CN109762823B - circ _1892, application thereof in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation - Google Patents
circ _1892, application thereof in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation Download PDFInfo
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Abstract
The invention belongs to the technical field of tumor molecular biology, and particularly relates to circ _1892, and application thereof in preparation of a nasopharyngeal carcinoma treatment preparation and a treatment preparation. The invention ensures that circ _1892 is over-expressed in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 by constructing an over-expression vector, then a Transwell cell experiment and a scratch healing experiment are carried out, and compared with a control group, the invasion and migration speed of over-expression group cells are obviously reduced; in contrast, when the expression of circ _1892 was down-regulated in the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 by RNA interference technique, the invasion and migration speed of the cells was significantly increased compared to the control group. Namely, circ _1892 can inhibit invasion and migration of nasopharyngeal carcinoma cells. The over-expression circ _1892 can delay the diffusion and metastasis of nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of tumor molecular biology, and particularly relates to a circular RNA circ _1892, a reagent for over-expressing the circular RNA circ _1892 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 doror) to attack a 5' end splice acceptor (splice) and an Alu region is 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 a circRNA (circRNA) 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, circrnas were found in human cells, and the number of circrnas known to date has reached over 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.
The nasopharyngeal carcinoma (NPC) belongs to the tumor of the head and neck, originating from the epithelial tissue of the nasopharynx, is generally developed at the posterior lateral crypt of the nasopharynx (Fossa of Rosenm ü ler), and the nasopharyngeal carcinoma cells are easy to invade the adjacent tissues and organs.
We detected a circ _1892 length of 378 bp. 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 _1892 with the size of 378bp and the relationship between the circ _1892 and nasopharyngeal darcinoma, and the circ _1892 and the nasopharyngeal darcinoma can be used as a nasopharyngeal darcinoma diagnosis marker and a treatment target.
The primary object of the present invention is to provide a novel circular RNA circ _1892, the sequence of which is shown in SEQ ID NO. 1.
The second purpose of the invention is to provide an application of a reagent for over-expressing circular RNA circ _1892 in preparing a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circular RNA circ _1892 is shown as SEQ ID NO. 1.
Further, the reagent for over-expressing the circular RNA circ _1892 comprises but is not limited to a plasmid vector for over-expressing the circular RNA circ _ 1892.
Further, the plasmid vector for overexpressing circular RNA circ _1892 is preferably constructed using pcDNA3.1 plasmid vector, but is not limited thereto.
Further, the preferable restriction enzyme sites for constructing the plasmid vector for overexpressing the circular RNA circ _1892 are Sacll and Clal, but are not limited to the two restriction enzyme sites.
Further, the reagent for over-expressing the circular RNA circ _1892 also comprises a negative control, namely an unloaded plasmid.
The third purpose of the invention is to provide an agent for treating nasopharyngeal carcinoma, which comprises an agent for over-expressing circular RNA circ _1892, wherein the sequence of the circular RNA circ _1892 is shown as SEQ ID NO. 1.
Further, the reagent for over-expressing the circular RNA circ _1892 comprises but is not limited to a plasmid vector for over-expressing the circular RNA circ _ 1892.
Further, the plasmid vector for overexpressing circular RNA circ _1892 is constructed using pcDNA3.1 plasmid vector, but is not limited to this plasmid vector.
Furthermore, the restriction enzyme sites of the plasmid vector for overexpression of circular RNA circ _1892 are Sacll and Clal, but are not limited to the two restriction enzyme sites.
Further, the reagent for over-expressing the circular RNA circ _1892 also comprises a negative control, namely an unloaded plasmid.
The present invention is not limited to only the overexpression vectors and negative controls provided above.
The therapeutic agent for nasopharyngeal carcinoma according to the present invention further comprises a reagent required for transfection of the overexpression vector.
In order to explore the role of circ _1892 in tumorigenesis and development, the invention constructs an overexpression vector according to the sequence of circ _1892, and transiently transfects the overexpression vector and the overexpression vector into HONE1, HNE2 and CNE2 cell lines in an empty load manner by using a liposome method lipofectamine3000 to overexpress circ _ 1892. After transfection, cells are collected after being cultured for 36 hours, the real-time fluorescence quantitative PCR technology is used for detecting the expression level of circ _1892 so as to detect the transfection efficiency of an over-expression vector, and meanwhile, the expression level of circ _1892 is detected, and the over-expression vector can be found to be capable of remarkably improving the expression level of circ _ 1892.
The present invention has confirmed the above conclusion through a number of experiments: namely, an agent overexpressing circ _1892 can be used for the preparation of a therapeutic agent for nasopharyngeal carcinoma. These tests included: in vitro overexpression of circ _1892 inhibits migration of nasopharyngeal carcinoma cells, and in vitro silencing of circ _1892 promotes migration of nasopharyngeal carcinoma cells; in vitro overexpression circ _1892 test shows that the inhibitor can inhibit nasopharyngeal carcinoma cell invasion, and in vitro silencing circ _1892 promotes nasopharyngeal carcinoma cell invasion.
The overexpression vector has a good overexpression effect. After ensuring that circ _1892 is overexpressed, Transwell chamber experiments and scratch healing experiments were carried out in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 overexpressing circ _1892, and compared with NC control group, the invasion migration speed of cells in overexpression group was significantly reduced, i.e. overexpression of circ _1892 inhibited the invasion migration of nasopharyngeal cancer cells. Namely, the over-expression of circ _1892 can delay the nasopharyngeal carcinoma diffusion and metastasis, 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 _1892 overexpression in nasopharyngeal carcinoma cell lines by qRT-PCR assay;
qRT-PCR assay for the effect of circ _1892 overexpression in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 pcdna3.1(+) group was normalized to 1, P <0.01, P <0.001 with β -actin as reference.
FIG. 3 shows the silencing efficiency of circ _1892siRNA in nasopharyngeal carcinoma cell lines detected by qRT-PCR;
the silencing efficiency of circ _1892siRNA in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 was measured by qRT-PCR, and the expression level analysis of circ _1892 normalized NC groups to 1, P <0.05, P <0.01, P <0.001 with β -actin as a reference.
FIG. 4 shows the qrT-PCR detection of circ _1892 overexpression efficiency in scratch test cells;
the overexpression efficiency of nasopharyngeal carcinoma cell strains HONE1, HNE2 and CNE2 used in the scratch test is detected by using a qRT-PCR test, the expression level analysis of circ _1892 takes β -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 _1892 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 _1892, scratched when the cell density reached 100%, 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 _ 1892;
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 the Student's t-test method,. P <0.05,. P <0.01,. P < 0.001.
FIG. 7 shows the interference efficiency of circ _1892 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 by qRT-PCR test, and the expression level analysis of circ _1892 was performed by using β -actin as a reference, and the NC groups were normalized to 1, P <0.05, P <0.01, and P < 0.001.
FIG. 8 is a graph of the effect of interference circ _1892 on nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 scratch healing experiments;
nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 were transfected with NC siRNA/circ _1892siRNA, 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 _ 1892;
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 the Student's t-test method,. P <0.05,. P <0.01,. P < 0.001.
FIG. 10 shows the overexpression efficiency of circ _1892 in a Transwell cell matrigel invasion assay by qRT-PCR detection;
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 _1892 expression level analysis was normalized to 1, P <0.05, P <0.01, P <0.001 using β -actin as a reference.
FIG. 11 is a graph showing the effect of overexpression of circ _1892 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 _1892, respectively, and on the respective control cells.
FIG. 12 is a statistical chart of Transwell cell matrigel invasion experiments overexpressing circ _ 1892;
each group was counted by randomly selecting 3 fields of cells, and each experiment was repeated three times and counted by Student's t-test method, and pcDNA3.1(+) group was normalized to 1, P <0.05, P <0.01, and P <0.001 using β -actin as a reference.
FIG. 13 shows interference efficiency of circ _1892 in a qRT-PCR assay for Transwell chamber matrigel invasion assay;
transwell cell matrigel invasion assay nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 interference efficiency was tested by qRT-PCR assay, and circ _1892 expression level analysis was normalized to 1, P <0.05, P <0.01, P <0.001 using β -actin as a reference.
FIG. 14 is a graph of the effect of interference circ _1892 on the ability of nasopharyngeal carcinoma cell lines HONE1 and HNE2 to invade;
transwell chamber matrigel invasion experiments were performed with HONE1, HNE2 and CNE2, which interfered with circ _1892, respectively, and with the respective control cells.
FIG. 15 is a statistical chart of a Transwell cell matrigel invasion assay that interferes with circ _ 1892;
the cells were counted in three random fields of 3 cells per group and counted in triplicate in Student's t-test, and the NC groups were normalized to 1, P <0.05, P <0.01, P <0.001, using β -actin as a reference.
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 swellings of the university of south ChinaStored in molecular genetics laboratory of the institute for tumor. 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 over-expression vector of the circular RNA is designed according to the sequence of the circRNA, the full-length sequence of the circ _1892 is put into an NEB cutter 2.0 online website for analysis, the restriction sites of Sacll and Clal are sites which do not exist in the full-length sequence of the circ _1892, and DNA restriction endonucleases exist in a pcDNA3.1 plasmid vector (purchased from Biotechnology engineering Co., Ltd.) simultaneously. An overexpression vector constructed accordingly.
(1)β-actin
An upstream primer: 5'-TCACCAACTGGGACGACATG-3', as shown in SEQ ID NO.2,
a downstream primer: 5'-GTCACCGGAGTCCATCACGAT-3', as shown in SEQ ID NO.3,
(2) circular RNA circ _1892 real-time quantitative PCR primer
An upstream primer: 5'-TTCTCAAATTGCAGCAGGAG-3', as shown in SEQ ID NO.4,
a downstream primer: 5'-CACCAATTTCTGGAGGGTACA-3', as shown in SEQ ID NO.5,
(3) amplification of circ _1892 full-length primer
An upstream primer: 5'-CCCATCGATAATAATTGTGTGCCCAAGAAGAT-3', as shown in SEQ ID NO.6,
a downstream primer: 5'-TCCCCGCGGCCATTCACCATTATCCAGAATTTTC-3', as shown in SEQ ID NO.7,
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 _1892 overexpression Effect in nasopharyngeal carcinoma cell lines
Firstly, we select the enzyme cutting sites, put the full-length sequence of circ _1892 into NEB cutter 2.0 online website 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 circ _1892, and DNA restriction enzymes which exist in pcDNA3.1 plasmid vector (purchased from Biotechnology and bioengineering GmbH) singly. An overexpression vector was constructed accordingly.
To examine the cyclization efficiency of circ _1892, we first expressed the constructed pcDNA3.1/circ _1892 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 _1892 overexpression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 by using a liposome method lipofectamine3000, and continuously culturing for 48 hours. Cells were collected and the expression level and cyclization efficiency of circ _1892 were determined by real-time fluorescent quantitative PCR. The qPCR result shows that the expression level of circ _1892 is significantly increased in the pcDNA3.1/circ _1892 overexpression plasmid group cells compared with the pcDNA3.1 empty plasmid group cells, and the expression fold is more than 50 times in HONE1, HNE2 and CNE2 cell lines, which is shown in FIG. 2, and the result has statistical significance.
Example 2: detection of Effect of silencing circ _1892 expression in nasopharyngeal carcinoma cell lines
According to the splicing site, a siRNA sequence of circ _1892 is designed, wherein the siRNA is a double-stranded RNA molecule with 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 _1892 in tumorigenesis development, we designed sirnas based on the splice site of circ _1892 and transiently transfected sirnas (see below) and siNC (see below) into HONE1, HNE2 and CNE2 cell lines using Hiperfect's reagent to silence the expression of circ _ 1892. After transfection, cells are collected after culturing for 48 hours, the expression level of circ _1892 is detected by using a real-time fluorescent quantitative PCR technology to detect the transfection efficiency of siRNA, and the expression level of circ _1892 after siRNA transfection is confirmed to be lower than 60% of the expression level of circ _1892 after siNC transfection. The results are shown in FIG. 3.
The siRNA used were as follows:
sense strand (5'-3') GAUAAUGGUGAAUGGAAUAUU, shown in SEQ ID NO.8,
the antisense strand (5'-3') UAUUCCAUUCACCAUUAUCUU, shown in SEQ ID NO.9,
negative control (siNC):
sense strand (5'-3') UUCUCCGAACGUGUCACGUUU, shown in SEQ ID NO.10,
antisense strand (5'-3') ACGUGACACGUUCGGAGAAUU, shown in SEQ ID NO. 11.
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 _1892 inhibits migration of nasopharyngeal carcinoma cells
After determining that the cyclic RNAcir _1892 has an inhibitory effect on the proliferation capacity of nasopharyngeal carcinoma cells, we performed a scratch experiment in the nasopharyngeal carcinoma cell lines to verify that circ _1892 has no effect 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 _1892 overexpression vectors by liposome method lipofectamine3000, and cultured for 48 hours. Cells were collected and the expression level and cyclization efficiency of circ _1892 were determined by real-time fluorescent quantitative PCR. After confirming the good effect of overexpression of circ _1892 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 cells of pcDNA3.1/circ _1892 overexpressing plasmid group was significantly reduced relative to the unloaded pcDNA3.1(+) plasmid group. The width difference of the scratch is large and has statistical significance. The above results show that overexpression of circ _1892 in nasopharyngeal carcinoma cell lines can inhibit the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to migrate in vitro. (results are shown in FIGS. 4,5 and 6)
In vitro silencing of circ _1892 promotes 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 _ 1892. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _1892 was measured by real-time fluorescent quantitative PCR to determine the transfection efficiency of siRNA. The results show that the siRNA has good silencing effect. After ensuring that circ _1892 was disturbed, we performed scratch experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _1892, 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 enhanced. The scratch width difference is obvious and has statistical significance. The above results show that silencing of the expression of circ _1892 in nasopharyngeal carcinoma cell lines can promote 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, circ _1892 can inhibit 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, wherein the cell concentration is adjusted to 20000 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 _1892 for inhibiting 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 _1892 on cell invasion capacity. We also transiently transfected endotoxin-free plasmids pcDNA3.1 and pcDNA3.1/circ _1892 over-expression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 using Lipofectamine3000 by liposome method, and continued culturing for 48 hours. Cells were collected and the expression level and cyclization efficiency of circ _1892 were determined by real-time fluorescent quantitative PCR. After determining the good effect of overexpression of circ _1892 overexpression plasmid, we seeded cells into matrigel-plated Transwell chambers and found that the number of cells invading the lower surface of the chamber was significantly less for the overexpressed plasmid group than for the unloaded group and that the trend of the three cell line results was consistent. 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 above results show that the over-expression of circ _1892 in nasopharyngeal carcinoma cell lines can inhibit the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to invade in vitro. (results are shown in FIGS. 10,11 and 12)
In vitro silencing of circ _1892 expression affecting nasopharyngeal carcinoma invasion
To investigate whether silencing circ _1892 could reverse the phenotypic change brought by overexpression of circ _1892, we performed Transwell cell matrigel invasion experiments in three cell lines, transiently transfecting circ _1892siRNA and NC with Hiperfect reagent into hore 1, HNE2 and CNE2 cell lines to silence the expression of circ _ 1892. After transfection, cells were collected by culturing for 48 hours, and the expression level of circ _1892 was measured by real-time fluorescent quantitative PCR to determine the transfection efficiency of siRNA. The results show that circ _1892siRNA has good silencing effect. After ensuring that circ _1892 is disturbed, we performed Transwell cell matrigel invasion experiments in nasopharyngeal cancer cell lines HONE1, HNE2 and CNE2 that silenced circ _1892, and the results showed that the siRNA group could observe significantly more tumor cells on the lower surface of the Transwell cell than the NC group, and the trend of the three cell line results was consistent. 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 silencing the expression of circ _1892 in nasopharyngeal carcinoma cell lines can promote the invasion capacity of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. The ring-shaped RNAcir _1892 can inhibit the invasion of nasopharyngeal carcinoma cells as proved by two directions, namely positive and negative directions. (see FIGS. 13,14,15 for results).
Sequence listing
<110> university of south-middle school
<120> circ _1892, application thereof in preparation of nasopharyngeal carcinoma treatment preparation and treatment preparation
<160>11
<170>SIPOSequenceListing 1.0
<210>1
<211>378
<212>RNA
<213> Intelligent (Homo sapiens)
<400>1
cuuuggucug ggucggccuc uaccuuugca cuuccuucgg agagcaucua agauuggaga 60
gguugauguc gagcaacaua cuuuggccaa auaccugaug gaacuaacua uguuggacua 120
ugacauggug cacuuuccuc cuucucaaau ugcagcagga gcuuuuugcu uagcacugaa 180
aauucuggau aauggugaau ggaauaauug ugugcccaag aagaugcugc agcugguugg 240
ugucacugcc auguuuauug caagcaaaua ugaagaaaug uacccuccag aaauugguga 300
cuuugcuuuu gugacugaca acacuuauac uaagcaccaa aucagacaga uggaaaugaa 360
gauucuaaga gcuuuaaa 378
<210>2
<211>20
<212>DNA
<213> Unknown (Unknown)
<400>2
<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
<210>5
<211>21
<212>DNA
<213> Unknown (Unknown)
<400>5
caccaatttc tggagggtac a 21
<210>6
<211>32
<212>DNA
<213> Unknown (Unknown)
<400>6
cccatcgata ataattgtgt gcccaagaag at 32
<210>7
<211>34
<212>DNA
<213> Unknown (Unknown)
<400>7
tccccgcggc cattcaccat tatccagaat tttc 34
<210>8
<211>21
<212>RNA
<213> Unknown (Unknown)
<400>8
gauaauggug aauggaauau u 21
<210>9
<211>21
<212>RNA
<213> Unknown (Unknown)
<400>9
uauuccauuc accauuaucu 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 (4)
1. The application of a reagent for over-expressing circular RNA circ _1892 in preparing a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circular RNA circ _1892 is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein said agent for overexpression of circular RNA circ _1892 comprises a plasmid vector for overexpression of circular RNA circ _ 1892.
3. The use according to claim 2, wherein the plasmid vector for overexpression of circular RNA circ _1892 is constructed using pcDNA3.1 plasmid vector.
4. The use according to claim 2, wherein the plasmid vector for overexpression of circular RNA circ _1892 is constructed with Sacll and Clal cleavage sites.
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