Application of PRR11-SKA2-miR301a-miR454 transcription unit in tumors

Abstract

The invention relates to an application of a PRR11-SKA2-miR301a-miR454 transcription unit in tumors, and particularly relates to an application of the The invention belongs to the technical field of biological medicine. The invention aims to solve the problems that the regulation and control mechanism of a PRR11-SKA2-miR301a-miR454 transcription unit in the generation and development of malignant tumors is not clear, and an effective treatment means for the transcription unit is lacked. The key point of the technical scheme lies in that the transcription unit comprises four genes, the transcription of the four genes is regulated and controlled by the same promoter, and sgRNA for inhibiting the expression level of the transcription unit is provided. In addition, the invention also provides four gene detection modes for prognosis prediction of lung adenocarcinoma patients. The main purpose of the invention is to provide a new strategy for the treatment of tumors, especially lung cancer, by regulating and controlling the transcription unit of the gene, and provide a new biomarker combination for the prognosis prediction of tumors at the same time, thus having important theoretical and practical significance.

Description

Technical Field

[0001] The present invention relates to applications of PRR11, SKA2, miR301a and miR454 genes and their transcriptional units CRISPRi-sgRNA in tumor treatment and prognosis prediction, especially in lung cancer treatment and prognosis prediction. Background Art

[0002] Globally, malignant tumors remain a leading threat to human life and health. In recent years, the number of new cases and deaths from malignant tumors in China has continued to rise. In 2022, new cases of malignant tumors in China reached 4.8247 million, and the total number of deaths reached 2.5742 million. Therefore, conducting research on malignant tumors and developing basic theories and applied research are of great theoretical and practical significance for elucidating the mechanisms of tumor development, preventing and treating tumors, and reducing the morbidity and mortality of malignant tumors.

[0003] The development of malignant tumors is an extremely complex biological phenomenon involving multiple factors, multiple genes, and multiple stages. The dysregulated expression of oncogenes and tumor suppressor genes, which in turn induces uncontrolled cell growth, plays a crucial role in tumor development and progression. Currently, a large number of new tumor-related genes remain to be discovered. Elucidating their functions will not only directly contribute to the mechanistic understanding of tumorigenesis but also potentially serve as effective biological targets for the development of novel tumor diagnostics and anti-tumor drugs.

[0004] The PRR11-SKA2-miR301a-miR454 transcription unit is a new tumor-associated transcription unit recently discovered by the applicant. The applicant has pioneered research into the biological functions and expression regulation mechanisms of this transcription unit both domestically and internationally. Regarding the biological functions of this transcription unit, the applicant's preliminary research results indicate that this gene participates in multiple biological processes, including cell proliferation, cell cycle, and cell carcinogenesis. It plays a particularly important role in the development and progression of lung cancer, and has significant application value in tumor treatment, particularly lung cancer, and prognosis prediction. Summary of the Invention

[0005] The present invention discovered a transcription unit involved in cell growth, proliferation, migration and cell cycle regulation, which contains four genes: PRR11, SKA2, miR301a and miR454. PRR11 and SKA2 are protein-coding genes, and their transcription start sites are no more than 1 kb apart and have opposite transcription directions. miR301a and miR454 are non-coding genes located within the first intron of SKA2. The transcriptional activities of these four genes are regulated by the same promoter (PRR11-SKA2 promoter), and it was found that inhibiting the transcriptional activity of the PRR11-SKA2 promoter can simultaneously inhibit the expression levels of PRR11, SKA2, miR301a and miR454, as well as the growth, proliferation, migration and tumorigenicity of tumor cells. Based on the above findings, the present invention was completed.

[0006] One objective of the present invention is to provide an sgRNA (CRISPRi-sgRNA#1-targeting sequence) that can inhibit the expression level of the PRR11-SKA2-miR301a-miR454 transcription unit. For sequence information, see SEQ ID NO: 1 in the sequence listing. The present invention also provides the use of the aforementioned sgRNA in the preparation of a drug for the treatment of lung cancer.

[0007] The present invention also provides a vector comprising the aforementioned sgRNA. The present invention also provides a non-human host cell comprising the vector. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned sgRNA, vector or host cell, and a pharmaceutically acceptable carrier.

[0008] Another object of the present invention is to provide four genetic testing methods for predicting the prognosis of patients with lung adenocarcinoma. The first is that detecting the expression level of miR454-5p alone can effectively predict the prognosis of patients with lung adenocarcinoma; the second is that detecting the expression levels of PRR11, SKA2, miR301a, and miR454 together can effectively predict the prognosis of patients with lung adenocarcinoma; the third is that detecting TP53 mutation status and the expression levels of PRR11, SKA2, miR301a, and miR454 together can effectively predict the prognosis of patients with lung adenocarcinoma; and the fourth is that detecting the expression levels of SKA2, E2F1, E2F7, and FOXM1 together can effectively predict the prognosis of patients with lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the PRR11-SKA2-miR301a-miR454 transcription unit structure.

[0010] Figure 2RNA levels of PRR11, SKA2, miR301a, and miR454 (A) and protein levels of PRR11 and SKA2 (B) in lung cancer cells after inhibition of the PRR11-SKA2-miR301a-miR454 transcription unit using CRISPRi technology.

[0011] Figure 3 The cell cycle progression of lung cancer cells after the expression of the PRR11-SKA2-miR301a-miR454 transcription unit was inhibited using CRISPRi technology.

[0012] Figure 4 To investigate the effect of inhibiting the expression of PRR11-SKA2-miR301a-miR454 transcription unit on the growth ability of lung cancer cells using CRISPRi technology.

[0013] Figure 5 To investigate the effect of inhibiting the expression of PRR11-SKA2-miR301a-miR454 transcription unit on the proliferation ability of lung cancer cells using CRISPRi technology.

[0014] Figure 6 To investigate the effect of inhibiting the expression of PRR11-SKA2-miR301a-miR454 transcription unit on the migration ability of lung cancer cells using CRISPRi technology.

[0015] Figure 7 To investigate the effect of inhibiting the expression of PRR11-SKA2-miR301a-miR454 transcription unit using CRISPRi technology on the colony formation ability of lung cancer.

[0016] Figure 8 To investigate the effect of inhibiting the expression of PRR11-SKA2-miR301a-miR454 transcription unit using CRISPRi technology on the tumorigenicity of lung cancer in vivo.

[0017] Figure 9 To separately detect the effect of miR454-5p expression level in predicting the prognosis of lung adenocarcinoma.

[0018] Figure 10 To jointly consider the effects of PRR11, SKA2, miR301a and miR454 expression levels in the prognosis prediction of lung adenocarcinoma.

[0019] Figure 11 To jointly consider the effects of TP53 mutation status and PRR11, SKA2, miR301a and miR454 expression levels in the prognosis prediction of lung adenocarcinoma.

[0020] Figure 12To jointly consider the effects of SKA2, E2F1, E2F7 and FOXM1 expression levels in the prognosis prediction of lung adenocarcinoma. DETAILED DESCRIPTION

[0021] The PRR11-SKA2-miR301a-miR454 transcription unit of the present invention is a new tumor-related transcription unit recently discovered by the applicant. The transcription unit contains four genes: PRR11, SKA2, miR301a and miR454. PRR11 and SKA2 are protein-coding genes. The transcription start sites of the two genes are no more than 1 kb apart and the transcription directions are opposite. MiR301a and miR454 are non-coding genes located in the first intron of SKA2. Figure 1 .

[0022] Using CRISPR interference (CRISPRi) technology, the expression levels of PRR11, SKA2, miR301a and miR454 in lung cancer cells were simultaneously and effectively inhibited. Figure 2 . Findings: Inhibition of the expression level of the PRR11-SKA2-miR301a-miR454 transcription unit led to cell cycle arrest and inhibition of growth, proliferation and migration in lung cancer cells. Figure 3-6 Cell plate clone formation analysis showed that the inhibition of PRR11-SKA2-miR301a-miR454 transcription unit expression by CRISPRi technology also significantly inhibited the clone formation ability of lung cancer cells. Figure 7 Nude mouse xenograft tumor formation analysis showed that inhibition of the PRR11-SKA2-miR301a-miR454 transcriptional unit expression by CRISPRi technology also significantly inhibited the growth of lung cancer xenograft tumors in nude mice. Figure 8 .

[0023] Bioinformatics analysis revealed that combined detection of PRR11, SKA2, miR301a, and miR454 expression levels can effectively predict the prognosis of patients with lung adenocarcinoma. Figure 9-11 By building a model and conducting bioinformatics analysis on the regulatory network genes of the PRR11-SKA2-miR301a-miR454 transcription unit, it was found that the combined detection of SKA2, E2F1, E2F7 and FOXM1 can also effectively predict the prognosis of patients with lung adenocarcinoma. Figure 12 .

[0024] By using the CRISPRi-sgRNA sequence of the present invention that can inhibit the expression of the PRR11-SKA2-miR301a-miR454 transcription unit, the growth and proliferation of tumor cells can be effectively inhibited, thereby achieving the purpose of treating tumors.

[0025] By using the multi-gene combination of the present invention for joint detection, the prognosis of patients with lung adenocarcinoma can be effectively predicted. Sequence Listing Example

[0026] Example 1: CRISPRi technology inhibits the expression of the PRR11-SKA2-miR301a-miR454 transcription unit and blocks lung cancer cell cycle progression, inhibits cell growth, proliferation, migration, and in vivo tumorigenesis

[0027] 1. Synthesis of CRISPRi-sgRNA

[0028] The DNA sequence 2 kb upstream of the first exon of PRR11 was obtained from the UCSC database. The DNA sequence between the first exon of PRR11 and the first exon of SKA2 was then imported into the sgRNA design website (http: / / www.rgenome.net / cas-designer / ) to select the appropriate sgRNA targeting sequence.

[0029] The two CRISPRi-sgRNA targeting sequences finally selected were:

[0030] CRISPRi-sgRNA#1 - targeting sequence: 5'-CTCCGTGCTAGTACGGCTTGCGG-3'

[0031] CRISPRi-sgRNA#4 - Targeting sequence: 5'-AACACACCTTCTTTCCCCTCCGG-3'

[0032] Selected negative control sgRNA targeting sequence (Mock-sgRNA): 5'-GGGCGAGGAGCTGTTCACCGGGG-3'

[0033] Complementary bases were added to the plasmid vector to construct the CRISPRi-sgRNA targeting sequence, resulting in the following oligos sequences:

[0034] CRISPRi-sgRNA#1-Oligos plus strand: 5'-CACCGCTCCGTGCTAGTACGGCTTG-3'

[0035] CRISPRi-sgRNA#1-Oligos negative strand: 5'-AAACCAAGCCGTACTAGCACGGAGC-3'

[0036] CRISPRi-sgRNA#4-Oligos plus strand: 5'-CACCGAACACACCTTTCTTTCCCCTC-3'

[0037] CRISPRi-sgRNA#4-Oligos negative strand: 5'-AAACGAGGGGAAAGAAGGTGTGTTC-3'

[0038] Mock-sgRNA-Oligos positive strand: 5'-CACCGGGCGAGGAGCTGTTCACCG-3'

[0039] Mock-sgRNA-Oligos negative strand: 5'-AAACCGGTGAACAGCTCCTCGCCC-3'

[0040] Shanghai Sangon Biotechnology Co., Ltd. was commissioned to synthesize the above-mentioned CRISPRi-sgRNA-Oligos sequence in vitro by chemical synthesis.

[0041] 2. Plasmid vector construction

[0042] The CRISPRi-sgRNA was cloned into the Lenti-dCas9-KRAB-PURO ivsgRNA plasmid using Thermo Fisher's BsmBⅠ endonuclease, and sequencing was used to verify the correctness of the plasmid sequence.

[0043] 3. Lentiviral packaging

[0044] 293 cells were seeded in 10 cm cell culture dishes and transfected with plasmids when the confluence reached approximately 80%. The transfection system consisted of 4.5 μg of PLP1 lentiviral packaging plasmid, 1.8 μg of PLP2 lentiviral packaging plasmid, 2.7 μg of VSVG lentiviral packaging plasmid, and 9 μg of the target plasmid (the target plasmids for silencing the PRR11 / SKA2 bidirectional transcriptional unit were Lenti-dCas9-KRAB_PRR11 / SKA2-sgRNA#1 and Lenti-dCas9-KRAB_PRR11 / SKA2-sgRNA#4; the target plasmid for the sgRNA mock group was Lenti-dCas9-KRAB_Mock sgRNA). The transfection reagent used was NEOFECT transfection reagent from Beijing Codein Technology. 24 hours after transfection, the 293 dish was supplemented with 10 mL of complete DMEM medium. 48 hours after transfection, the lentiviral particles were collected using a 100K ultrafiltration tube, aliquoted, and stored at -80°C.

[0045] 4. Lentiviral infection of target cells and screening of stable transfected strains

[0046] When the cell confluence reaches 30-40%, remove the lentiviral concentrate frozen at -80°C and add 1 μL of 5 μg / μL polybrene to the EP tube containing the lentiviral concentrate. Add 1 mL of complete culture medium to the EP tube containing the lentiviral concentrate and mix thoroughly with a pipette. Then, add the entire contents of the EP tube to a 6-well cell culture plate containing cells to be infected, discarding the top layer of culture medium. Continue incubating the cell culture plate in a 37°C, 5% CO2 incubator. 12 hours after infection, add 1 mL of complete culture medium to the cell culture plate containing the lentiviral concentrate. 48 hours after infection, digest the infected cells and replate them in a 6-well cell culture plate. When the cell confluence reaches 30%, add puromycine working solution (complete culture medium and puromycine, final concentration: 2 ng / mL) to the cell culture plate. After 72 hours of continuous screening with the puromycine working solution, discard the supernatant medium, digest the cells, and re-seed them in a 10 cm dish. After 24 hours of cell culture, harvest the protein and RNA from the cells or proceed with subsequent experiments.

[0047] 5. Total RNA Extraction and Quantitative PCR

[0048] (1) Total RNA was extracted using Invitrogen's Trizol reagent.

[0049] (2) Reverse transcription: Use TAKARA's PrimeScript™ RT reagent Kit with gDNAEraser. Refer to the kit instructions for specific methods.

[0050] (3) Quantitative PCR: Real-time fluorescence quantitative PCR was used to detect the expression level of the PRR11 gene in various tumor tissues. The quantitative PCR reagent SYBR® Premix Ex Taq™ (Perfect Real Time, TAKARA) was purchased from Takara Biotechnology (Dalian) Co., Ltd. The specific PCR was performed according to the product instructions with slight modifications. GAPDH was used as an internal control to detect the mRNA expression levels of the PRR11 and SKA2 genes, and U6 was used as an internal control to detect the expression levels of the miR301a and miR454 genes. The upstream and downstream primers for GAPDH were: upstream primer: 5'-ACCTGACCTGCCGTCTAGAA-3', downstream primer: 5'-TCCACCACCCTGTTGCTGTA-3'. PRR11_mat-mRNA upstream primer: 5'-GACTCCAAAGCTGTGCTTCC-3', PRR11_mat-mRNA downstream primer: 5'-CTGCATGGGTCCATCTTTTT-3', PRR11_pre-mRNA upstream primer: 5'-GCTCATAGCACACTTCGTTGC-3', PRR11_pre-mRNA downstream primer: 5'-ATGGAGCTGACAGTTCGGTG-3'; SKA2_mat-mRNA upstream primer: 5'-ACTTTGTATGCCCGCTTTAA-3', SKA2_mat-mRNA downstream primer: 5'-GCCGCAGTTTTCTCTTCTTT-3', SKA2_pre-mRNA upstream primer: 5'-ACAGTGGTTGAGGAATGGGT-3', SKA2_pre -mRNA downstream primer: 5'-TGCCTTTACCCTCAAGCTGA-3'; miR-301a_3p upstream primer: 5'-GGCTGGCAGTGCAATAGTATT-3', miR-301a_3p downstream primer: 5'-CGAGGAAGAAGACGGAAGAAT-3'; miR-301a_pre-miRNA upstream primer: 5'-ACTGCTAACGAATGCTCTGA-3', miR-301a_pre-miRNA downstream primer: 5'-CGAGGAAGAAGACGGAAGAAT-3'; miR-301a_pri-miRNA upstream primer: 5'-TCTGTCCAAAATGGCAACCCT-3', miR-301a_pri-miRNA downstream primer: 5'-GAATCAAAATTCTGGCGTGCA-3';miR-454_3p upstream primer: 5'-TTGGTTAGGCTAGTGCAATATTG-3', miR-454_3p downstream primer: 5'-CGAGGAAGAAGACGGAAGAAT-3'; miR-454_pre-miRNA upstream primer: 5'-TCTGTTTATCACCAGATCCT-3', miR-454_pre-miRNA downstream primer: 5'-CGAGGAAGAAGACGGAAGAAT-3'; miR-454_pri-miRNA upstream primer: 5'-TGCGCGATTTATCAGACACT-3', miR-454_pri-miRNA downstream primer: 5'-ACT TTTGCTTGTTTCTGCTACATACT-3'; TRIM37 upstream primer: 5'-ATGAGCGTGTTCGGGAAATTAG-3', TRIM37 downstream primer: 5'-TCACTCTTACTACAAGACCGCAA-3'; SMG8 upstream primer: 5'-AGCACCTCGAGATGATCCCT-3', SMG8 downstream primer: 5'-AAACTCAGGCTCGTGCTCTC-3'; U6 upstream primer: 5'-CTCGCTTCGGCAGCACA-3', U6 downstream primer: 5'-AACGCTTCACGAATTTGCGT-3'; quantitative PCR amplification reaction system consisted of: 10 µL SYBR® Premix Ex Taq™ (2×), 0.4 µL of 10 µmol / L upstream and downstream primer mixture, 2 µL of cDNA template, and double-distilled water to 20 µL. Quantitative PCR amplification was performed on a Bio-Rad MiniOpticon quantitative PCR instrument. The amplification program was denaturation at 95°C for 10 seconds, followed by 40 cycles of 95°C for 5 seconds, 58°C for 15 seconds, and 72°C for 15 seconds. The Ct value was then read. The relative quantitative expression level of the PRR11 gene was calculated as follows: Law.

[0051] The results showed that CRISPRi-sgRNA simultaneously and effectively inhibited the mRNA expression levels of PRR11, SKA2, miR301a and miR454 in lung cancer cells. Figure 2 -A.

[0052] 6. Preparation of Cell Protein Lysate and Western Blotting

[0053] (1) Preparation of cell protein lysate: Aspirate the culture medium from the culture dish, wash the cells twice with PBS, collect the cells with a cell scraper, and suspend them in RACK1 lysis buffer (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100, with an appropriate amount of protease inhibitor added before use). Place on ice for 30 min and sonicate to further disrupt the cells and DNA. Centrifuge at 14,000 rpm at 4°C for 10 min, and collect the supernatant as the cell lysate. The protein concentration in the cell lysate was determined using the Bradford assay. For specific methods, please refer to the instructions for use of the Bio-Rad Protein Assay kit from Bio-Rad.

[0054] (2) Western blotting: Take an appropriate amount of cell lysate, add SDS loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% β-mercaptoethanol, 0.01% bromophenol blue) and heat denature at 100°C for 5 min. Separate the proteins in the sample by 10% SDS-PAGE. Use the Tank method for transfer. For specific methods, refer to the instructions for use of Millipore's ImmobilonTM-P membrane. Block the transferred protein membrane with blocking solution (TBS-T containing 5% milk) at room temperature for 1 hour or at 4°C overnight. Wash the membrane three times with TBS-T, each for 5 minutes; add an appropriately diluted primary antibody (PRR11 antibody purchased from Abcam, GAPDH antibody purchased from Hangzhou Xianzhi Company), incubate at room temperature for 1 hour; wash the membrane three times with TBS-T; add a 1:2000 diluted secondary antibody conjugated to horseradish peroxidase (purchased from Cell Signaling Company), incubate at room temperature for 1 hour; wash the membrane three times with TBS-T. Finally, chemiluminescence was used for detection. For specific operations, please refer to the instructions of the ECL Western blotting detection reagents and analysis system kit of Amersham Company, and the KODAK imaging system was used for development.

[0055] (3) Results: See Figure 2 -B, The results showed that CRISPRi-sgRNA simultaneously and effectively suppressed the protein expression levels of PRR11 and SKA2 in lung cancer cells.

[0056] 7. Cell cycle detection

[0057] Digest the cells to be tested with trypsin to reduce them to single cells. Transfer the cell suspension to a 1.5 mL centrifuge tube and centrifuge at 700 rpm for 5 minutes. Discard the supernatant. Wash the cells twice with PBS, centrifuging at 700 rpm for 5 minutes each time. Add 1 mL of 75% ethanol and pipette to mix the suspension. Incubate at 4°C for 12 hours. Cell cycle analysis was performed using flow cytometry.

[0058] The results showed that CRISPRi-sgRNA effectively blocked the cell cycle progression of lung cancer. Figure 3 .

[0059] 8. Cell growth, proliferation and migration ability detection

[0060] (1) Cell growth ability test: 1×10 5 Cells were seeded into 6-well cell culture plates, with three wells per group. After seeding, the cells were cultured in a 37°C incubator with 5% CO2. After 48 or 72 hours of culture, the cells were counted using a cell counter.

[0061] The results showed that CRISPRi-sgRNA effectively inhibited the growth ability of lung cancer cells. Figure 4 .

[0062] (2) Cell proliferation and migration ability detection: An appropriate number of cells were inoculated into a 24-well cell culture plate so that the cell confluence reached an appropriate level 16 hours after inoculation (monitoring the cell confluence growth rate requires a confluence of 5-10%, and monitoring cell movement requires 8-15 cells in a single field of view). After inoculation, the cells were cultured in an incubator at 37°C and 5% CO2. The 24-well cell culture plate was placed in the JULI Stage Real-time Cell History Recorder instrument, and appropriate shooting parameters were set to start shooting. After shooting, the cell movement trajectory data was analyzed and plotted using Image-Pro 10 software. The cell confluence data was analyzed using JuLI STAT software.

[0063] The results showed that CRISPRi-sgRNA effectively inhibited the proliferation of lung cancer cells. Figure 5 , and migration capabilities, see Figure 6 .

[0064] (3) Plate clone formation analysis experiment: Grow cells to the logarithmic growth phase, digest the cells with trypsin until they become round, then add complete culture medium equal to the trypsin digestion solution, pipette to make a cell suspension, transfer to a cell centrifuge tube, centrifuge at 700 rpm for 5 minutes, and discard the supernatant. Add complete culture medium to resuspend the cells. Inoculate cells in 6-well plates with a cell density of 1000 cells per well. Set up three replicates for each experimental group. Change the medium every 3 days and observe the cell clones. After 7 days of culture, discard the culture medium, wash the cells 3 times with 1× PBS, and discard the supernatant. Add methanol to fix for 20 minutes. Discard the methanol and add 0.5% crystal violet solution to stain for 20 minutes. Discard the crystal violet and slowly rinse the cells with 1× PBS until the cell background is clear. Take pictures and count the clones with more than 50 cells.

[0065] The results showed that CRISPRi-sgRNA effectively inhibited the colony formation ability of lung cancer cells. Figure 7 .

[0066] 9. Nude mouse transplantation experiment of lung cancer cells: Prepare female nude mice aged 4-6 weeks. Grow the cells to be inoculated to the logarithmic growth phase. After trypsinizing the cells until they become round, add complete culture medium equal to the trypsin digestion solution. Use a pipette to blow into a cell suspension, transfer to a cell centrifuge tube, centrifuge at 700 rpm for 5 minutes, and discard the supernatant. Wash the cells twice with 1×PBS, centrifuge at 700 rpm for 5 minutes each time, and discard the supernatant. Dilute the cells with 1×PBS at 4°C to a concentration of 1×10 7 The tumor cell suspension was injected subcutaneously into the armpit of nude mice at a concentration of 100 μl / mouse, i.e. the number of tumor cells was 1×10 6 Every 7 days, measure the tumor size along the vertical axis of the tumor with a vernier caliper and keep a record. (Tumor volume calculation formula: 1 / 2 × length × width 2 At about 5 weeks, the nude mice were killed, the tumors were removed, weighed, and the data were recorded.

[0067] The results showed that CRISPRi-sgRNA effectively inhibited the tumorigenicity of lung cancer cells in vivo. Figure 8 .

[0068] Example 2: PRR11-SKA2-miR301a-miR454 transcription unit gene detection predicts the prognosis of lung adenocarcinoma patients

[0069] 1. Data preparation

[0070] Transcriptome and clinical data for lung adenocarcinoma samples (including patient TP53 mutation data) were downloaded from the TCGA database (https: / / cancergenome.nih.gov / ). A total of 527 lung adenocarcinoma transcriptome data samples, including mRNA and miRNA data, were downloaded and normalized. Finally, 421 samples with prognostic information were selected for prognostic analysis.

[0071] 2. Prognosis prediction

[0072] The optimal survival cutoff value was calculated based on the log-rank test, and lung adenocarcinoma patients were divided into high-risk and low-risk groups. Kaplan-Meier survival curves were then plotted for each gene, and the significance of overall survival between the high-risk and low-risk groups was assessed using the log-rank test. A polygenic model was constructed for PRR11, SKA2, miR301a, and miR454 using the stepwise regression method of multivariate Cox regression. A polygenic model was constructed for 29 genes associated with the regulatory network of the PRR11-SKA2-miR301a-miR454 transcription unit using the stepwise regression method of multivariate Cox regression.

[0073] The results show that:

[0074] Detection of miR454-5p expression level alone can effectively predict the prognosis of patients with lung adenocarcinoma. Figure 9 ; Combined consideration of PRR11, SKA2, miR301a and miR454 expression levels can effectively predict the prognosis of patients with lung adenocarcinoma, see Figure 10 ; Combined consideration of TP53 mutation status and PRR11, SKA2, miR301a and miR454 expression levels can effectively predict the prognosis of patients with lung adenocarcinoma, see Figure 11 ; Combined consideration of SKA2, E2F1, E2F7 and FOXM1 expression levels can also effectively predict the prognosis of patients with lung adenocarcinoma, see Figure 12 .

[0075] It should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the invention disclosed herein, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein are representative and exemplary of preferred embodiments and are not intended to be limiting of the scope of the present invention.

Claims

1. An sgRNA that inhibits the expression level of the PRR11-SKA2-miR301a-miR454 transcription unit, characterized in that The sgRNA includes the sequence shown in SEQ ID NO:

1.

2. A vector comprising the sgRNA according to claim 1.

3. A non-human host cell comprising the vector according to claim 2.

4. A pharmaceutical composition comprising a therapeutically effective amount of the sgRNA of claim 1, the vector of claim 2, or the host cell of claim 3, and a pharmaceutically acceptable carrier.

5. Use of the sgRNA according to claim 1, the vector according to claim 2, or the host cell according to claim 3 in the preparation of a medicament for treating lung cancer.

6. A gene test for predicting the prognosis of patients with lung adenocarcinoma, characterized in that: The gene detection includes the gene miR454-3p.

7. A gene detection combination for predicting the prognosis of patients with lung adenocarcinoma, characterized in that: The gene detection combination includes four genes: PRR11, SKA2, miR301a and miR454.

8. A gene detection combination for predicting the prognosis of patients with lung adenocarcinoma, characterized in that: The gene detection combination includes TP53 mutation status and four genes: PRR11, SKA2, miR301a and miR454.

9. A gene detection combination for predicting the prognosis of patients with lung adenocarcinoma, characterized in that: The gene detection combination includes four genes: SKA2, E2F1, E2F7 and FOXM1.

10. Use of the gene test or gene test combination according to claim 6, 7, 8 or 9 in the preparation of a diagnostic reagent or kit for predicting the prognosis of lung adenocarcinoma.

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