Application of RGS14 in preparing a kit for auxiliary diagnosis or prognosis of liver cancer and application of an RGS14 expression inhibitor in preparing a drug for treating liver cancer

Abstract

The present invention discloses the application of RGS14 in the preparation of a kit for the auxiliary diagnosis or prognosis of liver cancer and the application of an RGS14 expression inhibitor in the preparation of a drug for treating liver cancer. The present invention discovers for the first time that the expression of RGS14 in liver cancer is significantly increased compared with that in adjacent tissues, and the high expression of RGS14 is not conducive to the overall survival of liver cancer patients; and when RGS14 is knocked down, the colony formation and migration of liver cancer cells are significantly inhibited. The high expression of RGS14 is closely related to the poor prognosis of liver cancer patients, suggesting that RGS14 can be used as a prognostic marker for liver cancer, providing effective information for the prognostic evaluation and efficacy monitoring of liver cancer. The present invention also provides an RGS14 expression inhibitor siRNA, which has good interference effect when used for inhibiting the expression of RGS14 and has potential for clinical application.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of RGS14 in the preparation of a kit for the auxiliary diagnosis or prognosis of liver cancer and the application of an RGS14 expression inhibitor in the preparation of a drug for treating liver cancer. Background Art

[0002] Liver cancer is one of the major challenges in the global health field. Its incidence ranks sixth among all malignant tumors, and the mortality rate ranks fourth, making it the second leading cause of cancer-related deaths. Hepatocellular carcinoma (HCC) accounts for approximately 90% of liver cancer cases, showing invasive growth, metastasis, and resistance to standard treatments, resulting in extremely poor prognosis. In China, the five-year survival rate of liver cancer patients is only 14.1%, fully reflecting the severity of this disease. The progression of HCC is a complex process influenced by multiple innate and acquired factors. In recent years, researchers have made significant progress in understanding the mechanisms of HCC progression, including dysregulation of signaling pathways and remodeling of the extracellular matrix (ECM). However, although current treatment methods such as surgical resection combined with radiotherapy and chemotherapy are considered the most effective means for treating early-stage liver cancer, their efficacy is still very limited, and the postoperative recurrence rate is relatively high, resulting in extremely poor overall prognosis for liver cancer patients. This situation highlights the urgent need to better understand the molecular mechanisms of HCC and strengthen the research on new therapeutic targets for liver cancer in order to improve the prognosis of patients and increase the survival rate.

[0003] The regulator of G protein signaling (RGS) family consists of four subfamilies: RGS A / RZ, RGS B / R4, RGS C / R7, and RGSD / R12. Recent studies have shown that the dysregulation of RGS protein expression is closely related to the development of multiple cancer types. Among them, Rgs16 has been found to promote the exhaustion of anti-tumor CD8 T cells in tumors, becoming an important potential target for improving cancer treatment. At the same time, RGS17 is upregulated in patients with lung adenocarcinoma, prostate cancer, and hepatocellular carcinoma (HCC), indicating its importance in these cancers. These findings suggest that RGS proteins may serve as potential prognostic predictors and can be used as precision treatment targets for HCC. Therefore, it is particularly important to further study the functions and mechanisms of RGS proteins related to HCC.

[0004] In this context, regulator of G protein signaling 14 (RGS14), as an important multifunctional signaling protein in the RGSD / R12 family, its potential biological functions have not been fully studied. RGS14 has been confirmed to be related to learning and memory processes, cardiac remodeling, and ischemia-reperfusion injury. There is currently no report on its exact function in HCC. Summary of the Invention

[0005] To address the deficiencies in the prior art, the primary objective of the present invention is to provide the use of RGS14 in the preparation of a kit for the auxiliary diagnosis or prognosis of liver cancer.

[0006] Another objective of the present invention is to provide the use of an RGS14 expression inhibitor in the preparation of a drug for treating liver cancer.

[0007] Yet another objective of the present invention is an RGS14 expression inhibitor and its application.

[0008] The objective of the present invention is achieved through the following technical solutions: The use of RGS14 in the preparation of a kit for the auxiliary diagnosis or prognosis of liver cancer is based on the first discovery in the present invention that the expression of RGS14 in liver cancer is significantly higher than that in adjacent tissues, and high expression of RGS14 is not conducive to the overall survival of liver cancer patients.

[0009] The RGS14 mentioned above is the regulator of G-protein signaling 14, and the sequence of its encoding gene is as shown in GenBank NM_006480.

[0010] The use of RGS14 in the preparation of a kit for the auxiliary diagnosis or prognosis of liver cancer is to detect the expression level of RGS14 in the liver tissue to be tested. Compared with the expression level of RGS14 in normal liver tissue, the higher the expression level of RGS14, the greater the possibility of liver cancer; the higher the expression level of RGS14 in liver cancer tissue, the worse the prognosis.

[0011] The kit mentioned above contains reagents for detecting the expression level of RGS14 protein; preferably, it is an RGS14 antibody.

[0012] The use of an RGS14 expression inhibitor in the preparation of a drug for treating liver cancer is based on the further discovery in the present invention that knocking down the expression of RGS14 reduces the colony formation rate and cell proliferation rate of liver cancer cells.

[0013] The RGS14 expression inhibitor mentioned above includes molecules that interfere with the expression of RGS14; preferably, it is RGS14 siRNA.

[0014] An RGS14 expression inhibitor includes RGS14 siRNA; preferably, it includes siRNA with the following sequences:

[0015] Sense strand: 5'-GACCUGGUACUUCCAGAAUUUTT-3';

[0016] Antisense strand: 5'-AAAUUCUGGAAGUACCAGGUCTT-3'.

[0017] The above-mentioned RGS14 expression inhibitor is used in the preparation of a drug for treating liver cancer.

[0018] The present invention has the following advantages and effects compared with the prior art:

[0019] (1) The present invention first discovers that the expression of RGS14 in liver cancer is significantly increased compared with that in adjacent tissues (P < 0.001). The high expression of RGS14 is not conducive to the overall survival of liver cancer patients (P = 0.019). When knocking down RGS14, the colony formation and migration of liver cancer cell Huh7 are significantly inhibited. The high expression of RGS14 is closely related to the poor prognosis of liver cancer patients, suggesting that RGS14 can be used as a prognostic marker for liver cancer, providing effective information for the prognostic evaluation and efficacy monitoring of liver cancer. Therefore, RGS14 can be used as a new biological target for guiding the treatment of liver cancer.

[0020] (2) The present invention provides an siRNA for inhibiting the expression of RGS14, which has good interference effect when used for inhibiting the expression of RGS14 and has the potential for clinical application. Description of the Drawings

[0021] Figure 1 It is a result diagram of the expression of RGS14 in liver cancer analyzed by database and immunofluorescence; among them, A is the result of analyzing the expression level of RGS14 in liver cancer tissues and adjacent tissues by TCGA data; B is the result of detecting the expression level of RGS14 in liver cancer tissues and adjacent tissues by immunofluorescence; *** indicates P < 0.001, and ** indicates P < 0.01.

[0022] Figure 2 It is a result diagram of the influence of high expression of RGS14 on the survival of liver cancer patients analyzed by TCGA data; among them, A is the analysis result of the overall survival period, and B is the analysis result of the progression-free survival period.

[0023] Figure 3 It is a result diagram of detecting the protein level of RGS14 in the cell line with knocked-down RGS14; among them, A is the photo diagram, and B is the statistical result diagram.

[0024] Figure 4 It is a result diagram of the influence on the proliferation of Huh7 cells after knocking down RGS14; among them, A is the photo and statistical result of colony formation; B is the cell proliferation curve.

[0025] Figure 5 It is a result diagram that the migration ability of Huh7 cells significantly decreases after knocking down RGS14; among them, A is the number of migrated cells in each region; B is the number of invaded cells in each region. Detailed Embodiments

[0026] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0027] Embodiment 1

[0028] This example is used to illustrate the changes in the expression level of RGS14 in liver cancer by public database and immunofluorescence analysis.

[0029] (1) TCGA data analysis

[0030] Enter the TCGA official website (https: / / cancergenome.nih.gov), use the GDC client tool on the official website to download the HTSeq-FPKM data in liver cancer (LIHC) RNA-Seq, and obtain an expression matrix containing sample numbers and gene HTSeq-FPKM data. Use GraphPad Prism 8.0 to analyze the expression data of RGS14 in the HTSeq-FPKM matrix of liver cancer tissues and adjacent tissues and plot the graph. The statistical analysis method is the Mann-Whitney U test. The analysis results are as shown in Figure 1 A in it. It can be seen that the expression level of RGS14 in liver cancer tissues is significantly higher than that in adjacent tissues, P < 0.001.

[0031] (2) Immunofluorescence experiment

[0032] For immunofluorescence analysis, liver cancer tissues and adjacent tissues are cut, embedded in Tissue-Tek (Sakura Finetek), quickly frozen in isopentane, and stored at -80 °C. Prepare 10-μm liver cancer tissue sections and fix the tissue sections in 4% paraformaldehyde / PBS solution for 3 minutes. After fixation, rinse thoroughly with PBS to remove residual paraformaldehyde. Next, treat the tissue sections with 50% acetone, the treatment time with 100% acetone is 2 minutes, and then treat with 50% acetone again. Finally, rinse with PBS. After treating the tissue sections, first soak them in PBS for hydration for 10 minutes. Then aspirate the PBS and incubate with the primary antibody. Place the primary antibody, anti-RGS14 monoclonal antibody (clone number 1C1B2, diluted 1:200; purchased from Thermo Fisher Scientific, catalog number 67394-1-IG) diluted in PBS / 0.25% BSA in a wet box and incubate overnight at 4 °C. After incubation, aspirate the primary antibody and rinse with PBS 3 times, 5 minutes each time, to ensure the removal of unbound primary antibody. Next, use the fluorescently labeled secondary antibody Cy TM 3 AffiniPure TMIncubate with Donkey Anti-Mouse IgG(H+L) (diluted 1:200, catalog number 715-165-150, Jackson Immunoresearch) and Anti-Actin, α-Smooth Muscle-FITC antibody (SMA-FITC, clone: 1A4, diluted 1:200, catalog number: F3777-.2ML, purchased from Merck) for 60 minutes. At the same time, perform nuclear staining with DAPI (diluted 1:100), and note that light should be avoided throughout the process. Finally, rinse with PBS three times again, 5 minutes each time, and ensure that light is also avoided during this process. After all steps are completed, soak the sections in PBS for 10 minutes, mount with Fluoromount G, and perform subsequent photography with a confocal laser.

[0033] The results are as Figure 1 shown in B of

[0034] Figure 1 : DAPI is used for nuclear staining, SMA is used for blood vessel staining, the RGS14 antibody binds to RGS14, and it can be seen that RGS14 is highly expressed at the protein level in liver cancer tissues.

[0035] Example 2

[0036] This example is used to illustrate the relationship between the high expression of RGS14 analyzed by TCGA data and the prognosis of liver cancer patients.

[0037] TCGA data analysis process:

[0038] Enter the TCGA official website (https: / / cancergenome.nih.gov), use the GDC client tool to download the clinical data (clinical) of liver cancer (LIHC) to obtain the matrix of sample numbers and various clinical data. Extract the HTSeq-FPKM data of RGS14 in each sample, the survival status and survival days data of the samples. Use the Kaplan-Meier method to obtain the survival curve, and perform a log-rank test on the differences in the survival curves. P < 0.01 indicates statistical significance.

[0039] The results are as Figure 2 shown: The high expression of RGS14 is not conducive to the overall survival (P = 0.019) and progression-free survival (P = 0.02) of liver cancer patients.

[0040] Example 3

[0041] This embodiment provides an siRNA sequence for knocking down RGS14 in a cell line, specifically the Huh7 liver cancer cell line. The Huh7 liver cancer cells were purchased from Wuhan Punosai Life Science Co., Ltd. and cultured in DMEM medium containing 10% v / v fetal bovine serum at 37°C and 5% CO 2 under the following conditions.

[0042] 1. The construction method is as follows:

[0043] Design three siRNAs for negative control siRNA and interfering with RGS14 expression, specifically as follows:

[0044] Negative control siRNA sequence:

[0045] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3';

[0046] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3';

[0047] siRGS14-SEQ1 sequence:

[0048] Sense strand: 5'-GACCUGGUACUUCCAGAAUUUTT-3';

[0049] Antisense strand: 5'-AAAUUCUGGAAGUACCAGGUCTT-3';

[0050] siRGS14-SEQ2 sequence:

[0051] Sense strand: 5'-CUUGAUGAAGUUCGACAGCUATT-3';

[0052] Antisense strand: 5'-UAGCUGUCGAACUUCAUCAAGTT-3’;

[0053] siRGS14-SEQ3 sequence:

[0054] Sense strand: 5'-CCCACCUAGAACUCAGGAUAATT-3’;

[0055] Antisense strand: 5'-UUAUCCUGAGUUCUAGGUGGGTT-3'.

[0056] 2. Operate according to the instructions of the liposome kit. One day before transfection, seed Huh7 cells in an appropriate amount of growth medium without antibiotics, and make the cell confluence reach 80 - 90% at the time of transfection. Replace the cell medium (without double antibiotics and without serum) before transfection. Dilute siRNA with opti - mem, and dilute lip2000 (Invitrogen) with opti - mem as well. After mixing the two and standing for 2 - 3 minutes, mix them in the same tube. Avoid rough pipetting when mixing, and stand for 3 - 5 minutes. Then add the prepared transfection solution to the cell culture plate and gently shake to mix evenly. Replace the medium (change the serum - free medium to the complete medium) in a timely manner 6 hours after siRNA transfection of the cells. After successful transfection for 24 - 48 hours, take the cells out of the incubator for subsequent relevant detections.

[0057] 3. Detection of gene knockdown effect

[0058] For the total protein extracted from the cells, after separation by SDS - polyacrylamide gel using protein lysis buffer, immunoblot analysis was performed using the primary antibody against RGS14 (diluted 1:5000, catalog number 16258 - 1 - AP, purchased from proteintech) and the primary antibody against GAPDH (diluted 1:4000, catalog number HC301 - 01, purchased from TransGen). Each experiment was repeated three times.

[0059] The results are as Figure 3 shown: The efficiency of RGS14 silencing in the HuH7 cell line was detected by Western blot technology. It can be seen that compared with the control group cells, the siRGS14 - SEQ1 sequence can inhibit the protein expression of RGS14, indicating that RGS14 knockdown was successful in the cell line, and this method can screen out hepatocellular carcinoma cells with low expression of RGS14.

[0060] Experimental example 4

[0061] In this example, the cell line with RGS14 knockdown constructed in Example 3 was used to detect the effects of RGS14 on the proliferation, invasion and migration of hepatocellular carcinoma cells.

[0062] 1. The specific detection contents are as follows:

[0063] (1) Cell colony formation assay

[0064] Use high - glucose DMEM medium to prepare siNC - transfected Huh7 cells and siRGS14 - SEQ1 - transfected Huh7 cells into concentrations of 2×10 3A suspension of cells at a density of [X] cells / mL was added to a 6-well plate at a volume of 2 mL / well. After the cells grew into visible cell clusters, the culture medium was removed, and the cells were fixed with methanol for 15 minutes, then washed twice with PBS and air-dried. Subsequently, the cells were stained with 0.5% w / v crystal violet solution for 10 minutes, washed with ultrapure water, air-dried, and photographed.

[0065] (2) Cell proliferation assay

[0066] siNC-transfected Huh7 cells and siRGS14-SEQ1-transfected Huh7 cells were seeded into 96-well plates at a density of 2×10 3 cells / well and cultured under normal conditions for 5 days. At the end of each time point (1 day, 2 days, 3 days, 4 days, or 5 days), 10 μL of CCK-8 (MedChemExpress, HY-K0301) was added to each well, and then the plate was incubated at 37 °C for 2 hours. Cell viability was evaluated by measuring the absorbance at a wavelength of 540 nm to obtain the cell proliferation curve.

[0067] (3) Cell migration and invasion assays

[0068] To evaluate cell invasion and migration, cell culture inserts (pore size 8 μm, Corning) coated with / without Matrigel (BD Biosciences) were used. siNC-transfected Huh7 cells and siRGS14-SEQ1-transfected Huh7 cells were collected and suspended in serum-free medium. 5×10 3 cells were placed in the upper chamber of the cell culture insert. The lower chamber was filled with DMEM supplemented with 20% v / v FBS and incubated for 24 hours. After the cells migrated from the upper chamber to the lower chamber, the membrane was stained, and the number of migrated cells was quantified by examining five randomly selected areas on the lower membrane.

[0069] 2. Results:

[0070] The results of cell colony formation are shown in Figure 4 A, indicating that the number of clones formed by the cell line with knocked-down RGS14 was less than that of the control cell line; the results of the cell proliferation assay are shown in Figure 4 B, indicating that the proliferation rate of the cell line with knocked-down RGS14 was lower than that of the control cell line. Figure 4 This indicates that the expression of RGS14 has a significant effect on the proliferation of liver cancer cells.

[0071] Figure 5 The results of cell migration and invasion assays of the cell line with knocked-down RGS14 and the control cell line are shown. Figure 5It can be seen that after knocking down RGS14 in liver cancer cells, their migration and invasion abilities are significantly reduced, indicating that the migration and invasion of liver cancer cells are significantly correlated with the expression of RGS14. This shows that RGS14 is involved in the occurrence and development of liver cancer, and can be used as an indicator for clinical efficacy monitoring and prognosis evaluation of liver cancer, providing a strong basis for the diagnosis and targeted treatment of liver cancer and improving the survival rate of patients.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of RGS14 protein in the preparation of a kit for auxiliary diagnosis or prognosis of liver cancer.

2. The use according to claim 1, characterized in that: The reagent for detecting the expression amount of RGS14 protein includes RGS14 antibody.

3. Use of an RGS14 expression inhibitor in the preparation of a drug for treating liver cancer, characterized in that: The RGS14 expression inhibitor includes siRNA with the following sequence: Sense strand: 5′-GACCUGGUACUUCCAGAAUUUTT-3′; Antisense strand: 5′-AAAUUCUGGAAGUACCAGGUCTT-3′.

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