Application of RGS14 in preparation of kit for auxiliary diagnosis or prognosis of liver cancer and application of RGS14 expression inhibitor in preparation of medicine for treating liver cancer

Abstract

The invention discloses application of RGS14 in preparation of a kit for auxiliary diagnosis or prognosis of liver cancer and application of an RGS14 expression inhibitor in preparation of a medicine for treating liver cancer. It is found for the first time that RGS14 expression in liver cancer is remarkably improved compared with para-carcinoma tissue, and high RGS14 expression is not beneficial to overall survival of liver cancer patients; and when the RGS14 is knocked down, the clone formation and migration of the liver cancer cells are obviously inhibited. The high expression of the RGS14 is closely related to the poor prognosis of the liver cancer patient, which prompts that the RGS14 can be used as a prognosis marker of the liver cancer, and effective information is provided for prognosis evaluation and curative effect monitoring of the liver cancer. The invention also provides an RGS14 expression inhibitor siRNA which has a good interference effect when being used for inhibiting the expression of RGS14 and has clinical application potential.

Description

Technical Field

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

[0002] Liver cancer is one of the major challenges facing global health. Its incidence ranks sixth among all malignant tumors, and its mortality ranks fourth, making it the second leading cause of cancer-related death. Hepatocellular carcinoma (HCC) accounts for approximately 90% of liver cancer cases. It exhibits aggressive growth, metastasis, and resistance to standard treatment, resulting in a very poor prognosis. In my country, the five-year survival rate of liver cancer patients is only 14.1%, which fully reflects the severity of this disease. The progression of HCC is a complex process affected 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 treatments such as surgical resection combined with chemoradiotherapy are considered to be the most effective means of treating early liver cancer, their efficacy is still very limited, and the postoperative recurrence rate is high, resulting in an overall very poor prognosis for liver cancer patients. This situation highlights the urgent need to better understand the molecular mechanisms of HCC and strengthen research on new therapeutic targets for liver cancer in order to improve patient prognosis and increase survival rates.

[0003] The RGS (regulator of G protein signaling) family consists of four subfamilies: RGS A / RZ, RGS B / R4, RGS C / R7, and RGSD / R12. Studies in recent years have shown that dysregulated expression of RGS proteins is closely related to the development of various cancer types. Among them, Rgs16 was 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 therapeutic targets for HCC. Therefore, it is particularly important to further study the functions and mechanisms of RGS proteins associated with HCC.

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

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

[0006] Another object 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] Another object of the present invention is to provide an RGS14 expression inhibitor and its application.

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

[0009] The RGS14 is a G protein signal regulator 14, and the sequence of its coding gene is shown in GenBank NM_006480.

[0010] The application of RGS14 in the preparation of a kit for 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 comprises a reagent for detecting the expression level of RGS14 protein; preferably an RGS14 antibody.

[0012] The use of the RGS14 expression inhibitor in the preparation of drugs for treating liver cancer is based on the present invention's discovery that knocking down the expression of RGS14 reduces the cloning rate of liver cancer cells and reduces the cell proliferation rate.

[0013] The RGS14 expression inhibitor includes a molecule that interferes with the expression of RGS14; preferably, it is RGS14 siRNA.

[0014] An RGS14 expression inhibitor, comprising RGS14 siRNA; preferably comprising an siRNA having the following sequence:

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

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

[0017] Use of the above RGS14 expression inhibitor in the preparation of drugs for treating liver cancer.

[0018] Compared with the prior art, the present invention has the following advantages and effects:

[0019] (1) The present invention first discovered that RGS14 expression in liver cancer was significantly higher than that in adjacent tissues (P < 0.001), and that high expression of RGS14 was detrimental to the overall survival of liver cancer patients (P = 0.019). When RGS14 was knocked down, the cloning and migration of liver cancer cells Huh7 were significantly inhibited. High expression of RGS14 was 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 prognosis assessment and efficacy monitoring of liver cancer. Therefore, RGS14 can be used as a new biological target to guide the treatment of liver cancer.

[0020] (2) The present invention provides an RGS14 expression inhibitor siRNA, which has a good interference effect when used to inhibit the expression of RGS14 and has potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The following are the results of database and immunofluorescence analysis of RGS14 expression in liver cancer; A is the result of TCGA data analysis of RGS14 expression in liver cancer tissue and adjacent tissue; B is the result of immunofluorescence detection of RGS14 expression in liver cancer tissue and adjacent tissue; *** indicates P < 0.001, ** indicates P < 0.01.

[0022] Figure 2 This is the result of TCGA data analysis of the impact of high expression of RGS14 on the survival of liver cancer patients; among them, A is the analysis result of overall survival, and B is the analysis result of progression-free survival.

[0023] Figure 3 The graphs are the results of protein level detection of RGS14 in the cell lines with RGS14 knockdown; A is a photograph and B is a statistical result graph.

[0024] Figure 4 The figure shows the effect of knocking down RGS14 on Huh7 cell proliferation; A is the photo and statistical results of clone formation; B is the cell proliferation curve.

[0025] Figure 5 This is a graph showing the significant decrease in the migration ability of Huh7 cells after knocking down RGS14; A is the number of migrating cells in each region; B is the number of invading cells in each region. DETAILED DESCRIPTION

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

[0027] Example 1

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

[0029] (1) TCGA data analysis

[0030] Go to the TCGA official website (https: / / cancergenome.nih.gov), use the GDC client tool on the official website to download the HTSeq-FPKM data in the liver cancer (LIHC) RNA-Seq, and obtain the expression matrix containing the sample number 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 tissue and adjacent tissue and draw a graph. The statistical analysis method is the Mann-Whitney U test. The analysis results are shown in Figure 2. Figure 1 As shown in A, it can be seen that the expression level of RGS14 in liver cancer tissues was significantly higher than that in adjacent tissues, P < 0.001.

[0031] (2) Immunofluorescence experiment

[0032] For immunofluorescence analysis, HCC tissues and paracancerous tissues were cut, embedded in Tissue-Tek (Sakura Finetek), quickly frozen in isopentane, and stored at −80°C. 10 μm HCC sections were prepared and fixed in 4% paraformaldehyde / PBS solution for 3 minutes. After fixation, the sections were rinsed thoroughly with PBS to remove residual paraformaldehyde. Next, the sections were treated with 50% acetone, 100% acetone for 2 minutes, and 50% acetone again. Finally, they were rinsed with PBS. After the sections were treated, they were first hydrated by soaking in PBS for 10 minutes. Then the PBS was aspirated and the primary antibody was incubated. The primary RGS14 monoclonal antibody (clone 1C1B2, 1:200 dilution; purchased from Thermo Fisher Scientific, Cat. No. 67394-1-IG) diluted in PBS / 0.25% BSA was placed in a wet box and incubated overnight at 4°C. After incubation, remove the primary antibody by aspiration and rinse with PBS three times for 5 minutes each to ensure removal of unbound primary antibody. Next, use a fluorescently labeled secondary antibody Cy TM 3 AffiniPure TMDonkeyAnti-Mouse IgG (H+L) (1:200 dilution, catalog number 715-165-150, Jackson Immunoresearch) and Anti-Actin, α-Smooth Muscle-FITC antibody (SMA-FITC, clone: ​​1A4, 1:200 dilution, catalog number: F3777-.2ML, purchased from Merck) were incubated for 60 minutes. At the same time, DAPI (diluted 1:100) was used for nuclear staining. Note that light should be avoided during the entire process. Finally, rinse again with PBS 3 times, 5 minutes each time, and make sure to avoid light during this process. After all steps are completed, soak the slices in PBS for 10 minutes, use Fluoromount G for sealing, and take subsequent photos with laser confocal.

[0033] The results are as follows Figure 1 As shown in B: DAPI is used for cell nucleus staining, SMA is used for blood vessel staining, and RGS14 antibody binds to RGS14, showing that RGS14 is highly expressed at the protein level in liver cancer tissue.

[0034] Figure 1 The results showed that the expression levels of RGS14 at the mRNA and protein levels in liver cancer tissues were significantly higher than those in normal tissues (P < 0.01).

[0035] Example 2

[0036] This example is used to illustrate that TCGA data analysis shows that high expression of RGS14 is related to 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 of liver cancer (LIHC) (clinical) to obtain the matrix of sample number and clinical data. Extract the HTSeq-FPKM data of RGS14 in each sample, the survival status and survival days of the sample. The Kaplan-Meier method was used to obtain the survival curve, and the log-rank test showed that the difference in survival curves was statistically significant. P < 0.01 indicated statistical significance.

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

[0040] Example 3

[0041] This embodiment provides a siRNA sequence of a cell line for knocking down RGS14, and the cell line is specifically a Huh7 liver cancer cell line. Huh7 liver cancer cells were purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured in DMEM medium containing 10% v / v fetal bovine serum at 37°C and 5% CO2.

[0042] 1. The construction method is:

[0043] The negative control siRNA and three siRNAs designed to interfere with RGS14 expression are 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. Follow the instructions of the liposome kit. One day before transfection, plate Huh7 cells in an appropriate amount of growth medium without antibiotics, and make the cell confluency reach 80-90% during transfection. Replace the cell culture medium (without double antibody, without serum) before transfection, dilute siRNA with opti-mem, and dilute lip2000 (Invitrogen) with opti-mem. Mix the two together and let them stand for 2-3 minutes, then mix them in the same tube. Avoid rough blowing and let them stand for 3-5 minutes. Add the prepared transfection solution to the cell culture plate and shake it gently. Change the medium in time 6 hours after siRNA transfection (replace serum-free medium with complete medium). After 24-48 hours of successful transfection, remove the cells from the incubator for subsequent related tests.

[0057] 3. Gene knockdown effect detection

[0058] The total protein of the extracted cells and the protein lysate were separated by SDS-polyacrylamide gel and then analyzed by immunoblotting using primary antibodies against RGS14 (1:5000 dilution, catalog number 16258-1-AP, purchased from Proteintech) and primary antibodies against GAPDH (1:4000 dilution, catalog number HC301-01, purchased from TransGen). Each experiment was repeated three times.

[0059] The results are as follows Figure 3 As shown: Western blot technology was used to detect the efficiency of RGS14 silencing in the HuH7 cell line. 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 was successfully knocked down in the cell line. This method can be used to screen liver cancer cells with low expression of RGS14.

[0060] Experimental Example 4

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

[0062] 1. Specific test contents are:

[0063] (1) Cell clone formation experiment

[0064] DMEM high-glucose medium was used to prepare Huh7 cells transfected with siNC and Huh7 cells transfected with siRGS14-SEQ1 at a concentration of 2×10 3The suspension was added to a 6-well plate at a volume of 2 mL / well. After the cells grew to a size visible to the naked eye, the culture medium was removed, and the cells were fixed with methanol for 15 minutes, then washed twice with PBS and dried. Subsequently, 0.5% w / v crystal violet solution was added for staining for 10 minutes, washed with ultrapure water, and photographed after drying.

[0065] (2) Cell proliferation assay

[0066] Press 2×10 3 siNC-transfected Huh7 cells and siRGS14-SEQ1-transfected Huh7 cells were seeded in 96-well plates 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 CCK-8 (MedChemExpress, HY-K0301) was added to each well, followed by incubation at 37°C for 2 hours. Cell activity was assessed by measuring absorbance at a wavelength of 540 nm to obtain a cell proliferation curve.

[0067] (3) Cell migration and invasion assay

[0068] To evaluate cell invasion and migration, cell culture chambers (pore size 8 μm, Corning) coated with or 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 chamber. DMEM supplemented with 20% v / v FBS was added to the lower chamber across the membrane 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 clone formation are as follows Figure 4 As shown in A, it can be seen that the number of clones formed by the cell line with RGS14 knockdown is less than that of the control cell line; the results of the cell proliferation experiment are shown in Figure 4 As shown in B, it can be seen that the proliferation rate of the cell line with RGS14 knockdown is lower than that of the control cell line. Figure 4 It shows 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 RGS14 knockdown cell lines and control cell lines are shown. Figure 5It can be seen that after knocking down RGS14, the migration and invasion ability of liver cancer cells was 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 implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of RGS14 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 kit comprises a reagent for detecting the expression amount of RGS14 protein.

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

4. Application of RGS14 expression inhibitors in the preparation of drugs for the treatment of liver cancer.

5. The use according to claim 4, characterized in that: The RGS14 expression inhibitor includes molecules that interfere with the expression of RGS14.

6. The use according to claim 4, characterized in that: The molecule interfering with the expression of RGS14 is RGS14 siRNA.

7. An RGS14 expression inhibitor, characterized in that: The RGS14 expression inhibitor is characterized by comprising RGS14 siRNA.

8. The RGS14 expression inhibitor according to claim 7, characterized in that: The RGS14 expression inhibitor includes siRNA with the following sequence: Sense strand: 5′-GACCUGGUACUUCCAGAAUUUTT-3′; Antisense strand: 5′-AAAUUCUGGAAGUACCAGGUCTT-3′.

9. Use of the RGS14 expression inhibitor according to claim 7 or 8 in the preparation of a drug for treating liver cancer.

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