Application of miR-30a-5p and target gene thereof in diagnosis and treatment of hepatocellular carcinoma
By regulating the expression level of miR-30a-5p and using Ulva polysaccharide in combination, the toxic side effects of 5-fluorouracil were reduced. A diagnostic kit was developed using miR-30a-5p and Thbs2, which solved the problems of early detection of liver cancer and drug side effects, and achieved accurate diagnosis of liver cancer and relief of drug side effects.
Patent Information
- Application Number
- CN202511540782.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-01-30
AI Technical Summary
In the current technology, there are insufficient methods for early detection of liver cancer, and chemotherapy drugs such as 5-fluorouracil have serious toxic side effects, and there is a lack of effective biomarkers and drug screening methods.
By utilizing miR-30a-5p and its target gene Thbs2, the toxic side effects of 5-fluorouracil were reduced by regulating the expression level of miR-30a-5p. Ulva polysaccharide (ULP) was used in combination to screen for drugs that reduce toxic side effects. A diagnostic kit was developed by combining miR-30a-5p primers and the target gene Thbs2.
It significantly reduced the toxic side effects of 5-fluorouracil and provided a basis for the early diagnosis of liver cancer. Thbs2, as a potential biomarker, improved the accuracy of liver cancer diagnosis and the ability to alleviate drug side effects.
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Figure CN121428095A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of miR-30a-5p and a target gene thereof in diagnosis and treatment of hepatocellular carcinoma. BACKGROUND
[0002] Liver cancer is one of the cancer problems, and the disease burden situation for patients is grim. Among them, hepatocellular carcinoma (HCC) accounts for 85%-90% of all primary liver cancer. In the development process of cancer treatment, the importance of prevention and early detection has attracted more and more attention. Early detection can improve the success rate of cancer treatment, and with the continuous development of medical technology, many screening and diagnosis methods for cancer have been significantly improved. Gene detection can help understand the genetic status of patients, find people prone to liver cancer, and help early intervention; biomarker detection can detect liver cancer markers, and has a certain auxiliary role for the diagnosis of early liver cancer. Therefore, finding suitable biomarkers is of great significance for the diagnosis and treatment of liver cancer.
[0003] Green algae, as the most widely distributed macroalgae in the ocean, are rich in various pharmacologically active ingredients, including polysaccharides, polyphenolic compounds and polyunsaturated fatty acids. Among them, Ulva lactuca polysaccharides (ULP) have attracted attention due to their unique chemical structure and significant biological activity. Studies have shown that ULP not only has anti-tumor activity itself, but also can be combined with nano-drug carriers to improve the anti-cancer effect while significantly reducing the toxic side effects of chemotherapy drugs. Compared with traditional chemotherapy drugs 5-FU, ULP, as a natural source of bioactive substances, has the advantages of low toxicity, good selectivity, multi-target action, etc.
[0004] MicroRNA (miRNA) is a kind of short sequence, non-coding single-stranded small molecule RNA with a length of about 18-24 nt, which has important regulatory functions. Its generation process starts from the initial transcription product (Pri-miRNA) of long-chain RNA. Pri-miRNA is cleaved in the nucleus to form a miRNA precursor with a stem-loop structure; the precursor is then transported to the cytoplasm and further processed to form mature miRNA. In animal and plant cells, microRNAs play a role by cutting target mRNAs or inhibiting their transcription, and are widely involved in cell growth, tissue differentiation, tumor formation and other physiological and pathological processes. SUMMARY
[0005] Therefore, the purpose of the present application is to provide the application of miR-30a-5p and its target gene in the diagnosis and treatment of hepatocellular carcinoma.
[0006] To achieve the above technical purposes, the technical scheme adopted by the present application is: The application of miR-30a-5p in reducing the toxic side effects of anti-liver cancer drugs, wherein the Gene ID of miR-30a-5p in NCBI is 407029, and the anti-liver cancer drug is 5-fluorouracil.
[0007] The application is to reduce the toxic side effects of anti-liver cancer drugs by regulating and reducing the expression amount of miR-30a-5p.
[0008] The present application also provides the application of miR-30a-5p in screening drugs that can reduce the toxic side effects of 5-fluorouracil, wherein the Gene ID of miR-30a-5p in NCBI is 407029; and the drug can regulate and reduce the expression amount of miR-30a-5p.
[0009] The present application also provides a kit for screening drugs that can reduce the toxic side effects of 5-fluorouracil, which comprises primers for detecting the expression amount of miR-30a-5p, wherein the Gene ID of miR-30a-5p in NCBI is 407029, and the primers for miR-30a-5p comprise reverse transcription primers and forward detection primers, the sequence of the reverse transcription primers is shown in SEQ ID NO: 1, and the sequence of the forward detection primers is shown in SEQ ID NO: 2, and the specific sequence is as follows: SEQ ID NO: 1: 5-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTTCCAGT-3'; SEQ ID NO: 2: 5'-ACACTCCAGCTGGGTGTAAACATCCTCGAC-3'.
[0010] The present application also provides the application of the target gene of miR-30a-5p in preparing products for diagnosing or assisting in diagnosing liver cancer, wherein the target gene is Thbs2, and the Gene ID of Thbs2 in NCBI is 7058.
[0011] Compared with the prior art, the application has the beneficial effects that the application relates to the application of miR-30a-5p and its target gene in liver cancer, wherein the Gene ID of miR-30a-5p in NCBI is 407029; it is proved by experiments that the toxic side effects of the anti-liver cancer drug 5-fluorouracil can be significantly reduced by reducing the expression amount of miR-30a-5p, and this finding makes miR-30a-5p become a potential target for screening drugs for reducing the toxic side effects of 5-fluorouracil; when the target gene Thbs2 of miR-30a-5p is low in expression, the immunohistochemical (IHC) staining data is searched from the HPA database, and the expression level of Thbs2 is obtained from the protein aspect. Among them, Thbs2 is moderately stained in normal liver tissue, and Thbs2 is highly stained in liver cancer tissue, which shows that Thbs2 is expected to be used as a molecular marker for diagnosing liver cancer. The research not only provides an important basis for the early diagnosis, differential diagnosis, effective observation and alleviation of the side effects of anticancer drugs of liver cancer, but also provides a new idea for the precise diagnosis of liver cancer and the alleviation of the side effects of anticancer drugs from the molecular level, expands the cognition of the mechanism of the occurrence and development of liver cancer in the theoretical level, and also shows significant practical value and conversion potential in clinical application. BRIEF DESCRIPTION OF DRAWINGS
[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, a brief introduction will be given below to the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.
[0013] Figure 1 is a tumor tissue diagram of the mice in the Model group, the 5FU group and the 5FU+ULP group in Example 1; Figure 2 is a comparison of the tumor inhibition rates of the Model group, the 5FU group and the 5FU+ULP group in Example 1; Figure 3 is a schematic diagram of the difference in the expression levels of miRNA in the two (group) samples in Example 2 and the statistical significance of the difference; Figure 4 is a schematic diagram of the overall correlation pattern of the difference fold and the expression abundance of the two (group) samples in Example 2; Figure 5 is a comparison schematic diagram of the significant increase of the expression amount of miR-30a-5p in the 5-FU+ULP combined administration group compared with the 5-FU group in Example 3; Figure 6is a schematic diagram of the prediction of the miR-30a-5p target gene in Example 4; Figure 7 is a schematic diagram of the Thbs2 gene, which is significantly related to the occurrence and development of liver cancer among the target genes regulated by miR-30a-5p in Example 4; Figure 8 is a schematic diagram of the data showing that the expression of Thbs2 in hepatocellular carcinoma is higher than that in normal liver tissue, which is found by using the GEPIA database in Example 4; Figure 9 is a schematic diagram of the data showing that the RT-qPCR analysis results are consistent with the sequencing data in Example 4; Figure 10 is a schematic diagram showing the results of the immunohistochemical (IHC) staining data retrieved from the HPA database to obtain the expression level of Thbs2 from the protein perspective, which shows that Thbs2 is moderately stained in normal liver tissue, and Thbs2 is highly stained in liver cancer tissue in Example 4. DETAILED DESCRIPTION
[0014] The application will be further described below in conjunction with the drawings and examples. It is particularly pointed out that the following examples are only used to illustrate the application, but do not limit the scope of the application. Similarly, the following examples are only part of the examples of the application, not all examples, and all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of the application.
[0015] Example 1 Animal model construction In this embodiment, the hepatoma H22 cells were diluted with RPMI 1640 medium to 106 cells / mL, and then 0.2 mL was injected intraperitoneally into the left anterior axillary fossa of the mice to construct a H22 hepatoma tumor-bearing mouse model. Then the successfully modeled mice were randomly divided into groups (model group, normal group, 5FU group, 5FU+ULP group). Among them, the model group (i.e. Model group) and the normal group did not use any drugs; the 5FU group of mice was injected intraperitoneally with a single dose of 20 mg / kg / d of 5-fluorouracil; the 5FU+ULP group of mice was injected intraperitoneally with a single dose of 20 mg / kg / d of 5-fluorouracil, and orally administered with 300 mg / kg / d of Ulva lactuca polysaccharide (ULP); the ULP group of mice was orally administered with 300 mg / kg / d of Ulva lactuca polysaccharide. After 21 days of continuous administration, the mice were sacrificed by cervical dislocation, the thymus index and spleen index were determined, and the tumor tissues of the mice were collected. The collected mouse tumor tissues are shown in Figure 1 . From Figure 1It can be known from the above table that the growth of H22 tumor in the mice was significantly inhibited by 5FU. Taking the tumor weight of the model group as a reference, the tumor inhibition rates of the 5FU group and the combined administration group (5FU+ULP group) were calculated to be 62.01% and 74.63%, respectively. The results show that the ULP and 5FU can play a stronger anti-tumor activity when used in combination, and the anti-tumor activity is significantly stronger than that of 5FU alone. As shown in Figure 2 .
[0016] From Figure 2 It can be known from the above table that the growth of H22 tumor in the mice was significantly inhibited by 5FU. Taking the tumor weight of the model group as a reference, the tumor inhibition rates of the 5FU group and the combined administration group (5FU+ULP group) were calculated to be 62.01% and 74.63%, respectively. The results show that the ULP and 5FU can play a stronger anti-tumor activity when used in combination, and the anti-tumor activity is significantly stronger than that of 5FU alone. As shown in
[0017] Example 2 Differential expression miRNA screening and target gene prediction In the drug group (5FU group) and the combined administration group (5FU+ULP group), 3 mice were taken from each group, and the drug group (B1, B2, B3) and the combined administration group (E1, E2, E3) were cut and weighed about 50-100 mg of tissue, and then added to a mortar containing liquid nitrogen for grinding. The total RNA was extracted by Trizol method. After the sample was detected, the special structure of the small RNA 3' and 5' end was used to construct the small RNA library. The small RNA was added with a linker at both ends, and was reverse transcribed into cDNA. Then PCR amplification and PAGE gel electrophoresis were performed to construct the cDNA library. After the library was constructed, Qubit2.0 was used for preliminary quantification. Next, Agilent2100 was used to detect the insert size of the library, and then qPCR was used to quantify the effective concentration of the library. Finally, after the inspection was qualified, high-throughput sequencing was performed.
[0018] The sequencing data was quality controlled to ensure the reliability of information analysis. The following data was deleted: low-quality reads (sQ≤20, the number of bases accounting for more than 30% of the entire fragment); reads with indeterminate base information and a proportion greater than 10%; polyA / T / G / C; reads without 3' linker sequence; insert fragments and reads with 5' linker contamination. The 3' linker sequence was trimmed.
[0019] Bowtie was used to screen sRNAs, locating their lengths on reference sequences and analyzing their distribution. Within a specified range of sequences in the miRBase database, reads mapped to the reference sequences were compared to obtain detailed information on sRNA matching in the samples. Mouse ncRNA sequences were used to annotate the detected sRNAs. Next, repetitive sequences in tRNA, rRNA, snRNA, and snoRNA were removed. Finally, the exons and introns of the mRNA were aligned to identify sRNAs derived from mRNA degradation fragments.
[0020] The DESeq2 function in R was used to screen for significantly different miRNAs between the drug group and the combination therapy group. miRNAs with log2FoldChange < 0 were considered downregulated, while those with log2FoldChange < 0 were considered upregulated. The p-value must be < 0.05 during the screening process. A total of 209 differentially expressed miRNAs were identified. In the combination therapy group, 98 miRNAs were significantly upregulated compared to the drug group, and 111 miRNAs were significantly downregulated. Figure 3 As shown, the differences in miRNA expression levels between the two (groups) of samples, and the statistical significance of these differences. Figure 4 As shown, the overall correlation between the fold change and expression abundance of the two (groups) of genes can be further visualized. The changes in the expression profiles of the differentially expressed miRNAs suggest that Ulva prolifera polysaccharides may play a potential role in alleviating the toxicity of 5-FU by regulating specific miRNA molecular networks.
[0021] Example 3 RT-qPCR validation of differentially expressed miRNAs The differentially expressed miRNAs in Example 2 were verified by RT-qPCR.
[0022] The reverse transcription of miRNA was performed using a biomolecular miRNA first-strand cDNA synthesis (tailing method) kit. The reverse transcription steps, temperature, and program settings were followed according to the corresponding instructions and the operation was performed on a PCR multifunction instrument. The synthesized cDNA was diluted 50 times and stored at -20 ℃ for later use.
[0023] The primers for detecting miR-30a-5p expression include reverse transcription primers and forward detection primers. The sequence of the reverse transcription primer is shown in SEQ ID NO: 1, and the sequence of the forward detection primer is shown in SEQ ID NO: 2.
[0024] The cDNA after reverse transcription was taken as template RNA to prepare for RT-qPCR reaction. Eight connected tubes, ice box and sterilized gun head were prepared in advance. The miRNA fluorescent quantitative PCR kit (dye method) of Shengwo was used as the kit of miRNA. The steps and program setting of RT-qPCR reaction were carried out according to the kit instructions.
[0025] Finally, from the obtained miRNA consistent with the sequencing results, it was determined that the expression amount of miR-30a-5p in the 5-FU+ULP combined administration group was significantly higher than that in the 5-FU group Figure 5 ). Further support the reliability of the omics data.
[0026] Example 4 Target gene prediction of differentially expressed miRNA In order to systematically analyze the potential regulatory network of miR-30a-5p, the research adopts the joint strategy of multiple databases to predict the target genes. Based on the complementarity of miRNA target gene prediction algorithm, the target genes of miR-30a-5p are predicted through three different miRNA prediction websites Figure 6 ). The intersection target genes of the three databases are screened, and the results show that Thbs2 gene is significantly related to the occurrence and development of liver cancer among the target genes regulated by miR-30a-5p Figure 7 ).
[0027] Using GEPIA database, it is found that Thbs2 is higher in hepatocellular carcinoma than in normal liver tissue Figure 8 ), and the RT-qPCR analysis results are consistent with the sequencing data Figure 9 ). Further clarify that ULP can inhibit the expression of Thbs2 by regulating miR-30a-5p, so that the expression of Thbs2 tends to be normal liver tissue.
[0028] Further search for immunohistochemical (IHC) staining data from HPA database to obtain the expression level of Thbs2 from the protein perspective. The results show that Thbs2 is moderately stained in normal liver tissue, while Thbs2 is highly stained in liver cancer tissue Figure 10 ).
[0029] The above results suggest that ULP may play a role in relieving the side effects of 5-fluorouracil drugs by regulating multiple inflammation-related signaling pathways. ULP may reduce the toxic side effects of anti-liver cancer drug 5-fluorouracil by reducing the expression of miR-30a-5p. miR-30a-5p can be applied to screen drugs that can reduce the toxic side effects of 5-fluorouracil, and Thbs2 is expected to be a marker for liver cancer.
[0030] The above merely describes some embodiments of the present application, and is not intended to limit the protection scope of the present application, and any equivalent device or equivalent process transformation, or direct or indirect application in other related technical fields, which are made by using the content of the present application specification and drawings, are also included in the patent protection scope of the present application.
Claims
1. Application of miR-30a-5p in reducing toxic side effects of anti-liver cancer drugs, characterized in that: The Gene ID of the miR-30a-5p in NCBI is 407029, and the anti-liver cancer drug is 5-fluorouracil.
2. Use according to claim 1, wherein The expression amount of miR-30a-5p is regulated and reduced to reduce the toxic side effects of the anti-liver cancer drug.
3. Application of miR-30a-5p in screening of drugs capable of reducing side effects of 5-fluorouracil, characterized in that: The Gene ID of the miR-30a-5p in NCBI is 407029.
4. The use according to claim 3, wherein the compound is ###0002### The expression amount of miR-30a-5p is regulated and reduced.
5. A kit for screening a drug which can reduce the toxic side effects of 5-fluorouracil, characterized by: The primer for detecting the expression amount of miR-30a-5p, the sequence of the reverse transcription primer of miR-30a-5p is shown in SEQ ID NO: 1, and the sequence of the forward detection primer is shown in SEQ ID NO:
2. The Gene ID of the miR-30a-5p in NCBI is 407029.
6. Application of the target gene of miR-30a-5p in preparation of a product for diagnosing or assisting diagnosis of liver cancer, characterized in that: The target gene is Thbs2, and the Gene ID of Thbs2 in NCBI is 7058.
7. Use of the ulva polysaccharide in preparation of a drug for reducing the toxic side effects of 5-fluorouracil.