Application of olfactory domain family protein 4 (OLFM4) in colon cancer detection products

Abstract

The present invention provides a method for detecting / diagnosing colon cancer, specifically, detecting the specific length and sequence of the Poly(A)-tail of the OLFM4 gene in the patient's colon cancer stem cells by the PAL-SEQ technology, thereby achieving early diagnosis of colon cancer patients, having extremely high accuracy, and can be widely used in clinical practice.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2022115087657 filed with the State Intellectual Property Office on November 29, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of medical diagnosis, and more specifically, to an application of olfactory domain family protein 4 (OLFM4) in a colon cancer detection product. Background Art

[0004] Early screening of tumors plays an extremely important role in controlling and treating tumors. Early detection, early treatment and prevention of disease progression can significantly improve the survival rate and quality of life of patients. Poly(A)-tail was first discovered on RNA in the 1970s and is speculated to be related to translation, RNA formation and transport. Almost all newly synthesized mRNAs contain a polyadenylation signal (PAS), which participates in the synthesis of Poly(A)-tail by recruiting polysomes. Under the action of poly(A) polymerase (PAP), a Poly(A)-tail with 11-14 adenosines is formed downstream of PAS. Subsequently, nuclear poly(A) binding protein (PABPN) extends the newly formed short Poly(A)-tail to 200-250 adenosines. As research deepens, more and more evidence proves that Poly(A)-tail plays an important role in the occurrence and development of tumors.

[0005] Colorectal cancer (CRC) is one of the most common malignant tumors, with 1,096,601 new cases and 551,269 deaths each year, making it the second leading cause of cancer-related deaths worldwide. The treatment of colorectal cancer is mainly based on surgery and chemotherapy, combined with different treatment options such as immunotherapy or targeted therapy. However, these treatments have different disadvantages and limitations. The prognosis of CRC patients is closely related to the stage. The 5-year survival rate of stage 1 and 2 can reach 70%, while the 5-year survival rate of stage 4 is less than 10%. Therefore, there is an urgent need to identify new biomarkers for early diagnosis of colorectal cancer, improve the treatment effect of colorectal cancer, and improve the survival rate of colorectal cancer patients. Summary of the invention

[0006] In order to fill the gap in the prior art, the present invention provides an application of olfactory domain family protein 4 (OLFM4) in a colon cancer diagnosis product.

[0007] The first aspect of the present invention provides an application of olfactory domain family protein 4 (OLFM4) in the preparation of a colon cancer detection product.

[0008] In one embodiment, the product can be used to diagnose whether a patient has colon cancer by detecting the length of Poly(A)-tails of olfactory domain family protein 4 (OLFM4) mRNA in the patient.

[0009] In one embodiment, the product is a kit or a gene chip.

[0010] The second aspect of the present invention provides a colon cancer detection product, which can diagnose whether a patient has colon cancer by detecting the length of the Poly(A)-tails of the patient's olfactory domain family protein 4 (OLFM4) mRNA.

[0011] In one embodiment, the product is a kit.

[0012] Compared with the prior art, the present invention has the following outstanding progress:

[0013] Compared with the existing third-generation sequencing technology, this technical solution can sequence the full-length transcripts of colon cancer Poly(A)-tails through the patented PAL-seq technology, study the relationship between Poly(A)-tails, isoforms and non-coding regions at the overall RNA level, and obtain the specific lengths of different transcript isoforms (isoforms) and Poly(A)-tails of a certain gene.

[0014] This technical solution can detect the specific length and sequence of the Poly(A)-tail of the OLFM4 gene in colon cancer stem cells through the PAL-SEQ technology, thereby achieving early diagnosis of colon cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0016] Figure 1 This is a flow chart of the experiment in Example 1 of the present invention.

[0017] Figure 2 A shows that the expression of cell stemness-related genes LGR5, SOX9, MYC and SALL4 in colon cancer tissues is significantly higher than that in adjacent tissues; Figure 2 B shows the expression of OLFM4 in colon cancer and adjacent tissues (Tumor: colon cancer tissue; Adjacent: adjacent tissue); Figure 2 C shows the expression of OLFM4 in various colon cancer tissue types and different stages (Adjacent: adjacent tissue; LG: poorly differentiated cancer; HG: well-differentiated cancer; TIS: carcinoma in situ; Stage Ⅰ: stage 1; Stage Ⅱ: stage 2; Stage Ⅲ: stage 3; Stage Ⅳ: stage 4). It was observed that the expression of OLFM4 in well-differentiated cancer was higher than that in poorly differentiated cancer, and in the early stage of cancer (stages Ⅰ and Ⅱ) than in the late stage of cancer (stages Ⅲ and Ⅳ). Figure 2 D shows the relationship between OLFM4 expression level and the prognosis of colon cancer patients. It was observed that the prognosis of the low expression group was significantly better than that of the high expression group.

[0018] Figure 3 A shows the expression of OLFM4 mRNA level in colon cancer and adjacent tissues (Tumor: colon cancer tissue; Adjacent: adjacent tissue). It was observed that there was no significant difference in OLFM4 mRNA level between colon cancer and adjacent tissues. Figure 3 B shows the expression of OLFM4 in different cell types in colon cancer tissues, and it was observed that OLFM4 was mainly expressed in stem cells; Figure 3 C shows the localization of OLFM4 and CD133 in colon cancer tissues (Stroma: stroma; Cancer Nest: cancer nest). It was observed that OLFM4 and CD133 were co-localized in colon cancer tissues.

[0019] Figure 4 The relationship between the Poly(A)-tails of OLFM4 mRNA and colon cancer and adjacent tissues was shown: The results showed the expression of the Poly(A)-tails of OLFM4 mRNA in colon cancer and adjacent tissues. It was observed that the Poly(A)-tails of OLFM4 mRNA were only detected in the patient's colon cancer tissues with a length between 50-60nt, but not detected in adjacent tissues.

[0020] Figure 5 Displays a ROC curve plot. DETAILED DESCRIPTION

[0021] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0022] Example 1 Study on the high expression of OLFM4 in colon cancer stem cells and its relationship with colon cancer staging and prognosis

[0023] We collected 31 clinical colon cancer and adjacent tissue samples (patient samples were from the Affiliated Hospital of Guangdong Medical University, patients with stage II or III colon cancer, and informed consent was obtained from all patients) for RNA next-generation sequencing, mass spectrometry, and immunohistochemistry testing. The specific steps are as follows (process as shown in the figure). Figure 1 shown):

[0024] 1. RNA Extraction

[0025] Extract total RNA from tissues using the TroZol method. Take an appropriate amount of clean frozen tissue samples of qualified quality, grind them into powder in liquid nitrogen; add 1 ml TroZol and let stand at room temperature for 5 minutes, then add 200 μl chloroform, shake and mix thoroughly, let stand at room temperature for 5 minutes; centrifuge at 4°C 10,000 × g for 15 minutes, take the supernatant and transfer it to a new tube; add 200 μl isopropanol, let stand at room temperature for 10 minutes, centrifuge at 4°C 10,000 × g for 10 minutes; remove the supernatant, add 500 μl 75% ethanol, centrifuge at 4°C 10,000 × g for 5 minutes, remove the supernatant, repeat this step once; let stand at room temperature for 5-10 minutes, wait for 75% ethanol to completely evaporate, and add an appropriate amount of DEPC water to dissolve.

[0026] 2. RNA Next Generation Sequencing

[0027] 2.1 Sample testing

[0028] Agilent 2100 Bioanalyzer was used to detect the total RNA concentration, RIN value, 28S / 18S and fragment size.

[0029] 2.2 Library construction

[0030] 2.2.1 Purification of mRNA

[0031] Take a certain amount of total RNA sample, denature it at an appropriate temperature to open its secondary structure, and purify it using oligo-dT magnetic beads.

[0032] 2.2.2 Fragmentation

[0033] Add a fragmentation reagent to the mRNA obtained in the previous step, react at an appropriate temperature for a certain period of time, and fragment the mRNA.

[0034] 2.2.3 cDNA synthesis

[0035] Add the pre-prepared first-strand synthesis reaction system to the interrupted mRNA, synthesize the first-strand cDNA according to the corresponding program on the PCR instrument, prepare the second-strand synthesis reaction system, react at an appropriate temperature for a certain time, and synthesize the second-strand cDNA.

[0036] 2.2.4 End repair and adapter ligation

[0037] A reaction system is prepared, and the reaction is carried out at an appropriate temperature for a certain time to repair the double-stranded cDNA end, and an A base is added to the 3' end. A linker connection reaction system is prepared, and the reaction is carried out at an appropriate temperature for a certain time to connect the linker to the cDNA.

[0038] 2.2.5 PCR reaction and product recovery

[0039] Prepare the PCR reaction system and set the reaction program to amplify the ligation product.

[0040] 2.3 Library detection

[0041] The insert fragment range of the library was detected using Agilent 2100 Bioanalyzer (Agilent DNA 1000 Reagents), and the concentration of the library was quantified using ABI StepOnePlus Real-Time PCR System (TaqMan Probe).

[0042] 2.4 Sequencing

[0043] The qualified library was denatured into single strands by adding NaOH, and diluted to a certain concentration according to the expected data volume. The denatured and diluted library was added to the FlowCell, hybridized with the adapter on the FlowCell, and then bridge PCR amplification was completed on the cluster generation platform cBot. Finally, the prepared FlowCell was sequenced using the Illumina sequencing system SBS reagent.

[0044] 3. Liquid chromatography-mass spectrometry analysis

[0045] The detection system was Easy-nLC 1200 (Thermo Scientic, P / N LC140) and Orbitrap Exploris 480 (Thermo Scientic, P / N BRE725533) from Thermo Scientific. 3 μL of the sample was dissolved in 7 μL of mobile phase A (0.1% formic acid in water) and loaded. The peptides were trapped on a trapping column (PepMap C18, 100 μm x2 cm) at a flow rate of 10 μL / min for 3 minutes. The peptides were then separated by gradient elution chromatography on a nanoliter analytical column (PepMap C18, 75 μm x 25 cm). The separation gradient was from 5% to 30% of mobile phase B (0.1% formic acid in acetonitrile) within 70 minutes. The chromatographic flow rate was 200 nL / min and the column temperature was 55 degrees Celsius. The ion source spray voltage was 2.3 kV, the mass spectrometer heated capillary was set to 320 ° C, and the data-dependent mode was used to automatically switch between MS and MS / MS acquisition. The full scan MS used an orbitrap for primary scanning, with a scan range of m / z 350-1600 and a resolution of 120,000 (at m / z 200). The maximum ion introduction time was 50 ms, and the automatic gain control (AGC) was set to 5x105. Subsequently, the top 20 parent ions that met the cascade (MS / MS) fragmentation conditions were fragmented using higher energy collision dissociation (HCD) and scanned with orbitrap, with a scan resolution set to 17,500. The scan range was automatically controlled according to the mass-to-charge ratio of the parent ion, and the minimum scan range was fixed at m / z = 110. The maximum ion introduction time for MS / MS was 50 ms, the AGC control was set to 2.0x105, and the parent ion selection window was set to 1.6 Daltons. For ions with charge numbers of 2, 3, and 4, MS / MS acquisition was performed, and dynamic exclusion was set to perform one MS / MS for each precursor ion within 10 seconds, followed by exclusion for 40 seconds and 30% collision energy.

[0046] 4. Immunohistochemistry:

[0047] The primary antibodies used for validation studies included: rabbit anti-olfactory mediator protein 4 (Cat.#28432-1-AP, proteintech, 1:50), rabbit anti-β-tubulin (Cat.#, Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:200). Quantitative scoring was performed by estimating the percentage of immunopositively stained cells: 0, <10% cells; 1, 10-30% cells; 2, 30-50% cells; 3, 50-70% cells; and 4, >70% cells. Secondly, the staining intensity was scored by evaluating the average staining intensity of positive cells (0, none; 1, weak; 2, moderate; and 3, strong). The immunohistochemical data were statistically analyzed as described above to obtain MS results.

[0048] The results showed that the mRNA levels of stemness-related genes (LGR5, SOX9, MYC, and SALL4) in colon cancer tissues were significantly higher than those in adjacent tissues ( Figure 2 A), which suggests that tumor stem cells may play an important role in the development of colon cancer. To further verify our hypothesis, we used liquid chromatography-mass spectrometry to detect 10 pairs of colon cancer samples and found that the expression of OLFM4 protein in cancer tissues was significantly higher than that in adjacent tissues (see Figure 2 A), we then found through WB experiments that OLFM4 protein tends to be overexpressed in well-differentiated and early-stage colon cancer, but is reduced or absent in poorly differentiated and advanced colon cancer (see Figure 2 B) and is positively correlated with patient prognosis (see Figure 2 C).

[0049] Example 2 Study on the expression of OLFM4 in colon cancer stem cells

[0050] Single-cell sequencing:

[0051] 1. Prepare the sample into a single cell suspension, perform cell quality inspection, and then perform cell counting and cell activity determination, so that the cell activity is higher than 90% and the cell concentration is 700-1200 cells / μL.

[0052] 2. Take 75 μL of Master Mix + cell suspension, 40 μL of gel beads containing barcode information and 280 μL of oil droplets and add them to different chambers of Chromium Chip B respectively, and form oil droplet-encapsulated gel beads (Gel Bead-In-EMulsions, GEMs) through the microfluidic "double cross" cross system.

[0053] 3. Collect GEMs and mix them all. Gel beads automatically dissolve in each oil droplet to release a large number of Barcode primer sequences. Cell lysis releases mRNA. mRNA comes into contact with reverse transcriptase, poly dT reverse transcription primer on gel beads and dNTP substrate. Under the action of reverse transcriptase, reverse transcription reaction occurs to produce a cDNA strand with Barcode and UMI information for sequencing. The second strand synthesis is then completed by SMART amplification method.

[0054] 4. The cDNA fragmentation oil droplets were broken, and one strand of cDNA was purified by magnetic beads, and then PCR amplification was performed using cDNA as a template.

[0055] 5. After cDNA amplification, the cDNA is first broken into fragments of about 200-300bp using chemical methods. The cDNA fragments are screened by end repair and A addition. The P7 adaptor is connected to the Read2 sequencing primer, and the sample index is introduced through PCR amplification. Finally, the fragments are screened to construct a cDNA library containing P5 and P7 adapters.

[0056] 6. Perform sequencing on an Illumina sequencer. After sequencing is complete, perform data analysis.

[0057] Based on the above findings, we further analyzed the second-generation sequencing (RNA-seq) data, first using bowtie2 for sequence alignment, then using htseq for reading statistics, and finally using edgeR for normalization and differential expression analysis. We found that there was no significant difference in the mRNA level of the OLFM4 gene between cancer and adjacent tissues ( Figure 3 A). Based on the above experimental data, the expression of OLFM4 mRNA and protein levels in colon cancer tissues is inconsistent, which aroused our interest. In order to clarify whether the expression of OLFM4 gene is heterogeneous in cancer tissues, we used single-cell sequencing technology to detect a colon cancer tissue sample and found that OLFM4 mRNA is mainly expressed in colon cancer stem cells ( Figure 3 B). We then performed IF experiments on paraffin sections of colon cancer tissues, and the results showed that OLFM4 and CD133 co-localized (see Figure 3 C). The above results indicate that OLFM4 is mainly expressed in colon cancer stem cells.

[0058] Example 3 Study on the length of OLFM4 mRNA Poly(A)-tail in colon cancer

[0059] PALso-seq sequencing:

[0060] 1. Terminal extension: Select extension primers, prepare extension system, and perform terminal extension.

[0061] 2. USER enzyme digestion: Add USER enzyme to digest the extension product from the previous step.

[0062] 3. RNA purification: Use RNA Clean&ConcentratorTM-5 to purify the intermediate product after USER digestion.

[0063] 4. Reverse transcription and template switching: SuperScipt II reverse transcriptase and oligo sequence TSO were used for reverse transcription and template switching.

[0064] 5. PCR amplification: The reverse transcribed cDNA is amplified by PCR.

[0065] 6. PCR purification: Use DNA Clean&ConcentratorTM-5 or AMPure XP Product magnetic beads to purify the PCR product to obtain the cDNA library.

[0066] 7. SMRT adapter connection: Connect the SMRTbell circular adapter to construct the SMRT library, and then purify it with magnetic beads for subsequent sequencing.

[0067] 8. Sequencing: The samples were sequenced on the PacBio SequelII third-generation sequencing platform.

[0068] In summary, we believe that there may be some special regulatory mechanism between the RNA level and protein level expression of OLFM4 in colon cancer stem cells, which affects the translation efficiency of OLFM4 gene RNA-protein. The reason may be related to the Poly(A)-tail located at the 3' end of the OLFM4 gene mRNA. Therefore, we used PALso-seq technology to detect the length and sequence of the Poly(A)-tail of the OLFM4 gene transcript ENST00000219022.3 in 15 groups of colon cancer samples (cancer and adjacent to cancer).

[0069] We found that Poly(A)-tail was only detected in colon cancer tissues (the numbers in the tissue sample number represent the patient number, T represents cancer tissue, and adj represents adjacent tissue) and the length was between 50-60 nt, but not in adjacent tissues (see Figure 4 ), and we further measured the specific sequence (Table 1) and average length (Table 2) of Poly(A)-tail in colon cancer tissues.

[0070] Table 1

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Table 2

[0077]

[0078]

[0079] Example 4 Diagnostic function of Poly(A)-tail for colon cancer

[0080] In order to evaluate the diagnostic function of Poly(A)-tail for colon cancer, we used the receiver operating characteristic curve (ROC curve) ROC curve and AUC to evaluate the effectiveness of the indicator (Poly(A)-tail). The ROC curve is a curve that reflects the relationship between sensitivity and specificity. The horizontal axis X-axis is 1-specificity, also known as the false positive rate; the vertical axis Y-axis is called sensitivity, also known as the true positive rate (sensitivity). The area under the curve (Area Under Curve, AUC) is often used to evaluate disease diagnosis. The AUC value range is generally between 0.5 and 1. The closer the AUC is to 1, the better the diagnostic effect of the variable in predicting the outcome. The Poly(A) data of the gene OLFM4 were analyzed by R language, and the results were visualized using ggplot2. The results showed that the AUC was 1 ( Figure 5 ), indicating that the length of the Poly(A) tail of the gene OLFM4 has a good diagnostic value for colon cancer.

[0081] Based on the above findings, we believe that by analyzing the length and sequence of the Poly(A)-tail of the OLFM4 gene in colon cancer stem cells, a more accurate prediction of the patient's staging and prognosis can be made.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of a preparation for detecting the length of Poly (A)-tails of olfactory domain family protein 4 (OLFM4) mRNA in the preparation of colon cancer detection products.

2. The use according to claim 1, characterized in that The product is a kit or a gene chip.

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