CN109847064B - Application of circ _3480 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation - Google Patents

Abstract

The invention belongs to the technical field of tumor molecular biology, and particularly relates to application of circ _3480 in preparation of a nasopharyngeal carcinoma treatment preparation and a treatment preparation. As the siRNA has good silencing effect, after ensuring that the circ _3480 is interfered, MTT experiments are carried out in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 which silence the circ _3480, compared with an NC group, the proliferation speed of the cells in the siRNA group is obviously reduced, namely the silencing of the circ _3480 inhibits the proliferation of the nasopharyngeal cancer cells. Namely, the inhibition of circ _3480 can treat nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospect.

Description

Application of circ _3480 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation

Technical Field

The invention belongs to the technical field of tumor molecular biology, and particularly relates to a reagent for inhibiting circular RNA circ _3480 and application thereof in preparation of a nasopharyngeal carcinoma treatment preparation.

Background

Circular RNA (circular RNA) is a research hotspot, and circular RNA is mainly derived from exonic regions of protein coding genes and can also be formed by intronic regions, UTR regions, intergenic regions, non-coding RNA sites and antisense sites of known transcripts. The CircRNA is a non-coding RNA molecule formed by reverse splicing of pre-mRNA (pre-mRNA) without a 5 'terminal cap and a 3' terminal poly (a) tail and covalently bonded to form a circular structure.

The process of CircRNA formation can be divided into two major mechanisms, exonic circularization (exon circularization) and intron circularization (intron circularization). Jeck et al propose that exon-derived circRNAs (exonic circular RNAs) can be divided into two forming modes of lasso-driven circularization (large-driving-circularization) and intron-paired driven circularization (intron-driving-circularization), lasso-driven circularization is that the 3 'end of an exon is used as a splice donor (splice donor) to attack a 5' end splice acceptor (splice acceptor), Alu regions are covalently combined to form a lasso structure, and the lasso structure is internally spliced and then an intron is cut to form the circRNAs; intron pairing-driven circularization is the complementary pairing of two intron bases to form a circular structure, and then the intron is cut off to form a circRNA. In fact, the intron itself may be circularized, and circular RNA (circular intracellular RNA) derived from the intron may be formed. The CircRNA is a non-coding RNA molecule which is formed by reversely splicing precursor mRNA and does not have a 5 'terminal cap and a 3' terminal poly (A) tail and is in a closed ring structure formed by covalent bonds. Has the characteristics of high stability, conservation, specificity and high content.

CircRNA was first discovered in 1976 in RNA viruses, and subsequently Hsu MT et al found the presence of CircRNA in monkey kidney cytoplasm using electron microscopy. In 1996, circRNA was found in human cells, and with the development of a new generation of RNA-seq sequencing technology, more and more circRNAs were found in recent years, and the number of circRNAs known so far has reached more than thirty thousand. CircRNA is no longer considered to be a wrong RNA transcript, but rather rises as a glaring star in noncoding RNA studies. More novel circRNA is screened and verified as a biomarker for tumor diagnosis and prognosis and application thereof, can be well protected in the patent field as soon as possible, and can remarkably improve the international competitiveness of China in the technical field.

Nasopharyngeal carcinoma (NPC) belongs to a tumor of the head and neck, originating from the epithelial tissue of the nasopharynx. Generally, the disease occurs in the posterolateral crypt (Fossa of rosenmuller) of the nasopharynx, where nasopharyngeal carcinoma cells invade adjacent tissues and organs. Because the occurrence and development of nasopharyngeal carcinoma are multi-stage complex gene regulation processes caused by accumulated familial heredity and somatic genetic mutation and epigenetic mutation, the activation and silencing of protooncogenes and cancer suppressor genes, the epigenetic regulation of ncRNA participation and the like are involved. Therefore, the specific molecular mechanism of occurrence and development of nasopharyngeal carcinoma is not clear, and the curative effect of the traditional radiotherapy and chemotherapy is not significant due to tumor heterogeneity and individual difference. Therefore, research on occurrence and development mechanisms of the nasopharyngeal carcinoma and the research on the nasopharyngeal carcinoma by the circRNA, further diagnosis of the nasopharyngeal carcinoma and control on proliferation, invasion and metastasis of the nasopharyngeal carcinoma are hot spots of research.

We detected a circ _3480 of 425bp in length in RNA-seq of nasopharyngeal carcinoma cells 5-8F. Experiments show that the circular RNA is related to the occurrence and development of nasopharyngeal carcinoma, and can be used as a nasopharyngeal carcinoma diagnosis marker and a therapeutic target.

Disclosure of Invention

The invention discovers a circ _3480 with the size of 425bp from the RNA-seq data of 5-8F cells, discovers the relation between the circ _3480 and nasopharyngeal carcinoma, and can be possibly used as a diagnostic marker and a therapeutic target of the nasopharyngeal carcinoma.

The first purpose of the invention is to provide application of a preparation for inhibiting circular RNA circ _3480 in preparation of a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circular RNA circ _3480 is shown as SEQ ID NO. 1.

Further, the preparation for inhibiting the circular RNA circ _3480 comprises siRNA.

Further, the siRNA is as follows:

sense strand (5'-3') CUGGAUCAACAAGAUAACAUU

Antisense strand (5'-3') UGUUAUCUUGUUGAUCCAGUU.

Further, the reagent for inhibiting circ _3480 also comprises a negative control:

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU

Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.

The second purpose of the invention is to provide an agent for treating nasopharyngeal carcinoma, which comprises an agent for inhibiting circular RNA circ _3480, wherein the sequence of the circular RNA circ _3480 is shown in SEQ ID NO. 1.

Further, the preparation for inhibiting the circular RNA circ _3480 comprises siRNA.

Further, the siRNA is as follows:

sense strand (5'-3') CUGGAUCAACAAGAUAACAUU

Antisense strand (5'-3') UGUUAUCUUGUUGAUCCAGUU.

Further, the reagent for inhibiting circ _3480 also comprises a negative control:

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU

Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.

However, the present invention is not limited to the above-provided siRNA and negative control.

The nasopharyngeal cancer therapeutic preparation further comprises an agent required for transfection of siRNA.

Currently, siRNA has been developed as an important tool for gene function studies. To explore the role of circ _3480 in tumorigenesis development, we designed a pair of sirnas based on the splice site of circ _3480, and transiently transfected sirnas and siNC into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circ _ 3480. After transfection, cells are collected after being cultured for 36 hours, the expression level of the circ _3480 is detected by using a real-time fluorescent quantitative PCR technology to detect the transfection efficiency of the siRNA, and meanwhile, the expression level of the circ _3480 is detected, and the designed siRNA is found to be capable of obviously inhibiting the expression level of the circ _ 3480.

The present invention has confirmed the above conclusion through a number of experiments: namely, an agent for inhibiting circ _3480 can be used for preparing a therapeutic agent for nasopharyngeal carcinoma. These tests included: in vitro overexpression circ _3480 test shows that the proliferation of nasopharyngeal carcinoma cells can be promoted, and in vitro silencing circ _3480 test shows that the proliferation of nasopharyngeal carcinoma cells can be inhibited; in vitro overexpression of circ _3480 promotes the migration of nasopharyngeal carcinoma cells, and in vitro silencing of circ _3480 inhibits the migration of nasopharyngeal carcinoma cells; in vitro overexpression of circ _3480 promotes invasion of nasopharyngeal carcinoma cells, and in vitro silencing of the expression of circ _3480 inhibits invasion of nasopharyngeal carcinoma.

The siRNA has good silencing effect. After ensuring that circ _3480 is interfered, we performed MTT experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3480, the proliferation rate of cells in the siRNA group was significantly slower compared to the NC group, i.e. silencing circ _3480 inhibited proliferation of nasopharyngeal cancer cells. Namely, the inhibition of circ _3480 can treat nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospect.

Drawings

FIG. 1 is a graph showing the qRT-RCR detection of the expression level of circ _3480 in nasopharyngeal carcinoma tissues and nontumor rhinitis epithelial tissues;

analysis of the expression level of circ _3480 non-tumor nasopharyngeal epithelial tissue was normalized to 1 with β -actin as a reference, N was non-tumor nasopharyngeal epithelial tissue, and the number of samples was 12; t is nasopharyngeal carcinoma tissue, the number of samples is 28, n is the number of samples, the T test is adopted, and P is less than 0.05, so that the statistical significance is achieved.

FIG. 2 is a map of an overexpression vector.

FIG. 3 shows the effect of circ _3480 overexpression in nasopharyngeal carcinoma cell lines as measured by qRT-PCR;

qRT-PCR assay circ _3480 overexpression in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 pcdna3.1(+) panel was normalized to 1, P <0.05, P <0.01, P <0.001 with β -actin as reference.

FIG. 4 shows the silencing efficiency of circ _3480siRNA in nasopharyngeal carcinoma cell lines detected by qRT-PCR technique;

qRT-PCR to test the silencing efficiency of circ _3480siRNA in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2; circ _3480 expression level analysis NCsiRNA groups were normalized to 1, ns representing no significance, P <0.05, P <0.01, P <0.001, with β -actin as a reference.

FIG. 5 is a graph showing the effect of in vitro overexpression of circ _3480 on nasopharyngeal carcinoma cell proliferation;

qrt-PCR to detect circ _3480 overexpression efficiency in nasopharyngeal carcinoma cell lines HONE1, HNE2, and CNE 2; d-f. the cells were tested for cell proliferation by performing MTT experiments, and the circ _3480 expression level analysis normalized pcdna3.1(+) group to 1, ns for no significance, P <0.05, P <0.01, P <0.001, with β -actin as a reference.

FIG. 6 is a graph of the effect of in vitro silencing of circ _3480 on nasopharyngeal carcinoma cell proliferation;

qrt-PCR assay for circ _3480 silencing efficiency in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2; d-f. the cells were tested for cell proliferation by performing MTT experiments, and the circ _3480 expression level analysis normalized pcdna3.1(+) group to 1, ns for no significance, P <0.05, P <0.01, P <0.001, with β -actin as a reference.

FIG. 7 is a graph of the qrT-PCR detection of circ _3480 overexpression efficiency in scratch test cells;

the overexpression efficiency of nasopharyngeal carcinoma cell strains HONE1, HNE2 and CNE2 used in the scarification experiment is detected by using a qRT-PCR experiment, and the expression level analysis of circ _3480 takes beta-actin as a reference, and the pcDNA3.1(+) group is normalized to 1, P <0.05, P <0.01 and P < 0.001.

FIG. 8 is a graph showing the effect of overexpression of circ _3480 on nasopharyngeal carcinoma cells HONE1, HNE2, and CNE2 scratch healing experiments;

c. nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 were transfected pcDNA3.1(+) No-load, pcDNA3.1 (+)/circ-3480, scratched when the cell density reached 100%, and photographed at 0,12 and 24 hours.

FIG. 9 is a statistical chart of cell scratch experiments for nasopharyngeal carcinomas HONE1, HNE2, and CNE2 overexpressing circ _ 3480;

a-c, randomly selecting 6 visual fields in each group to measure the scratch width, standardizing the scratch width of 0 hour to 1, and making a statistical chart; each experiment was repeated three times and counted using Student's t-test method. P <0.05, P <0.01, P < 0.001.

FIG. 10 shows the interference efficiency of circ _3480 in scratch test cells measured by qRT-PCR; (ii) a

a-c. nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 used in the scratch test were tested for interference efficiency using qRT-PCR test, and the analysis of circ _3480 expression levels was performed with β -actin as a reference, and NC siRNA groups were normalized to 1,. P <0.05,. P <0.01,. P < 0.001.

FIG. 11 is a graph of the effect of interference circ _3480 on nasopharyngeal carcinoma cells HONE1, HNE2, and CNE2 scratch healing experiments;

nasopharyngeal carcinoma HONE1, HNE2 and CNE2 cells were transfected with NC siRNA/circ _3480siRNA, scored after the cell density reached 100%, and photographed at 0,12 and 24 hours.

FIG. 12 is a statistical chart of nasopharyngeal carcinoma HONE1, HNE2 and CNE2 cell scratch experiments that interfere with circ _ 3480;

a-c, randomly selecting 6 visual fields in each group to measure the scratch width, standardizing the scratch width of 0 hour to 1, and making a statistical chart; each experiment was repeated three times and counted using Student's t-test method. P <0.05, P <0.01, P < 0.001.

FIG. 13 shows the overexpression efficiency of circ _3480 in the qRT-PCR detection of Transwell cell matrigel invasion assay;

transwell cell matrigel invasion assay the overexpression efficiency of nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 was tested by qRT-PCR assay, and the circ _3480 expression level analysis was normalized to 1 for pcdna3.1(+) group, P <0.05, P <0.01, P <0.001, with β -actin as a reference.

FIG. 14 is a graph showing the effect of overexpression of circ _3480 on the invasive potential of the nasopharyngeal carcinoma cell lines HONE1, HNE2, and CNE 2;

the Transwell chamber matrigel invasion experiments were performed on HONE1, HNE2 and CNE2 overexpressing circ _3480, respectively, and on the respective control cells.

FIG. 15 is a statistical chart of a Transwell cell matrix gel invasion assay overexpressing circ _ 3480;

randomly selecting 3 cell fields in each group for cell counting, and making a statistical chart; each experiment was repeated three times and counted using Student's t-test method; the pcdna3.1(+) panel was normalized to 1, P <0.05, P <0.01, P <0.001 with β -actin as a reference.

FIG. 16 shows the interference efficiency of circ _3480 in a qRT-PCR assay for Transwell chamber matrigel invasion assay;

nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 used in transwell stromal gel invasion assay interference efficiency was measured by qRT-PCR assay, and the assay for circ _3480 expression levels was normalized to 1, P <0.05, P <0.01, P <0.001 using β -actin as a reference.

FIG. 17 is a graph of the effect of interference circ _3480 on the invasive potential of the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2;

the Transwell chamber matrigel invasion experiments were performed on HONE1, HNE2 and CNE2, which interfered with circ _3480, respectively, and on the respective control cells.

FIG. 18 is a statistical chart of a Transwell cell matrigel invasion assay interfering with circ _ 3480;

randomly selecting 3 cell fields in each group for cell counting, and making a statistical chart; each experiment was repeated three times and counted using Student's t-test method; with β -actin as a reference, NC groups were normalized to 1, P <0.05, P <0.01, P < 0.001.

Detailed Description

The following detailed description is intended to further illustrate the invention without limiting it.

The normal inflammatory nasopharyngeal epithelial tissue and the nasopharyngeal carcinoma tissue specimen used by the invention are from the first-diagnosis patient treated by the affiliated tumor hospital of the university of Central and south China, and are not treated by radiotherapy, chemotherapy and operation. After fresh nasopharyngeal carcinoma tissues are collected, the tissues are immediately put into a liquid nitrogen tank for storage, and then relevant clinical data of all patients are collected, and all experimental tissue samples are collected and authorized by the ethical committee of the university of the south China and approved by the patients.

The three nasopharyngeal cancer cell lines of HONE1, HNE2 and CNE2 used in the invention are all stored in molecular genetic laboratories of the institute of tumor research of the university of Zhongnan. The cell culture conditions were: RPMI1640 liquid medium containing 10% Fetal Bovine Serum (FBS) and 1% double antibody (penicillin, streptomycin), 37 deg.C95% humidity, 5% CO₂The constant temperature incubator with the concentration grows by adhering to the wall.

The design of the primer of the circular RNA is different from that of the linear RNA primer, the primer is designed according to two sides of a splicing site, is designed on a Primer3.0 website on line, and the final primer synthesis work is finished by sending an electronic mail order and entrusting the Changsha synthesis department of Ongken biology company.

(1)β-actin

An upstream primer: 5'-TCACCAACTGGGACGACATG-3', the sequence is shown in SEQ ID NO. 2;

a downstream primer: 5'-GTCACCGGAGTCCATCACGAT-3', and the sequence is shown in SEQ ID NO. 3.

(2) Circular RNA circ _3480 real-time quantitative PCR primer

An upstream primer: 5'-CAACGTTCAGTGCCTCGAAA-3', the sequence is shown in SEQ ID NO. 4;

a downstream primer: 5'-CCTGCTCATACTGGTCAACG-3', and the sequence is shown in SEQ ID NO. 5.

(3) Amplification circ _3480 full-length primer

An upstream primer: 5'-CCATCGATGGATAACATCCCTGAGGACCTC-3', the sequence is shown in SEQ ID NO. 6;

a downstream primer: 5'-TCCCCGCGGGGACTTGTTGATCCAGAACACCA-3', and the sequence is shown in SEQ ID NO. 7.

In the invention, in order to specifically knock down the circular RNA without influencing the linear gene expression of the circular RNA, siRNA is designed according to splicing sites, and targeted silencing circ _3480 is realized.

circ _3480siRNA sequence:

a sense strand (5'-3') CUGGAUCAACAAGAUAACAUU, the sequence is shown in SEQ ID NO. 8;

the antisense chain (5'-3') UGUUAUCUUGUUGAUCCAGUU, the sequence is shown in SEQ ID NO. 9.

Negative control:

a sense strand (5'-3') UUCUCCGAACGUGUCACGUUU, the sequence of which is shown in SEQ ID NO. 10;

the antisense chain (5'-3') ACGUGACACGUUCGGAGAAUU, the sequence is shown in SEQ ID NO. 11.

The test results of the invention are all analyzed by statistics: the t-test was used to evaluate the difference between the two groups. Chi-square test is used to assess differences in gene expression or lack thereof with respect to clinical parameters such as sex, age, tumor stage, clinical staging and metastasis. Survival analysis was performed using the Kaplan-Meier assay. p <0.05 was used to indicate statistical significance, and all p values were tested using a two-sided test. Statistical analysis was performed using SPSS 13.0 and Graphpad 5.0 software.

Example 1: expression of circ _3480 in nasopharyngeal carcinoma tissues and cells

1. According to the standard sample collection protocol, 40 nasopharyngeal cancer patient tissue samples were collected from tumor hospitals in Hunan province. All cases were first-diagnosed patients in head and neck surgery in tumor hospitals in Hunan province (time interval: 2016 1 month to 2016 11 months). 28 cases of nasopharyngeal carcinoma and 12 cases of nasopharyngeal inflammation (excluding tumor diseases, inactive infectious diseases, serious immune diseases and other serious diseases) are diagnosed by a pathology department.

Complete personal information and clinical data including name, gender, age, clinic number, hospitalization number, pathology type, case stage, EBV infection and the like are recorded in the collection process, and detailed Excel electronic form screenshot is shown. All samples are collected and approved by the patient, and the patient signs a written agreement with the patient to establish a specimen bank with complete data.

2. RNA extraction of nasopharyngeal carcinoma or normal inflammatory nasopharyngeal tissue

(1) Preparation work: cleaning mortar with detergent, and soaking in 3% hydrogen peroxide (H)₂O₂) Washing for more than 4 hours, washing with distilled water for several times, covering mortar with tinfoil paper (for uniform heating and preventing pollution when taking out), and oven-drying at 180 deg.C for more than 8 hours. And after the drying time is reached, closing the drying oven, taking out the mortar when the temperature of the drying oven is reduced to the room temperature, and storing in a clean area.

(2) Grinding by liquid nitrogen: adding liquid nitrogen into a mortar for precooling, then clamping and taking the nasopharynx tissue preserved in the freezing storage tube, quickly grinding, continuously adding a small amount of liquid nitrogen while grinding, and grinding again until the nasopharynx tissue is ground into powder. According to the optimal ratio, 1ml Trizol is added to every 50-100mg of normal or nasopharyngeal carcinoma sample (NPC). During our experiment, one nasopharyngeal carcinoma tissue sample is about 200mg, so 2ml Trizol is needed. Further grinding, mixing, and placing in refrigerator at 4 deg.C for 5-10min to allow complete tissue lysis. The lysate was transferred to a 2ml Tube when it had melted to a pink liquid. Each sample can be separated into 2 tubes and stored at-80 ℃.

(3) And (3) water phase separation: to 1000. mu.l of tissue lysate containing trizol was added 200. mu.l of 4 ℃ pre-cooled chloroform, and the mixture was mixed by shaking for about 30 seconds. After leaving in a low-temperature atmosphere for 5 minutes, centrifugation was carried out for 25 minutes (12,000rpm, 4 ℃). After centrifugation, the liquid in the tube was observed to separate into 3 layers, RNA was present in the upper transparent layer, the middle layer was a membranous white precipitate, and the lower layer was pink. Therefore, the upper aqueous phase containing RNA was further pipetted into 1.5ml of Tube and gently pipetted using a 100. mu.l gun to avoid as much as possible the aspiration into the middle and lower layers of material, which would cause RNA contamination.

(4) RNA precipitation: adding isopropanol with the volume of 1:1 equal to that of approximately 500 mu l into the supernatant, gently inverting and mixing the mixture up and down for a plurality of times, placing the mixture at the temperature of 20 ℃ in a refrigerator for 30 minutes, centrifuging the mixture for 30 minutes (4 ℃,12,000rpm) to see that RNA precipitates exist at the bottom of a tube, sucking the mixture by using a 100 mu l pipette gun, and discarding the supernatant to keep the RNA precipitates as much as possible.

(5) RNA washing: 1ml of 75% ethanol prepared with enzyme-free water was added to each tube of RNA pellet sample, and the centrifuge tube was gently inverted upside down to wash the RNA pellet. Then, the mixture was centrifuged for 5 minutes (4 ℃,7,600rpm), the supernatant was discarded as much as possible by using a 100. mu.l pipette gun, and the mixture was dried at room temperature for 10 to 15 minutes.

(6) RNA re-lysis and storage: each tube was filled with 15-30. mu.l DECP water and stored at-80 ℃.

3. Total RNA extraction from cells

Preparation work: after sterilization, the test table and the pipette are wiped with 75% alcohol before the test is started, wherein the sterile RNase-free water, 75% ethanol (prepared without RNase), chloroform, isopropanol, 1 XPBS, an enzyme-free tip and an EP tube are precooled to 4 ℃ by a high-speed low-temperature centrifuge.

(1) Taking cells of RNA to be extracted, and washing the cells twice by using 1 XPBS or D-hanks;

(2) adding 500 mul Trizol lysate into each hole of a 12-hole plate, lysing for 1-2 minutes at room temperature, gently blowing down cells by using a pipette gun, gently turning upside down for 10 times, and standing for 5 minutes at room temperature;

(3) adding 100 μ l chloroform (1 ml Trizol:0.2ml chloroform: 0.5ml isopropanol), shaking vigorously for 15-30s, and standing on ice for 5 min;

(4)4℃，12000rpm/20min；

(5) putting the upper water phase into a precooled Tube, adding 250 mul of isopropanol, and uniformly mixing the mixture with a vortex mixer or a pipette (the temperature is 20 ℃ below zero is more than 1 hour);

(6) at 4 ℃, 12000rpm/30min, and discarding the supernatant;

(7) adding 1ml of 75% ethanol (precooling), and mixing uniformly;

(8) 7600rpm/5min at 4 ℃; discarding the supernatant, and repeating the steps 8 and 9;

(9) flashing off for 10s, sucking up the supernatant as much as possible, and inverting and drying for 10 minutes;

(10) 20-30. mu.l DEPC was added and the RNA concentration and OD measured.

4. Reverse transcription PCR reaction of gene circRNA

(according to the manual of the instructions of 5 × All-In-OneRTMasterMix (with AccuRTGenomiccDNAremovalkit) (# G492) of abm Co.)

The following reaction system is configured:

the reverse transcription PCR reaction program is as follows:

25℃ 10min，

42℃ 15min，

85℃ 5min。

after the reaction is finished, the product is stored at-20 ℃ for later use.

5. Real-time fluorescent quantitative PCR

The reverse transcription reaction product was diluted 5 times and then the following reaction system was configured according to the instruction manual of EvaGreen qPCR MasterMix (MasterMix-R) from abm:

the reaction program on the real-time fluorescent quantitative PCR machine is as follows: (Cycle X39)

After the reaction is completed by the Bio-RadIQ5 real-time fluorescence quantitative PCR instrument, the gene is labeled with the reference gene beta-actin with 2-^ΔΔThe CT value indicates the relative expression level of the target gene, and the difference in expression of the gene is determined. P values were calculated using unpaired t-test.

As a result: a plurality of nasopharyngeal carcinoma tissues and normal inflammatory nasopharyngeal epithelial tissues are collected, and the expression condition of circ _3480 is detected by utilizing a qRT-PCR technology. The results showed that circ _3480 was significantly highly expressed in 28 nasopharyngeal carcinoma tissues compared to 12 normal inflammatory nasopharyngeal epithelium tissues, and circ _3480 expression level was about 7 times higher than normal inflammatory nasopharyngeal epithelium in nasopharyngeal carcinoma group, and the difference between the two groups of data was statistically significant (P ═ 0.0006), and the results are shown in fig. 1. Therefore, the circRNA circ _3480 is highly expressed in nasopharyngeal carcinoma tissues, and the circRNA circ _3480 may have important biological functions for the occurrence and development of nasopharyngeal carcinoma.

Example 2: detection of circ _3480 overexpression Effect in nasopharyngeal carcinoma cell lines

First we selected the cleavage site and put the full length circ _3480 sequence into NEB cutter 2.0 on-line website for analysis, showing that the ClaI and SacII cleavage sites are sites that are not present in the full length circ _3480 sequence, and DNA restriction enzymes that are present in pcDNA3.1 plasmid vector (available from Invitrogen). Thereby constructing an overexpression vector; FIG. 2 is a map of the over-expression vector.

To examine the cyclization efficiency of circ _3480, we first expressed the constructed pcDNA3.1/circ _3480 eukaryotic over-expression vector in nasopharyngeal carcinoma cells. And (3) inoculating third and fourth generation nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 with good growth condition into a 12-well plate, when the cell fusion degree reaches 60-80%, transiently transfecting endotoxin-free plasmids pcDNA3.1 empty vectors and pcDNA3.1/circ _3480 overexpression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 by using a liposome method lipofectamine 3000, and continuously culturing for 48 hours. Collecting cells, and detecting the expression level and the cyclization efficiency of circ _3480 by using a real-time fluorescent quantitative PCR technology. The qPCR results showed that the expression level of circ _3480 was significantly increased in the pcDNA3.1/circ _3480 overexpressing plasmid group cells compared to the pcDNA3.1 empty plasmid group cells, and the expression fold was 20-fold or more in the HONE1, HNE2 and CNE2 cell lines, as shown in FIG. 3, the results were statistically significant.

Example 3: detection of Effect of silencing circ _3480 expression in nasopharyngeal carcinoma cell lines

According to the splicing sites, an SI sequence of circ _3480 is designed, and siRNA is a double-stranded RNA molecule which has the length of 21-25 nucleotides, can be complementarily combined with homologous RNA, and specifically degrades target RNA, thereby inhibiting the expression of the target RNA. Currently, siRNA has been developed as an important tool for gene function studies. To explore the role of circ _3480 in tumorigenesis development, we designed siRNA based on the splice site of circ _3480 and transiently transfected siRNA and siNC (blank) into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circ _ 3480. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3480 was measured by real-time fluorescent quantitative PCR to determine the transfection efficiency of siRNA, confirming that the transfection efficiency was reduced to below 50%. The results are shown in FIG. 4.

Example 4: in vitro overexpression of circ _3480 to promote proliferation of nasopharyngeal carcinoma cells

Firstly, instantaneously transfecting endotoxin-free plasmids pcDNA3.1 and pcDNA3.1/circ _3480 overexpression vectors with lipofectamine 3000 by using a liposome method to nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2, continuously culturing for 48 hours, and carrying out MTT (methyl thiazolyl tetrazolium) experiment after ensuring that the expression level of circ _3480 in the nasopharyngeal carcinoma cell lines is increased by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction) to verify the influence of the overexpression vectors on the cell proliferation. Based on the detection results from the first to the fifth days, we found that there was a significant difference in cell proliferation between the pcDNA3.1 empty plasmid group and the pcDNA3.1/circ _3480 overexpressed plasmid group, and that overexpression of circ _3480 promoted proliferation of nasopharyngeal carcinoma cells under in vitro culture conditions. (see FIG. 5 for results)

Example 5: in vitro silencing of circ _3480 inhibits proliferation of nasopharyngeal carcinoma cells

siRNA and siNC (blank) were transiently transfected into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circ _ 3480. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3480 was measured by real-time fluorescent quantitative PCR to examine the transfection efficiency of siRNA. The results show that the siRNA has good silencing effect. After ensuring that circ _3480 was disturbed, we performed MTT experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3480, verifying its effect on cell proliferation. Based on the detection results of the first to fifth days, the proliferation speed of the cells in the siRNA group is obviously reduced compared with that in the NC group, namely, the silent circ _3480 inhibits the proliferation of the nasopharyngeal carcinoma cells. Through the verification of the positive direction and the negative direction, we can say that the circ _3480 has the promotion effect on the proliferation of nasopharyngeal carcinoma cells under the in vitro culture condition. (results are shown in FIG. 6)

Example 6: cell scratch healing migration experiment:

(1) a cell illumination table: a Tip head of 1000 mul/10 mul, D-Hank's sterilized at high temperature and high pressure, a ruler, a pipette gun of 1000 mul/10 mul, a marker pen and the like, and the components are sterilized by alcohol and then placed in an ultra-clean bench for ultraviolet irradiation for 30 minutes;

(2) respectively transfecting siRNA and NC groups or transfection plasmids when the cells grow to about 50-70%;

(3) scratching is started the next day after the cells grow over the bottom of the flat plate: the 10 mul gun head is perpendicular to the bottom of the 6-hole plate than a ruler to perform cross or # -shaped scratch quickly without inclination, and the force is consistent, so as to ensure that scratch broadband is as same as possible;

(4) the culture solution is removed by suction, and the cells are washed by D-hanks for 3 times, so that the broken cells caused by scratches are washed away as much as possible;

(5) adding 1640 culture medium of 1% double-antibody 2% fetal bovine serum;

(6) taking a picture to record the width of the scratch beside the cross at the moment, and recording the width as 0 h;

(7) putting the 6-hole plate back to the incubator for culture, taking out the 6-hole plate at intervals of 12 hours, and taking the position of the picture taken when 0 hour is taken, wherein the position is marked as 12 hours;

(8) the same position was again photographed at 24h intervals until the scratch healed, all pictures were collated and statistical analysis was performed.

In vitro overexpression of circ _3480 promotes migration of nasopharyngeal carcinoma cells

After determining that the cyclic RNAcirc _3480 has the effect of promoting the proliferation capacity of nasopharyngeal carcinoma cells, scratch experiments are carried out in the nasopharyngeal carcinoma cell lines to verify that the circ _3480 has no influence on the migration of the nasopharyngeal carcinoma cell lines. The nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 were transiently transfected with endotoxin-free plasmids pcDNA3.1 and pcDNA3.1/has _ circ _3480 overexpression vectors by liposome method lipofectamine 3000, and cultured for 48 hours. Collecting cells, and detecting the expression level and the cyclization efficiency of circ _3480 by using a real-time fluorescent quantitative PCR technology. After confirming the good effect of overexpression of circ _3480 overexpression plasmid, we performed cell scratch healing experiments on nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2. Scratch healing experiments were confirmed at various time points (0 h, 12h, 24h for HONE 1; 0h, 12h, 24h for HNE 2; 0h, 12h, 24h for CNE 2) in these cells: the migration ability of the cells of pcDNA3.1/circ _3480 overexpressing plasmid set was significantly enhanced relative to the unloaded pcDNA3.1(+) plasmid set. The width difference of the scratch is large and has statistical significance. The above results show that overexpression of circ _3480 in nasopharyngeal carcinoma cell lines can promote the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to migrate in vitro. (results are shown in FIGS. 7,8 and 9)

In vitro silencing of circ _3480 inhibits migration of nasopharyngeal carcinoma cells

siRNA and NC were transiently transfected into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circ _ 3480. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3480 was measured by real-time fluorescent quantitative PCR to examine the transfection efficiency of siRNA. The results show that the siRNA has good silencing effect. After ensuring that circ _3480 was disturbed, we performed scratch experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3480, verifying its effect on cell migration. Scratch healing experiments were confirmed at various time points (0 h, 12h, 24h for HONE 1; 0h, 12h, 24h for HNE 2; 0h, 12h, 24h for CNE 2) in these cells: compared with the NC group, the migration capacity of the siRNA group cells is obviously weakened. The scratch width difference is obvious and has statistical significance. The above results show that silencing the expression of circ _3480 in nasopharyngeal carcinoma cell lines can inhibit the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to migrate in vitro. As proved by verification of the positive direction and the negative direction, the circ _3480 can promote the migration of nasopharyngeal carcinoma cells. (results are shown in FIGS. 10,11 and 12)

Example 7: cell transwell invasion assay:

(1) preparing matrigel: the BDmatrigel glue frozen at-20 ℃ is placed in a refrigerator at 4 ℃ to be melted into liquid state one day in advance, a tip head and an EP pipe for releasing the glue are placed at-20 ℃ overnight, so that the Matrigel glue cannot be solidified too fast when being paved on the next day;

(2) matrix glue dilution: BDMatrigel gum: adding 20 mul of matrigel into 160 mul of 1640 culture medium, blowing and mixing evenly, wherein the serum-free culture medium is 1: 8;

(3) adding diluted matrix glue into a transwell chamber of 100 mu l, sucking out 80 mu l along the edge, sequentially paving the matrix glue and putting the matrix glue into an incubator at 37 ℃ for incubation for 2-3 hours, and when the glue paving layer is white, indicating that the liquid Matrigel glue is solid;

(4) digesting the transfected experimental cells, washing with a serum-free medium for 2 times, suspending the cells with a serum-free medium, and counting the cells, wherein the cell concentration is adjusted to 2,0000 cells per 200 μ l;

(5) adding 800 μ l of 1640 medium containing 20% FBS to the lower chamber, and placing the 24-well plate in the chamber while tilting at an angle of 45 ° to avoid air bubbles between the chamber and the liquid surface during placement in the chamber;

(6) 200 mul of the cell suspension with the uniform count is added into each chamber, and the 24-well plate is placed back into the 37 ℃ incubator and incubated for about 24-48 hours according to the cell state and the cell invasion speed.

(7) The 24-well plate is taken out and washed twice by PBS or D-hanks, soaked and washed for 10 minutes by 4 percent paraformaldehyde and washed 3 times by clear water.

(8) Dyeing: dripping 0.1% crystal violet to the bottom of the transwell chamber, standing at room temperature for 5-10min, washing with PBS for 2-3 times, and carefully wiping off the matrix glue on the chamber with cotton swab;

(9) 800. mu.l of distilled water was added to a 24-well plate, about 200. mu.l of distilled water was added to the upper chamber of the transwell, and observation was performed under an inverted microscope, photographs were taken of different fields, counted using image J software, and the significance of the difference was statistically analyzed.

In vitro overexpression of circ _3480 to promote invasion of nasopharyngeal carcinoma cells

We performed Transwell cell matrigel invasion experiments on nasopharyngeal carcinoma cell lines HONE1, HNE2, and CNE2 to observe the effect of overexpression of circ _3480 on cell invasion capacity. We also transiently transfected endotoxin-free plasmids pcDNA3.1 and pcDNA3.1/circ _3480 overexpression vectors with lipofectamine 3000 to nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 by liposome method, and continued culturing for 48 hours. Collecting cells, and detecting the expression level and the cyclization efficiency of circ _3480 by using a real-time fluorescent quantitative PCR technology. After confirming that the overexpression of circ _3480 overexpression plasmid was good, we seeded the cells into matrigel-plated Transwell chamber and found that the number of cells invading the lower surface of the chamber was significantly greater for the overexpressed plasmid group than for the unloaded group and that the trend of the results was consistent for both cell lines. 3 pictures were taken randomly and the cell numbers were recorded, with significant differences between the two individual data in each cell line and statistical significance. The results show that the over-expression of circ _3480 in nasopharyngeal carcinoma cell lines can promote the invasion capacity of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. (the results are shown in FIGS. 13,14 and 15)

In vitro silencing of circ _3480 expression affects nasopharyngeal carcinoma invasion

To investigate whether silencing circ _3480 could reverse the phenotypic change brought by overexpression of circ _3480, we performed Transwell cell matrix gel invasion experiments in three cell lines, transiently transfected circ _3480siRNA and NC with Hiperfect reagent into the hore 1, HNE2 and CNE2 cell lines to silence the expression of circ _ 3480. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3480 was measured by real-time fluorescent quantitative PCR to examine the transfection efficiency of siRNA. The results show that the circ _3480siRNA has good silencing effect. After ensuring that circ _3480 was disrupted, we performed a Transwell cell matrigel invasion assay in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3480, and the results showed that the number of tumor cells observable under the Transwell cell in the siRNA group was significantly less than in the NC group, and the trend of the results was consistent for both cell lines. The number of cells was recorded by randomly taking 6 pictures, and the two data sets in each cell line were statistically significant. The results show that the expression of circ _3480 in nasopharyngeal carcinoma cell lines can inhibit the invasion capacity of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. The two directions show that the ring-shaped RNAcirc _3480 can promote the invasion of nasopharyngeal carcinoma cells. (see FIGS. 16,17,18 for results).

Sequence listing

<110> university of south-middle school

Application of <120> circ _3480 in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 425

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 1

agugcccaca uggcaauggu ggaugcccug augauggccu acacugugga gaugaucagc 60

aucgagaagg ugguggccag ugucaagcgc uucucaacgu ucagugccuc gaaagaacuu 120

ccguacgacc ucgaggaugc caugguguuc uggaucaaca agauaacauc ccugaggacc 180

ucagagaccc uuucuacguu gaccaguaug agcaggagca cauuaagccg ccuguuauca 240

agcuucuccu guccagcgag cuguacugcc gugucugcag ccucauccug aaaggggacc 300

agguggccgc cuuacaggga caccagucug ucauccaggc ccugucccgg aaagggaucu 360

augugaugga gagugaugac acccccguga cagaguccga ccucagucgc gcacccauaa 420

aaaug 425

<210> 2

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 2

tcaccaactg ggacgacatg 20

<210> 3

<211> 21

<212> DNA

<213> Unknown (Unknown)

<400> 3

gtcaccggag tccatcacga t 21

<210> 4

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 4

caacgttcag tgcctcgaaa 20

<210> 5

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 5

cctgctcata ctggtcaacg 20

<210> 6

<211> 30

<212> DNA

<213> Unknown (Unknown)

<400> 6

ccatcgatgg ataacatccc tgaggacctc 30

<210> 7

<211> 32

<212> DNA

<213> Unknown (Unknown)

<400> 7

tccccgcggg gacttgttga tccagaacac ca 32

<210> 8

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 8

cuggaucaac aagauaacau u 21

<210> 9

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 9

uguuaucuug uugauccagu u 21

<210> 10

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 10

uucuccgaac gugucacguu u 21

<210> 11

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 11

acgugacacg uucggagaau u 21

Claims (1)

1. The application of siRNA in preparing a preparation for treating nasopharyngeal carcinoma is characterized in that the siRNA is as follows:

sense strand (5'-3') CUGGAUCAACAAGAUAACAUU

Antisense strand (5'-3') UGUUAUCUUGUUGAUCCAGUU.

Images