CN111118007B

CN111118007B - Application of long non-coding RNA in preparation of medicine for treating cervical cancer

Info

Publication number: CN111118007B
Application number: CN202010038787.6A
Authority: CN
Inventors: 傅士龙; 许叶涛; 孙玮
Original assignee: Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
Priority date: 2020-01-14
Filing date: 2020-01-14
Publication date: 2022-11-25
Anticipated expiration: 2040-01-14
Also published as: CN111118007A

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to application of RP11-7K24.3 in preparation of medicines for diagnosing cervical cancer and treating target spots. The down regulation of RP11-7K24.3 in the tumor tissue of a cervical cancer patient is related to the generation and development of cervical cancer, the RP11-7K24.3 with low expression level is closely related to the pathogenesis of the cervical cancer, the invasion, migration, lymphatic vessel formation and the like of the cervical cancer cells of the cervical cancer patient are influenced by changing the expression of RP11-7K24.3, and the down regulated expression of RP11-7K24.3 can promote the invasion, migration and lymphatic vessel formation of the cervical cancer cells.

Description

Application of long non-coding RNA in preparation of medicine for treating cervical cancer

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of long non-coding RNA in preparation of a medicine for treating cervical cancer.

Background

Cervical cancer is one of the most common gynecological malignancies worldwide, with about 500000 newly diagnosed cases worldwide each year, of which about 260000 die from cervical cancer. In recent years, the incidence and mortality of cervical cancer has generally decreased in many countries due to the wide availability of TCT smear screening tests. However, prognosis remains poor in patients with advanced cervical cancer, with 5-year survival rates in the advanced stage almost as low as 15%. Therefore, there is an urgent need to find new prognostic markers to better understand the molecular mechanisms involved in the development of cervical cancer. Despite the current development of medical treatments, the overall patient incidence remains high. With the rapid development of sequencing technology and molecular biology, gene diagnosis and molecular targeted therapy become a hot spot problem in cervical cancer treatment. Therefore, the study of the molecular mechanisms involved in the development and metastasis of cervical cancer is crucial to the development of specific diagnostic methods and personalized therapeutic strategies.

In the past decade, large-scale human genomics studies have been driven by rapid-emerging high-throughput sequencing-based gene expression analysis techniques and bioinformatics, leading to the discovery of non-coding RNAs. Only 2% of the coding in the human genome is transcribed into proteins, while the vast majority is transcribed into non-coding RNAs, including small ribonucleic acids, long non-coding RNAs (lncRNAs), and pseudogenes. Recently, the role of mirnas in various aspects of cellular processes has been demonstrated, however, functional studies of lncRNAs are not very thorough. The GENECODE research group new data in the ENCODE project revealed thousands of lncRNAs, but only some of them were biologically functional. Interestingly, these lncRNAs are involved in the regulation of a variety of cellular processes through chromatin remodeling, including recombinant stem cell pluripotency, parental imprinting and tumor cell spreading and metastasis as well as epigenetic modification and adsorption of miRNAs.

Recently, a number of studies have shown that abnormal lncRNAs expression exerts different biological mechanisms involved in the development of a variety of human diseases. For example, upregulated SPRY4-IT1 inhibits the ability of preeclamptic trophoblasts to proliferate, migrate and vascularize by binding to the HUR. lncRNA ROR promotes the demethylation of the promoter region H3K9 of TESC to be involved in tumorigenesis by inhibiting methyltransferase G9A. In addition, AOC4P inhibits epithelial-mesenchymal transition (EMT) by binding to and promoting the degradation of vimentin, thereby inhibiting hepatocellular carcinoma metastasis. These findings suggest that lncRNAs play a crucial role in the development of human diseases, especially cervical cancer. Therefore, finding more lncRNAs related to cervical cancer and researching the biological functions and mechanisms of the lncRNAs have important significance for better understanding the molecular biology of the occurrence and development of the cervical cancer.

RP11-7K24.3 is a length of 2027nt of lncRNA, the sequence of which is shown in SEQ ID No. 1, which is located on human chromosome 6. We found that RP11-7K24.3 is expressed in a lower amount in the tumor tissues of patients with cervical cancer than in the tissues adjacent to the cervical cancer. After overexpression or knock-out of RP11-7K24.3, the role of RP11-7K24.3 in the development and progression of cervical cancer was investigated and the function of RP11-7K24.3 in the relevant target genes in tumour cells from patients with cervical cancer was investigated.

Disclosure of Invention

The invention aims to provide application of RP11-7K24.3 in diagnosing cervical cancer and preparing a medicament for treating the cervical cancer.

The invention relates to a long-chain non-coding RNA, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The application of a long-chain non-coding RNA in the preparation of a medicine for treating cervical cancer;

the application of a long non-coding RNA in the preparation of a reagent for diagnosing cervical cancer;

a primer for detecting RP11-7K24.3, which comprises the following components: 2 RP11-7K24.3F CTGAAGGCACCCTTTCCT, 3 RP11-7K24.3R ACGTTTACAGGGAGTTGG;

an siRNA interfering with RP11-7K24.3, as shown in sequences 4 and 5, namely: 4 si-RP11-7K24.3 # CATACTCTATCCTCCCAACAA for SEQ ID NO, 5 si-RP11-7K24.3 # GCTCTGAAATGTTACAAC for SEQ ID NO;

a kit comprising the primer;

a pharmaceutical composition comprising the long non-coding RNA;

a plasmid comprising the above pharmaceutical composition;

the application of the primer in preparing a reagent for diagnosing cervical cancer;

the application of the pharmaceutical composition in preparing a medicine for treating cervical cancer.

The pharmaceutical composition also comprises auxiliary materials. The auxiliary materials comprise: (lip 2000, opti-mem broth, PBS phosphate buffered saline).

Technical scheme

The differential expression of RP11-7K24.3 in clinical tissues is screened by qPCR, and the expression level of RP11-7K24.3 in cervical cancer tumor tissues is found to be lower than that in cervical cancer side tissues. Guessing: whether RP11-7K24.3 is involved in the pathogenesis of cervical cancer.

Then, the internationally recognized SiHa and HLEC-P3 are selected as experimental research objects, an interference sequence of RP11-7K24.3 is designed, and the incidence process of cervical carcinoma diseases is simulated after the interference sequence is transferred into cells by taking lip2000 as a transfection vector. By detecting the functions of the cells such as migration and invasion capacity, lymphatic network formation and the like after the interference sequence is transferred into the cells. Thus proving that the knocking down of the expression of RP11-7K24.3 in the cervical cancer cells SiHa and HLEC-P3 influences the functions of the cells and induces or accelerates the pathogenesis process of the cervical cancer disease. On the contrary, RP11-7K24.3 plasmid was constructed, and the function of RP11-7K24.3 in cervical carcinoma cells SiHa, HLEC-P3 was verified positively and negatively.

The relevant downstream target genes of RP11-7K24.3, which may be involved in cell function (such as migration, invasion and lymphangiogenesis), were detected by transcriptome sequencing, followed by preliminary investigations into the regulatory mechanisms of RP11-7K24.3.

The various reagents required for the transfection process,

(1) lip2000, a versatile lipofection reagent, is suitable for transfection of DNA, RNA and oligonucleotides, and has high transfection efficiency for most eukaryotic cells. The unique formulation allows for direct addition to the medium without affecting transfection efficiency by the presence of serum, thereby allowing the RP11-7K24.3 interference sequence to be transferred into the cell.

(2) The Opti-mem culture medium containing HEPES 2400mg/L sodium bicarbonate, hypoxanthine, thymine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1mg/L was used as an adjuvant for transfection reagents, which are not harmful to cells themselves, and were better and more efficiently transferred into cells to achieve the intended purpose.

(3) PBS phosphate buffered saline (phosphate buffer saline) generally acts as a solvent to solubilize the protective agent. It is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na ₂ HPO ₄ 、KH ₂ PO ₄ NaCl and KCl due to Na ₂ HPO ₄ And KH ₂ PO ₄ They have secondary dissociation, and the pH value range of buffering is wide; while NaCl and KCl mainly act to increase the salt ion concentration. Excluding its own effect on the subjects.

Tissue collection

20 pairs of patients who receive cervical cancer operation treatment in national hospitals of Jiangsu province and women and children health care hospitals of Jiangsu province from 2017 to 2018 are collected, and cervical cancer tumor tissues and paracancer normal tissues of the patients suffering from cervical cancer are diagnosed. And record clinical features: including patient age, history of smoking, systolic pressure, diastolic pressure, tumor marker, tumor size, and invasion depth. Tissue samples were collected either in liquid nitrogen for the first time or stored at-80 ℃ until RNA extraction. The study was approved by the ethics committee of the university of medical, nanjing. Written informed consent was obtained from all patients.

Cell lines

Cervical cancer (SiHa) was selected from the shanghai cell research institute. SiHa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100mg/ml streptomycin. 5% CO ₂ And culturing at 37 ℃ in an incubator by a conventional method. The culture medium is replaced every 1-2 days, and the cells are passaged when the cell fusion degree reaches 80% -90%. All cell lines were verified by DNA analysis of short tandem repeats.

RNA extraction and quantitative PCR analysis

Total RNA was isolated using Trizol reagent according to the instructions for use of the reagent. Reverse transcription was performed using TaKaRa Prime Script kit (TaKaRa, dalian, china). The reverse transcription kit reverse transcribes 0.8. Mu.g of total RNA to a final volume of 20. Mu.l. And (4) analyzing results: analyzing the specificity and the amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. And (5) obtaining a Ct value according to the amplification curve, and analyzing the relative expression quantity of the target gene by adopting a relative quantity method and an internal reference GAPDH. The calculation formula is as follows: 2^ (-. DELTA.Ct), and Δ Ct = Ct gene-Ct control.

Plasmid construction

Synthesizing full-length human RP11-7K24.3 cDNA, inserting it into eukaryotic expression vector pCDNA3.1 to construct RP11-7K24.3 over-expression vector plasmid, transforming the plasmid into colibacillus, shaking, culturing, selecting monocloning bacterium, culturing and amplifying.

Cell transfection (Small interference transfection)

si-RP11-7K24.3 used for transfection. The interference sequences and the random control (si-NC) of RP11-7K24.3 were purchased from Invitrogen (Invitrogen, calif., USA). Cells Hela were plated per well 2×10 ⁵ Planting the cells on a 6-hole culture plate, and after the cells adhere to the wall, removing the original culture medium 6h before transfection, and replacing the original culture medium with a double-antibody-free culture medium; diluting 10 μ L liposome in 240 μ L OPTI-MEM, gently blowing, mixing, and incubating at room temperature for 5 min; diluting 100pmol siRNA, si-NC in 250 μ L OPTI-MEM, pipetting, mixing, and incubating at room temperature for 5 min; mixing the incubated liposome with siRNA or plasmid diluent, and gently whipping and uniformly mixing. Incubating at room temperature for 20 min; the mixture was dropped into a 6-well plate containing 1.5 mL of OPTI-MEM, and gently mixed. 37 ℃ and 5% CO ₂ After the cultivation in the incubator is continued for 6h, the complete culture medium is replaced. 36 h after transfection, collecting cells to extract RNA or protein for real-time quantitative RT-PCR or immunoblot analysis.

Nanoparticle synthesis

Carboxylic acid ligand synthesis-0.1 mol carboxylate precursor molecule was added with NaOH in a 1.1 molar ratio and dissolved in 80 ml deionized water and stirred vigorously in an ice bath. Acryloyl chloride (0.11 mol) was added dropwise to 15ml of tetrahydrofuran, and the reaction pH was maintained at 7.5 to 7.8 with 1m NaOH solution. Then acidified with 1M HCl solution and reacted overnight. The organic layers were collected, dried over sodium sulfate and the solvent was removed by rotary evaporation to give a white powder. Polymer synthesis — monomers B7 and B8 were dissolved in anhydrous dimethyl sulfoxide (DMSO) at a 0.8 molar ratio, and the ratio of total ethylene/amine was 2.2:1 was added to the final monomer so that the final concentration was 150 mg/mL, and the reaction solution was stirred at 90 ℃ overnight. Subsequently, the polymer was reacted with monomer E1 (final concentration in DMSO of 0.2 m) for 2 hours at room temperature. The resulting E1 polymer was purified by two ether washes, after which the polymer was dissolved in 200 mg/ml DMSO and capped with a carboxylic acid ligand (final concentration in DMSO of 0.2M) for 2 hours at room temperature. The resulting carboxylated polymer was further purified by ether precipitation and the remaining solvent was removed in a vacuum chamber. The polymer was dissolved in dimethyl sulfoxide at 100mg/ml and stored in disposable aliquots at-20 ℃ with desiccant. Nanoparticle characterization-nanoparticles were prepared by dissolving the polymer and protein separately in 25 mm sodium acetate (NaAC, pH 5), mixing the two solutions in a 1. At room temperature for 10 minutes. To prepare nanoparticles encapsulating CRISPR-RNPs, sgRNA-RP11-7k24.3 and Cas9 protein were first mixed together in a 2; RNPs were then mixed with the polymer at a volume ratio of 1. The nanoparticles were diluted in 150 mM Phosphate Buffer (PBS) with 1. Hydrodynamic diameter was measured by dynamic light scattering on a Malvern Zetasizer Pro (Malvern panaltytic). BSA (30 w/w) encapsulated nanoparticles 1.8 mg/ml were prepared at polymer concentration and 25 mm NaAc was added. 30 microliters of nanoparticles were added to a 400 square grid carbon coated TEM grid and incubated for 20 minutes. The grid was then rinsed with ultra pure water and dried completely before imaging.

Cell transfection (Small interference transfection)

Seeded at 15000 cell density per well in 96 well tissue culture plates. And performing transfection operation after the cells grow to 50% -60%. Protein-encapsulated nanoparticles were prepared as described above, and 20 microliters of nanoparticles per well were added to serum containing complete cell culture medium and incubated with cells for 4 hours. In the FITC-BSA uptake experiment, the nanoparticle/media mixture was removed within 4 hours, cells were washed three times with PBS, and uptake was assessed by flow cytometry using BD Accuri C6. Nanoparticle uptake was quantified by normalizing the geometric mean fluorescence of the treated wells to that of the untreated control wells. For all other transfection experiments, fresh complete medium was changed after 4 hours of incubation with nanoparticles. Efficiency was assessed 3 days after transfection.

Cell migration and invasion assay

Transwell cells with a pore size of 8 μm were placed in the 24-well plates. Cell invasion assay, the upper face of the bottom membrane of a Transwell chamber was coated with 50mg/l BD Matrigel 1 dilution 6, the coated chamber was placed in a 24-well plate and incubated in an incubator for 6h. Digesting the cells, terminating digestion, centrifuging to remove the culture solution, washing with PBS for 1-2 times, and resuspending with serum-free culture medium containing BSA. Adjusting cell density to 3x10 ⁵ . Add 300. Mu.l of cell suspension to the Transwell chamber. 24 orifice lower chamber add700 μ l of a medium containing 10% FBS was put into an incubator and cultured for 24 hours. Cell migration experiments, adjusting cell density to 3x104. Cell suspension 300. Mu.l was taken and added to a Transwell chamber. 700 μ l of the medium containing 10% FBS was added to the 24-well lower chamber and placed in an incubator for conventional culture for 24 hours. The cell was taken out, the matrix and the cells in the upper chamber were wiped off with a cotton swab, and the stained cells on the outer bottom surface of the cell were stained with 0.1% crystal violet, and the stained cells attached to the upper and lower chamber sides of the basement membrane of the Transwell cell were photographed and counted using an inverted microscope.

LncRNA sequencing

200ug of cervical cancer tumor tissue and tissue beside the cancer are treated by Trizol to collect cells, the cells are sent to a Beijing gene detection mechanism for implementation, illumina is selected for subsequent experiments, and corresponding data are obtained and processed.

Lymphatic network formation experiment

Firstly, 50 mu L of matrigel is added into a 96-well plate and put into an incubator at 37 ℃ for more than 30 min. Preparing single cell suspension of cervical cancer cell strain HLEC-P3 cells from cervical cancer cell supernatant transfected with si-RNA control, si-RP11-7K24.3 # and si-RP11-7K24.3 # or negative plasmid and RP11-7K24.3 # plasmid, adjusting cell concentration to 2 × 105/mL, and adding into the pores with matrigel. The number of lymphangiogenesis was observed after 2-3 h.

Data processing

The experimental data were analyzed using the SPSS17.0 software, expressed as mean ± standard error of three experiments, and the differences between groups were tested using the two-tailed Student's T test, the rank-sum test and the chi-square test. Subsequent reuse of multi-factor analysis with P <0.05 in single factor analysis.

Compared with the prior art, the invention has the technical effects that:

in this study, we have discovered a novel lncRNA RP11-7k24.3 and demonstrated its down-regulation of expression in cervical cancer tumor tissue. The expression of the down-regulated RP11-7K24.3 has close relation with the pathogenesis of cervical carcinoma tumor. Knock-down of RP11-7K24.3 promotes cell migration and invasion, and lymphangiogenesis effects.

Our studies found for the first time that RP11-7k24.3 expression was down-regulated in cervical cancer tumor tissues and cells. The cervical cancer tumor cell SiHa knockdown of the expression of RP11-7K24.3 shows a cell promoting function, and the over-expression of RP11-7K24.3 shows cell migration inhibition and reduction of cell lymphatic vessel formation. Furthermore, the method is simple. Our findings will further enrich the pathogenesis of cervical cancer and facilitate lncRNA-directed diagnosis and treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1, RP11-7K24.3, is downregulated in cervical cancer tumor tissue.

1A analysis by lncRNA sequencing revealed that RP11-7K24.3 expression was down-regulated in cervical cancer tumor tissue (n = 4).

1B-1C the results of the IncRNA sequencing, RP11-7K24.3, were verified by RT-PCR.

FIG. 2, RP11-7K24.3 expression in cervical carcinoma tumor tissue correlates with tumor tissue size, metastasis, and lymphoid metastasis.

2A RP11-7k24.3 is significantly lower expressed in patients with larger cervical carcinoma tumors (n = 30).

2B RP11-7k24.3 is expressed significantly less in cervical cancer metastatic tumor tissues (n = 30).

2C RP11-7k24.3 is expressed significantly less in cervical carcinoma lymphoid metastatic tumor tissues (n = 30).

FIG. 3RP11-7K24.3 inhibits cervical cancer cell migration and invasion.

3A inhibits the expression of RP11-7K24.3 after transfection of RP11-7K24.3 interference sequences in SiHa.

3B increased migration and invasion capacity of cervical cancer cells was found after transfection of RP11-7K24.3 interference sequences in SiHa.

3C RP11-7K24.3 plasmid (pcDNA3.1 + RP 11-7K24.3) was transfected in SiHa and the expression of RP11-7K24.3 was upregulated.

3D reduced migration and invasion capacity of cervical cancer cells was found after transfection of RP11-7K24.3 plasmid in SiHa.

FIG. 4 RP11-7K24.3 inhibits cervical cancer lymphangiogenesis.

4A inhibits the expression of RP11-7K24.3 after transfection of an RP11-7K24.3 interference sequence in HLEC-P3.

4B cervical cancer lymphangiogenesis was found to be increased by transfecting RP11-7K24.3 interference sequences in HLEC-P3.

4C the expression of RP11-7K24.3 was upregulated after transfection of the RP11-7K24.3 plasmid (pcDNA3.1 + RP 11-7K24.3) in HLEC-P3.

4D was transfected into RP11-7K24.3 plasmid in HLEC-P3 and detected to find that the lymph vessel generation capability of cervical cancer is reduced.

Detailed Description

The invention is further illustrated by the following examples, without restricting the invention thereto.

The experimental procedures for the examples without specifying specific conditions are carried out essentially according to the conditions and methods described in molecular cloning instructions (3 rd edition) written by Sambrook, J et al, molecular cloning: molecular cloning, 3 rd. Huang-Peyer et al, scientific Press 2002.8, or according to the conditions and methods suggested by the suppliers of the materials, other techniques not described in detail corresponding to standard procedures well known to the person skilled in the art.

The material of the invention: the cell lines and culture media mentioned in this application are commercially available or otherwise publicly available, and are by way of example only and not exclusive to the present invention, and may be replaced by other suitable means and biological materials, respectively.

Example 1 measurement of RP11-7K24.3 expression in tissues and cells

Grinding 0.1 g tissue with liquid nitrogen to obtain powder or 1-5 × 10 ⁷ The cells were discarded from the medium and rinsed 2 times with pre-cooled PBS. Adding 1 ml of Trizol lysate, blowing and beating uniformly by an enzyme-free gun head, standing for 5min, and transferring the lysate into a pre-marked centrifuge tube with 1.5 ml of no enzyme. Centrifuging at 4 deg.C for 5min at 7500 g, collecting supernatant, adding 1/5 volume of chloroform, mixing by inversion for 30s, and standing for 2 min. Centrifuge at 12000 g for 15min at 4 ℃. The solution was divided into three layers (aqueous phase-white precipitate-red organics) and the aqueous layer was transferred to a new 1.5 ml centrifuge tube, with as little white precipitate as possible. Adding isopropanol with the same volume, slightly reversing and mixing evenly,standing for 5-10 min. Centrifuge at 12000 g for 10 min at 4 ℃. The supernatant was aspirated off, 1 ml of 75% ethanol (ready to use) was added, and the RNA pellet was washed. Centrifuge at 7500 g for 5min at 4 deg.C, and discard the supernatant. Removing 75% of alcohol as much as possible, and air drying at room temperature for about 15 min. The RNA pellet was dissolved in RNase-free water (20-25. Mu.l).

The concentration of RNA was determined by UV absorbance assay. RNA concentration and purity were determined using an ultraviolet spectrophotometer, with the RNA dissolved DEPC water being zeroed prior to measurement. The reading at 260 nm is 1, which represents 40 ng/. Mu.l, the ratio of A260/A280 of the RNA solution is used for detecting the purity of the RNA, and the ratio ranges from 1.8 to 2.1, which indicates that the requirements are met. Agarose gel electrophoresis identified the integrity of the RNA. 1 percent of agarose gel is prepared. The agarose was dissolved by heating, cooled and 1. Mu.l of ethidium bromide (EB, 10 mg/ml) was added. Shaking, pouring gel, condensing gel, placing in electrophoresis tank, soaking in 1 × TAE buffer solution, and balancing for 10 min. And (4) spotting. 5 Xnucleic acid electrophoresis loading buffer was mixed with the samples as 1. 80 V constant voltage electrophoresis for 50 min. After the electrophoresis was finished, the results were observed on a gel imager.

Tris-acetate (TAE) buffer formulation (1L) 50 ×:

242 g of 2M Tris base

1M acetic acid 57.1 mL glacial acetic acid (17.4M)

100 mM EDTA 200 mL 0.5 M EDTA (pH8.0)

Deionized water to 1L

Real-time quantitative PCR

The total RNA of SiHa cells in cervical cancer tumor tissue and tumor paracancer normal tissue specimens and reverse transcription reaction were performed using TaKaRa PrimeScript kit (Dalianbao bioengineering Co., ltd.).

The reverse transcription reaction system is as follows:

5 × PrimeScript Buffer（for Real Time） 4 μl

Total RNA (1 μg/μL) 1 μl

2. Mu.l of Random or Oliga dT

Primescript RT enzyme Mix 1μl

RNase Free dH ₂ O to 20. Mu.l

The reverse transcription reaction conditions were as follows: 15min at 37 ℃ (reverse transcription reaction); 5sec at 85 ℃ (inactivation reaction of reverse transcriptase). Primer sequences were designed based on the gene sequences provided by Genebank, QPCR using the 7300 PCR system (Applied Biosystems, warrington, UK). The cDNA samples were amplified using a three-part PCR standard protocol.

Reaction system:

SYBR Premix Ex Taq 2 μl

F primer 0.4 μl

R primer 0.4 μl

ROX 0.4 μl

cDNA 1 μl

ddH ₂ O 5.8 μl

the reaction conditions are as follows:

95℃ 30 s

95℃ 5 s

60℃ 34 s

68℃ 45 s

and (4) analyzing results: analyzing the specificity and the amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. And (5) obtaining a Ct value according to the amplification curve, and analyzing the relative expression quantity of the target gene by adopting a relative quantity method and an internal reference GAPDH. The calculation formula is as follows: 2^ (-. DELTA.Ct), and Δ Ct = Ct gene-Ct control.

The primers for RP11-7K24.3 were as follows:

Primer F 5’-CTGAAGGCACCCCTTTTCCT-3’， SEQ ID NO：2，

Primer R 5’-ACGTTTACAGGGGAGTTGGG-3’， SEQ ID NO：3。

the results show that the expression of lncRNA RP11-7K24.3 in cervical cancer tumor tissues is reduced compared with the expression in tumor side tissues. The expression level of 30 pairs of cervical cancer tumor tissues is detected by real-time quantitative PCR compared with the expression level of RP11-7K24.3 in tumor side tissues. The results showed that the expression of RP11-7K24.3 was reduced at 80% (24/30) in cervical cancer versus tumor para-cancer (fold >1.5, P < 0.05) (FIG. 1). It is suggested that RP11-7K24.3 may play an important role in the diagnosis, development and treatment of cervical cancer diseases.

Example 2 Effect of RP11-7K24.3 on SiHa invasion and migration of cervical carcinoma tumor cells

An important aspect in the pathogenesis of cervical cancer tumor cell infiltration and metastasis. SiHa was used as the subject and the RP11-7K24.3 interference sequence or RP11-7K24.3 plasmid was transfected using lip2000 as the vector to down-or up-regulate the expression of RP11-7K24.3. The effect of RP11-7K24.3 knockdown on SiHa cell migration and invasiveness was studied using the transwells assay. In contrast, overexpression of RP11-7K24.3 had an effect on SiHa cell migration and invasiveness. The results show that compared with the cells of a control group, the cells with the knocked-down RP11-7K24.3 promote the SiHa migration capacity and the invasion capacity of the cervical cancer cells; after high expression of RP11-7K24.3, siHa migration and invasion of cervical carcinoma cells were inhibited compared with those of control group (FIG. 3). It can be seen that the low expression of RP11-7K24.3 promotes the migration and invasion capacity of cervical cancer cells and induces the occurrence and development of cervical cancer diseases.

Example 3 Effect of RP11-7K24.3 on lymphangiogenesis of cervical cancer tumor cells

The lymphatic metastasis of tumor cells is an important aspect of the pathogenesis of cervical cancer. HLEC-P3 was used as a subject to down-regulate or up-regulate the expression of RP11-7K24.3 by transfecting an RP11-7K24.3 interference sequence or an RP11-7K24.3 plasmid using lip2000 as a vector. The influence of RP11-7K24.3 knock-down on the lymphangiogenesis ability of HLEC-P3 cells was studied using a lymphangiogenesis assay. In contrast, overexpression of RP11-7K24.3 had an effect on the lymphangiogenic capacity of HLEC-P3 cells. The results show that the cervical cancer cell HLEC-P3 lymphangiogenesis is promoted after the RP11-7K24.3 is knocked down compared with the cells in the control group, and the cervical cancer cell HLEC-P3 lymphangiogenesis capacity is inhibited after the RP11-7K24.3 is highly expressed compared with the cells in the control group (figure 4). It can be seen that the low expression of RP11-7K24.3 promotes the lymphangiogenesis ability of cervical cancer cells, and induces the generation and development of cervical cancer diseases.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

SEQUENCE LISTING

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> long non-coding RNA and application thereof in diagnosis/treatment of cervical cancer

<130> 2020

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 2027

<212> DNA

<213> Homo sapiens

<400> 1

tgtgtagaca tagaataaga tttattagag aaacttgtgg gcagaaaaac agcaaagtga 60

ctgggttctc tgaaccctcc ttatctcata gaggtggaat tggccagact gagctggaag 120

caccaagata agcgcttagg gaggaggatc ccccacaacc ccctgcccat tctaggttat 180

ttaatttttt ttcttttttt tttagagaca gtatctcgct ctgtcaccca ggctggagtg 240

cagtggtgtg atctcggctg actgcaacct ccacctcccg agttcaaacg attctcctgc 300

ctcagcctcc ccagtagctg ggattacagg cgtgcacaac cacagctggc taatttttgt 360

atttttagta gagacagggt tttgccatgt tggtgaggcc ggtctccaat tcctggcctc 420

aagtgattcg cctgcctcag cctcccacag tgctgggatt acaggtgtga gctacacagg 480

attacagatg tgcccggcca tcagtgtaca cgtttacagg ggagttggga ggagggtagg 540

aaatgcctgg tccactacat taccctagga gcttcctggt cgaggaaaag gggtgccttc 600

agagctcagc cactgctatt aaccagaagc tctgtgtgat cttcccttga actaacttgg 660

gataatattt gtccttcaag gatcatgaaa tccttggagg tttgccctcc aaaccttctc 720

tccactccca tgctcaagta tctctcacca ccttcaccaa agtccactcc caagcagtca 780

tcacactggg actccttcag ggtaaaacca gccaggacac caggcttcca gctagagcac 840

agggtttctc aacctcagta ctaccgacat ttagctagat ttgttgtggg aggctgccct 900

gtacattgta ggacatttag tagcaccctt ggcctctacc tgctgaatgc ccttactatc 960

cccctaggat gtcaatgaaa aaatatctcc agatcttcca gatatcacca aatatcctct 1020

gggaagcaaa attggccctg gataagaacc attaaagtca actaagacac cagggggctg 1080

catattcaga aactaaagtg agactggtag aaaactgggg caacagagat tttttttttt 1140

ttttgagacg gagtcttgtt ctgtccccta ggctggagtg cagtgtcgcg atctcgactc 1200

actgcaacct ctgtgtcctg ggttcaagcg attctcctgc ctcagcctcc tgagtagctg 1260

ggattacagg cacccaccac cacacgcggc taattttttc ttttgtattt ttagtagaaa 1320

tggggctagt ttctactcct gacttcaaat gatccacccg cctcaacttc ccaaagtgct 1380

gggattatag gcatgagcca ccgtgcccgg ctgagattta ttcccccctc aaatcagaat 1440

acatcttcac tgtttcttct gtctaatttc ctcaagctag actataggaa ttaagacagt 1500

gttgtaacat ttcagagccc cggagcccaa ctccctgcat gacaaccctg gctctctggc 1560

ttgctagctc tgtgaccttg gacaagttac atagcctttg ataataaaag tgcctgcctc 1620

ttggggttgt tgggaggata gtatgaaata atatgcgtaa agtgcctagg atcgtgcctg 1680

gtacctaata agctgttgca ggacaggctc cccaggagac tgaggtttgt gtgcagcaag 1740

tttgctggga gtgccctcaa gaagcacact ggcatgggag agggagaagt gagcacaggg 1800

aaaagctggg ctgcctgcac ttgcaatgga ggggcgccct ggtctgcaag gaccttcaga 1860

gttgtcccgc attgagggga gggacttagt gaatgtgagc tgcctctagg gtgtgggcgt 1920

gaccttgggt gaggtggctc tcccgggaag gacagttcct ggggagggac tgccagctgc 1980

tggctggggg tgtgaaagct tcagtcctgg aaagggagag ggtcagg 2027

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

ctgaaggcac cccttttcct 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

acgtttacag gggagttggg 20

Claims

1. The application of the long non-coding RNA in preparing the medicine for treating cervical cancer is characterized in that the long non-coding RNA is RP11-7K24.3, and the nucleotide sequence of the long non-coding RNA is SEQ ID NO:1.

2. the use of a pharmaceutical composition comprising the long non-coding RNA of claim 1 in the preparation of a medicament for the treatment of cervical cancer.

3. The use of claim 2, wherein the pharmaceutical composition further comprises an adjuvant.

4. Use of a nanoformulation comprising the long non-coding RNA of claim 1 in the preparation of a pharmaceutical composition for the treatment of cervical cancer.