Innovative screening methods and applications of key molecules in the occurrence and development of intrahepatic cholangiocarcinoma

Abstract

The present invention provides an innovative screening method and application of key molecules in the occurrence and development of intrahepatic cholangiocarcinoma. The present invention first performs transcriptome sequencing on intrahepatic cholangiocarcinoma tissue and its corresponding adjacent tissue. Subsequently, according to the difference multiples between the sequencing results of the tumor and the adjacent tissue, several long non-coding RNAs are selected for subsequent cell phenotype screening experiments. According to the results of the screening experiment, the lncRNA that can reduce the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells is selected as the key molecule in the occurrence and development of intrahepatic cholangiocarcinoma. Further, in vivo and in vitro experiments confirmed that targeting key molecules can inhibit the occurrence and development of ICC, and also verified the feasibility and effectiveness of the key molecule mining strategy in the occurrence and evolution mechanism of intrahepatic cholangiocarcinoma of the present invention. The present invention aims to propose an innovative screening strategy for the discovery of key molecules in the occurrence and development of ICC, in order to provide useful ideas for early diagnosis and precise treatment of ICC.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an innovative screening method and application of key molecules in the occurrence and development of intrahepatic cholangiocarcinoma. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC), as a malignant tumor originating from the intrahepatic bile duct epithelium, is highly invasive and accounts for 10% to 20% of primary liver cancer, ranking second after hepatocellular carcinoma (HCC). Based on the different anatomical locations, cholangiocarcinoma is subdivided into distal cholangiocarcinoma, hilar cholangiocarcinoma, and ICC. ICC specifically refers to the type that occurs above the proximal secondary bile duct of the liver, accounting for approximately 8% to 20% of all cholangiocarcinoma cases. The uniqueness of ICC is that although it originates from biliary epithelial cells, it shares similar liver microenvironment characteristics with HCC, making its research not simply classified as traditional cholangiocarcinoma. In recent years, the incidence of ICC has been rising year by year worldwide, showing obvious racial and regional differences, especially in Southeast Asia and East Asia. ICC often has an insidious onset and lacks specific clinical manifestations. Most patients are already in the middle and late stages of the disease when diagnosed, often accompanied by complex conditions such as multiple intrahepatic lesions, hilar lymph node metastasis, and even abdominal metastasis. Surgical resection combined with lymph node dissection is considered the preferred treatment for ICC, but it is only applicable to about 30% to 40% of patients, and the postoperative recurrence rate remains high, with a 5-year survival rate of less than 33%. For non-surgical candidates, comprehensive treatment strategies mainly focus on systemic chemotherapy and local radiotherapy. Although gemcitabine combined with cisplatin is a first-line treatment option, its overall efficacy is limited. Radiotherapy is mostly used for palliative treatment of patients with locally advanced disease, but it is often accompanied by complications such as radiation gastroenteritis. With the advancement of medicine, the rise of targeted therapy and immunotherapy has opened up new avenues for the treatment of ICC. Although related clinical trials and studies are underway, the ideal therapeutic effect has not yet been achieved. Therefore, in-depth exploration of the molecular pathogenesis of ICC and the discovery of new biomarkers and molecular targets are of vital significance for the early diagnosis and precise treatment of ICC.

[0003] Non-coding RNA, a type of RNA molecule that cannot directly encode proteins, accounts for more than 90% of the transcription products of the human genome. Among them, long noncoding RNA (lncRNA), specifically non-coding RNA transcripts with a length of more than 200 nucleotides, accounts for 80% of the total non-coding RNA. They play a pivotal role in various functions of cells and exert their far-reaching influence by intervening in multiple levels of regulatory processes, including transcription, post-transcription, translation, post-translation, and epigenetic regulation. Summary of the invention

[0004] The present invention takes the screening of key non-coding RNA as the entry point, aiming to lay a solid foundation for the subsequent exploration of the screening of important RNA molecules in the progression of ICC, such as mRNA, miRNA, and circRNA, and strives to open up an innovative screening path for the excavation of key molecules in the occurrence and evolution mechanism of intrahepatic cholangiocarcinoma, aiming to propose innovative screening strategies for the discovery of key molecules in the occurrence and development of ICC, in order to provide useful ideas for the early diagnosis and precise treatment of ICC.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An innovative screening method for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma, including:

[0007] Obtain intrahepatic cholangiocarcinoma tissues and their corresponding adjacent tissues for transcriptome sequencing, and then screen lncRNAs that are differentially expressed in intrahepatic cholangiocarcinoma tissues and their corresponding adjacent tissues based on transcriptome sequencing;

[0008] According to the differential expression direction of the lncRNA obtained by screening, a corresponding method is designed to carry out a cell phenotype screening experiment in intrahepatic cholangiocarcinoma cells, wherein the cell phenotype screening experiment includes proliferation and / or invasion ability detection; according to the screening experiment results, the lncRNA that can affect the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells is selected as the key molecule in the occurrence and development of intrahepatic cholangiocarcinoma.

[0009] Furthermore, the screening of lncRNAs differentially expressed in intrahepatic cholangiocarcinoma tissues and their corresponding adjacent tissues based on transcriptome sequencing specifically includes:

[0010] Based on the lncRNA database, identify and annotate lncRNAs with known sequences in transcriptome sequencing results;

[0011] Screening of lncRNAs that are differentially expressed in intrahepatic cholangiocarcinoma tissues and their corresponding adjacent tissues in transcriptome sequencing.

[0012] Furthermore, according to the differential expression direction of the lncRNA obtained by screening, a corresponding method is designed to conduct a cell phenotype screening experiment in intrahepatic cholangiocarcinoma cells, specifically:

[0013] If the differential expression direction of the screened lncRNA is up-regulated in intrahepatic cholangiocarcinoma tissue, a corresponding knockdown method is designed to conduct a cell phenotype screening experiment in intrahepatic cholangiocarcinoma cells.

[0014] Furthermore, the knockdown method includes using siRNA and shRNA.

[0015] Furthermore, it also includes selecting lncRNAs that can affect the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells based on the screening experiment results to further conduct in vivo and in vitro experiments to confirm the key role of lncRNAs that can affect the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells in the occurrence and development of ICC.

[0016] An innovative screening system for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma, used to implement an innovative screening method for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma, comprising:

[0017] LncRNA screening module, used to obtain intrahepatic cholangiocarcinoma tissue and its corresponding adjacent tissues for transcriptome sequencing, and then screen lncRNAs with differential expression in intrahepatic cholangiocarcinoma tissue and its corresponding adjacent tissues based on transcriptome sequencing;

[0018] The cell phenotype screening experiment module is used to design a corresponding method to perform a cell phenotype screening experiment in intrahepatic cholangiocarcinoma cells based on the lncRNA highly expressed in intrahepatic cholangiocarcinoma obtained by screening, and the cell phenotype screening experiment includes proliferation and / or invasion ability detection; based on the screening experiment results, the lncRNA that can affect the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells is selected as the key molecule in the occurrence and development of intrahepatic cholangiocarcinoma.

[0019] An application of the key molecules obtained by screening the innovative screening method for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma includes:

[0020] Study the mechanism of occurrence and development of intrahepatic cholangiocarcinoma;

[0021] Preparation of medicine for treating intrahepatic bile duct cancer.

[0022] Furthermore, the drugs for treating intrahepatic cholangiocarcinoma are prepared as siRNA and shRNA of ABHD11-AS1, and the target sequences of siRNA and shRNA are the same.

[0023] Furthermore, the siRNA is one of SEQ ID NO.1-SEQ ID NO.3, and the shRNA used in the animal experiment has the same target sequence as SEQ ID NO.3.

[0024] The beneficial effect of the present invention is that the present invention proposes an innovative screening strategy for the discovery of key molecules in the occurrence and development of ICC, in order to provide useful ideas for the early diagnosis and precise treatment of ICC. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of the innovative screening strategy for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma of the present invention;

[0026] Figure 2 It is a volcano plot of lncRNAs differentially expressed between ICC and adjacent tissues in the transcriptome sequencing results of 19 patients. The figure shows the distribution of ABHD11-AS1 in the volcano plot;

[0027] Figure 3 This is a diagram of the proliferation phenotype results of the siRNA library screening in step 2 of the present invention, and only the phenotype screening results of the 15 siRNAs with the most obvious differences are shown in the figure;

[0028] Figure 4 This is a diagram of the invasion phenotype results of the siRNA library screening in step 2 of the present invention, and only the phenotype screening results of the 15 siRNAs with the most obvious differences are shown in the figure;

[0029] Figure 5 The knockout efficiency of the top 15 siRNAs with the most significant phenotypic differences in the cell phenotype screening experiment in step 2 of the present invention;

[0030] Figure 6 This is a result diagram of the effects of three different siRNAs targeting ABHD11-AS1 on the proliferation ability of ICC cell lines (RBE, HuCCT1 and 9810) in the cell phenotype screening experiment in step 2 of the present invention;

[0031] Figure 7 This is a result diagram of the effects of three different siRNAs targeting ABHD11-AS1 on the invasion ability of ICC cell lines (RBE, HuCCT1 and 9810) in the cell phenotype screening experiment in step 2 of the present invention;

[0032] Figure 8 This is a flow chart of the animal experiment used in step 3 of the present invention;

[0033] Fig. 9 This is a statistical graph of weight changes of two groups of mice during the entire experimental process of the animal experiment in step 3 of the present invention;

[0034] Fig.10 is a comparison chart of the cancer sizes of the two groups at the end of the animal experiment in step 3 of the present invention (16 days after treatment);

[0035] Fig.11 This is the growth curve of the two groups of tumor PDX during the entire experimental process of the animal experiment in step three of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments.

[0037] The present invention provides an innovative screening method for key molecules in the occurrence and development of intrahepatic cholangiocarcinoma, such as Figure 1 As shown, the following steps are included:

[0038] Step 1: Obtain intrahepatic cholangiocarcinoma tissue and its corresponding adjacent tissues for transcriptome sequencing, and then screen lncRNAs with differential expression in intrahepatic cholangiocarcinoma tissue and its corresponding adjacent tissues based on transcriptome sequencing;

[0039] In a specific embodiment, the present invention first performed transcriptome sequencing on 19 pairs of intrahepatic cholangiocarcinoma tissues and their corresponding adjacent tissues. After completing sequencing and data preprocessing, lncRNAs with known sequences were identified and annotated using lncRNA databases such as GENCODE, NONCODE, and LncRNADisease. Subsequently, based on the difference in sequencing results between tumors and adjacent tissues, the top 79 lncRNAs with upregulated differences were selected for subsequent cell phenotype screening experiments. The specific implementation steps are as follows:

[0040] 1) Library construction and sequencing: RNA sequencing library construction kit was used to prepare RNA-seq libraries of non-ribosomal transcripts by removing ribosomal RNA or selecting polyadenylic acid (poly(A)) enrichment methods. Subsequently, the library was sequenced on the Illumina PE150 platform to generate paired-end sequencing fragments.

[0041] 2) Quality control: FastQC was used to assess the quality of raw sequencing reads. Trimmomatic was used to remove adapters and low-quality bases to ensure that only high-quality sequencing fragments were retained for subsequent analysis.

[0042] 3) Alignment: The trimmed sequence fragments were aligned to the human reference genome (hg38) using the alignment tool HISAT2 that supports splicing recognition. The alignment process uses default settings to identify known splicing junctions and accurately map the sequence fragments to the transcriptome.

[0043] 4) Assembly and quantification: StringTie is used to assemble the aligned sequencing fragments into transcripts. This tool can reconstruct full-length isoforms from RNA-seq data to identify known and novel transcripts. At the same time, HTSeq is used to quantify the aligned sequencing fragments to estimate the transcript expression level, and normalize by considering the sequencing depth and gene length, and finally obtain the expression level per million transcripts.

[0044] 5) Identification of known lncRNAs: After completing transcriptome sequencing and data preprocessing, lncRNA databases such as GENCODE, NONCODE, and LncRNADisease are used to identify and annotate lncRNAs by aligning known sequences.

[0045] Subsequently, according to the difference in fold between the sequencing results of tumor and adjacent tissue, Figure 2 As shown, the top 79 lncRNAs with up-regulated differences were selected for subsequent cell phenotype screening experiments.

[0046] Step 2: Based on the differential expression direction of the lncRNA obtained by screening, a corresponding method is designed to perform a cell phenotype screening experiment in intrahepatic cholangiocarcinoma cells, wherein the cell phenotype screening experiment includes proliferation and / or invasion ability detection; based on the screening experiment results, lncRNA that can affect the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells is selected.

[0047] In a specific embodiment, as described above, the top 79 lncRNAs with up-regulated differences were selected, and then, in the cell phenotype screening experiment, the siRNA library (three siRNAs for each lncRNA) was used, as shown in Table 1, and the proliferation and invasion ability were detected.

[0048] Table 1 Specific sequences of siRNA library (only 9 sequences are shown)

[0049]

[0050] The proliferation capacity was tested as follows:

[0051] Cell Counting Kit (CCK-8) (Yeasen, China) was used to detect cell proliferation. Cells were seeded in 96-well plates and transfected with siRNA. After culturing the cells for a specific period of time according to the experimental purpose, CCK-8 solution was added at a ratio of 10 vol%. Subsequently, the mixture was incubated under CO 2 The mixture was further incubated in an incubator for 1-2 hours, and then the absorbance OD value was measured at a wavelength of 450 nm using a microplate reader.

[0052] The invasive ability test method is as follows:

[0053] First, a layer of diluted Matrigel (Yeasen, China) was pre-coated on the upper chamber of the Transwell chamber (Corning, USA) and incubated in a constant temperature incubator for 4 hours. Next, the digested and centrifuged cells were resuspended in serum-free DMEM medium. After that, approximately 3×10 4 cells, and add 600 μL of DMEM medium containing serum to the lower chamber. After the cells invaded for 24-48 hours, the cells in the 24-well plate were gently washed with PBS to remove the detached cells and fixed with 4 vol% paraformaldehyde for 15 minutes. Next, the cells in the upper and lower chambers were stained with crystal violet staining solution, gently washed with PBS to remove excess dye, and the upper chamber was gently wiped to remove the cells, leaving only the invading cells in the lower chamber. Afterwards, images were taken using an inverted microscope.

[0054] The proliferation phenotype results of siRNA library screening are as follows Figure 3 As shown in the figure, only the screening results of 15 siRNAs with the most obvious differences in proliferation phenotypes are shown. The invasion experiment results of the above 15 siRNAs are shown in Figure 4 The five siRNAs that showed the most significant differences in the invasion assay were si-ABHD11-AS1_001, si-HIF1A-AS2_002, si-H1FX-AS1_002, si-ABHD11-AS1_002, and si-ABHD11-AS1_003. The knockdown effects of the above 15 siRNAs are shown in Figure 5 As shown, various siRNAs have good knockdown effects. The above lncRNAs that have expression differences and can affect the phenotype of ICC cells may be key molecules in the occurrence and development of intrahepatic cholangiocarcinoma.

[0055] In order to further confirm that the screened lncRNA is a key molecule, step three can also be included, using in vivo and in vitro experiments to confirm the key role of lncRNA that can reduce the proliferation and / or invasion ability of intrahepatic cholangiocarcinoma cells in the occurrence and development of ICC, and at the same time verifying the feasibility and effectiveness of the key molecule mining strategy in the occurrence and evolution mechanism of intrahepatic cholangiocarcinoma proposed in the present invention.

[0056] In a specific embodiment, in order to reduce the impact of siRNA off-target, ABHD11-AS1 is finally anchored as a key molecule in the occurrence and development of intrahepatic cholangiocarcinoma. The distribution of ABHD11-AS1 in the volcano map is as follows: Figure 2 shown. Figure 6-Figure 7The figure shows the effect of siRNA corresponding to ABHD11-AS1 on the proliferation and invasion abilities of three intrahepatic cholangiocarcinoma cells. Using three different siRNAs targeting ABHD11-AS1 can inhibit the proliferation and invasion of these three cholangiocarcinoma cell lines (RBE, HuCCT1 and 9810) to varying degrees, among which si-ABHD11-AS1_003 showed the most stable and significant inhibitory effect.

[0057] In this embodiment, if Figure 8 As shown, a patient-derived tumor xenograft model was used to explore the role of ABHD11-AS1 in ICC progression through in vivo experiments. The model mice were divided into two groups, and then the mice were injected peritumorally every four days. The two groups were injected with lentivirus expressing sh-ABHD11-AS1 or negative control lentivirus (sh-ABHD11-AS1 has the same target sequence as SEQ ID NO.3). After 16 days of treatment, the mice were euthanized ( Figure 8 ). During the experimental timeline, the weight and tumor changes of mice were observed. Figure 9-11 As shown, there was no significant difference in body weight between the two groups of mice ( Fig. 9 However, the tumor volume of mice treated with sh-ABHD11-AS1 was significantly smaller than that of the control group ( Figure 10-11 ). This indicates that ABHD11-AS1 is a key molecule in the occurrence and development of intrahepatic cholangiocarcinoma and can be used to study the related mechanisms of the occurrence and development of intrahepatic cholangiocarcinoma. In addition, the proliferation of intrahepatic cholangiocarcinoma cells can be inhibited by inhibiting the expression of ABHD11-AS1, and drugs that inhibit the expression of ABHD11-AS1 can be used to fight intrahepatic cholangiocarcinoma.

[0058] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Use of siRNA of ABHD11-AS1 in the preparation of a drug for treating intrahepatic cholangiocarcinoma, characterized in that: The siRNA is one of SEQ ID NO.1-SEQ ID NO.3.

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