CN111808951B

CN111808951B - Application of CTC-246B18.10 and related product and pharmaceutical composition thereof

Info

Publication number: CN111808951B
Application number: CN202010514635.9A
Authority: CN
Inventors: 李正美; 赵强; 邱建峰; 侯坤; 石丽婷; 赵慧慧; 路伟钊
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences; Second Affiliated Hospital of Shandong First Medical University
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences; Second Affiliated Hospital of Shandong First Medical University
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2023-03-28
Anticipated expiration: 2040-06-08
Also published as: CN111808951A

Abstract

The invention discloses application of CTC-246B18.10 and related products and pharmaceutical compositions thereof, and particularly relates to application of CTC-246B18.10 in diagnosis and treatment of liver cancer. The invention also provides a liver cancer diagnosis product capable of detecting the expression level of CTC-246B18.10 and a liver cancer treatment pharmaceutical composition capable of inhibiting the expression level of CTC-246B18.10.

Description

Application of CTC-246B18.10 and related product and pharmaceutical composition thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to application of CTC-246B18.10 and related products and pharmaceutical compositions thereof.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies and is characterized by strong invasiveness and high mortality, and ranks the second cause of cancer-related death (Ether, cidon. Systematic treamtent of hepatocellular carcinoma: panel, present and future [ J ]. World Journal of Hepatology,2017,9 (18): 797-807.). Symptoms of liver cancer include lumps or pain under the right side of the costal cage, ascites, cellulitis, susceptibility to bruising, weight loss, and physical weakness. Hepatitis B Virus (HBV), hepatitis C Virus (HCV), alcoholic and non-alcoholic cirrhosis, aflatoxins, obesity, etc. are currently thought to be associated with the development of liver cancer. The pathogenesis of liver Cancer is not completely understood, and studies suggest that it is associated with genetic mutations, liver Cancer stem cells, microenvironment, non-coding RNAs, metabolic abnormalities, etc. (Farazi P a, depinho R a. Hepatocellular environment [ J. Many patients are staged to middle and late stages when the initial diagnosis is confirmed, and some patients have already developed metastasis and miss the best diagnosis and treatment opportunity, so that the prognosis effect is quite poor. In the last decades, along with the progress of science and technology, the immunotherapy and gene therapy of tumors have made great progress, so it is very important to deeply research the gene spectrum of liver cancer cells and search new therapeutic targets.

Long non-coding RNAs are RNAs with the length of more than 200 nucleotides and without protein translation function, and are widely expressed in transcriptional regulation. Currently, incRNAs are mainly classified into five types, namely antisense IncRNA, intron IncRNA, lincRNA, promoter-related IncRNA and UTR-related IncRNA. LncRNA has spatiotemporal specificity, and expression patterns behave differently in different tissues. Compared with microRNA, lncRNA is longer in length and has similar mRNA structure. LncRNA can be combined with microRNA, mRNA and protein, and plays an important regulation and control role in cells. Currently, lncRNA is related to biological activities such as gene transcription, epigenetic regulation, protein coding genes, chromatin organization, and the like. With the progress of research, lncRNA has been proved to be closely related to the development and metastasis of many tumors. Therefore, the discovery that more long-chain non-coding RNA related to diseases has a non-negligible effect on recognizing the occurrence and development of tumors and providing corresponding prevention measures.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide a biomarker related to the occurrence and development of liver cancer and application of the biomarker in diagnosis and treatment of liver cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent for detecting CTC-246B18.10 in preparation of a product for diagnosing liver cancer.

Further, the reagent for detecting CTC-246B18.10 comprises a substance capable of binding to CTC-246B18.10 nucleic acid.

Further, the substance is a probe or primer directed to CTC-246B18.10.

Further, the primer sequence is shown in SEQ ID NO. 1-2.

The invention provides a product for diagnosing liver cancer, which contains a reagent for detecting CTC-246B18.10.

Further, the product comprises a chip, a carrier or a kit.

Further, the substance is a probe or primer aiming at CTC-246B18.10.

Further, the primer sequence is shown in SEQ ID NO. 1-2.

Further, the product also contains a specification which describes the use and the use method of the product, and the use is selected from any one of the following: 1) Assessing the risk of liver cancer; 2) Diagnosing liver cancer; the method of use includes the step of detecting the nucleic acid of CTC-246B18.10 using nucleic acid sequencing, amplification or hybridization techniques.

The invention provides application of CTC-246B18.10 in preparing a pharmaceutical composition for treating liver cancer or inhibiting tumor cell proliferation/invasion.

Further, the pharmaceutical composition includes an agent that specifically inhibits the transcription of CTC-246B18.10.

Further, the agent is selected from a nucleic acid molecule, a carbohydrate, a small molecule compound or an interfering lentivirus.

Further, the nucleic acid molecule includes, but is not limited to, an antisense oligonucleotide, a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

Further, the nucleic acid molecule is selected from the group consisting of small interfering RNA.

The invention provides a pharmaceutical composition for treating liver cancer or inhibiting tumor cell proliferation/invasion, which comprises a reagent for specifically inhibiting CTC-246B18.10 transcription.

Further, the agents include nucleic acid molecules directed against CTC-246B18.10, nucleic acid constructs/cells/lentiviruses containing the nucleic acid molecules.

The invention provides application of CTC-246B18.10 in screening candidate drugs for treating liver cancer, and if substances to be screened can specifically reduce the level of CTC-246B18.10, the substances to be screened are candidate drugs for treating liver cancer.

The invention provides application of CTC-246B18.10 in construction of a calculation model for predicting liver cancer.

Drawings

FIG. 1 is a graph showing the detection of the expression of CTC-246B18.10 gene in hepatocellular carcinoma tissue by QPCR.

FIG. 2 is a ROC plot of CTC-246B18.10 as a detection variable.

Detailed Description

Through a large number of experiments and repeated researches, the inventor of the invention finds that CTC-246B18.10 is up-regulated in liver cancer tissues, and the tumor cell proliferation can be inhibited by down-regulating CTC-246B18.10 gene expression, thereby achieving the purposes of diagnosing and treating tumors.

The CTC-246B18.10 gene is located on chromosome 19, and its sequence can be referred to, for example, ENST00000599274.1. In the present invention, when a gene which is differentially expressed is referred to, different transcripts of the gene are also included in the present invention, and a mutant form thereof or a fragment thereof is also included in the present invention.

It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level. The present invention may utilize any method known in the art for determining gene expression, including but not limited to nucleic acid sequencing, amplification, and hybridization techniques.

Chip, kit and carrier

The invention provides products including (but not limited to) preparations, chips or kits for detecting the expression level of CTC-246B18.10 gene. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown by CTC-246B18.10.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be labeled directly or indirectly, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

The kit comprises a reagent for detecting the CTC-246B18.10 gene, and one or more substances selected from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The carrier comprises a substrate and an oligonucleotide probe which is fixed on the substrate and aims at CTC-246B18.10; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass slide, a silica gel wafer, a micro magnetic bead, or the like.

Calculation model

The invention provides application of CTC-246B18.10 in preparation of a calculation model for predicting hepatocellular carcinoma. As the skilled artisan will appreciate, the measurement of two or more markers may be used to improve the diagnostic question in the survey. The biochemical markers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual cut-offs for each marker, or a combination thereof.

In the present invention, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the gene and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Pharmaceutical composition

Based on the discovery of the inventor, the invention provides application of CTC-246B18.10 in preparing a pharmaceutical composition for treating hepatocellular carcinoma, wherein the pharmaceutical composition comprises an inhibitor of CTC-246B18.10.

In preparing the pharmaceutical compositions of the present invention, the active ingredient is typically mixed with, or diluted by, an excipient or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may also include wetting agents, emulsifying agents, preservatives (such as methyl and propyl hydroxybenzoates), sweetening agents, and the like.

The application of the pharmaceutical composition provides a method for treating tumors, in particular to a method for preventing or treating tumors in a subject, which comprises the step of administering an effective dose of the pharmaceutical composition to the subject.

When the pharmaceutical composition is used for preventing or treating tumors in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited.

As used herein, a "small interfering RNA" is a double-stranded small RNA molecule consisting of a first and second strand that are perfectly complementary, processed by Dicer (an enzyme of the RNAase III family that is specific for double-stranded RNA). The first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is identical to a target sequence in the CTC-246B18.10 gene, or a sequence that hybridizes to the target sequence under high stringency conditions. The length of the first strand and the second strand of the double-stranded RNA are both 15-27 nucleotides; preferably, each 19-23 nucleotides in length; more preferably, each 19, 20 or 21 nucleotides in length. siRNA is a major member of siRISC, triggering silencing of the target gene to which it is complementary. RNA interference (RNAi) refers to the phenomenon of specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsRNA), resulting in silencing of expression of a target gene and the corresponding loss of a functional phenotype.

As used herein, the shRNA, i.e., a small hairpin RNA or short hairpin RNA (shRNA), is a segment of RNA sequence having a tight hairpin loop (light hairpin turn), which comprises a sense strand segment, an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and is commonly used for expression of RNA interference-silenced target genes. Wherein the sequences of the sense and antisense strands are complementary and the sequence of the sense strand fragment is identical to 15-27 contiguous nucleotide sequences in the CTC-246B18.10 gene, preferably the sense strand fragment is identical to 19-23 contiguous nucleotide sequences in the CTC-246B18.10 gene; more preferably, the sense strand fragment is identical to 19, 20 or 21 consecutive nucleotide sequences in CTC-246B18.10 gene, or a sequence that hybridizes to each of the above sequences under conditions of high stringency. The hairpin structure of the shRNA can be cleaved into siRNA by cellular machinery, and the siRNA is then bound to an RNA-induced silencing complex (RISC) which is capable of binding to and degrading the gene of interest.

The small interfering RNA of the invention can be a chemically synthesized double-stranded RNA; it may also be a double-stranded RNA expressed by a vector or an expression frame in which the expression of a small interfering RNA in mammalian cells is regulated using, for example, an RNA polymerase III promoter including a U6 promoter of human or murine origin and a human H1 promoter, and an RNA polymerase III terminator.

The small interfering RNA of the invention can be composed of a single small interfering RNA acting on a target sequence, or can be composed of a plurality of small interfering RNAs acting on a plurality of target sequences of a gene or target sequences on a plurality of genes; the target sequence can be the genome sequence of CTC-246B18.10 gene or the cDNA sequence of CTC-246B18.10 gene. .

The sirnas of the invention can be screened by the methods disclosed in the examples herein or by methods known in the art.

The first strand in the siRNA of the present invention and the sense strand in the shRNA have the same sequence as the target sequence, and have at least 10 (preferably at least 15, and more preferably at least 18) consecutive identical nucleotide sequences as compared to the siRNA target sequence, or a sequence that hybridizes to the target sequence under high stringency conditions.

In the present invention, the nucleic acid construct is meant to include a replication system and sequences capable of being transcribed in a given target cell. It can be obtained by cloning a fragment encoding the shRNA described herein into a known vector. Further, the nucleic acid construct is a lentiviral vector. After the lentiviral vector is packaged into infectious virus particles through viruses, the cells are infected, the shRNA is further transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the CTC-246B18.10 gene.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 detection of expression of CTC-246B18.10 in hepatocellular carcinoma

1. Sample collection

The cancer tissues of 27 patients with primary hepatocellular carcinoma and corresponding tissue samples beside the cancer (liver tissues with the distance of more than or equal to 5cm from the tumor margin) are respectively collected, and the matched cancer tissues are pathologically verified to be hepatocellular carcinoma. The specimen is cleaned by a large amount of normal saline in the operation, then is immediately subpackaged, is frozen by liquid nitrogen and is put into a refrigerator at minus 80 ℃ for freezing storage.

2. Preparation and Mass analysis of RNA samples

Extraction of tissue total RNA Using TRIZOL method

1) Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1min; standing at room temperature for 10min to completely decompose nucleoprotein.

2) Adding 200 μ l chloroform (chloroform), covering the tube, shaking vigorously for 15s, and standing at room temperature for 10min.

3) Centrifuge at 11000rpm for 15min at 4 ℃.

4) Transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after the mixture was inverted and mixed, the mixture was left standing at room temperature for 10min.

5) Centrifuge at 11000rpm for 15min at 4 ℃.

6) The liquid was carefully aspirated off with a gun, the precipitate was left at the bottom of the tube, 1ml of 75% ethanol was added, the mixture was shaken on a shaker for 5s, and the precipitate was washed once.

7) Centrifuge at 8000rpm for 5min at 4 deg.C.

8) Carefully removing the supernatant, drying the precipitate for 10min, and adding appropriate amount of water to dissolve the precipitate for 10min.

9) And detecting the concentration of the RNA, and identifying the yield and purity of the RNA.

3. QPCR detection

1) Primer design

Designing primers according to the gene sequences of CTC-246B18.10 and GADPH, wherein the specific primer sequences are as follows:

CTC-246B18.10 gene:

the forward primer is 5'-CCTTTAGGGTGGGAAGAGAA-3' (SEQ ID NO. 1);

the reverse primer is 5'-CAGCAACAACTCCCGTTTA-3' (SEQ ID NO. 2).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 3);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID No. 4).

2) Reverse transcription reaction

Using FastQ μ ant cDNA first Strand Synthesis kit (cat # KR 106) to perform IncRNA reverse transcription, genomic DNA reaction was first removed, 5 XgDNA B μ ffer 2.0 μ l, total RNA 1 μ g, and RNase Free ddH were added to a test tube ₂ O to make the total volume 10. Mu.l, heating in a water bath at 42 ℃ for 3min, and adding 10 Xfast RT B. Mu.ffer 2.0. Mu.l, RT Enzyme Mix 1.0. Mu.l, FQ-RT Primer Mix 2.0. Mu.l, RNase Free ddH ₂ O5.0. Mu.l, mixing, adding into the test tube, mixing to give 20. Mu.l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3min.

3) QPCR amplification assay

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP 205) and the experimental procedures were performed according to the product instructions.

A20. Mu.l reaction was used: 2 XSuperReal PreMix Plus 10. Mu.l, forward and reverse primers (10. Mu.M) 0.6. Mu.l each, 5 XROX Reference Dye ^△ 2. Mu.l, DNA template 2. Mu.l, sterilized distilled water 4.8. Mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was: 95 ℃ 15min, (95 ℃ 10s,55 ℃ 30s,72 ℃ 32 s) x 40 cycles.

4) Screening for cDNA template concentration

Mixing cDNA of each sample, diluting the cDNA by 10 times gradient (10 times, 100 times, 1000 times, 10000 times and 100000 times) by taking the cDNA as a template, taking 2 mu l of each diluted sample as the template, respectively amplifying by using a target gene primer and an internal reference gene primer, simultaneously carrying out melting curve analysis at 60-95 ℃, and screening the concentration of the template according to the principle of high amplification efficiency and single peak of the melting curve.

From the dissolution curve, it can be seen that when 10-fold dilution of cDNA was performed, the amplification efficiency of PCR was high and the single peak of the dissolution curve was good.

5) Sample Real Time PCR detection

After 10-fold dilution of cDNA of each sample, 2 μ l of cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. Simultaneously performing dissolution curve analysis at 60-95 deg.C, and determining target band by dissolution curve analysis and electrophoresis, 2 ^-ΔΔCT The method is used for relative quantification.

4. As a result, the

The QPCR results are shown in FIG. 1, and compared with the precancerous tissue, the expression of CTC-246B18.10 is up-regulated in the hepatocellular carcinoma tissue, and the difference is statistically significant (P < 0.05).

Specifically, there were 25 samples showing significant upregulation in CTC-246B18.10, 23 of which were cancer tissue samples and 2 of which were paracancerous tissue samples. The CTC-246B18.10 has higher specificity and sensitivity when being applied to diagnosis of hepatocellular carcinoma.

Example 2 validation of the diagnostic potency of CTC-246B18.10

1. Data collection

The expression profile data of lncRNA were downloaded from TCGA database, which included 371 liver cancer tissues and 50 paracarcinoma tissues.

2. ROC curve analysis

And (3) analyzing the working characteristics of the subject of lncRNA CTC-246B18.10 by using a pROC package in the R language, calculating a two-term accurate confidence space, and drawing an ROC curve.

3. As a result, the

The ROC analysis results are shown in fig. 2, from which it can be seen that CTC-246B18.10 has a higher area under the curve (AUC = 0.894) as a detection variable, indicating that the diagnosis of hepatocellular carcinoma using CTC-246B18.10 has higher sensitivity and specificity.

Example 3 functional validation of CTC-246B18.10

1. Cell culture

The human hepatoma cell line HepG2 was purchased from the Shanghai cell bank, and the cell lines were each determined by CO 5% at 37 ℃ in DMEM medium containing 10% fetal bovine serum and 1%P/S ₂ Cultured in an incubator. Cell growth was observed daily and fluid changed every other day.

2. Transfection

2.1 Synthesis of siRNA

The interference siRNA-CTC-246B18.10 against CTC-246B18.10 was designed and synthesized by Shanghai Jima pharmaceutical technology, inc., and the control was universal siRNA-NC.

2.2 transfection

The experiment was divided into 3 groups, control (HepG 2), negative control (siRNA-NC) and experimental (siRNA-CTC-246B18.10). Transfection was performed according to the instructions of the lipofectamine 2000 transfection reagent from Invitrogen. The method comprises the following specific steps:

1) The hepatoma cell line HepG2 was added to DMEM medium containing 10% fetal bovine serum and was left at 37 ℃ with 5% CO ₂ Cultured in an incubator. When the cells were grown to 80-90%, the serum components in the cell flask were removed by washing 3 times with PBS, and then 1-2mL of a 0.02% EDTA-containing digestive solution containing 0.25% pancreatin was added to the cells when the cells were grown to confluenceDigestion was stopped by adding 1mL of complete medium when single cells became present.

2) Transferring the cells into a centrifuge tube, placing into a normal temperature centrifuge, centrifuging at 1000g/min for 5min to collect cell precipitate, and resuspending the cells to 1 × 10 with complete culture medium ⁵ Per mL, pipetting 500. Mu.L, inoculating into 24-well plate, placing at 37 deg.C, 5% ₂ Was cultured overnight in an incubator.

3) Lipofectamine 2000 was added to the OPTI-MEM culture medium, and the mixture was incubated at room temperature for 5min.

4) siRNA is added into the OPTI-MEM culture solution and mixed evenly.

5) Mixing the diluted 3) and 4) uniformly, and standing at room temperature for 20min.

6) Adding the mixture into 24-well plate of serum-free medium-cultured cells, shaking gently, mixing, adding 37 deg.C and 5% CO ₂ After culturing for 6h in the incubator, the cell culture solution is replaced by fresh complete culture medium for continuous culture.

3. QPCR detection of the expression level of CTC-246B18.10 in cells

After 48h of transfection and culture of each group of cells, total RNA of the cells was extracted by Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method of example 1.

4. CCK-8 method for detecting cell proliferation capacity

1) After 24h of transfection, each group of cells was washed 3 times with PBS to remove serum components inside the cells, then the cells were sufficiently digested with trypsin, centrifuged at 1000rpm for 5min to collect the cells, the supernatant was discarded and resuspended in 1ml of DMEM medium containing 10% fetal bovine serum, and the cells were counted by a cell counting plate.

2) Cells were diluted to 1X 10 with DMEM medium containing 10% fetal bovine serum ⁴ mL, then 200 μ L cells were added to each well of a 96-well plate (5 replicate wells were seeded with each cell).

3) To the other wells, which were not plated with cells around the 96-well plate, 200. Mu.L of PBS was added.

4) Placing the 96-well plate inoculated with tumor cells at 37 ℃ and 5% ₂ After culturing for 72h, 10 mul CCK-8 is added, and the mixture is put into an incubator to be incubated for 1h.

5) The plate was removed and absorbance at a wavelength of 450nm was measured using a microplate reader.

5. Cell migration assay

1) 24h after transfection of each group of cells, the cells were washed 3 times with PBS to remove serum components inside the cells, then the cells were digested with trypsin, the cell suspension was transferred to a 15mL centrifuge tube, and then placed in a room temperature centrifuge and centrifuged at 1000rpm for 5min to collect the cells. The supernatant was discarded and the cells were resuspended in 1ml of DMEM medium without fetal bovine serum and counted using a cell counting plate. Cells were diluted to 1X 10 with DMEM medium without fetal bovine serum ⁶ Per mL, suction 1X 10 ⁵ One cell was added dropwise to the upper chamber of the Transwell chamber.

2) Add 600. Mu.L of complete medium containing 10% serum to the lower chamber of the Transwell chamber, then place the upper chamber into the well plate, at 37 ℃ and 5% CO ₂ The cells are cultured in the cell culture box for 24 hours.

3) The chamber was removed, the medium in the chamber was gently discarded, washed 2 times with PBS, and fixed in 75% ethanol fixative for 15min.

4) Cells in the upper chamber of the Transwell were gently wiped off with a cotton swab and the chamber was stained in crystal violet for 15min.

5) The chamber was removed, washed 3-5 times with PBS to remove excess cell stain, and then observed under a microscope.

6. Cell invasion assay

Matrigel was diluted with serum-free medium 1, 100 μ L was spread in a chamber and incubated overnight at 4 ℃. The remaining steps were the same as in the cell migration experiment.

7. Statistical analysis

All data were obtained from three independent experiments, shown by Mean SD. Differences between groups were analyzed using paired sample t-test, and all results were analyzed using GraphPad Software, with p <0.05 indicating statistical differences.

8. Results

1) The expression level of CTC-246B18.10 (0.240 + -0.06557) was significantly decreased after transfection of siRNA-CTC-246B18.10 in the experimental group (control vs experimental group, P value =0.0025,. Times.) compared to the control group (P value =0.0025,. Times.) and the expression level of CTC-246B18.10 (0.93 + -0.05686) in the negative control transfected with siRNA-NC was not significantly changed (control vs negative control group, P value =0.1794, ns), with the expression level of CTC-246B18.10 in the control group (HepG 2) being defined as 1, and the transfection results showed that compared to the control group, the expression level of CTC-246B18.10 (0.240 + -0.06557) was significantly decreased (control vs negative control group, P value =0.1794, ns)

2) The cell proliferation test results showed that the cell proliferation activity (OD value: 1.176 ± 0.0757), cell proliferation activity of experimental group (OD value: 0.628 ± 0.06181) are significantly reduced (negative control vs experimental group, P < 0.0001:), which indicates that CTC-246B18.10 affects the proliferation of hepatoma cells, and suggests that CTC-246B18.10 can be used as a molecular target for the treatment of hepatoma.

3) The cell migration experiment result shows that compared with the cell transmembrane number (167.3 +/-11.5) of the negative control group, the cell transmembrane number of the experimental group is remarkably reduced (78 +/-8.185), the difference has statistical significance (a negative control group vs experimental group, P = 0.0156), the change of the expression level of CTC-246B18.10 can change the migration capacity of the cell, and the CTC-246B18.10 can be applied to the treatment of liver cancer metastasis.

4) The cell invasion experiment result shows that compared with the cell transmembrane number (125.3 +/-9.609) of a negative control group, the cell transmembrane number of the experimental group is remarkably reduced (68.33 +/-10.02), the difference has statistical significance (a negative control group vs experimental group, P =0.0369, x), the cell invasion capacity can be changed by changing the expression level of CTC-246B18.10, and the CTC-246B18.10 can be applied to treatment of liver cancer infiltration.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Shandong first medical university (Shandong province medical science institute)

The Second Affiliated Hospital of Shandong First Medical University

<120> use of CTC-246B18.10 and related products and pharmaceutical compositions thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cctttagggt gggaagagaa 20

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagcaacaac tcccgttta 19

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aatcccatca ccatcttcca g 21

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagccccagc cttctccat 19

Claims

1. The application of the reagent for specifically inhibiting the transcription of CTC-246B18.10 in preparing the medicine composition for treating liver cancer or inhibiting the proliferation/invasion of liver cancer tumor cells.

2. Use according to claim 1, wherein the agent is selected from the group consisting of nucleic acid molecules, nucleic acid constructs/cells/lentiviruses containing nucleic acid molecules, small molecule compounds.

3. The use according to claim 2, wherein the nucleic acid molecule is selected from the group consisting of: antisense oligonucleotides, small interfering RNA or short hairpin RNA.

4. The use according to claim 3, wherein the nucleic acid molecule is selected from the group consisting of small interfering RNA.

The application of CTC-246B18.10 in screening candidate drugs for treating liver cancer is characterized in that if substances to be screened can specifically reduce the expression level of CTC-246B18.10, the substances to be screened are the candidate drugs for treating the liver cancer.