Use of tRNA derivative tRF-His-008 in diagnosis and treatment of renal cancer

Abstract

This invention discloses the application of the tRNA fragment tRF-His-008 in the diagnosis and treatment of renal cell carcinoma. Quantitative real-time PCR revealed that tRF-His-008 expression was significantly low in renal cell carcinoma tissues, and its expression level in renal cell carcinoma cells was significantly lower than that in the human renal cortical proximal tubule epithelial cell line HK2. Furthermore, the expression level of tRF-His-008 in exosomes from the blood of renal cell carcinoma patients was positively correlated with its expression level in renal cell carcinoma tissues. Overexpression of tRF-His-008 significantly inhibited the proliferation, migration, and invasion of renal cell carcinoma. Targeted inhibition of tRF-His-008 with an inhibitor significantly promoted the proliferation, migration, and invasion of renal cell carcinoma cells. tRF-His-008 plays an important role in the diagnosis and treatment of renal cell carcinoma and can be used as a diagnostic biomarker and therapeutic agent for renal cell carcinoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of tumor diagnosis and biological medicine engineering, and relates to a new use of an endogenous tRNA derivative, in particular to application of tRNA derivative tRF-His-008 in diagnosis and treatment of renal cancer. BACKGROUND

[0002] Renal cell carcinoma (RCC) is the most common type of kidney cancer, originating from the renal cortex, accounting for about 85% of primary kidney cancer, and accounting for more than 4% of all adult malignancies. It is a common malignant tumor of the urinary system. The American Cancer Society estimates that there will be 79,000 new cases of kidney cancer in the United States in 2022, and 13,920 cases of kidney cancer will die. The most common pathological type of RCC is clear cell renal cell carcinoma (ccRCC), accounting for 75-80% of all RCC. Early ccRCC patients undergoing partial nephrectomy or radical resection can obtain good prognosis, and the 5-year survival rate is more than 90%. However, ccRCC lacks typical clinical symptoms in the early stage, and one-fourth of patients are found to have distant metastasis at the time of initial diagnosis. Metastatic ccRCC is not sensitive to chemotherapy and radiotherapy, and the 5-year overall survival rate is only 12%. At present, advanced ccRCC mainly relies on targeted drug therapy and immunotherapy, but patients will develop drug resistance after 6 to 15 months of targeted therapy. Therefore, the mechanism of metastatic ccRCC is a hot issue in current tumor research, and in-depth study of the metastasis mechanism of ccRCC can provide new ideas for the diagnosis and treatment of advanced metastatic ccRCC, and has important significance for improving its prognosis.

[0003] Transfer RNA (tRNA) derived fragments (tRF) are a research hotspot of small non-coding RNA (sncRNA) in recent years, which can affect the biological behavior of various cancer cells. Moreover, some studies have found that some tRFs may be potential cancer diagnostic indicators and therapeutic targets.

[0004] Exosomes are nanoscale (30-150 nm) extracellular vesicles surrounded by a lipid bilayer membrane. Exosomes are produced by the endosome pathway and can be released into body fluids by most types of cells. Exosomes carry a large number of specific proteins and functional DNA, mRNA, miRNA, and circular RNA, etc., and are involved in cell communication, cell migration, pro-angiogenesis, and other physiological processes in the body, and are closely related to the occurrence and progression of various diseases including tumors. Exosomes are distributed in peripheral blood, urine, saliva, breast milk, ascites, amniotic fluid, and other body fluids, and each component of exosomes can be both a marker for disease diagnosis and a specific target for disease treatment.

[0005] The present application discovers that tRNA-derived fragment (tRF-His-008) is significantly lowly expressed in kidney cancer tissue by high-throughput sequencing of 4 pairs of paired kidney cancer and paracancer tissues and verification in 21 pairs of paired kidney cancer and paracancer tissues, and the relationship between tRF-His-008 and kidney cancer and the role of tRF-His-008 in kidney cancer metastasis are discovered for the first time. The present application confirms that overexpression of tRF-His-008 inhibits kidney cancer proliferation and metastasis through functional experiments, and inhibition of tRF-His-008 expression can promote kidney cancer proliferation and metastasis. Moreover, tRF-His-008 exists in kidney cancer cell culture medium and kidney cancer patient blood exosomes. The present application provides a diagnostic marker and therapeutic drug for kidney cancer, and has important clinical application value. SUMMARY

[0006] An object of the present application is to provide an application of tRF-His-008 in diagnosis and treatment of kidney cancer, and to determine that tRF-His-008 is a tRF with down-regulated expression in cancer tissues and blood exosomes of kidney cancer patients, and to apply tRF-His-008 to diagnosis and treatment of kidney cancer.

[0007] Another object of the present application is to provide a kit for diagnosing kidney cancer disease.

[0008] A third object of the present application is to provide a drug for treating kidney cancer.

[0009] To achieve the above object, the technical scheme of the present application is as follows:

[0010] Application of tRF-His-008 as a diagnostic marker in preparation of a reagent for diagnosing kidney cancer, wherein the nucleotide sequence of tRF-His-008 is shown in SEQ ID No. 1.

[0011] Kidney cancer can be diagnosed by detecting the expression level of tRF-His-008 in blood exosomes of a subject.

[0012] A kit for diagnosing kidney cancer contains primers for specifically amplifying tRF-His-008. The kit can use a fluorescent quantitative PCR kit; further, the primers for specifically amplifying tRF-His-008 are as follows: the sequence of RT primer is SEQ ID No. 2, the sequence of PCR upper primer is SEQ ID No. 3, and the sequence of lower primer is SEQ ID No. 4.

[0013] Application of tRF-His-008 as a therapeutic drug in preparation of a drug for treating kidney cancer.

[0014] A therapeutic agent for renal cell carcinoma, said agent comprising a nucleic acid, a biologically active functional fragment, or a variant having the sequence shown in SEQ ID No. 1. Further, said agent includes a pharmaceutically acceptable carrier or excipient, including chitosan, cholesterol, liposomes, and nanoparticles.

[0015] Beneficial effects:

[0016] This invention is the first to discover that tRF-His-008 plays an important role in the diagnosis and treatment of renal cell carcinoma, and can be used as a biomarker for the diagnosis of renal cell carcinoma and a therapeutic target.

[0017] This invention utilizes quantitative real-time PCR to discover that tRF-His-008 expression is significantly reduced in renal cell carcinoma tissue. Furthermore, the expression level of tRF-His-008 in the exosomes of renal cell carcinoma patients is positively correlated with its expression level in renal cell carcinoma tissue. Therefore, detecting the expression level of tRF-His-008 in the exosomes of subjects' blood can achieve non-invasive and rapid diagnosis of renal cell carcinoma.

[0018] This invention reveals that the expression level of tRF-His-008 in renal cell carcinoma cells is significantly lower than that in adjacent normal tissue. Overexpression of tRF-His-008 significantly inhibits the proliferation, migration, and invasion of renal cell carcinoma cells; conversely, inhibition of tRF-His-008 significantly promotes the proliferation, migration, and invasion of renal cell carcinoma cells. These findings demonstrate the importance of tRF-His-008 in tumor growth and metastasis and suggest the feasibility of using tRF-His-008 mimics for renal cell carcinoma treatment. Attached Figure Description

[0019] Appendix Figure 1 Comparison of tRF-His-008 expression levels in 21 pairs of renal cell carcinoma tissues and adjacent normal tissues;

[0020] Appendix Figure 2 To compare the correlation between tRF-His-008 expression in exosomes from the blood of 10 renal cell carcinoma tissues and corresponding renal cell carcinoma patients;

[0021] Appendix Figure 3 The relative expression levels of tRF-His-008 in human renal cortical proximal tubule epithelial cell lines and renal cancer cell lines;

[0022] Appendix Figure 4 The expression levels of tRF-His-008 in renal cell carcinoma cells after transfection with tRF-His-008 mimics and inhibitors;

[0023] Appendix Figure 5 Figure showing the effects of interference and overexpression of tRF-His-008 on the proliferation of renal cell carcinoma cells;

[0024] Appendix Figure 6Figure showing the effects of interference and overexpression of tRF-His-008 on the migration and invasion abilities of renal cell carcinoma cells; Detailed Implementation

[0025] This invention designs specific stem-loop primers to amplify tRF-His-008 and uses quantitative real-time PCR to detect the differential expression of the tRF-His-008 gene in renal cell carcinoma (RCC) tissues and paired normal tissues. The results show that the expression level of tRF-His-008 in RCC tissues is significantly lower than that in adjacent normal tissues. Furthermore, the abundance of tRF-His-008 in exosomes from the blood of RCC patients is positively correlated with its abundance in RCC tissues. Therefore, a kit for detecting changes in tRF-His-008 expression can be developed, allowing for the diagnosis of RCC by detecting the expression level of tRF-His-008 in the exosomes of patients' blood.

[0026] Next, in vitro and in vivo functional studies were conducted on tRF-His-008. By transfecting renal cell carcinoma cell lines with tRF-His-008 mimics and inhibitors, the expression level of tRF-His-008 in renal cell carcinoma cells was successfully adjusted. Functional experiments showed that overexpression of tRF-His-008 significantly inhibited cell proliferation, migration, and invasion; conversely, inhibition of tRF-His-008 significantly promoted the proliferation, migration, and invasion of renal cell carcinoma cells. Therefore, tRF-His-008 can be used as a therapeutic agent in the preparation or screening of drugs for the treatment of renal cell carcinoma.

[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Analysis of tRF-His-008 expression levels in renal cell carcinoma tissues and paired normal tissues

[0029] I. Materials

[0030] All tissue specimens were obtained from patients pathologically diagnosed with renal cell carcinoma between January 2019 and May 2022. Twenty-one pairs of renal cell carcinoma tissues and their paired normal tissues were selected and grouped / numbered.

[0031] II. Methods

[0032] 1) RNA extraction: Take 30 mg of tissue sample and put it into 500 μL RNA lysis buffer (YiShan Biotechnology), add Jingxin grinding beads, put it into a homogenizer for homogenization, and then extract RNA according to the YiShan tissue RNA rapid extraction kit. Then use NanoDrop ND-1000 nucleic acid quantification instrument to quantify the purity and concentration of the extracted RNA. Agarose quality control ensures the integrity of the extracted RNA.

[0033] 2) RNA pretreatment: The extracted total RNA was demethylated using the Arraystar RNA pretreatment kit (rtStar tRF&tiRNAPretreatment kit) to improve cDNA synthesis.

[0034] 3) cDNA synthesis: The Novizan miRNA first-strand cDNA synthesis kit was used. st The stem-loop cDNA Synthesis Kit (by stem-loop) reverse transcribes pretreated total RNA to synthesize cDNA.

[0035] 4) Real-time quantitative PCR: Specific primers were designed based on the nucleic acid sequences of tRF-His-008 and U6. PCR reactions were performed using Kangwei Century UltraSYBR Mixture. The upstream and downstream primers for tRF-His-008 were SEQ ID NO.3 and SEQ ID NO.4, respectively, and the upstream and downstream primers for U6 were SEQ ID NO.5 and SEQ ID NO.6, respectively. The reaction system is shown in the table below:

[0036] Table 1 PCR reaction system

[0037] Reagents Amount used (μL) 2x UltraSYBR Mixture 5 Forward primer, 10 μM 1 Reverse primer, 10 μM 1 Template DNA 2 ddH2O 1 Total 10

[0038] After thoroughly mixing the above components, follow this procedure: 95℃ for 10 min pre-denaturation, 40 cycles: 95℃ for 15 s, 60℃ for 30 s. Determine the specificity of the reaction based on the dissolution curve, using Formula 2. -△△Ct The relative expression level of tRF-His-008 was calculated, and the results are shown in [the table below]. Figure 1 .Depend on Figure 1 The results showed that tRF-His-008 expression was significantly downregulated in cancer tissues in 21 pairs of clinical renal cell carcinoma tissue specimens.

[0039] Example 2: Detection of tRF-His-008 expression level in exosomes of blood from patients with renal cell carcinoma

[0040] 1) Blood exosome extraction from renal cell carcinoma patients: Blood was collected from patients undergoing radical nephrectomy for renal cell carcinoma. After centrifugation at 3000g for 10 minutes, the supernatant was obtained. 50ml of the blood supernatant was centrifuged at 100000g for 70 minutes. The precipitate obtained after centrifugation was resuspended in PBS buffer and filtered through a 0.22μM filter.

[0041] 2) Detection of tRF-His-008 expression level in exosomes: The filtered product was then processed using RNA extraction, cDNA synthesis and real-time quantitative PCR as described in Example 1 to obtain the relative expression level of tRF-His-008 in exosomes.

[0042] 3) The correlation between urinary exosomes and tRF-His-008 expression in renal cell carcinoma tissue was calculated using Graphpad Prism 8.0.2 software. The results are shown in [Figure number missing]. Figure 2 .like Figure 2 As shown, the expression level of tRF-His-008 in the blood exosomes of renal cell carcinoma is positively correlated with the expression level of tRF-His-008 in renal cell carcinoma tissue, with a correlation coefficient of 0.86, indicating a significant difference in the data.

[0043] Example 3: Detection of tRF-His-008 expression in renal cancer cells and human renal cortical proximal tubule epithelial cell lines.

[0044] I. Materials

[0045] Renal cell carcinoma cell lines 7860, A498, OSRC2, and human renal cortical proximal tubular epithelial cell line HK2 were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0046] II. Methods

[0047] The relative expression levels of tRF-His-008 in each cell were obtained by RNA extraction, cDNA synthesis, and real-time quantitative PCR as described in Example 1. The results are shown in [Figure 1]. Figure 3 Compared with the human renal cortical proximal tubule epithelial cell line HK2, tRF-His-008 was significantly downregulated in the three renal cell carcinoma cell lines.

[0048] Example 4: Construction of stable cell lines with tRF-His-008 knockdown and overexpression

[0049] 1) 786-O and OS-RC-2 cell lines were infected with GV281-NC and GV281-tRF-His-008-mimic lentiviruses, respectively, and selected with 5 μg / mL puromycin to establish stable tRF-His-008-overexpressing and blank control clear cell carcinoma cell lines. 7860 and OSRC2 cell lines were infected with GV281-NC / inhibitor and GV281-tRF-His-008-inhibitor lentiviruses, respectively, and selected with 5 μg / mL puromycin to establish stable tRF-His-008-knockdown and blank control clear cell carcinoma cell lines.

[0050] 2) Validation of interference and overexpression efficiency:

[0051] The cell lines selected after puromycin screening were collected, and the relative expression level of tRF-His-008 was detected using RNA extraction, cDNA synthesis, and real-time quantitative PCR as described in Example 1. The results... Figure 4 It is evident that infection with tRF-His-008-inhibitor virus can significantly reduce tRF-His-008 expression in cells, while infection with tRF-His-008-mimic lentivirus can significantly increase tRF-His-008 expression levels.

[0052] Example 5: Determination of renal cell carcinoma proliferation capacity after knockdown or overexpression of tRF-His-008

[0053] 1) The tRF-His-008 knockdown and overexpression stable cell lines (7860, OSRC2) obtained in Example 4 were digested into single-cell suspensions one day in advance, counted, and the cell concentration was adjusted to 20,000 cells / ml. They were then seeded into 96-well plates, 100 μL per well, i.e., 2,000 cells per well.

[0054] 2) When the cells adhered to the cell wall, CCK8 reagent was added at different time points (1, 2, 3, 4, 5 days) at a ratio of 1:10, that is, 100 μL of culture medium was added to 10 μL of detection solution.

[0055] 3) After incubation at 37℃ for 2 hours, the absorbance at 450nm was measured using an ELISA reader.

[0056] 4) Figure 5 This is a schematic diagram of cell growth curves after knockdown and overexpression of tRF-His-008 in renal cell carcinoma cells. Figure 5 It can be seen that knocking down tRF-His-008 expression in renal cell carcinoma cells accelerates proliferation, while overexpression of tRF-His-008 inhibits proliferation.

[0057] Example 6: Effects of knockdown or overexpression of tRF-His-008 on the migration and invasion ability of renal cell carcinoma cells

[0058] Renal cell carcinoma cells were seeded into transwell chambers at a density of 100 μL per well (without FBS). 0.6 ml of complete culture medium containing 10% FBS was added to the lower chamber to stimulate cell migration. After culturing in a cell culture incubator for 24 hours, the air medium was discarded, and the cells were fixed with tissue fixative at room temperature for 15 minutes, stained with 0.1% crystal violet for 10 minutes, rinsed with water, and the supernatant of unmigrated cells was gently wiped away with a cotton swab. The cells were observed under a microscope, and four fields of view were photographed for cell counting. For the cell invasion assay, 50 μL of Matrigel gel was added to the upper chamber of the transwell chamber, and the rest of the steps were basically the same as above. The experiment was independently repeated three times. Cell migration and invasion counts were statistically analyzed using ImageJ software, and a t-test was performed. **P < 0.01 was considered statistically significant, and ***P < 0.001 was considered highly statistically significant. The results showed that knockdown of tRF-His-008 significantly enhanced the migration and invasion abilities of renal cell carcinoma cells, while overexpression of tRF-His-008 inhibited the migration and invasion of renal cell carcinoma cells. Figure 6 ).

Claims

1. The application of a reagent for detecting tRF-His-008 in the preparation of reagents for diagnosing renal cell carcinoma, characterized in that, The nucleotide sequence of tRF-His-008 is shown in SEQ ID No.

1. The expression level of tRF-His-008 in kidney tissue or blood exosomes is used to diagnose kidney cancer.

2. The application of tRF-His-008 in the preparation of drugs for treating renal cell carcinoma, and its characteristics. Treatment of renal cell carcinoma by overexpression of tRF-His-008, the nucleotide sequence of which is shown in SEQ ID No.

1.

3. A drug for treating renal cell carcinoma, characterized in that, The drug contains a vector that overexpresses a nucleic acid with the sequence shown in SEQ ID No.

1.

4. The drug according to claim 3, characterized in that, The drug includes pharmaceutically acceptable carriers or excipients.

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