CN108866198B

CN108866198B - Application of marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma

Info

Publication number: CN108866198B
Application number: CN201811010361.9A
Authority: CN
Inventors: 董东; 石小峰
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2018-08-31
Filing date: 2018-08-31
Publication date: 2020-08-11
Anticipated expiration: 2038-08-31
Also published as: CN108866198A

Abstract

The invention discloses an application of a marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma. The invention firstly discovers that the expression level of LOC105376380 in rectal adenocarcinoma is obviously up-regulated, and simultaneously, the expression level of LOC105376380 is reduced by designing siRNA of the lncRNA, so that the proliferation, migration and invasion of rectal adenocarcinoma cells can be changed.

Description

Application of marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma

Technical Field

The invention belongs to the field of biological medicines, and relates to application of a marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma.

Background

Rectal cancer is one of the most common malignancies, with morbidity and mortality in the third place worldwide, with about 130 million cases per year. Although the cause of the onset of rectal cancer is still unclear, it has been determined to be closely related to dietary factors, genetic predisposition, parasites, pre-cancerous diseases. The occurrence and development process of the rectal cancer comprises a series of changes of transcription level, posttranscriptional level and epigenetics, but the mechanism of the occurrence and development of the rectal cancer is not fully elucidated so far, and the treatment of the rectal cancer does not achieve the expected effect, so that the intensive research on the pathogenesis of the rectal cancer and the research on a new diagnosis method and a new treatment means are key problems for overcoming the rectal cancer.

With the development of biology, lncRNA, which was previously considered "noise", was found to play an important role in the development of disease. lncRNA, generally referred to as a class of non-coding RNAs with a molecular weight greater than 200nt, is defined by its function as an RNA molecule that may function in either the initial or spliced transcript. Most lncRNA molecules are transcribed by RNA polymerase II and are polyadenylated.

With the intensive research on lncRNA, it is found that lncRNA is produced at a specific stage of growth and development of organisms, and has tissue and time specificity. Recent studies also show that lncRNA participates in various life activity processes such as genome imprinting, X chromosome inactivation, chromosome modification and telomere biology. The lncRNA can also regulate the expression of genes at the transcription level, the post-transcription level and the epigenetic level, and is involved in the occurrence and the development of various diseases of human beings, such as malignant tumors, coronary heart diseases, spinocerebellar ataxia 8, Alzheimer diseases, diabetes and the like. In recent years, more and more reports on the correlation between lncRNA and tumors show that lncRNA is an important link in the process of tumorigenesis and development, so that research on the effect of lncRNA in rectal adenocarcinoma is of great significance for the disclosure of the pathogenesis of rectal adenocarcinoma and clinical diagnosis and treatment.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide an lncRNA marker related to rectal adenocarcinoma, and the lncRNA marker is applied to clinic, so that diagnosis and treatment of rectal adenocarcinoma are realized, effective intervention is performed, and the survival rate and the survival quality of patients are improved.

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

the invention provides an application of LOC105376380 in preparing a product for diagnosing rectal adenocarcinoma.

Further, the product comprises a reagent for detecting the expression level of LOC105376380 in the sample.

Further, the reagent comprises a reagent for detecting the expression level of LOC105376380 by RT-PCR, real-time quantitative PCR, in-situ hybridization and chip technology.

The invention provides a product for diagnosing rectal adenocarcinoma, which comprises a chip or a kit, wherein the preparation, the chip or the kit comprises a reagent for detecting the expression level of LOC 105376380.

Furthermore, the reagent for detecting the expression level of LOC105376380 in the chip comprises a probe for specifically recognizing LOC105376380 gene; the reagent for detecting the expression level of LOC105376380 in the kit comprises a primer for specifically amplifying the LOC105376380 gene or a probe for specifically recognizing the LOC105376380 gene.

Further, the primer sequence of the specific amplification LOC105376380 gene is shown in SEQ ID No. 1-2.

The invention provides another application of LOC105376380 in preparing a pharmaceutical composition for treating primary rectal adenocarcinoma, rectal adenocarcinoma metastasis or rectal adenocarcinoma invasion.

Further, the pharmaceutical composition comprises an inhibitor of LOC 105376380. The inhibitor is selected from: an interfering molecule having LOC105376380 or a transcript thereof as a target sequence and capable of inhibiting LOC105376380 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is siRNA. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting a rectal adenocarcinoma cell line with transfection reagents respectively, selects siRNA with the best interference effect, and further verifies the correlation between lncRNA and the occurrence and development of rectal adenocarcinoma by cell level experiments.

Preferably, the sequence of the siRNA is shown as SEQ ID NO. 7-14;

more preferably, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

As an alternative of the present invention, the inhibitor of LOC105376380 may also be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA can inhibit gene expression through an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

The invention provides a pharmaceutical composition for treating rectal adenocarcinoma, which comprises an inhibitor of LOC 105376380. The inhibitor is selected from: an interfering molecule having LOC105376380 or a transcript thereof as a target sequence and capable of inhibiting LOC105376380 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is siRNA.

Preferably, the sequence of the siRNA is shown as SEQ ID NO. 7-14;

more preferably, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes (but is not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as simple syrup, glucose solution, starch solution, cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption accelerators quaternary ammonium compounds, sodium lauryl sulfate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, and the like.

In the present invention, the pharmaceutical composition may be prepared using various additives, such as buffers, stabilizers, bacteriostats, isotonizing agents, chelating agents, pH controlling agents, and surfactants.

The pharmaceutical compositions of the invention can also be used in combination with other drugs for the treatment of rectal adenocarcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

The invention also provides a method for screening a candidate drug for treating rectal adenocarcinoma, which comprises the following steps:

treating a system expressing or containing the LOC105376380 gene with a substance to be screened; and

detecting the expression of LOC105376380 gene in the system;

wherein, if the substance to be screened can inhibit the LOC105376380 gene level, the substance to be screened is a candidate drug for treating rectal adenocarcinoma.

The system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

The candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed against the LOC105376380 gene or its upstream or downstream genes.

When a compound isolated by the screening method of the present invention is administered as a drug to a human or other mammal, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees, the isolated compound can be administered directly or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug may be orally administered as sugar-coated tablets, capsules, elixirs and microcapsules, as desired; or as a sterile solution or suspension in water or any other pharmaceutically acceptable liquid for parenteral administration in the form of an injection. For example, the compounds may be mixed together in unit dosage forms (unit dose) as required for generally accepted modes of pharmaceutical administration, with pharmaceutically acceptable carriers or media including, but not limited to, sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients (exipient), vehicles (vehicle), preservatives, binders and the like. Depending on the content of the active ingredient in these preparations, an appropriate amount to be administered within a prescribed range can be obtained.

In the present invention, "marker", "biomarker", "genetic marker" may be used generically to refer to a molecular indicator having a specific biological property, biochemical characteristic or aspect, which may be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition.

In the present invention, the gene for transcription of LOC105376380 is located in the 5-band of region 1 of human No.10 chromosome, and LOC105376380 in the present invention includes wild type, mutant type or a fragment thereof. In the examples of the present invention, the nucleotide sequence of a representative transcribed LOC105376380 gene is shown in the present International public nucleic acid database GeneBank for LOC105376380 gene (XR _ 930610.2).

The present invention may utilize any method known in the art to determine the expression level of a gene. 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.

Some methods of detection or quantification of lncRNA levels are known in the art and are all suitable for use in the methods provided herein to measure levels of biomarkers. Exemplary methods include, but are not limited to, northern blots (northern blots), ribonuclease protection assays, and PCR-based methods. When the biomarker is a lncRNA molecule, the lncRNA sequence or fragment thereof can be used to prepare a probe that is at least partially complementary. The probes can then be used to detect lncRNA sequences in the sample using any suitable assay, such as PCR-based methods, northern blotting (northern blotting) or dipstick assay (dipstick assay).

The assay method may vary depending on the type of lncRNA information desired. Exemplary methods include, but are not limited to, Northern blots (Northern blots) and PCR-based methods (e.g., qRT-PCR). The method of qRT-PCR and the like can also accurately quantify the amount of lncRNA in the sample.

In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine tumor development and select a treatment regimen.

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.

In the present invention, the term chip also includes solid supports including plastic articles, microparticles, membrane supports, and the like. The plastic products can be combined with antibodies or protein antigens through a non-covalent or physical adsorption mechanism, and the most common plastic products are small test tubes, small beads and micro reaction plates made of polystyrene; the micro-particles are microspheres or particles polymerized by high molecular monomers, the diameter of the micro-particles is more than micron, and the micro-particles are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and the combination capacity is large; the membrane carrier comprises microporous filter membranes such as a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane and the like.

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, as the skilled person will know. Preferably, the measured concentrations of the marker 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.

Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be readily correlated with, for example, an individual's risk for rectal adenocarcinoma or with other intentional diagnostic uses that are helpful in assessing patients with rectal adenocarcinoma, in light of the underlying diagnostic question. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal humans, individuals at risk for rectal adenocarcinoma, patients with rectal adenocarcinoma, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) logarithmic regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a logarithmic function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing rectal adenocarcinoma is selected from DA (i.e., linear, quadratic, rule-discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

The invention has the advantages and beneficial effects that:

the invention firstly discovers that the differential expression of LOC105376380 is related to the occurrence and development of rectal adenocarcinoma, and whether a patient has the rectal adenocarcinoma can be judged by detecting the expression level of LOC 105376380.

The invention discovers for the first time that the change of LOC105376380 expression level can cause the change of proliferation, invasion and metastasis of rectal adenocarcinoma cells, and accordingly, the rectal adenocarcinoma cell can be applied to the treatment of the rectal adenocarcinoma.

The invention discloses a method for screening candidate drugs for treating rectal adenocarcinoma, which judges whether a substance to be screened is a candidate drug by detecting whether the substance to be selected can reduce the expression level of LOC 105376380.

Drawings

FIG. 1 is a graph showing the detection of the expression of LOC105376380 gene in rectal adenocarcinoma tissue by QPCR;

FIG. 2 is a graph showing the effect of detecting silencing of LOC105376380 by siRNA;

FIG. 3 is a diagram showing the effect of the CCK8 method for detecting LOC105376380 on the proliferation of rectal adenocarcinoma cells;

FIG. 4 is a graph of the effect of LOC105376380 on migration of rectal adenocarcinoma as tested by scratch test;

FIG. 5 is a graph of the effect of LOC105376380 on rectal adenocarcinoma cell invasion using a Transwell chamber.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with rectal adenocarcinoma

1. Sample collection

3 samples of normal epithelial tissue beside carcinoma of rectum and tissue of carcinoma of rectum were collected, all cases received no chemotherapy or radiotherapy before surgery, no other neoplastic diseases, autoimmune diseases and serious chronic diseases, normal epithelial tissue beside carcinoma was taken 5cm from the upper edge of tumor, all patients had known informed consent, and had passed the consent of the tissue ethics committee.

2. Preparation of RNA samples

Tissue RNA extraction was performed using a tissue RNA extraction kit from QIAGEN, and the specific procedures were performed according to the instructions.

3. Total RNA quantitation and purity analysis

The RNA extracted above was subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN value was determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

4. Construction of cDNA library

1) Removal of rRNA

Removing ribosomal RNA from the total RNA using a Ribo-Zero kit;

2) fragmented RNA

For the complete RNA sequence, metal ions are utilized to randomly break the RNA into small fragments of about 200 bp.

3) Reverse Synthesis of cDNA

Constructing a cDNA library by utilizing an Illumina Truseq RNA samplePrep Kit, reversely synthesizing single-strand cDNA by utilizing a random primer and taking lncRNA as a template under the action of reverse transcriptase, and replacing dTTP in dNTPs reagents to ensure that a base in a second strand of the cDNA contains A/U/C/G when carrying out double-strand synthesis.

4) Connection adapter

The sticky End of the double stranded cDNA is made blunt by adding End Repair Mix, followed by an A base at the 3' End for ligation to the Y-shaped adaptor.

5) UNG enzyme digestion of cDNA double strand

The second strand of the cDNA was digested with UNG enzyme, so that only the first strand of the cDNA was contained in the library.

5. Sequencing

2X 150bp sequencing was performed using the Illumina X-Ten sequencing platform.

6. High throughput transcriptome sequencing data analysis

1) Carrying out trim on 5 'and 3' sections of reads by using cutadapt, wherein bases with the mass of less than 20 are removed from trim, and more than 10% of reads with N are deleted;

2) tophat aligns to the reference genome. The reference genome version used was grch38.p7, fasta and gff files downloaded from NCBI;

3) the expression quantity of the lncRNA is quantified and outputted in a standardized way by cuffquant; the expression quantity of the lncRNA is quantified and outputted in a standardized way by cuffquant;

4) comparing the expression difference of lncRNA of a control group and a disease group by cuffdiffiff, and screening the standard of the lncRNA with the difference: p _ value<0.05，|log₂FC|>1。

7. Results

The results show that the expression level of LOC105376380 is significantly increased in rectal adenocarcinoma tissue compared to paracancerous normal epithelial tissue, suggesting that LOC105376380 may be applied as a molecular target in the diagnosis of rectal adenocarcinoma.

Example 2QPCR sequencing verification of differential expression of the LOC105376380 Gene

1. Large sample QPCR validation was performed on LOC105376380 gene differential expression. 45 cases of the normal tissue beside the rectal adenocarcinoma and the tissue of the rectal adenocarcinoma were selected in the same manner as in example 1.

2. RNA extraction

RNA samples were extracted using QIAGEN's tissue RNA extraction kit, and the specific procedures are described in the specification.

3、QPCR

1) Reverse transcription reaction

Using FastQ μ ant cDNA first strand synthesis kit (cat # KR106) to carry out IncRNA reverse transcription, genomic DNA removal reaction was first performed, 5 × g of DNA B μ ffer 2.0 μ l, total RNA1 μ g, and RNase Free ddH were added to a test tube₂O to a total volume of 10. mu.l, heating in a water bath at 42 ℃ for 3min, and mixing 10 × Fast RT B. mu.ffer 2.0. mu.l, RT enzymeMix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3 min.

2) Primer design

QPCR amplification primers were designed based on the coding sequences of LOC105376380 gene and GAPDH gene in Genebank and synthesized by Bomader Biotech. The specific primer sequences are as follows:

LOC105376380 gene:

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

the reverse primer was 5'-GGTTCAGACAGAAGACAA-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).

3) QPCR amplification assay

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

A20. mu.l reaction was used:

2 × SuperReal PreMix Plus 10. mu.l, forward and reverse primers (10. mu.M) 0.6. mu.l each, 5 × ROXReference 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 ℃ 32s) x 40 cycles, 95 ℃ 15s, 60 ℃ 60s, 95 ℃ 15 s).

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 RealTime 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^-ΔΔCTThe method is used for relative quantification.

4. Results

The QPCR result is shown in figure 1, and compared with the normal tissues beside the rectal adenocarcinoma, LOC105376380 is up-regulated in the rectal adenocarcinoma tissues, and the difference is statistically significant (P < 0.05); suggesting that LOC105376380 can be used as a target for molecular diagnosis of rectal adenocarcinoma.

Example 3 silencing of LOC105376380 Gene

1. Cell culture

Human rectal glandCancer cell line HRC-99 is prepared by culturing RPMI1640 medium containing 10% calf serum and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days, passaged by conventional digestion with 0.25% trypsin containing EDTA, and cells in logarithmic growth phase were taken for experiment.

2. Design of siRNA

Designing siRNA aiming at the sequence of LOC105376380 gene, wherein the designed siRNA sequence is shown as follows:

sequence of negative control siRNA-NC:

sense strand: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.5),

antisense strand: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 6);

siRNA1：

sense strand: 5'-UGGAAAAGGAGGAAAGAGGUG-3' (SEQ ID NO.7),

antisense strand: 5'-CCUCUUUCCUCCUUUUCCAAG-3' (SEQ ID NO. 8);

siRNA2：

sense strand: 5'-AAUUACCUCGGCAAAGACCCU-3' (SEQ ID NO.9),

antisense strand: 5'-GGUCUUUGCCGAGGUAAUUAA-3' (SEQ ID NO. 10);

siRNA3：

the sense strand is 5'-UAUAGACAGAAAAUGGAAGUG-3' (SEQ ID NO.11),

the antisense strand is 5'-CUUCCAUUUUCUGUCUAUAAA-3' (SEQ ID NO.12)

siRNA4：

The sense strand is 5'-UCAUUUGAAAUGAAUUGAGUU-3' (SEQ ID NO.13),

the antisense strand is 5'-CUCAAUUCAUUUCAAAUGAAA-3' (SEQ ID NO.14)

3. Transfection

Pressing the cells to 1 × 10⁴Perwell into 24-well cell culture plates at 37 ℃ with 5% CO₂Cells were cultured in an incubator for 24h and transfected in RPMI1640 medium without double antibody containing 10% FBS according to the instructions of lipofectin 2000 (purchased from Invitrogen).

The experiment was divided into a blank control group (HRC-99), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2, siRNA3, siRNA4), wherein the siRNA of the negative control group had no homology with the sequence of LOC105376380 gene at a concentration of 20 nM/well and was transfected separately at the same time.

4. QPCR detection of transcription level of LOC105376380 Gene

1) Extraction of Total RNA from cells

Total RNA in cells was extracted using QIAGEN cell RNA extraction kit, the detailed steps are described in the specification.

2) The reverse transcription procedure was as in example 2.

3) The QPCR amplification procedure was as in example 2.

5. Results

The results are shown in fig. 2, compared with the HRC-99 and transfected unloaded siRNA-NC groups, the experimental group (siRNA 1-4) can reduce the level of LOC105376380, wherein the effect of siRNA1 is the most significant, and siRNA1 is selected for subsequent experiments.

Example 4 Effect of LOC105376380 on proliferation of rectal adenocarcinoma cells

1. Rectal adenocarcinoma cells HRC-99 were inoculated into 6-well plates and cultured until the cell density reached 85% -90%, and siRNA1 was transfected using liposome 2000. Replacing the new culture medium after the culture in the serum-free culture medium for 4-6 h.

2. After siRNA1 transfection, the cells of the interference group and the cells of the control group are digested for 24h, and the HRC-99 cell suspension after transfection and each control group (100 mu.l/well) are inoculated in a 96-well plate, wherein the inoculation density is 5 × 10⁴And L. The plates were pre-incubated in an incubator (37 ℃ C., 5% CO)₂)。

3. To each well was added 10. mu.l of CCK8 solution.

4. The culture plate is placed in an incubator to be cultured for 1-4 h.

5. Absorbance at 490nm was measured using a microplate reader.

6. Results

As shown in fig. 3, compared with the control group, the cell proliferation in the experimental group was significantly inhibited, suggesting that LOC105376380 can be a molecular target of rectal adenocarcinoma.

Example 5 scratch test for detecting proliferation and migration of rectal adenocarcinoma cells after siRNA transfection

1. The HRC-99 cells are paved in a six-well plate, when the cell density reaches 85% -90%, siRNA1 is transfected by Lipo2000, and the culture medium is replaced after the cells are cultured for 4-6h in a serum-free manner.

2. After the bottom of the culture plate is uniformly covered by the rectal adenocarcinoma cells, a 1ml gun head is aligned to the orifice plate to keep a vertical angle for scratching the cells, so that the width of each scratch is the same as far as possible.

3. Removing the cell stock culture solution, washing the pore plate by using a phosphate buffer solution, washing away fragments formed by cell scratches, adding a serum-free culture medium, photographing and reserving data.

4. And placing the culture plate in a cell culture box for culture, taking out the culture plate after culturing for 4-6h, photographing again, recording data, and calculating the healing rate of the scratch.

5. Results

The results are shown in fig. 4, the score healing rate of the siRNA1 group is significantly lower than that of the siRNA-NC group and the blank control group, the difference is statistically significant (P <0.05), which indicates that LOC105376380 affects the migration of rectal adenocarcinoma cells, and suggests that LOC105376380 can be used as a molecular target for treating rectal adenocarcinoma.

Example 6Matrigel invasion assay

1. HRC-99 cells for colorectal cancer are inoculated in a 6-well plate and cultured, and when the cell density reaches 85% -90%, siRNA1 is transfected by Lipo 2000. The culture medium is replaced after the culture is carried out for 4 to 6 hours in the serum-free culture medium.

2. After the interference group and the control group are cultured for 24h, the mixture is subjected to trypsinization centrifugation, and is resuspended by using a serum-free medium, and the cell density is adjusted to 5 × l0⁵Mu.l of basal medium cell suspension without fetal bovine serum was added to a Transwell chamber in which Matrigel matrix gel had been laid, and 500. mu.l of medium containing 10% fetal bovine serum was added to the lower chamber of a 24-well plate, and the cells were cultured for 24 hours.

3. Cells were stained with DAPI after the end of the culture. The cell of the chamber is rinsed 2 times with PBS and then placed in DAPI working solution for staining for 5-20min at room temperature. Rinsed 2 times with PBS, placed under a fluorescent microscope for observation and counted.

4. Results

The results are shown in fig. 5, compared with the control group, the number of transmembrane cells in the experimental group is reduced, which indicates that the invasion capacity of the cells is obviously reduced after the expression level of LOC105376380 is reduced (P <0.05), and suggests that LOC105376380 can be used as a potential molecular target for treating the invasion of rectal adenocarcinoma.

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> Beijing, the deep biometric information technology GmbH

<120> use of marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. Use of a reagent for detecting the expression level of LOC105376380 in a sample, characterized in that it is used for the preparation of a product for the diagnosis of rectal adenocarcinoma.

2. The use according to claim 1, wherein the reagents comprise reagents for detecting the expression level of LOC105376380 by RT-PCR, real-time quantitative PCR, in situ hybridization, chip technology.

3. The use according to claim 1, wherein the product comprises a chip or kit, wherein said chip or kit comprises reagents for detecting the expression level of LOC 105376380.

4. The use according to claim 3, wherein the reagent for detecting the expression level of LOC105376380 in the chip comprises a probe specifically recognizing LOC105376380 gene; the reagent for detecting the expression level of LOC105376380 in the kit comprises a primer for specifically amplifying the LOC105376380 gene or a probe for specifically recognizing the LOC105376380 gene.

5. The application of claim 4, wherein the primer sequence for specifically amplifying the LOC105376380 gene is shown in SEQ ID No. 1-2.

Use of an inhibitor of LOC105376380 for the preparation of a pharmaceutical composition for the treatment of rectal adenocarcinoma, characterized in that said inhibitor reduces the expression level of LOC 105376380.

7. The use of claim 6, wherein the inhibitor is siRNA.

8. The use of claim 7, wherein the siRNA has the sequence shown in SEQ ID No. 7-14.

9. The use of claim 8, wherein the siRNA has a sequence as shown in SEQ ID No. 7-8.