CN109971760B

CN109971760B - circ _3234, application thereof in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation

Info

Publication number: CN109971760B
Application number: CN201910275579.5A
Authority: CN
Inventors: 曾朝阳; 郭灿; 熊炜; 范春梅; 赵梦瑶; 熊芳; 张立身; 莫勇真; 彭苗; 李征; 李小玲; 李桂源
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2019-04-08
Filing date: 2019-04-08
Publication date: 2021-04-06
Anticipated expiration: 2039-04-08
Also published as: CN109971760A

Abstract

The invention belongs to the technical field of tumor molecular biology, and particularly relates to circ _3234, and application thereof in preparation of a nasopharyngeal carcinoma treatment preparation and a treatment preparation. As the siRNA has good gene silencing effect, after ensuring that the circ _3234 in the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 is silenced by siRNA interference, performing a Transwell cell experiment and a scratch healing experiment, compared with a control group, the invasion and migration speed of the cells in the siRNA group is obviously reduced, namely the silencing circ _3234 inhibits the invasion and migration of the nasopharyngeal carcinoma cells, and correspondingly, a vector for over-expressing the circ _3234 is also constructed, and after over-expressing the circ _3234 in the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, the invasion and migration speed of the cells is obviously accelerated. Namely, the inhibition of circ _3234 can delay the diffusion and the metastasis of nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospects.

Description

circ _3234, application thereof 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 circular RNA circ _3234, a reagent for inhibiting the expression of the circular RNA circ _3234, and application of the reagent 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 recent years more and more circrnas have been found, 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 found to be used 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 _3234 of 377bp in length. 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 circ _3234 with the size of 377bp, discovers the relationship between the circ _3234 and nasopharyngeal darcinoma, and can be used as a diagnostic marker and a therapeutic target of the nasopharyngeal darcinoma.

The primary object of the present invention is to provide a novel circular RNA circ _3234, the sequence of which is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide application of an agent for inhibiting the expression of circular RNA circ _3234 in preparing a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circular RNA circ _3234 is shown as SEQ ID NO. 1.

Further, the agent for inhibiting the expression of circular RNA circ _3234 includes but is not limited to siRNA.

Further, the siRNA is preferably as follows:

sense strand (5'-3') CCUCAGAGCCUGAACUGGCUU

The antisense strand (5'-3') GCCAGUUCAGGCUCUGAGGUU,

but are not limited to the specific siRNAs mentioned above.

Further, the reagent for inhibiting the expression of circ _3234 also comprises a negative control:

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU

The antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU,

but are not limited to the specific negative controls described above.

The third purpose of the invention is to provide an agent for treating nasopharyngeal carcinoma, which comprises an agent for inhibiting the expression of circular RNA circ _3234, wherein the sequence of the circular RNA circ _3234 is shown as SEQ ID NO. 1.

Further, the siRNA is preferably as follows:

sense strand (5'-3') CCUCAGAGCCUGAACUGGCUU

The antisense strand (5'-3') GCCAGUUCAGGCUCUGAGGUU,

but are not limited to the specific siRNAs mentioned above.

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU

The antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU,

but are not limited to the specific negative controls described above.

The therapeutic agent for nasopharyngeal carcinoma according to the present invention further comprises an agent required for transfection of siRNA.

Currently, siRNA has been developed as an important tool for gene function studies. In order to explore the role of circ _3234 in tumorigenesis development, the invention designs a pair of siRNAs according to the splicing site of circ _3234, and transiently transfects the siRNAs and siNC (control) into HONE1, HNE2 and CNE2 cell lines by using Hiperfect reagent to silence the expression of circ _ 3234. After transfection, cells are collected after being cultured for 36 hours, the expression level of the circ _3234 is detected by using a real-time fluorescent quantitative PCR technology to detect the transfection efficiency of siRNA, and meanwhile, the expression level of the circ _3234 is detected, and the designed siRNA is found to be capable of obviously inhibiting the expression level of the circ _ 3234.

The present invention has confirmed the above conclusion through a number of experiments: namely, an agent inhibiting the expression of circ _3234 can be used for preparing a therapeutic agent for nasopharyngeal carcinoma. These tests included: in vitro overexpression of circ _3234 promotes the migration of nasopharyngeal carcinoma cells, and in vitro silencing of the expression of circ _3234 inhibits the migration of nasopharyngeal carcinoma; in vitro over-expression of circ _3234 promotes the invasion of nasopharyngeal carcinoma cells, and in vitro silencing of circ _3234 inhibits the invasion of nasopharyngeal carcinoma cells.

The siRNA has good silencing effect. After ensuring that the circ _3234 is interfered, the invention carries out Transwell chamber experiments and scratch healing experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 of which the circ _3234 is silenced, compared with an NC control group, the invasion and migration speed of the cells in the siRNA group is obviously reduced, namely, the silencing circ _3234 inhibits the invasion and migration of the nasopharyngeal cancer cells. Namely, the inhibition of circ _3234 can delay the diffusion of nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospects.

Drawings

FIG. 1 is a map of an overexpression vector.

FIG. 2 shows the effect of circ _3234 overexpression in nasopharyngeal carcinoma cell lines as measured by qRT-PCR;

qRT-PCR assay for the effect of circ _3234 overexpression in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 pcdna3.1(+) groups were normalized to 1, P <0.05, P <0.01, P <0.001 with β -actin as a reference.

FIG. 3 shows the silencing efficiency of circ _3234siRNA in nasopharyngeal carcinoma cell lines detected by qRT-PCR technique;

qRT-PCR to examine the silencing efficiency of circ _3234siRNA in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2; circ _3234 expression level analysis NC groups were normalized to 1 with β -actin as a reference, ns representing no significance, P <0.05, P <0.01, P < 0.001.

FIG. 4 shows the qrT-PCR detection of circ _3234 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 _3234 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. 5 is a graph showing the effect of overexpression of circ _3234 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-3234, and after the cell density reached 100%, they were scratched and photographed at 0,12 and 24 hours.

FIG. 6 is a statistical chart of cell scratch experiments for nasopharyngeal carcinomas HONE1, HNE2, and CNE2 overexpressing circ _ 3234;

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. 7 shows the interference efficiency of circ _3234 in scratch test cells detected 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 _3234 expression levels was normalized to 1, P <0.05, P <0.01, P <0.001 using beta-actin as a reference.

FIG. 8 is a graph of the effect of interference circ _3234 on nasopharyngeal carcinoma cells HONE1, HNE2, and CNE2 scratch healing experiments;

nasopharyngeal carcinoma HONE1, HNE2 and CNE2 cells were transfected with NC siRNA/circ _3234siRNA, scored after the cell density reached 100%, and photographed at 0,12 and 24 hours.

FIG. 9 is a statistical chart of nasopharyngeal carcinoma HONE1, HNE2 and CNE2 cell scratch experiments that interfere with circ _ 3234;

FIG. 10 shows the overexpression efficiency of circ _3234 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 expression level analysis of circ _3234 was performed with β -actin as a reference, and the pcdna3.1(+) group was normalized to 1, # P <0.05, # P <0.01, # P < 0.001.

FIG. 11 is a graph showing the effect of overexpression of circ _3234 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 _3234, respectively, and on the respective control cells.

FIG. 12 is a statistical chart of a Transwell cell matrigel invasion assay overexpressing circ _ 3234;

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. 13 shows the interference efficiency of circ _3234 in the qRT-PCR detection of Transwell cell 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 expression level analysis of circ _3234 was performed with β -actin as a reference, and NC groups were normalized to 1,. P <0.05,. P <0.01,. P < 0.001.

FIG. 14 is a graph of the effect of interference circ _3234 on the invasive potential of the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2;

transwell chamber matrigel invasion experiments were performed with HONE1, HNE2, and CNE2, which interfered with circ _3234, respectively, and with the respective control cells.

FIG. 15 is a statistical chart of a Transwell cell matrigel invasion assay interfering with circ _ 3234;

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 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% diabody (penicillin, streptomycin), 37 deg.C, 95% 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', SEQ ID NO. 2;

a downstream primer: 5'-GTCACCGGAGTCCATCACGAT-3', SEQ ID NO. 3.

(2) Circular RNA circ _3234 real-time quantitative PCR primer

An upstream primer: 5'-CAACAATCAGATGGCACCAG-3', SEQ ID NO. 4;

a downstream primer: 5'-AATTCTTCCAAGCCCCTTTG-3', SEQ ID NO. 5.

(3) Amplification circ _3234 full-length primer

An upstream primer: 5'-CCCATCGATAACTGGCATCAAATACTCACAAC-3', SEQ ID NO. 6;

a downstream primer: 5'-TAACCGCGGCAGGCTCTGAGGAGAACAG-3', SEQ ID NO. 7.

In the invention, siRNA is designed according to splicing sites and targeted silencing circ _3234 is carried out in order to specifically knock down circular RNA without influencing linear gene expression of the circular RNA.

circ _3234siRNA sequence:

sense strand (5'-3') CCUCAGAGCCUGAACUGGCUU, SEQ ID NO.8,

antisense strand (5'-3') GCCAGUUCAGGCUCUGAGGUU, SEQ ID NO. 9.

Negative control:

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU, SEQ ID NO.10,

antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU, 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. 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: detection of circ _3234 overexpression Effect in nasopharyngeal carcinoma cell lines

Firstly, we select the enzyme cutting sites, put the full-length sequence of the circ _3234 into an online website of NEB cutter 2.0 for analysis, and show that the enzyme cutting sites of Sacll and Clal are sites which do not exist in the full-length sequence of the circ _3234, and DNA restriction enzymes which exist singly in a pcDNA3.1 plasmid vector (purchased from Biotechnology Engineers, Inc.). An overexpression vector was constructed accordingly, see FIG. 1.

To examine the cyclization efficiency of circ _3234, we first expressed the constructed pcDNA3.1/circ _3234 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 _3234 overexpression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 by using a liposome method lipofectamine 3000, and continuously culturing for 48 hours. The cells were collected and the expression level and cyclization efficiency of circ _3234 were determined by real-time fluorescent quantitative PCR. The qPCR results showed that the expression level of circ _3234 was significantly increased in the pcDNA3.1/circ _3234 over-expressing plasmid group cells compared to the pcDNA3.1 empty plasmid group cells, and the expression fold was 300-fold or more in the HONE1, HNE2 and CNE2 cell lines, as shown in FIG. 2, the results were statistically significant.

Example 2: detection of Effect of silencing circ _3234 expression in nasopharyngeal carcinoma cell lines

According to the splicing site, an siRNA sequence of circ _3234 is designed, wherein the 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 so as to inhibit 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 _3234 in tumorigenesis development, we designed siRNA based on the splice site of circ _3234, and transiently transfected siRNA and siNC (blank control) into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circ _ 3234. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3234 was measured by real-time fluorescent quantitative PCR to determine the transfection efficiency of siRNA, confirming that the expression level of circ _3234 after siRNA transfection was lower than 60% of the expression level of circ _3234 after siNC transfection. The results are shown in FIG. 3.

Example 3: 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: performing cross or # -shaped scratch on the 10 microliter gun head perpendicular to the bottom of the 6-hole plate more quickly than a ruler without inclination, wherein the force is consistent so as to ensure that the scratch width is as same as possible;

(4) the culture solution is sucked and washed by D-hanks for 3 times, and 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 _3234 promotes migration of nasopharyngeal carcinoma cells

After determining that the cyclic RNAcir _3234 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 _3234 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 _3234 overexpression vectors by liposome method lipofectamine 3000, and cultured for 48 hours. The cells were collected and the expression level and cyclization efficiency of circ _3234 were determined by real-time fluorescent quantitative PCR. After confirming the good effect of overexpression of circ _3234 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 _3234 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 _3234 in nasopharyngeal carcinoma cell lines can promote the migratory ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. (results are shown in FIGS. 4,5 and 6)

In vitro silencing of circ _3234 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 _ 3234. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3234 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 _3234 was disturbed, we performed scratch experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3234, 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 _3234 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 _3234 can promote the migration of nasopharyngeal carcinoma cells. (results are shown in FIGS. 7,8 and 9)

Example 4: 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, adjusting the cell concentration to 20,000 cells per 200. mu.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 _3234 for promoting invasion of nasopharyngeal carcinoma cells

We performed Transwell cell matrigel invasion experiments in nasopharyngeal carcinoma cell lines HONE1, HNE2, and CNE2 to observe the effect of overexpression of circ _3234 on cell invasion capacity. We also transiently transfected endotoxin-free plasmids pcDNA3.1 and pcDNA3.1/circ _3234 over-expression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 using Lipofectamine 3000 by liposome method, and continued culturing for 48 hours. The cells were collected and the expression level and cyclization efficiency of circ _3234 were determined by real-time fluorescent quantitative PCR. After confirming that the overexpression of circ _3234 overexpression plasmid was good, we seeded the cells into matrigel-plated Transwell chamber and found that the number of cells that over-expressed the plasmid group invaded the lower surface of the chamber was significantly greater than the number of cells that did not reach the chamber, and the trend of the results was consistent for the three 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 _3234 in the nasopharyngeal carcinoma cell line can promote the invasion capacity of the nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. (results are shown in FIGS. 10,11 and 12)

In vitro silencing of circ _3234 expression affecting nasopharyngeal carcinoma invasion

To investigate whether silencing circ _3234 could reverse the phenotypic change associated with overexpression of circ _3234, we performed Transwell cell matrigel invasion experiments in two cell lines, transiently transfecting circ _3234siRNA and NC with Hiperfect reagent into hore 1, HNE2 and CNE2 cell lines to silence the expression of circ _ 3234. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _3234 was measured by real-time fluorescent quantitative PCR to examine the transfection efficiency of siRNA. The results show that the circ _3234siRNA has good silencing effect. After ensuring that circ _3234 was disrupted, we performed a Transwell cell matrigel invasion assay in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _3234, 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 _3234 in nasopharyngeal carcinoma cell lines can inhibit the invasion capacity of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. The ring-shaped RNAcir _3234 can promote the invasion of nasopharyngeal carcinoma cells as proved by two directions. (see FIGS. 13,14,15 for results).

Sequence listing

<110> university of south-middle school

<120> circ _3234, application thereof in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 377

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 1

agcauugauc ugguagccuu gcuccagaag ccuguuccuc acagucaagc cucagaagcc 60

aacuccuuug aaacuuccca acagcagggc uuuggccaag cccuugucuu cacaaauucg 120

caacacaaca aucagauggc accagggacu ggcagcucca cugccgucaa cuccuguucu 180

ccucagagcc ugaacuggca ucaaauacuc acaacauagc ucaggaucug ucaaacaaaa 240

guucuuaugg acucaaaggg gcuuggaaga auucugugga agaguggaca acagaagacu 300

ggacugaaga ucuuucugaa acaaaggucu ucacugccuc aucugcucca gcagagaauc 360

acaucuuacc ugggcaa 377

<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

caacaatcag atggcaccag 20

<210> 5

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 5

aattcttcca agcccctttg 20

<210> 6

<211> 32

<212> DNA

<213> Unknown (Unknown)

<400> 6

cccatcgata actggcatca aatactcaca ac 32

<210> 7

<211> 28

<212> DNA

<213> Unknown (Unknown)

<400> 7

taaccgcggc aggctctgag gagaacag 28

<210> 8

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 8

ccucagagcc ugaacuggcu u 21

<210> 9

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 9

gccaguucag gcucugaggu 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. The application of an agent for inhibiting the expression of circular RNA circ _3234 in preparing a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circular RNA circ _3234 is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the agent that inhibits the expression of circular RNA circ _3234 comprises siRNA.

3. The use of claim 2, wherein the siRNA is as follows:

sense strand (5'-3') CCUCAGAGCCUGAACUGGCUU

Antisense strand (5'-3') GCCAGUUCAGGCUCUGAGGUU.

4. The use according to claim 2, wherein the agent that inhibits the expression of circ _3234 further comprises a negative control:

sense strand (5'-3') UUCUCCGAACGUGUCACGUUU

Antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU.