Application of Yarrowia guilliermondii as action target of hepatocellular carcinoma medicine and hepatocellular carcinoma progress evaluation

Abstract

The invention relates to the technical field of biology, in particular to application of Yarrowia guilliermondii serving as an action target of a hepatocellular carcinoma medicine and application of the Yarrowia guilliermondii in hepatocellular carcinoma progress evaluation. The medicine for treating the hepatocellular carcinoma is characterized in that the medicine for treating the hepatocellular carcinoma comprises the following action target points: Meyerozyma guilliermondii (Meyerozyma guilliermondii). In the prior art, research on action targets of hepatocellular carcinoma drugs is generally concentrated in bacteria, fungi are taken as research objects in the prior art, but the Michelia guilliermondii provided by the invention is a kind of fungi, so that a foundation is laid for research on the fungi as the action targets of the hepatocellular carcinoma drugs; the invention provides a novel research direction of an action target of a hepatocellular carcinoma drug.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of Meyer yeast as a target of hepatocellular carcinoma drugs and in the evaluation of hepatocellular carcinoma progression. Background Art

[0002] Tumor cells or tissues in patients with hepatocellular carcinoma have significant heterogeneity, and existing diagnostic and treatment technologies have not yet achieved ideal survival expectations. Therefore, it is of great significance to discover new progression-related markers and possible therapeutic targets to promote precision treatment. Studies have shown that microorganisms are a new component of the tumor microenvironment and can promote or inhibit tumor progression through a variety of mechanisms. Most studies on intratumoral microorganisms focus on bacteria, and there are few studies on fungi in hepatocellular carcinoma.

[0003] Fluorescence in situ hybridization (FISH) is a type of nucleic acid molecular hybridization technology. Based on the principle of base complementarity, a known fluorescently labeled single-stranded nucleic acid is used as a probe. After denaturation, annealing, and renaturation, it specifically binds to an unknown single-stranded nucleic acid in the material to be tested to form a detectable hybrid double-stranded nucleic acid. Microbial FISH detection uses the relatively stable rRNA in microorganisms as the target of analysis, and designs probes based on rRNA sequences for hybridization. It identifies and quantitatively analyzes the distribution of microorganisms and characteristic microorganisms to be detected, and provides information such as microbial morphology, spatial distribution, and the number of cells in the organism. The entire process includes: baking slices - dewaxing and hydration - digestion - dehydration - hybridization - washing after hybridization - DAPI nuclear staining - microscopic examination and photography, which has been widely used in clinical practice.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Meyer yeast quailmonas as a target for hepatocellular carcinoma drugs and in the assessment of hepatocellular carcinoma progression. The present invention detects the abundance of the intratumoral fungus Meyer yeast quailmonas, and finds that the abundance of Meyer yeast quailmonas in hepatocellular carcinoma tumor tissue is significantly higher than that in adjacent cancer tissue. In vivo mouse experiments verify that the intratumoral fungus Meyer yeast quailmonas can significantly promote the progression of hepatocellular carcinoma, and can be used as a progression marker and therapeutic target for hepatocellular carcinoma. Based on the above conclusions, the present invention further designs a FISH probe for detecting Meyer yeast quailmonas in tissues to serve clinical detection applications.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a target of a hepatocellular carcinoma drug, wherein the target of the hepatocellular carcinoma drug comprises: Meyerozyma guilliermondii.

[0008] In the prior art, the research on the target of hepatocellular carcinoma drugs is usually focused on bacteria, and there is little prior art that takes fungi as the research object. The Meyer yeast proposed in the present invention is a fungus, which lays a foundation for the research on fungi as the target of hepatocellular carcinoma drugs and provides a new research direction for the target of hepatocellular carcinoma drugs.

[0009] Furthermore, the hepatocellular carcinoma drug regulates the abundance of Meyer yeast in hepatocellular carcinoma tissue.

[0010] In the prior art, when studying the action targets of hepatocellular carcinoma drugs, the focus is usually on the changes in microorganisms in the intestine, and little attention is paid to the changes in microorganisms in tumor tissues. The present invention uses the cancerous tissue and paracancerous tissue of surgical samples of hepatocellular carcinoma patients as experimental objects, and detects the Guillermo Mayer yeast in the two tissues respectively. It is found that the abundance of Guillermo Mayer yeast in cancerous tissue is significantly higher than that in paracancerous tissue, and proposes a research idea of ​​using the changes in microorganisms in tumor tissues as the action targets of hepatocellular carcinoma drugs.

[0011] Another aspect of the present invention also relates to the use of the target of the hepatocellular carcinoma drug in the preparation of a drug for treating hepatocellular carcinoma.

[0012] According to the abundance changes of Guillermo Mayer yeast in tumor tissues, the Guillermo Mayer yeast in tumors can be used as a target for hepatocellular carcinoma drugs. Based on this conclusion, research can be conducted to obtain drugs for the treatment of hepatocellular carcinoma with good therapeutic effects and strong specificity.

[0013] Another aspect of the present invention also relates to the use of Meyer yeast in preparing a product for evaluating the progression of hepatocellular carcinoma.

[0014] Through research, it can be found that the abundance of Guillermo Mayer yeast in stem cell cancer tissues and normal tissues changes significantly. By evaluating the abundance of Guillermo Mayer yeast in stem cell cancer tissues, the progression of stem cell cancer can be evaluated.

[0015] Another aspect of the present invention also relates to a FISH probe, the nucleotide sequence of the FISH probe is shown as SEQ ID No. 1, and one or both ends of the nucleotide sequence of the FISH probe are labeled with a fluorescent group.

[0016] Further, SEQ ID No. 1: GGGCGAATGGTTAGCCAGAAGGAAAG.

[0017] The FISH probe is designed based on the silva138.1 database, which is one of the most complete databases of fungal 18S sequences. According to all common sequences in the target 18S sequence library, excluding non-specific regions, all possible FISH probe sequences are listed, and the probe with the highest coverage of the target 18S sequence library and the least matching with the non-target sequence library is selected, that is, the probe with the highest specificity.

[0018] The FISH probe is used to detect Meyer yeast guilliermondii, can specifically mark M. guilliermondii in tissues, has the advantages of high coverage and good specificity, and can determine the presence and quantity of Meyer yeast guilliermondii according to the intensity and position of the detected fluorescent signal, thereby evaluating the abundance of the strain.

[0019] Furthermore, when both ends of the nucleotide sequence of the FISH probe are labeled with fluorescent groups, the types of the fluorescent groups are the same.

[0020] Furthermore, the fluorescent group includes, but is not limited to, at least one of Cy5, FITC, Texas Red, FAM, TET, CALGold 540, JOE, HEX, TAMRA, ROX, CY3 or VIC.

[0021] Furthermore, the FISH probe is used in conjunction with a positive probe during use.

[0022] Furthermore, the positive probe includes but is not limited to: MY1574 probe. This positive probe can detect all fungi and serves as a positive control in the present invention.

[0023] Furthermore, the sequence of the MY1574 probe is shown as SEQ ID No.2.

[0024] Further, SEQ ID No. 2: TCCTCGTTGAAGAGC.

[0025] Furthermore, one or both ends of the nucleotide sequence of the MY1574 probe are labeled with a second fluorescent group.

[0026] Furthermore, when the two ends of the nucleotide sequence of the MY1574 probe are labeled with a second fluorescent group, the type of the second fluorescent group is the same.

[0027] Furthermore, the second fluorescent group includes, but is not limited to, at least one of FITC, Cy3, Texas Red, FAM, TET, CALGold 540, JOE, HEX, TAMRA, ROX, CY5 or VIC.

[0028] Another aspect of the present invention also relates to the use of the FISH probe in preparing a product for evaluating the progression of hepatocellular carcinoma.

[0029] The FISH probe is used to detect Meyer yeast quaternaria and exhibits good specificity and high coverage. The use of the FISH probe to detect changes in Meyer yeast quaternaria in cancer tissues can further evaluate the progression of hepatocellular carcinoma.

[0030] Furthermore, the hepatocellular carcinoma progression assessment includes: the abundance of Myersinia guilliermonas yeast in hepatocellular carcinoma tissue is positively correlated with the degree of hepatocellular carcinoma progression.

[0031] When the abundance of Mycobacterium quaternaris in hepatocellular carcinoma tissue increases, it indicates that the hepatocellular carcinoma has progressed more severely, which is helpful for clinical preliminary judgment of the development degree of hepatocellular carcinoma and selection of more accurate diagnosis and treatment plans.

[0032] Another aspect of the present invention also relates to the use of the FISH probe in detecting Meyer yeast.

[0033] The FISH probe can be used to detect Meyer yeast in a sample, and the object of detection is not specifically limited. It is applicable to biological samples that need to be detected routinely in the field, including but not limited to hepatocellular carcinoma tissue.

[0034] Another aspect of the present invention also relates to a kit, comprising the FISH probe.

[0035] The kit can be used to detect Meyer yeast quaternaria, and can also be used to judge and evaluate the progress of hepatocellular carcinoma.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention detects the abundance of the intratumoral fungus Mycobacterium quaternum and finds that the abundance of Mycobacterium quaternum in hepatocellular carcinoma tumor tissue is significantly higher than that in adjacent tissues. In vivo mouse experiments verify that the intratumoral fungus Mycobacterium quaternum can significantly promote the progression of hepatocellular carcinoma and can be used as a progression marker and therapeutic target for hepatocellular carcinoma. Based on the above conclusions, the present invention further designs a FISH probe for detecting Mycobacterium quaternum in tissues to serve clinical detection applications.

[0038] (2) The present invention proposes to use Meyer yeast as a target for hepatocellular carcinoma drugs. In the prior art, research on the target of hepatocellular carcinoma drugs is usually focused on bacteria, and there is little prior art that uses fungi as research objects. Meyer yeast is a fungus. The present invention lays a foundation for the research on fungi as the target of hepatocellular carcinoma drugs and provides a new research direction for the target of hepatocellular carcinoma drugs.

[0039] (3) The present invention uses cancerous tissue and paracancerous tissue of surgical samples of hepatocellular carcinoma patients as experimental objects, detects Guillermo Mayer yeast in the two tissues respectively, and finds that the abundance of Guillermo Mayer yeast in cancerous tissue is significantly higher than that in paracancerous tissue, and proposes a research idea of ​​using the changes in microorganisms in tumor tissue as the target of hepatocellular carcinoma drugs. In the prior art, when studying the targets of hepatocellular carcinoma drugs, the focus is usually on the changes in microorganisms in the intestine, and little attention is paid to the changes in microorganisms in tumor tissues.

[0040] (3) The present invention proposes to use the target of the hepatocellular carcinoma drug for the preparation of a drug for treating hepatocellular carcinoma. According to the abundance changes of the Guillermo Mayer yeast in tumor tissues, the Guillermo Mayer yeast in tumors can be used as the target of the hepatocellular carcinoma drug. Based on this conclusion, research can be conducted to obtain a drug for treating hepatocellular carcinoma with good therapeutic effects and strong specificity.

[0041] (4) The FISH probe provided by the present invention is designed based on the silva138.1 database, which is one of the most complete databases of fungal 18S sequences. According to all the common sequences in the target 18S sequence library, excluding the non-specific regions therein, all possible FISH probe sequences are listed, and the probe with the highest coverage of the target 18S sequence library and the least match with the non-target sequence library is selected, that is, the probe with the highest specificity.

[0042] (5) The FISH probe provided by the present invention is used to detect M. guilliermondii and can specifically mark M. guilliermondii in tissues. It has the advantages of high coverage and good specificity. According to the intensity and position of the detected fluorescent signal, the presence and quantity of M. guilliermondii can be determined, thereby evaluating the abundance of the bacterial species.

[0043] (6) The present invention uses the FISH probe to detect Meyer yeast in quaternary yeast, showing good specificity and high coverage. The use of the FISH probe to detect changes in Meyer yeast in cancer tissues can further evaluate the progression of hepatocellular carcinoma.

[0044] (7) The FISH probe provided by the present invention can be used to detect Mayerella guillermonas in a sample, without any specific limitation on the object of detection, and is applicable to biological samples that need to be detected routinely in the art, including but not limited to hepatocellular carcinoma tissue.

[0045] (8) The kit provided by the present invention can be used to detect Mayer yeast and can also be used to determine and evaluate the progression of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 The LEfSe provided in the embodiment of the present invention analyzes the differential fungi in hepatocellular carcinoma tissue and adjacent tissue;

[0048] Figure 2 The M. guilliermondii provided in the embodiments of the present invention promotes the progression of mouse hepatocellular carcinoma;

[0049] Figure 3 The fluorescence in situ hybridization (FISH) method provided in the embodiment of the present invention is used to detect M. guilliermondii in hepatocellular carcinoma tissue. DETAILED DESCRIPTION

[0050] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0051] Example 1 Detection of the abundance of Meyer yeast in hepatocellular carcinoma tissue

[0052] In this example, surgical specimens from 52 patients with hepatocellular carcinoma were collected and divided into cancerous tissue and adjacent tissue. DNA was extracted to amplify ITS2 and sequenced for analysis. Figure 1As shown, the abundance of Meyerozyma guilliermondii (M. guilliermondii) in cancer tissues was significantly higher than that in adjacent paracancerous tissues.

[0053] Example 2 Verification of Meyer yeast promoting the progression of hepatocellular carcinoma

[0054] Construction of a mouse model of hepatocellular carcinoma induced by hydrodynamic injection of plasmids: 15 μg c-myc-PT3EF1a, 15 μg PX330-p53 and 5 μg pCMV(CAT)T7-SB100 plasmids were fully dissolved in 2 mL of 0.9% NaCl solution and the mixed solution was injected into 7-week-old C57BL / 6 male mice through the tail vein within 7 seconds. Two weeks after plasmid injection, the liver was inoculated with about 10 7 cfu of M.guilliermondii were inoculated once every 3 days. The mice were killed after being intervened with bacteria and PBS for about 3 weeks, and the liver weight was observed and counted.

[0055] M. guilliermondii and PBS were inoculated into the liver of a mouse model of hepatocellular carcinoma induced by hydrodynamic injection of plasmids. The mice were killed at the end of the experiment. It was observed with the naked eye that compared with the PBS group, the tumor progression of mice in the M. guilliermondii intervention group was obvious ( Figure 2 A); There was no difference in body weight between the two groups of mice ( Figure 2 B), while the liver weight of mice in the M. guilliermondii intervention group ( Figure 2 C) Liver weight to body weight ratio ( Figure 2 D) was greater than that in the PBS group. In conclusion, M. guilliermondii can significantly promote the progression of hepatocellular carcinoma, and the intratumoral fungus M. guilliermondii can be used as a tumor progression marker and therapeutic target.

[0056] Example 3 Design of FISH probes for detecting Monmeyer yeast

[0057] The probe design of this embodiment adopts the database of silva138.1 (released on August 27, 2020, currently the latest version). The entire database contains 58,790 representative sequences of eukaryotic 18S, which is one of the most complete databases of fungal 18S sequences. The name of the target species is Meyerozyma guilliermondii, there are 15 representative sequences of 18S belonging to the target species (hereinafter referred to as the target 18S sequence library), 319 species of Debaryomycetaceae, and 510,493 representative sequences belonging to non-target species (hereinafter referred to as the non-target sequence library). According to all the common sequences in the target 18S sequence library, the non-specific regions therein are excluded, all possible FISH probe sequences are listed, and the probe with the highest coverage of the target 18S sequence library and the least match with the non-target sequence library is selected, that is, the probe with the highest specificity. The final designed FISH probe sequence for detecting M. guilliermondii is shown in SEQ ID No. 1, with fluorescent group Cy5 labeled at both ends; the positive probe is MY1574, the sequence is shown in SEQ ID No. 2, with fluorescent group FITC labeled at both ends.

[0058] SEQ ID No. 1: GGGCGAATGGTTAGCCAGAAGGAAAG.

[0059] SEQ ID No. 2:TCCTCGTTGAAGAGC.

[0060] Example 4 Detection of M. guilliermondii in tissues using FISH probes

[0061] In this example, the FISH probe for detecting M. guilliermondii in liver cancer tissue obtained in Example 3 and the positive probe MY1574 were used to detect fungi in liver cancer tissue. The specific steps are as follows:

[0062] 1. Paraffin blocks of liver cancer tissue were cut into 3 μm thick sections with glue, and the sections were baked at 65℃ for 2 hours. At room temperature, the sections were immersed in xylene solution twice, each for 10 minutes; immersed in 100% ethanol twice, each for 5 minutes; immersed in 95% ethanol and 75% ethanol for 2 minutes each; immersed in distilled water for 3 minutes; and dried.

[0063] 2. Soak the humidifying strip in the Termo Brite hybridization instrument and preheat the instrument to 37°C; determine the 200μg / ml lytic enzyme digestion time (about 12 minutes) and the required amount (about 50ul) according to the thickness of each slice, place it in the preheated hybridization instrument, and start program "1": 37°C, 15 minutes; take out the slices on time and immediately put them in distilled water to remove the lytic enzyme activity; wash them twice with 2×SSC at room temperature, 5 minutes each time; dry the slices at room temperature, add 10μl DAPI; wash them twice with 2×SSC, 5 minutes each time. Soak them in 75% ethanol, 95% ethanol and 100% ethanol at room temperature for 2 minutes each, and dry the slices naturally.

[0064] 3. Mix 2 μl FISH probe, 1 μl positive probe and 37 μl hybridization solution (BersinBio TM

[0065] Vortex thoroughly to mix, apply about 10 μl of the probe mixture to the hybridization area of ​​the slice, cover with a siliconized coverslip, be careful to prevent bubbles, and seal the four sides of the coverslip with rubber glue; place the slice on the hybridizer and start program "2": denaturation at 73℃ for 8min, hybridization at 37℃ for 16-24h.

[0066] 4. Carefully remove the rubber glue, wash the tissue slices in 2×SSC for 5 minutes, and remove them from the siliconized coverslip; wash the tissue slices in 25% formamide / 2×SSC preheated at 53°C twice, 5 minutes each time; wash them in 0.1% NP-40 / 2×SSC preheated at 42°C twice, 5 minutes each time; wash them in 0.5×SSC preheated at 42°C once, 5 minutes; wash them in 0.2×SSC preheated at 42°C once, 5 minutes.

[0067] 5. Dry the slices naturally in the dark at room temperature, add 20 μl DAPI for staining for 10 min, then wash twice with 1× PBS for 5 min each time; add 50 μl 30% glycerol buffer, cover with a large coverslip, and place the hybridized tissue slices on an Olympus upright fluorescence microscope to observe and collect images.

[0068] Figure 3 It is clearly shown that the FISH probe for detecting M. guilliermondii provided by the present invention can specifically mark M. guilliermondii in tissues, indicating that the FISH probe designed by the present invention has high coverage and good specificity, and can accurately detect M. guilliermondii in tissues.

[0069] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. The target of hepatocellular carcinoma drug, characterized in that: The target of the hepatocellular carcinoma drug includes: Meyerozyma guilliermondii.

2. The target of the hepatocellular carcinoma drug according to claim 1, characterized in that: The hepatocellular carcinoma drug regulates the abundance of Meyer yeast in hepatocellular carcinoma tissue.

3. Use of the target of the hepatocellular carcinoma drug as claimed in claim 1 or 2 in the preparation of a drug for treating hepatocellular carcinoma.

4. Application of Meyer yeast in the preparation of hepatocellular carcinoma progression assessment products.

5. FISH probe, characterized in that The nucleotide sequence of the FISH probe is shown in SEQ ID No. 1, and one or both ends of the nucleotide sequence of the FISH probe are labeled with a fluorescent group.

6. The FISH probe according to claim 5, characterized in that When both ends of the nucleotide sequence of the FISH probe are labeled with fluorescent groups, the types of the fluorescent groups are the same; And / or, the fluorescent group includes at least one of Cy5, FITC, Texas Red, FAM, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3 or VIC.

7. Use of the FISH probe according to claim 5 or 6 in preparing a product for evaluating the progression of hepatocellular carcinoma.

8. The use according to claim 7, characterized in that: The hepatocellular carcinoma progression assessment includes: the abundance of Mycobacterium guilliermonensis in hepatocellular carcinoma tissue is positively correlated with the degree of hepatocellular carcinoma progression.

9. Use of the FISH probe according to claim 7 or 8 in detecting Meyer yeast.

10. A kit, characterized in that Comprising the FISH probe according to claim 7 or 8.

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