Use of pqlc2 gene in preparation of medicine for treating liver cancer and diagnostic kit

Abstract

The application of PQLC2 gene in the preparation of drugs for treating liver cancer and diagnostic kits belongs to the field of biological medicine. The present application finds that the expression of PQLC2 in liver cancer tissue is obviously higher than that in normal tissue through statistical analysis of the expression of PQLC2 in liver cancer tissue and normal liver tissue of hepatocellular carcinoma patients in TCGA database. And the expression vector interfering with PQLC2 gene is used to prove that the expression level of PQLC2 gene can inhibit the proliferation and migration of liver cancer cells through in vitro cell function experiment. The real-time fluorescent quantitative PCR experiment shows that the mRNA level of genes CCND1, MMP14, p21 and BIM related to the regulation of cell cycle and apoptosis is significantly changed after the expression of PQLC2 gene is interfered, which confirms that PQLC2 can promote the occurrence and development of liver cancer from the molecular level of gene, and indicates that PQLC2 can be used as a target for clinical treatment of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of the PQLC2 gene in the preparation of drugs or diagnostic kits for the treatment of liver cancer. Background Technology

[0002] Liver cancer ranks 6th in incidence and 4th in mortality among cancers worldwide, remaining a global health challenge. Primary liver cancer mainly includes two types: hepatocellular carcinoma (HCC) originating from hepatocytes and intrahepatic cholangiocarcinoma (ICC) originating from bile ducts, with HCC being the predominant type, accounting for approximately 90% of liver cancers. The difficulty in treating HCC stems from its often asymptomatic early stages, making it undetectable through existing screening methods. Diagnosis often occurs in the middle to late stages, causing patients to miss opportunities for treatment such as liver resection or transplantation. Even with radical treatment, the risk of recurrence and metastasis remains high, making systemic therapy particularly important. Liver cancer is also the third leading cause of cancer death in my country. Molecularly, the specific causes of HCC are related to specific gene alterations. Hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as cirrhosis, are major causes of HCC, associated with mutations in the TERT promoter and TP53. Other factors that may contribute to HCC include excessive alcohol consumption, diabetes, aflatoxin exposure, and possibly non-alcoholic fatty liver disease (NAFLD).

[0003] Currently, treatment for HCC primarily relies on targeted therapies. However, many early-stage target selections lacked both excellent selectivity and manageable side effects. With a deeper understanding of key genes and signaling pathways in the development and progression of liver cancer, highly selective and low-toxicity targeted drugs have become a focus of liver cancer research and treatment. The discovery and dynamic quantification of prognostic and treatment predictive biomarkers are crucial for the early detection of HCC, the development of novel treatment methods, and the monitoring of tumor progression. Therefore, it is necessary to find new, stable, and reliable targets to improve prognosis and provide accurate predictive and diagnostic indicators.

[0004] PQLC2, a membrane protein containing PQ loop repeat 2, is an amino acid transporter on the lysosome. It is characterized by seven transmembrane helices and a highly conserved proline-glutamine dipeptide repeat motif. It primarily transports cationic amino acids such as lysine, histidine, and arginine, regulating cellular amino acid metabolism and serving as an important therapeutic target for cysteine ​​disorders caused by lysosomal dysfunction. Current research indicates that the PQLC2 gene is highly expressed in cancer cells from various cancer patients, participating in glucose homeostasis and insulin signaling, regulating cellular glucose uptake, and significantly correlated with Akt and ERK phosphorylation levels, thus promoting tumor cell development. However, the functions of PQLC2 and its target proteins are not fully understood; therefore, research on PQLC2 could contribute to the development of potential novel targets for cancer therapy.

[0005] This invention first detects the expression level of the PQLC2 gene in liver cancer tissue, and then studies the effect of downregulation of PQLC2 expression on the expression levels of related proliferation and migration genes and tumor occurrence and development. The aim is to provide new drug targets for the treatment of liver cancer, which has potential value for improving the diagnosis rate of liver cancer and improving the survival status of liver cancer patients. Summary of the Invention

[0006] Objectives of the Invention: One objective of this invention is to provide the application of the PQLC2 gene in the preparation of drugs for treating liver cancer. Another objective of this invention is to provide the application of the PQLC2 gene in the preparation of diagnostic kits for liver cancer.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] 1. Application of PQLC2 gene (NCBI Gene ID: 54896) in the preparation of diagnostic kits for liver cancer.

[0009] 2. Application of the PQLC2 gene in the preparation of drugs for the treatment of liver cancer. The drugs are designed with the PQLC2 gene as a target.

[0010] 3. The inhibitor of the PQLC2 gene is a small interfering RNA (siPQLC2), with the sequence: 5'-GGAUCUCCUACUCUCUGUUTT-3'.

[0011] 4. Application of PQLC2 gene inhibitors in the preparation of drugs for inhibiting liver cancer.

[0012] 5. Genes that interfere with PQLC2 expression include knocking out or silencing the PQLC2 encoding gene, inhibiting or reducing the transcriptional and translational functions of PQLC2, and one or more genes that interfere with the function of the PQLC2 protein; the vector is a viral vector or a non-viral gene silencing vector.

[0013] 6. The viral vector is an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a herpesvirus vector.

[0014] 7. The non-viral gene silencing vector is a CRISPR / Cas9 system gene knockout vector, an RNAi system gene silencing vector, or a vector modified based thereon.

[0015] Beneficial Effects: This invention demonstrates through extensive experimental data that PQLC2 plays a crucial role in the development of liver cancer. Specific siRNA sequences were used to efficiently inhibit PQLC2 gene expression in human hepatocellular carcinoma cell lines. Cell proliferation assays, cell scratch assays, and real-time quantitative PCR showed that knocking down PQLC2 gene expression significantly inhibited the proliferation and migration of liver cancer cells, indicating that PQLC2 can serve as a diagnostic biomarker for liver cancer. Therefore, the PQLC2 gene has broad applications in the preparation of drugs for treating liver cancer and in liver cancer diagnostic kits. Attached Figure Description

[0016] Figure 1 This is a graph showing the differential expression of PQLC2 in hepatocellular carcinoma tissues and normal tissues from patients with hepatocellular carcinoma in the TCGA database.

[0017] Figure 2 This study analyzed the changes in intracellular PQLC2 gene mRNA levels in the HCCLM3 hepatocellular carcinoma cell line after transfection with a small interfering RNA (siPQLC2) targeting PQLC2. 18S rRNA was used as an internal control gene for PQLC2.

[0018] Figure 3 For real-time quantitative PCR analysis: A: Changes in intracellular CCND1 gene mRNA levels 448 hours after HCCLM3 hepatocellular carcinoma cells were transfected with siPQLC2; B: Changes in intracellular MMP14 gene mRNA levels 448 hours after HCCLM3 hepatocellular carcinoma cells were transfected with siPQLC2; C: Changes in intracellular p21 gene mRNA levels 448 hours after HCCLM3 hepatocellular carcinoma cells were transfected with siPQLC2; D: Changes in intracellular BIM gene mRNA levels 448 hours after HCCLM3 hepatocellular carcinoma cells were transfected with siPQLC2.

[0019] Figure 4MTT cell proliferation assay: Cell proliferation of HCCLM3 hepatocellular carcinoma cell line transfected with siPQLC2 within 96 hours.

[0020] Figure 5 For cell scratch assay; the healing status of HCCLM3 liver cancer cells transfected with siPQLC2 within 72 hours. Detailed Implementation

[0021] The interference with PQLC2 gene expression described in this invention includes knocking out the PQLC2 coding gene, interfering with the transcription or translation of the PQLC2 gene, and interfering with the entire biological process of PQLC2 protein function. Although the specific mechanism of interference is not yet fully understood, it does not prevent the realization of "interference".

[0022] In some specific embodiments, the drug may be combined with one or more pharmaceutically acceptable adjuvants, including but not limited to granules, buffers, surfactants and other recognized pharmaceutical adjuvants.

[0023] In some specific embodiments, the drug may be formulated as, but is not limited to, microinjection formulations or transfection-suitable formulations, which may be prepared according to conventional methods in the pharmaceutical field.

[0024] The main materials used in this invention and their specific sources are shown in the table below. Unless otherwise specified, the cell lines and reagents used in this invention can be obtained commercially. The invention will be further described below through specific embodiments. However, it should be understood that the method is merely exemplary and not a limitation of the invention. Materials similar to or the same as the types, models, performance, or functions of the cells, instruments, and reagents listed below can be used in the implementation of this invention.

[0025] Unless otherwise specified, the methods in the following examples are all ordinary methods.

[0026] Main materials:

[0027]

[0028] I. Differential expression analysis of PQLC2 in hepatocellular carcinoma patients and normal tissues from the TCGA database

[0029] Genomic statistical analysis of PQLC2 mRNA expression levels in hepatocellular carcinoma (HCC) tissues and normal tissues from HCC patient samples in the online TCGA database was performed using the Xena functional genomics browser, and the significant differences were calculated. The samples included 46 normal tissue samples and 350 HCC tissue samples.

[0030] Results: Compared with expression in normal liver tissue, the expression level of PQLC2 was significantly upregulated in hepatocellular carcinoma tissue (P<0.0001). This indicates that PQLC2 is highly expressed in hepatocellular carcinoma tissue and is a potential factor promoting liver cancer development.

[0031] II. Analysis of PQLC2 expression levels in liver cancer

[0032] 1. Cell Culture and Seeding Plates

[0033] Human hepatocellular carcinoma cell line HCCLM3 was cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, and streptomycin. Cells were cultured at 37°C under 5% (v / v) CO2 conditions. Cells were sporadically increased at a rate of 1.0 × 10⁶ cells / year. 5 Seeds were administered at a concentration of 100 μL / mL in 96-well cell culture plates for MTT assay to determine cell proliferation; then, at a concentration of 2 × 10⁶ cells / mL, were added to each well. 5 The cells were seeded at a concentration of 1.5 × 10⁶ cells / mL in 24-well plates, 500 μL per well, for cell scratch assay to determine cell migration ability; 5 A concentration of 1000 RNA molecules per mL was seeded in 6-well plates, 2 mL per well, for RNA extraction.

[0034] 2. Cell transfection

[0035] When HCCLM3 cells adhered and grew to cover 60%-70% of the bottom area of ​​a 10cm disc, siRNA was transfected into the cells. A si Negative Controll negative control group and a siPQLC2 treatment group were set up for transfection. The transfection concentration was 50 nM, and the transfection reagent used was Lipofectamine 2000. The transfection method was performed according to the manufacturer's instructions. Before transfecting with siRNA, the cell culture medium was replaced with incomplete medium, and after 6-8 hours, it was replaced with complete medium for continued culture.

[0036] 3. RNA extraction

[0037] After transfection and culturing cells for 48 hours, the culture medium was discarded, and 1 mL of Trizol reagent was added to lyse the cells. The lysed cells were transferred to a 1.5 mL centrifuge tube, and 100 μL of BCP solution was added for extraction. The mixture was vortexed for 15 seconds and then allowed to stand for 8 minutes. The cells were centrifuged at 12000 g for 15 minutes at 4°C. 500 μL of the supernatant was transferred to a new centrifuge tube, and 500 μL of isopropanol was added. After standing for 10 minutes, the cells were centrifuged at 12000 g for 10 minutes at 4°C to obtain RNA precipitate. The supernatant was collected, and the RNA was washed twice with 500 μL of 75% (v / v) DEPC ethanol solution. The supernatant was discarded, and the cells were air-dried at room temperature for 40 minutes. Depending on the amount of RNA, 15–30 μL of nuclease-free water was added, and the cells were dissolved in a 55°C metal bath for 10 minutes. The concentration of the RNA sample and the OD260 and OD280 absorbance values ​​were measured using a Nano Drop 2000 spectrophotometer. Generally, an A260 / A280 ratio between 1.8 and 2.0 is considered a good quality for RNA samples. After mixing the RNA sample, centrifuge and store at -80°C for long-term storage.

[0038] 4. Reverse transcription of cDNA

[0039] Using the Vazyme reverse transcription kit, load 3000 ng of RNA, 1 μL each of 20 μM Oligo(dT) and primers, and add nuclease-free water to a total volume of 10 μL into the corresponding PCR eight-row tubes. Incubate at 65°C for 10 min. Following the kit instructions, add 5×HiScript II Select qRTSuperMix and nuclease-free water, incubate at 50°C for 15 min, then at 85°C for 5 sec. After the reaction is complete, store at -80°C.

[0040] 5. Detection of intracellular PQLC2 gene mRNA levels

[0041] Using cDNA as a template, PQLC2 forward and reverse primers and 2×SYBR Select Master Mix were added for real-time quantitative PCR. Control group samples were serially diluted to serve as the standard curve. The reaction conditions were 50℃ for 2 min; 95℃ for 10 min, 95℃ for 1 s, 60℃ for 1 min, repeated for 40 cycles. Relative quantification was performed based on the standard curve, corrected using the internal reference gene 18S rRNA, and differences in PQLC2 content among different samples were compared. The 18S forward primer used was as described in Sequence Listing ID NO: 1; the 18S reverse primer was as described in Sequence Listing ID NO: 2; the PQLC2 forward primer was as described in Sequence Listing ID NO: 3; and the PQLC2 reverse primer was as described in Sequence Listing ID NO: 4.

[0042] Result: As Figure 2 As shown in the results, real-time quantitative PCR revealed that the mRNA level of PQLC2 in the siPQLC2 transfected group was knocked down by more than 80% compared with the control group (P<0.001), proving that siPQLC2 had been successfully transfected into cells and caused a decrease in PQLC2 content. This result indicates that the expression level of PQLC2 in liver cancer cells can be reduced by exogenous interference.

[0043] III. Interference with the PQLC2 gene can inhibit the expression of genes related to proliferation and migration in liver cancer cells.

[0044] HCCLM3 cells were seeded into 6-well plates and transfected after cell adhesion. A siNC negative control group and a siPQLC2 interference group were set up. RNA was extracted 48 h after transfection, its concentration was measured, cDNA was reverse transcribed, and then real-time quantitative PCR was performed. The levels of CCND1, MMP14, p21, and BIM mRNA were compared between the two groups using the 18S internal reference gene for correction. The 18S forward primer used is as described in sequence listing ID NO: 1; the 18S reverse primer is as described in sequence listing ID NO: 2; the CCND1 forward primer used is as described in sequence listing ID NO: 5; the CCND1 reverse primer is as described in sequence listing ID NO: 6; the MMP14 forward primer used is as described in sequence listing ID NO: 7; the MMP14 reverse primer is as described in sequence listing ID NO: 8; the p21 forward primer used is as described in sequence listing ID NO: 9; the p21 reverse primer is as described in sequence listing ID NO: 10; the BIM forward primer used is as described in sequence listing ID NO: 11; the BIM reverse primer is as described in sequence listing ID NO: 12.

[0045] Result: As Figure 3 As shown in Figure A, the mRNA level of CCND1 was inhibited in the PQLC2 interference group compared with the control group (P < 0.001), indicating that PQLC2 knockdown can downregulate the expression of CCND1 in cells, thereby blocking the cell cycle from G1 phase to S phase and inhibiting cell proliferation. Figure 3 As shown in Figure B, the mRNA level of MMP14 was significantly increased in the PQLC2 interference group compared with the control group (P < 0.01), indicating that PQLC2 knockdown can promote the expression of MMP14 in cells and inhibit cell invasion and migration. Figure 3 As shown in Figure C, the p21 mRNA level was significantly lower in the PQLC2 interference group compared to the control group (P < 0.0001), indicating that PQLC2 knockdown can downregulate p21 expression in cells, inhibit cell cycle progression and DNA replication, thereby inhibiting cell proliferation. Figure 3As shown in Figure D, the mRNA level of BIM was significantly lower in the PQLC2 interference group compared with the control group (P < 0.001), indicating that PQLC2 knockdown can downregulate the expression of BIM in cells, promote apoptosis, and thus inhibit cell proliferation.

[0046] IV. Interference with the PQLC2 gene can inhibit the proliferation of liver cancer cells.

[0047] Healthy HCCLM3 cells were seeded into 96-well plates and transfected after cell adhesion as described in Example 2. A siNC negative control group and a siPQLC2 interference group were set up. 24 hours after transfection, 10 μL of MTT was added. MTT reacts with succinate dehydrogenase in live cells to reduce it to a blue-purple crystalline substance called formazan. After 4 hours of sufficient reaction, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. The absorbance at 450 nm was measured using a microplate reader and recorded as the 0h group. Measurements were then taken every 24 hours, and the OD values ​​at 0h, 24h, 48h, 72h, and 96h were obtained. Cell proliferation curves were plotted based on the OD values.

[0048] Result: As Figure 4 As shown, at 96 h, the growth rate of the siPQLC2 transfected group was significantly lower than that of the control group (P<0.01); at 96 h, it was clearly observed that the number of cells in the siPQLC2 transfected group was significantly reduced compared to the control group, indicating that cell proliferation was inhibited, while the siNC control group cells still grew rapidly. These results indicate that knocking down the PQLC2 gene can effectively inhibit the proliferation of liver cancer cells.

[0049] V. Interference with the PQLC2 gene can inhibit the migration of liver cancer cells.

[0050] Healthy HCCLM3 cells were seeded into 24-well plates. After cell adhesion was achieved as described in Example 2, transfection was performed. Wells were randomly divided into a siNC negative control group and a siPQLC2 interference group. A scratch assay was performed 24 hours later: a straight line was drawn on a monolayer of cells using a 200 μL sterile pipette tip. The cells were washed twice with PBS to remove detached cells, and the medium was replaced with complete culture medium. A smooth scratch location was selected and marked under a 4x microscope and photographed as 0h. The cells were then incubated at 37°C. After 72 hours, the cells were removed, the medium was replaced, and photographs were taken at the same location to observe and measure the healing rate of the scratch area, thereby assessing the migration and repair capabilities of each group of cells.

[0051] Result: As Figure 5As shown, at 72 hours after transfection, cells transfected with siNC had completely healed, while the scratches on cells transfected with siPQLC2 had not completely healed, showing a significant difference. This result indicates that knocking down the PQLC2 gene can effectively inhibit the migration ability of liver cancer cells.

[0052] Statistical analysis: All data are the average of three parallel independent experiments. Standard deviation analysis was performed. P < 0.05 was considered statistically significant and was marked as *; P < 0.01 was marked as **; P < 0.001 was marked as ***; P < 0.0001 was marked as ****.

[0053] The scope of protection sought by this invention is not limited to the description of the specific embodiments.

[0054] sequence list

[0055]

Claims

1. The application of PQLC2 gene inhibitors in the preparation of drugs for inhibiting liver cancer, characterized in that, The inhibitor is a small interfering RNA siPQLC2, with the sequence: 5'-GGAUCUCCUACUCUCUGUUTT-3'.

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