requiring further investigation using other medical methods; no significant change in relative expression level compared to the control indicated no risk of HCC metastasis. The control sample included samples from healthy individuals. The preferred type of test sample was liver tissue.

[0030] In this invention, the reagents for detecting the expression level of SLC6A12 protein preferably include antibodies against SLC6A12 protein. There are no particular limitations on the type of antibody against SLC6A12 protein in this invention; any antibody known in the art can be used. The technique for detecting the expression level of SLC6A12 protein preferably includes Western blotting. There are no particular limitations on the method for Western blotting detection in this invention; any Western blotting detection protocol known in the art can be used. When preparing the diagnostic kit, it is preferable to also include other reagents involved in Western blotting detection, such as PVDF membrane, TBST, BeyoECL Plus chromogenic solution, 5% BSA, and secondary antibody. The method for diagnosing liver cancer invasion and metastasis using the kit is the same as that for qPCR diagnostic kits, and will not be elaborated here.

[0031] This invention provides the use of reagents for knocking out or reducing the expression of the SLC6A12 gene and / or SLC6A12 protein, reagents for inhibiting the biological function of SLC6A12 protein, or reagents for reducing γ-aminobutyric acid (GABA) cellular uptake in the preparation of drugs for the prevention and / or treatment of liver cancer metastasis.

[0032] In this invention, the reagent for knocking out or reducing the expression level of the SLC6A12 gene preferably includes at least one of the following: small interfering RNA, sgRNA, a gene editing vector containing the sgRNA, shRNA, and a recombinant lentiviral vector or recombinant lentivirus containing the shRNA. This invention does not impose any special limitations on the design methods of the small interfering RNA, sgRNA, and shRNA; design principles for interfering RNA well known in the art can be used. In an embodiment of this invention, a verification experiment was conducted on the effect of knocking out SLC6A12 gene expression on liver cancer metastasis using mice as subjects. The small interfering RNA preferably includes hSLC6A12#1-siRNA with a nucleotide sequence as shown in SEQ ID NO:3 and / or hSLC6A12#2-siRNA with a nucleotide sequence as shown in SEQ ID NO:4. The shRNA preferably includes shSLC6A12 with a nucleotide sequence as shown in SEQ ID NO:5. This invention does not impose any special limitations on the construction methods of the recombinant lentiviral vector or recombinant lentivirus containing the shRNA; construction methods for recombinant lentiviral vectors or recombinant lentiviruses well known in the art can be used. The drug is preferably a gene therapy drug. The present invention does not impose any special restrictions on the preparation method of the gene drug; any gene drug preparation method well known in the art can be used.

[0033] In this invention, the reagent for inhibiting the biological function of the SLC6A12 protein preferably includes an inhibitor of the SLC6A12 protein. The SLC6A12 protein inhibitor refers to a substance capable of affecting the activity of the SLC6A12 protease, such as a compound that binds to the active site of the SLC6A12 protease. This invention does not impose any particular limitation on the type of SLC6A12 protein inhibitor; any SLC6A12 protein inhibitor well-known in the art can be used. The dosage form of the drug is preferably an injection, powder, or dispersant. This invention does not impose any particular limitation on the preparation method of the drug; any preparation method for a drug dosage form well-known in the art can be used.

[0034] This invention does not impose any special restrictions on the types of reagents used to reduce γ-aminobutyric acid (GABA) uptake by cells; any reagents known in the art that reduce the content of GABA in the body can be used.

[0035] The following detailed description, in conjunction with embodiments, illustrates the application of SLC6A12 provided by the present invention as a diagnostic biomarker and / or therapeutic target for liver cancer metastasis, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Clinical patient testing

[0038] Using tumor tissues from 10 pairs of liver cancer patients (both orthotopic and metastatic), RNA and protein were extracted, and the expression level of SLC6A12 was detected by qPCR and Western Blot. Simultaneously, tissue sections were prepared for immunohistochemical staining of SLC6A12. The specific experimental steps are as follows:

[0039] 1. Total RNA extraction and real-time PCR reaction

[0040] 1.1 Total RNA Extraction from Tissue. Total RNA was extracted from tumor tissue according to the instructions of the Beijing Tiangen Total RNA Extraction Kit (centrifuge column type). The tissue was ground in liquid nitrogen. 1 ml of lysis buffer RZ was added to every 100 mg of tissue, and the cells were homogenized using a homogenizer for 5 min. 200 μl of chloroform was added, and the mixture was vigorously shaken for 15 s, incubated for 3 min, and then centrifuged at 12000 g for 10 min. The upper aqueous phase was collected, and 0.5 volume of anhydrous ethanol was added. After mixing thoroughly, the mixture was transferred to a CR3 centrifuge column and centrifuged at 12000 g for 1 min at 4 °C. 500 μl of RD Buffer was added to remove protein, and the mixture was centrifuged at 12000 g for 1 min at 4 °C. Wash twice with Washbuffer after centrifugation at 12000 g for 1 min at 4 °C. After another empty centrifugation, 30 μL of DEPC water was added to dissolve the RNA, and the mixture was eluted by centrifugation at 12000 g for 2 min at 4 °C. The RNA concentration was measured using Nanodrop, and A was recorded. 260nm / A 280nm The volume required for cDNA synthesis was calculated, and cDNA was synthesized using a cDNA synthesis kit. Then, it was amplified using a quantitative real-time PCR kit to detect mRNA expression levels. The reverse transcription system was as follows: 1 μg total RNA, 2 μl 5×gDNA buffer, and DEPC water to a final volume of 10 μl. The mixture was incubated at 42℃ for 3 min, then cooled to room temperature. The above reaction system was then mixed with 2 μl 10×King RT buffer, 1 μl FastKing RT Enzyme Mix, 2 μl FQ-RT Primer Mix, and 5 μl RNase-Free ddH2O. The reaction conditions were 42℃ for 15 min, followed by 95℃ for 3 min to obtain cDNA. The quantitative PCR reaction system consisted of: 10 μl 2×SYBR Green Mix, 1 μl cDNA template, 2 μl qPCR primers (5 μM), and 7 μl RNase-Free ddH2O. The qPCR primers were:

[0041] hSLC6A12-F: 5'-CGGAGGTGGAGCCTTCTTC-3' (SEQ ID NO: 1);

[0042] hSLC6A12-R: 5'-CTGGTGTATTGGCCCAACG-3' (SEQ ID NO: 2).

[0043] RT-qPCR reaction: Add to the PCR reaction mixture and mix well for amplification. RT-qPCR amplification conditions: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 15 s, annealing / extension at 60℃ for 1 min, 35 cycles.

[0044] 2. Protein extraction, concentration determination, and Western Blot analysis

[0045] Western blotting is a technique that separates proteins of different molecular weights on a gel using electrophoresis, then transfers them to a PVDF (Polyvinylidene fluoride) membrane, where specific antibodies bind to antigens to obtain the expression information of the target protein.

[0046] The specific experimental steps are as follows: Weigh the tissue, add 200 μl of RIPA lysis buffer (with protease inhibitor) for every 20 mg of tissue to extract total protein, homogenize using a homogenizer, lyse on ice for 30 min, and then centrifuge at 12000 rpm for 10 min at 4℃ to obtain the total protein of the cells.

[0047] The total cellular protein concentration was determined using the BCA method. BCA working solution was prepared by mixing A:B in a 50:1 ratio and adding the mixture. The mixture was incubated at 37°C for 30 min, and the absorbance was measured at 562 nm to calculate the protein concentration. The sample was then mixed with 5× protein loading buffer at a 4:1 ratio and incubated in a metal bath for 10 min to denature the protein.

[0048] Western Blot Experiment: A 10% separating gel and a 5% stacking gel were used. After the gels solidified, samples were loaded for electrophoresis. Electrophoresis was performed at 80V for 30 min, then reduced to 120V until the gel reached its maximum value and electrophoresis was stopped. After electrophoresis, the membrane was transferred. The PVDF membrane was soaked in methanol for 2 min. After assembly, pre-cooled transfer buffer (4℃) was added, and transfer was performed at 350mA for 120 min. After transfer, the membrane was blocked with 5% BSA for 1 h, followed by primary antibody incubation. The antibody was diluted 1:1000 with TBST and incubated overnight at 4℃. After primary antibody incubation, the PVDF membrane was washed with 1×TBST buffer and incubated with secondary antibody (1:2000 dilution) for 1 h. The PVDF membrane was then removed, washed three times with 1×TBST solution, and then developed. Color development was performed using an Image Quant LAS 4000ECL gel imaging system. BeyoECLPlus color developer was mixed at a ratio of A:B = 1:1 and added to the surface of the PVDF film. The instrument parameters were set for exposure and color development.

[0049] 3. Immunohistochemistry

[0050] 3.1 Embedding and Sectioning: Place the tissue to be embedded in paraffin wax, arrange them neatly, and freeze to solidify the paraffin. Remove the embedded tissue from the mold and section it using a paraffin microtome.

[0051] 3.2 The paraffin slices were baked in a 60℃ constant temperature oven for 120 minutes;

[0052] 3.3 Dewaxing and hydration: Xylene (10 min) → Xylene (10 min) → Anhydrous ethanol (5 min × 2 times) → 95% ethanol (5 min × 2 times) → 90% (5 min) → 85% ethanol (5 min) → 80% ethanol (5 min) → 75% ethanol (5 min);

[0053] 3.4 Antigen retrieval: After dewaxing, rinse twice with clean water for 5 minutes each time, then soak in 3% H2O2 for 10 minutes to remove endogenous catalase. Then discard the H2O2, wash twice with distilled water, add citrate buffer, and microwave for 5 minutes (medium heat), generally just until boiling. Cool to room temperature, then microwave again and cool to room temperature. The purpose of this cooking is to expose the antigen sites.

[0054] 3.5 Serum blocking: After cooling to room temperature, discard the citrate buffer, wash twice with water, and place the slide in PBS for 5 min, wash twice, wipe the PBS around the tissue dry, immediately add serum to block some non-specific sites, and then place in a 37°C incubator for half an hour. The serum is diluted 10 times (900 μl PBS and 100 μl serum blocking solution).

[0055] 3.6 Add primary antibody: Remove the slide from the incubator, wipe the serum around the tissue on both the reverse and front sides of the slide dry with absorbent paper, add primary antibody. If performing a control experiment, add PBS to the control tissue. After adding primary antibody, store in a 4°C refrigerator overnight.

[0056] 3.7 Add secondary antibody: Remove the slide from the refrigerator, wash it three times in PBS for 5 minutes each time, wipe the PBS around the tissue dry, add the secondary antibody, and then place it in a 37°C incubator for half an hour.

[0057] 3.8 Add chromogenic agent: Remove the slide from the incubator, wash it three times in PBS for 5 minutes each time, wipe the PBS around the tissue dry, and then add DAB chromogenic agent.

[0058] 3.9 Hematoxylin counterstaining: After rinsing the developed slides with water for a period of time, immerse them in hematoxylin for staining. For animal tissues, this is usually done in half a minute.

[0059] 3.10 Dehydration: After rinsing the counterstained slides in water, place the slides in the following order: 70% ethanol - 80% ethanol - 90% ethanol - 95% ethanol - 100% ethanol - 100% ethanol - xylene - xylene. Let each reagent soak for 2 minutes, then finally immerse in xylene and move to a fume hood.

[0060] 3.11 Mounting: Apply a drop of neutral resin next to the tissue, then cover it with a coverslip. First, place one side flat, then gently place the other side down to avoid air bubbles. After mounting, place the slide in a fume hood to dry.

[0061] The results showed that, compared to in situ tumors, the mRNA and protein levels of SLC6A12 were significantly increased in metastatic tumors. Figure 1 ).

[0062] Example 2

[0063] Immunohistochemical detection technology was used to detect tissue microarrays of commercially available clinical in situ and metastatic liver cancer, and the tissue staining was scored.

[0064] Immunohistochemical detection

[0065] 1. Antigen retrieval: After dewaxing, rinse twice with clean water for 5 minutes each time, add 3% H2O2 and soak for 10 minutes to remove endogenous catalase. Then discard the H2O2, wash twice with distilled water, add citrate buffer, and microwave for 5 minutes (medium heat), generally just until boiling. Cool to room temperature, then microwave again and cool to room temperature. The purpose of boiling is to expose the antigen sites.

[0066] 2. Serum blocking: After cooling to room temperature, discard the citrate buffer, wash twice with water, and place the slide in PBS for 5 min, wash twice, wipe the PBS around the tissue dry, and immediately add serum to block some non-specific sites. Then place in a 37°C incubator for half an hour. The serum is diluted 10 times (900 μl PBS: 100 μl serum blocking solution).

[0067] 3. Add primary antibody: Remove the slide from the incubator, wipe the serum around the tissue on both the reverse and front sides of the slide with absorbent paper, add primary antibody, and if performing a control experiment, add PBS to the control tissue. After adding primary antibody, store in a refrigerator at 4°C overnight.

[0068] 4. Add secondary antibody: Take the slide out of the refrigerator, wash it three times in PBS for 5 minutes each time, wipe the PBS around the tissue dry, add the secondary antibody, and then place it in a 37°C incubator for half an hour.

[0069] 5. Add chromogenic agent: Remove the slide from the incubator, wash it three times in PBS for 5 minutes each time, wipe the PBS around the tissue dry, and then add DAB chromogenic agent.

[0070] 6. Hematoxylin counterstaining: After rinsing the developed slides with water for a period of time, immerse them in hematoxylin for staining. For animal tissues, this is usually done in half a minute.

[0071] 7. Dehydration: After rinsing the counterstained slides in water, place the slides in the following order: 70% ethanol - 80% ethanol - 90% ethanol - 95% ethanol - 100% ethanol - 100% ethanol - xylene - xylene. Let each reagent soak for 2 minutes, then finally immerse in xylene and move to a fume hood.

[0072] 8. Mounting: Apply a drop of neutral resin next to the tissue, then cover it with a coverslip. First, lay one side flat, then gently lower the other side to avoid air bubbles. After sealing, place the slide in a fume hood to dry.

[0073] See results Figure 2 Based on immunohistochemical staining of SLC6A12, the samples were divided into three cohorts: high, intermediate, and low expression. Results showed that compared to primary HCC tumors (P), metastatic tumors (M) had a higher proportion of high SLC6A12 expression. Furthermore, the proportion of high SLC6A12 expression was even higher in liver cancer samples with higher clinical stages.

[0074] Example 3

[0075] Animal-level validation of differential expression of SLC6A12

[0076] Animal-based validation of the relationship between SLC6A12 expression level and liver cancer cell metastasis

[0077] 1. Lentiviral Packaging: 293T cells were seeded in 10cm culture dishes at a density of 80-90%. After cell adhesion, transfection was performed. The plasmid expressing luciferin-GFP (4 μg), helper plasmid VSVG (3 μg), and psPAX2 (2 μg) were mixed with 200 μl of Opti-MEM, while simultaneously... Mix 3000 μl of Opti-MEM with 200 μl of Opti-MEM, let stand for 15 min, then mix the two together, let stand for 15 min, and then add dropwise to a cell culture dish. Add 2 ml of Opti-MEM, and after 6 h, add complete culture medium and incubate in an incubator. After 72 h, collect the culture supernatant, filter it through a 0.45 μm filter membrane, and then directly infect cells.

[0078] 2. Cell infection and establishment of stable cell lines: Add 1 ml of collected virus and 1 ml of culture medium to the cells to be infected. Adding 2 μl of 4 μg / ml polybrene simultaneously can enhance infection efficiency. After 24 hours, replace with fresh culture medium and perform flow cytometry screening for GFP protein. Expand the culture.

[0079] 3. Inoculation of mice with stable cell lines: 5 × 10 6 Huh7 cells (all expressing luciferase) were injected into the liver of mice to generate orthotopic tumors. Mice were then injected intraperitoneally every other day with either the SLC6A12 transporter metabolite GABA (50 mg / kg) or a solvent, for 12 weeks, after which the orthotopic tumors metastasized to the lungs or peritoneum. RNA and protein were extracted from the orthotopic and metastatic tumors, and the expression level of SLC6A12 was detected using qPCR and Western blotting methods described in Example 1. Simultaneously, immunohistochemical staining of SLC6A12 was performed on tissue sections prepared according to Example 1.

[0080] The results showed that, compared to in situ tumors, the protein level of SLC6A12 was significantly increased in metastatic tumors. Figure 3 ).

[0081] Example 4

[0082] Knocking down SLC6A12 inhibits the metastasis and invasion of HCC cell lines.

[0083] The mRNA expression profiles of GABA transporter were detected in Huh7 and MHCC97H HCC cells, followed by knockdown of SLC6A12 in both cells. The specific experimental steps are as follows:

[0084] 1. Total RNA extraction and real-time PCR reaction

[0085] 1. Total RNA was extracted from Huh7 and MHCC97H cells. cDNA was synthesized using a cDNA synthesis kit and then amplified using a quantitative real-time PCR kit to detect mRNA expression levels. The qPCR primers are as follows:

[0086] SLC6A1: 5'-ACAATGTCTACAGGGACTCCAT-3' (SEQ ID NO:8); and 5'-CCTCTGGGTATGCCAGGAAC-3' (SEQ ID NO:9);

[0087] SLC6A11: 5'-CTGATTCCCTACGTGGTGTTTT-3' (SEQ ID NO: 10); and 5'-CACCTGTGTTGCATAGCCAAT-3' (SEQ ID NO: 11);

[0088] SLC6A12: 5'-CGGAGGTGGAGCCTTCTTC-3' (SEQ ID NO: 1); and 5'-CTGGTGTATTGGCCCAACG-3' (SEQ ID NO: 2);

[0089] SLC6A13: 5'-TCTCAGGCACAACCAGTAATGG-3' (SEQ ID NO: 12); and 5'-CCTAAGCCAATGATCTCCCCA-3' (SEQ ID NO: 13).

[0090] The results showed that only the SLC6A12 gene was highly expressed in Huh7 and MHCC97H cells.

[0091] 2. Establishment of transient cell lines

[0092] Huh7 and MHCC97H cells in good growth condition were seeded into 6-well plates. After 24 hours, cell confluence reached 70%–80%. The target siRNA was then injected with... RNAiMAX was added to 0.2 ml of Opti-MEM medium at a volume ratio of 1:1.5 and incubated at room temperature for 5 min. The two solutions were then mixed separately. After 15 min, 400 μl of the mixture was added to the corresponding wells of a 6-well plate and gently shaken. After incubation for 24 h, GABA was added to a final concentration of 100 mM. After 48 h of incubation, the protein was collected for further analysis.

[0093] The specific sequence of the target siRNA is as follows:

[0094] hSLC6A12#1siRNA:GCCAAUGGACCAACAAGAUTT (SEQ ID NO: 3);

[0095] hSLC6A12#2siRNA:CCUCACCUGUCAUGGAAUUTT(SEQ ID NO:4);

[0096] The results showed that SLC6A12 knockdown inhibited the metastasis and invasion of HCC cell lines. Furthermore, supplementation with GABA, a transport metabolite of SLC6A12, after SLC6A12 knockdown could restore the reduced effect of SLC6A12 on metastasis and invasion. Figure 4 ).

[0097] Example 5

[0098] Knocking down SLC6A12 inhibits spontaneous metastasis in a mouse model of orthotopic hepatocellular carcinoma.

[0099] 1. Construction method of shRNA plasmid

[0100] 1) The DNA sequence corresponding to the shRNA that knocks down SLC6A12 gene expression is as follows:

[0101] shSLC6A12: 5'-CGAGTCATATTTGAATGTCTA-3' (SEQ ID NO: 5).

[0102] The upstream and downstream fragments were synthesized based on the DNA sequence corresponding to the shRNA. The upstream and downstream fragments of the shRNA were dissolved in water to 10 μM and mixed. The system (50 μl) is shown in Table 1.

[0103] Table 1. shRNA hybridization system

[0104]

[0105] Annealing reaction conditions are shown in Table 2.

[0106] Table 2 Annealing reaction conditions

[0107]

[0108] 2) Enzyme digestion

[0109] The PCR-annealed mixture and the plko.1 empty vector were digested with restriction endonucleases AgeI and EcoRI for 4 hours.

[0110] 3) Connection

[0111] The digested PCR mixture and the plko.1 empty vector were ligated. Ligation was performed overnight at 16°C. The ligation volume (15 μl) is shown in Table 3.

[0112] Table 3 Connection System

[0113]

[0114]

[0115] 4) Transformation

[0116] The competent Trans5α cells were mixed with the ligation product, incubated on ice for 30 min, heat-shocked at 42°C for 50 s, and then incubated on ice again for 2 min. 500 μl of LB medium was added. After incubation on a shaker at 37°C for 1 h, 200 μl of the mixture was spread onto LB agar plates containing ampicillin and incubated at 37°C for 16 h.

[0117] 5) Pick single clones and place them in liquid LB medium containing ampicillin, and shake at 37°C for 16 hours.

[0118] 6) Plasmid mini-preparation

[0119] The bacterial culture was collected by centrifugation, and plasmids were extracted using the Tiangen Biotech rapid mini-prep kit. After concentration detection and enzyme digestion identification, the samples were sent to the company for sequencing. Plasmid samples that perfectly matched the target gene were used for subsequent experiments.

[0120] 2. Lentiviral Packaging: 293T cells were seeded in 10cm culture dishes at a density of 80-90%. After cell adhesion, transfection was performed. shRNA plasmid (4μg), helper plasmid VSVG (3μg), and psPAX2 (2μg) were mixed with 200μl Opti-MEM, and simultaneously... Mix 3000 ml of Opti-MEM with 200 ml of Opti-MEM and let stand for 15 min. Then mix the two together, let stand for 15 min, and add the mixture dropwise to a cell culture dish. Add 2 ml of Opti-MEM, and after 6 h, add complete culture medium and incubate in an incubator. After 72 h, collect the culture supernatant, filter it through a 0.45 μm filter membrane, and then directly infect cells.

[0121] 3. Cell infection and establishment of stable cell lines: 1 ml of collected virus and 1 ml of culture medium were added to the cells to be infected. Adding 2 μl of 4 μg / ml polybrene simultaneously enhanced infection efficiency. After 24 hours, the medium was replaced with fresh medium, and puromycin (2 mg / ml) was added for selection. After 7 days, Western blot was used to assess infection efficiency. The culture was then expanded.

[0122] 4. Animal Model Establishment Using Stable Cell Lines: Huh7 cells stably expressing control shRNA or SLC6A12 shRNA (both expressing luciferase) were injected into the liver of mice to generate orthotopic tumors. Mice were then intraperitoneally injected every other day with GABA (a transport metabolite of SLC6A12) or a solvent at a dose of 50 mg / kg for 12 weeks. The orthotopic tumors metastasized to the lungs or peritoneum. Bioluminescent imaging was used to monitor tumors throughout the animals, and the number of metastatic tumors was counted. Simultaneously, immunohistochemical staining was performed on sections of lung metastatic tumor tissue.

[0123] The results showed that knocking down SLC6A12 inhibited the metastasis of hepatocellular carcinoma (HCC) cell lines, and supplementation with GABA, a transport metabolite of SLC6A12, after knocking down SLC6A12 could restore the effect of SLC6A12 on reducing metastatic invasion. Figure 5 ).

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting SLC6A12 gene mRNA level and / or SLC6A12 protein level in the preparation of diagnostic reagents for liver cancer metastasis.

2. The application according to claim 1, characterized in that, Reagents for detecting SLC6A12 gene mRNA levels include qPCR detection primers for the SLC6A12 gene; The qPCR detection primers for the SLC6A12 gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, Reagents for detecting SLC6A12 protein levels include antibodies against SLC6A12 protein.

4. The use of an agent for reducing SLC6A12 gene expression in the preparation of a drug for inhibiting liver cancer metastasis, wherein the agent for reducing SLC6A12 gene expression is at least one of the following: small interfering RNA, shRNA, and a recombinant lentiviral vector or recombinant lentivirus containing said shRNA; The small interfering RNA is selected from hSLC6A12#1-siRNA with nucleotide sequence as shown in SEQ ID NO:3 and / or hSLC6A12#2-siRNA with nucleotide sequence as shown in SEQ ID NO:4; The shRNA is a nucleotide sequence such as shSLC6A12 as shown in SEQ ID NO:5.

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