CN114921550A - Application of circSTX6 in preparation of nasopharyngeal carcinoma diagnosis and/or prognosis and treatment preparation and diagnosis and treatment preparation - Google Patents

Abstract

The invention belongs to the technical field of tumor molecular biology, and particularly relates to application of circSTX6 in preparation of a nasopharyngeal carcinoma diagnosis and/or prognosis and treatment preparation, and a diagnosis and treatment preparation. The differential expression of circSTX6 in nasopharyngeal carcinoma is found for the first time, and the circSTX6 can be used as a diagnosis or prognosis molecular marker and can also be a potential target for nasopharyngeal carcinoma treatment. According to the invention, an antisense oligonucleotide (ASO) is adopted to design a sequence aiming at a splicing site at the head-to-tail connection part of a circular RNA to interfere circSTX6, and a matrigel invasion experiment and a scratch healing experiment are carried out in nasopharyngeal carcinoma cell lines HNE2, CNE2 and HONE1, so that compared with an NC group, the invasion and migration capacity of cells in the ASO group is obviously weakened, namely the circSTX6 is silenced to inhibit the invasion and transfer of nasopharyngeal carcinoma cells, namely the circSTX6 is inhibited to treat the nasopharyngeal carcinoma, and the antisense oligonucleotide has profound clinical significance and important popularization and application prospects.

Description

Application of circSTX6 in preparation of nasopharyngeal carcinoma diagnosis and/or prognosis and treatment preparation and diagnosis and treatment preparation

Technical Field

The invention belongs to the technical field of tumor molecular biology, and particularly relates to application of circSTX6 in preparation of a nasopharyngeal carcinoma diagnosis and/or prognosis and treatment preparation, and a diagnosis and treatment preparation.

Background

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and the neck in south China, and has obvious regional aggregation and excellent human race. The pathogenesis factors of nasopharyngeal carcinoma mainly comprise genetic factors, EB virus infection, environmental factors and the like. Most of nasopharyngeal carcinomas are non-keratinized squamous cell carcinomas, which have high malignancy degree and can cause cervical lymph node metastasis in early stage. At present, the treatment methods of nasopharyngeal carcinoma mainly comprise radiotherapy, combined chemotherapy, surgical treatment and the like, although the 5-year overall survival rate of an early NPC patient is as high as 95%, the recurrence rate of nasopharyngeal and cervical lymph nodes is 8.6% -23.7%. This is because the generation and development of NPC involve complicated gene regulation and multi-stage processes, the molecular mechanism is not clear, and the therapeutic effect of conventional radiotherapy is not very ideal due to the heterogeneity of tumor and individual differences caused by radiotherapy resistance. Therefore, it is of great importance to study therapeutic targets and diagnostic molecular markers for treating nasopharyngeal carcinoma.

Circular RNA (Circular RNA) is mainly derived from the exonic region of a protein-coding gene, and may be formed of an intron region, a UTR region, an intergenic region, a non-coding RNA site, and an antisense site of a known transcript. The CircRNA is a non-coding RNA molecule formed by reverse splicing of a precursor (mRNA pre-mRNA) without a 5 'terminal cap and a 3' terminal poly (a) tail and forming a circular lasso structure by covalent bonds.

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 the exon-derived circRNAs (exonic circRNAs) can be divided into two forming modes of lasso-drive-circularization and intron-pairing drive-circularization, wherein the lasso-drive circularization is that the 3 'end of an exon is used as a splice donor (splice acceptor) to attack a 5' end splice acceptor (splice acceptor), 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 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 in a covalent bond form. 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.

The invention detects a circular RNA circSTX6 with the length of 391 bp. Experiments show that the circular RNA is highly expressed in nasopharyngeal carcinoma and can promote the invasion and metastasis of the nasopharyngeal carcinoma, and the circular RNA can be used as a nasopharyngeal carcinoma diagnosis or prognosis marker and a treatment target.

Disclosure of Invention

The invention discovers a ring-shaped RNAcircSTX6 with the size of 391bp, discovers the relation between the ring-shaped RNAcircSTX6 and nasopharyngeal carcinoma, and can be used as a nasopharyngeal carcinoma diagnosis or prognosis marker and a treatment target.

The first purpose of the invention is to provide the application of circSTX6 in preparing a nasopharyngeal carcinoma diagnosis and/or prognosis preparation, wherein the sequence of the circSTX6 is shown as SEQ ID NO. 1.

Further, the nasopharyngeal carcinoma diagnosis and/or prognosis preparation comprises a reagent for detecting the expression quantity of circSTX6 by PCR or in situ hybridization.

Furthermore, the reagent for detecting the expression level of the circSTX6 by PCR or in situ hybridization comprises:

circular RNA circSTX6 real-time quantitative PCR primer

An upstream primer: 5'-GGCTGGACAATGTGATGAAG-3', as shown in SEQ ID NO. 2;

a downstream primer: 5'-AGTTCTGGCTGCCACTGTCT-3', as shown in SEQ ID NO. 3;

or amplifying circSTX6 full-length primer

An upstream primer: 5'-CCATCGATGACATGAAAGATCAGATGTCAACTTCAT-3', as shown in SEQ ID NO. 4;

a downstream primer: 5'-TCCCCGCGGCACTGGTCATATGAGATACTTTTGCAAG-3', as shown in SEQ ID NO. 5.

circSTX6 in situ hybridization probe sequence: 5'-TGATCTTTCATGTCCACTGGTCATATGAG-3', as shown in SEQ ID NO. 6.

The diagnostic and/or prognostic reagents of the present invention include, but are not limited to, the primer and probe sequences described above.

The second purpose of the invention is to provide a nasopharyngeal carcinoma diagnosis and/or prognosis preparation, which comprises a reagent for detecting the expression quantity of circSTX6 by PCR or in situ hybridization, wherein the sequence of circSTX6 is shown in SEQ ID NO. 1.

Furthermore, the nasopharyngeal carcinoma diagnostic preparation comprises the primer or the in situ hybridization probe.

According to the invention, the expression level of circSTX6 in the nasopharyngeal carcinoma clinical tissue is detected through qRT-RCR, the fact that the circSTX6 is obviously up-regulated in the nasopharyngeal carcinoma tissue compared with the non-tumor nasopharyngeal epithelium tissue is found, the result is related to the prognosis survival of a patient, and the result has statistical significance, so that the circSTX6 can be used as a marker for auxiliary diagnosis or prognosis of the nasopharyngeal carcinoma, and a new detection way is provided for diagnosis and prognosis of the nasopharyngeal carcinoma.

The third purpose of the invention is to provide the application of the reagent for inhibiting the expression of circSTX6 in the preparation of the preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circSTX6 is shown in SEQ ID NO. 1.

Further, the agent for inhibiting the expression of circSTX6 comprises an antisense oligonucleotide.

Further, the antisense oligonucleotide aso (antisense oligonucleotide):

sense strand (5'-3') CAUAUGACCAGUGGACAUGATT, shown in SEQ ID NO. 7;

antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT, shown in SEQ ID NO. 8.

The fourth purpose of the invention is to provide a preparation for treating nasopharyngeal carcinoma, which comprises an agent for inhibiting the expression of circSTX6, wherein the sequence of circSTX6 is shown in SEQ ID NO. 1.

Further, said agent that inhibits the expression of circSTX6 comprises an antisense oligonucleotide, preferably said antisense oligonucleotide:

sense strand (5'-3') CAUAUGACCAGUGGACAUGATT

Antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT.

The present invention is not limited to the specific ASO described above.

Further, the reagent for inhibiting the expression of circSTX6 also comprises a negative control:

sense strand (5'-3') GAGAACGGGAUAGCAUCGACTT, shown in SEQ ID NO. 9;

antisense strand (5'-3') GUCGAUGCUAUCCCGUUCUCTT, shown in SEQ ID NO. 10;

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

Currently, ASO has developed into an important tool for gene function studies. To explore the role of circSTX6 in tumorigenesis development, the present invention designed a pair of ASOs based on the splice site of circSTX6, transiently transfected ASOs and siNC (control) into HNE2, CNE2 and HONE1 cell lines using Hiperfect reagent to silence the expression of circSTX 6. After transfection, cells were collected by culturing for 36 hours, and the expression level of circSTX6 was detected by real-time fluorescent quantitative PCR to detect the transfection efficiency of ASO, and it was found that the designed ASO significantly suppressed the expression level of circSTX 6.

The present invention has confirmed the above conclusion through a number of experiments: namely, the agent for inhibiting the expression of circSTX6 can be used for preparing a nasopharyngeal carcinoma treatment preparation. These tests included: in vitro overexpression circSTX6 test shows that the polypeptide can promote the invasion and migration of nasopharyngeal carcinoma cells, and in vitro silencing circSTX6 can inhibit the invasion and migration of nasopharyngeal carcinoma cells.

Because ASO has good silencing effect, the invention adopts splicing sites at the head-to-tail joint of circular RNA to design ASO to interfere with circSTX6 (only circRNA is silenced, no influence is caused to linear RNA), scratch healing experiments and matrigel invasion experiments are carried out in nasopharyngeal carcinoma cell lines HNE2, CNE2 and HONE1, compared with NC (control) group, the invasion and migration capacity of ASO group cells is obviously weakened, namely the silent circSTX6 inhibits the invasion and transfer of nasopharyngeal carcinoma cells. Namely, the inhibition of circSTX6 can treat nasopharyngeal carcinoma, and has profound clinical significance and important popularization and application prospect.

Drawings

FIG. 1 screening identification of circSTX6 and detection of the expression level of circSTX6 in nasopharyngeal carcinoma clinical tissues by qRT-RCR; A. the biogenic analysis shows that the expression of circSTX6 is remarkably up-regulated in nasopharyngeal carcinoma tissues by analyzing the nasopharyngeal carcinoma tissue sequencing data of GSE68799 in a GEO database; B.N is non-tumor nasopharyngeal epithelial tissue, and the number of samples is 16 cases; t is nasopharyngeal carcinoma tissue, the number of samples is 26, n is the number of samples, all the samples adopt T test, and p is less than 0.05, which has statistical significance.

FIG. 2. confirmation by database alignment and Sanger sequencing that circSTX6 is formed by reverse splicing of exons 4 to 7 of the STX6 gene and has a size of 391 nt; circstx6 is formed by splicing together the exons 4-7 of STX6 end-to-end, E denotes exon, and underlined sequences denote the end-to-end linker sequence; schematic of circstx6 formation; c. peak plot of sequencing results, black arrows indicate the head-to-tail connection therefrom;

FIG. 3. detection of circSTX6 expression in nasopharyngeal carcinoma cell lines. NP69 is an immortalized normal nasopharyngeal epithelial cell, the remainder being nasopharyngeal cancer cell lines as a reference.

FIG. 4. detection of tolerance of circSTX6 to RNase R. After RNase R treatment, relative RNA levels of circSTX6 and STX6 in nasopharyngeal carcinoma cells were determined using qRT-PCR.

FIG. 5 stability of circSTX6 was tested. After actinomycin D treatment, the relative RNA levels of circSTX6 and STX6 in nasopharyngeal carcinoma cells at different time points were measured using qRT-PCR.

FIG. 6 RNA-fish assay detects subcellular localization of circSTX 6. circSTX6 was localized mostly in the cytoplasm.

FIG. 7 recombinant plasmid map of circSTX6 overexpression vector.

FIG. 8.qRT-PCR technique to detect the overexpression efficiency of circSTX6 overexpression plasmid and the silencing efficiency of circSTX6 ASO in nasopharyngeal carcinoma cell lines. qRT-PCR detection of the overexpression efficiency of circSTX6 plasmid in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, analysis of circSTX6 expression level with beta-actin as reference; qRT-PCR assay silencing efficiency of circSTX6 ASO in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, analysis of circSTX6 expression level with β -actin as reference. C. Expression of linear RNA STX6 was tested after transfection of ASOs in the nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, ns representing meaningless p <0.05 p <0.01 p < 0.001.

FIG. 9. Effect of in vitro overexpression/silencing circSTX6 on nasopharyngeal carcinoma cell proliferation. A. Transient transfection of pcDNA3.1(+) CircRNA Mini Vector, circSTX6 overexpression plasmids with Neofect into nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, and MTT assay to examine cell proliferation capacity after further 24 hours of culture, normalized pcDNA3.1(+) CircRNA Mini Vector group to 1, ns represents no significance,. p <0.05,. p <0.01,. p < 0.001. B. siNC, circSTX6 ASO were transiently transfected with Hiperfect in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, and after 24 hours of culture, MTT experiments were performed to examine cell proliferation capacity, and siNC groups were normalized to 1, ns representing no significance, p <0.05, p <0.01, p < 0.001.

FIG. 10. Effect of over-expression/silencing circSTX6 on nasopharyngeal carcinoma cell invasion. A. Simulating cell crossing of matrix barrier by matrix gum invasion assay, after 24 hours of transfection of pcDNA3.1(+) CircRNA Mini Vector, CircSTX6 overexpression plasmid in HONE1, HNE2 and CNE2 cells, the effect of overexpression of CircSTX6 on nasopharyngeal carcinoma cell invasion was examined by matrix gum invasion assay, where the lower panel is a statistical plot of cell counts (B) and pcDNA3.1(+) CircRNA Mini Vector is normalized to 1, p <0.05, p <0.01, p < 0.001. C. Cells were simulated to cross the matrix barrier using matrigel invasion assay and the effect of silencing circSTX6 on nasopharyngeal carcinoma cell invasion was examined using matrigel invasion assay 24 hours after transfection of siNC, circSTX6 ASO in HONE1, HNE2 and CNE2 cells, where the right panel is a statistical plot of cell number (D) and siNC is normalized to 1, <0.05, <0.01, < 0.001.

FIG. 11. Effect of over-expression/silencing circSTX6 on the scratch healing ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE 2. A. After transfection of pcdna3.1(+) CircRNA Mini Vector, circSTX6 in HONE1, HNE2 and CNE2 cells, scratches were photographed at different time points according to the cell healing rate after the cell density reached 100%, and statistical plots (B) of scratch width were placed below to label pcdna3.1(+) CircRNA Mini Vector as 1, <0.05, <0.01, < 0.001. A. After transfection of siNC, circSTX6 ASO in HONE1, HNE2 and CNE2 cells, after the cell density reached 100%, scratches were scored and photographed at different time points according to the cell healing rate, and statistical plots (C) of scratch width were placed below, and siNC was labeled as 1, p <0.05, p <0.01, p < 0.001.

FIG. 12 circSTX6 is highly expressed in nasopharyngeal carcinoma tissue and is associated with a poor prognosis for the patient. Circstx6 expression was verified by in situ hybridization experiments in paraffin-embedded tissue sections from 95 NPC patients and 25 normal tissues (nasopharyngeal epithelium, NPE). High circstx6 expression is associated with a shortened overall survival of patients with nasopharyngeal carcinoma.

Detailed Description

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

The nasopharyngeal cancer cell lines such as HNE2, CNE2 and HONE1 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 of 10% Fetal Bovine Serum (FBS) and 1% diabody (penicillin, streptomycin), 37 deg.C, 95% humidity, 5% CO ₂ The constant temperature incubator with the concentration grows by adhering to the wall.

The design of the circular RNA primer is different from that of the linear RNA primer, and the circular RNA primer and the linear RNA primer are designed according to two sides of a splicing site, are designed on a Primer3.0 website on line, and finally are synthesized by the Changsha synthesis department of Ongkeke biology company.

(1)β-actin

An upstream primer: 5'-TCACCAACTGGGACGACATG-3', respectively; as shown in SEQ ID NO. 11;

a downstream primer: 5'-GTCACCGGAGTCCATCACGAT-3', respectively; as shown in SEQ ID NO. 12;

(2) circular RNA circSTX6 real-time quantitative PCR primer

An upstream primer: 5'-GGCTGGACAATGTGATGAAG-3'

A downstream primer: 5'-AGTTCTGGCTGCCACTGTCT-3'

(3) Full-length primer for amplifying circSTX6

An upstream primer: 5'-CCATCGATGACATGAAAGATCAGATGTCAACTTCAT-3'

A downstream primer: 5'-TCCCCGCGGCACTGGTCATATGAGATACTTTTGCAAG-3'

In the invention, in order to specifically knock down the circular RNA without influencing the linear gene expression thereof, ASO is designed according to splicing sites, and circSTX6 is targeted and silenced.

circSTX6 ASO sequence:

sense strand (5'-3') CAUAUGACCAGUGGACAUGATT

Antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT.

Negative control:

sense strand (5'-3') GAGAACGGGAUAGCAUCGACTT

Antisense strand (5'-3') GUCGAUGCUAUCCCGUUCUCTT.

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 non-expression in clinical parameters such as sex, age, tumor stage, clinical stage and metastatic status. 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 7.0 software.

Example 1: screening and identification of circSTX6

In the invention, a set of RNA-seq data which contains 4 normal nasopharyngeal tissue samples and 41 nasopharyngeal cancer tissue samples and is numbered as GSE68799 of 45 clinical samples in total is downloaded from a GEO database. By analyzing the set of nasopharyngeal carcinoma tissue RNA-seq data, 8884 circRNAs were identified, and then the data were analyzed differentially using SAM software, 178 circRNA molecules with significant differences between normal nasopharyngeal carcinoma tissue and nasopharyngeal carcinoma tissue were obtained, among which hsa _ circ _23135(circSTX6) was found among the up-regulated circular RNA molecules in nasopharyngeal carcinoma tissue (FIG. 1).

Example 2: sanger sequencing demonstrated that circular RNA was formed

To demonstrate that circSTX6 forms circular RNA rather than linear, the qRT-PCR product in fig. 1B was recovered and sent to sanger sequencing (engine). The sequences returned by the company are compared by using DNASTAR software, and peak images are looked up by using chromas software to judge the sequencing quality. The results show that circSTX6 is indeed formed by head-to-tail cyclization of exons 4-7 of the parent gene STX 6. Circstx6 is formed by splicing exons 4-7 of STX6 end to end, E represents exon, and underlined sequences represent end-to-end linker sequences; schematic representation of circrna formation; c. the peak pattern of the sequencing results, the black arrows indicate the head-to-tail progression from this point (see FIG. 2).

Example 3: expression of circSTX6 in nasopharyngeal carcinoma cells

1. 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 mu L Trizol lysate into each hole of a 12-hole plate, performing room temperature lysis for 1-2 minutes, gently blowing down cells by using a pipette gun, slightly reversing the upper part and the lower part for 10 times, and standing for 5 minutes at room temperature;

3) adding 100. mu.L of chloroform (1 mL of chloroform: 0.2mL of isopropanol: 0.5 mL), shaking vigorously for 15-30s, and standing on ice for 5 minutes;

4)4℃，12000rpm/20min；

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

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

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

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 to measure the RNA concentration and OD value.

2. Reverse transcription PCR reaction of circRNA

(operation was carried out according to the instruction manual of 5 × All-In-OneRTMasterMix (WithAccuRTGenomiccDNAmovalKit) (# G492) of abm Co.)

The following reaction system is prepared:

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.

3. 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 a Bio-RadIQ5 real-time fluorescence quantitative PCR instrument, the gene is marked with the reference gene beta-actin, the relative expression quantity of the target gene is displayed by a 2-delta-CT value, and the expression difference of the gene is judged. P-values were calculated using unpaired t-test.

As a result: the expression of circSTX6 in nasopharyngeal carcinoma cells was significantly higher than that of normal nasopharyngeal epithelial cell NP69 (fig. 3). Therefore, circSTX6 is highly expressed in nasopharyngeal carcinoma cell line, and circSTX6 may have important biological function for the development of nasopharyngeal carcinoma, so that antisense oligonucleotide can be used for treating nasopharyngeal carcinoma.

Example 4: RNase R digestion assay

1 reaction System

RNase R digestion reaction system

2 reaction conditions

37℃，10-30min。

Note: 1) the digestion time can be properly prolonged along with the increase of RNA, most of linear RNA can be digested in 10-30min generally, and the abundance of the linear RNA detected by PCR is reduced by hundreds of times. Digestion for more than 1h is not necessary as too long may result in digestion of a small number of poorly tolerated circrnas. 2) After incubation, the cells can be purified and recovered, or downstream experiments can be directly carried out after enzyme inactivation is carried out at 70 ℃ for 10 min.

3 purification and recovery

Digested RNA can be digested using phenol: chloroform: extracting isoamyl alcohol (25:24:1, V: V) solution, and then precipitating and recovering by using ethanol; or purifying and recovering by using an RNA purification column and magnetic beads. The qRT-PCR detects circSTX6 and linear mRNA STX6 (see figure 4).

Note: phenol: chloroform: the solution of isoamyl alcohol (25:24:1, V: V) is preferably ready for use and may be replaced by Trizol Reagent without reagents.

Example 5: actinomycin D treatment experiment

In order to test the stability of circRNA and linear RNA, nasopharyngeal carcinoma cells are inoculated into a 12-well plate at the density of about 50%, actinomycin D with the final concentration of 1 mu g/mL is added after the cells are attached to the plate, the cells are treated for 0, 8, 16 and 24 hours respectively, RNA of the cells is extracted, reverse transcription is carried out to form cDNA, and the expression of circSTX6 and STX6 mRNA is detected by qRT-PCR with 18s as internal reference (figure 5).

Example 6: RNA-fish experiment for detecting intracellular localization of circSTX6

Since ASO functions mainly in the cytoplasm, examining the localization of circSTX6 can determine whether the expression of circSTX6 can be well interfered with. The RNA-FISH is used for detecting the proportion of circSTX6 expressed in nucleus and cytoplasm, and the circSTX6 is mainly positioned in cytoplasm. (see fig. 6).

RNA-fish step:

1) climbing sheets: inoculating adherent cells into a 24-well plate (with the holes being internally provided with slide glass in advance) according to 1000 cells/hole, and placing the plate in an incubator for overnight culture;

2) the medium was aspirated and washed twice with pre-heated PBS at 37 ℃ for 5min each time;

3) fixing: adding 200 μ L of 4% paraformaldehyde into each well, and fixing at room temperature for 15 min;

4) membrane rupture: removing the fixative, adding 200 μ L of 0.25% TritonX-100 (dissolved in PBS) per well, and treating at room temperature for 15 min;

5) washing with PBS twice for 5 min;

6) pre-hybridization: preparing a wet box, adding 20mL of 20% glycerol at the bottom of the hybridization box to keep the temperature, adding 20 mu L of prehybridization liquid into each slice, incubating at 37 ℃ in a constant temperature box for 2-4h, sucking redundant liquid, and not washing;

7) digacine-labeled circSTX6 RNA probe was diluted with hybridization solution, and 20. mu.L of hybridization solution (concentration typically 4. mu.M/8. mu.M, volume 15-20. mu.L) was added to each section and incubated overnight (more than 16h) in an incubator at 37 ℃;

8) washing a hybrid sheet: washing twice with 2XSSC at water temperature of about 30-37 ℃ for 5min each time; washing once with 0.5XSSC for 5 min; washing with 0.2XSSC for 5min, and re-washing with 0.2XSSC for 5min if necessary;

9) dropping sealing liquid at 37 deg.C for 30min, sucking excessive liquid, and washing;

10) dropwise adding biotinylated mouse anti-digoxin antibody, washing with 0.5Mx PBS for four times at 37 deg.C for 60min or room temperature for 2h, each time for 5 min;

11) adding fluorescent secondary antibody (1:200) dropwise, washing with 0.5M PBS for 5min three times at 37 deg.C for 60 min;

12) adding DAPI working solution, and dyeing for 10min in dark;

13) washing with 0.5M PBS for 5min three times;

14) and (6) sealing and shooting.

Example 7: detection of circSTX6 overexpression effect in nasopharyngeal carcinoma cell line

Firstly, selecting enzyme cutting sites, putting a circSTX6 full-length sequence into an NEB cutter 2.0 online website for analysis, and displaying that the ClaI and SacII enzyme cutting sites are sites which do not exist in the circSTX6 full-length sequence and DNA restriction enzymes which exist in pcDNA3.1(+) circum Mini Vector plasmid Vector (purchased from Biotechnology and bioengineering companies) singly. Cloning of the circSTX6 full length sequence into pcdna3.1(+) CircRNA Mini Vector plasmid was empty and fig. 7 is a mapping of the overexpression Vector.

To test the cyclization efficiency of circSTX6, we first overexpressed the constructed pcDNA3.1(+) circRNA Mini Vector/circSTX6 eukaryotic overexpression Vector in nasopharyngeal carcinoma cells. And (2) 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 an endotoxin-free plasmid pcDNA3.1(+) CircRNA Mini Vector empty Vector and a circSTX6 overexpression Vector with Neofect to the nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2, continuously culturing for 36h, and detecting the expression level and cyclization efficiency of the circSTX6 by using a real-time fluorescent quantitative PCR technology. The qPCR results showed a significant increase in the expression level of circSTX6 in cells transfected with the circSTX6 overexpressing plasmid group compared to pcdna3.1(+) CircRNA Mini Vector empty plasmid group cells, with statistical significance as a result (see figure 8A).

Example 8: effect test of silencing circSTX6 expression in nasopharyngeal carcinoma cell lines

Antisense oligonucleotides (ASOs) are a class of molecules that inhibit the expression of a target gene by sequence-specifically binding to the gene DNA or mRNA. ASO is a hybrid of single-stranded DNA and RNA, and acts via RNase-H, an enzyme that is also found in the nucleus, and thus can interfere with both nuclear and cytoplasmic genes. ASOs design sequences complementary to the target sequence for the splice site, thereby avoiding interference with the expression of linear RNA. Currently, ASO has developed into an important tool for gene function studies. To explore the role of circSTX6 in tumorigenesis development, we designed ASOs based on the splice site of circSTX6, transiently transfected ASOs and siNC (blank control) into HONE1, HNE2, and CNE2 cell lines using Hiperfect's reagent to silence the expression of circSTX 6. After the transfection, the cells were collected by culturing for another 36 hours, and the expression level of circSTX6 was measured by real-time fluorescent quantitative PCR to examine the transfection efficiency of ASO, confirming the circSTX6 knock-out effect (see fig. 8B). However, when the linear primers were designed based on the sequence of circSTX6, the expression of linear RNA was not knocked down by ASO as detected by real-time fluorescence quantitative PCR, indicating that ASO is specifically silencing the expression of circSTX6 (see fig. 8C).

Antisense oligonucleotide in this example:

sense strand (5'-3') CAUAUGACCAGUGGACAUGATT

Antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT.

Blank control:

sense strand (5'-3') GAGAACGGGAUAGCAUCGACTT

Antisense strand (5'-3') GUCGAUGCUAUCCCGUUCUCTT.

Example 9: MTT assay for detecting cell proliferation

We first transfected sinC, ASO circSTX6 with Hiperfect or transiently transfected nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 with endotoxin-free plasmids pcDNA3.1(+) circRNA Mini Vector and circSTX6 overexpression Vector with Neofect, cultured for 24h, and then performed MTT experiment to verify the effect on cell proliferation. As a result, it was found that ASO circSTX6 significantly inhibited the proliferation of the three cell lines, whereas overexpression of circSTX6 significantly promoted the proliferation of the three cell lines (FIG. 9)

1) Preparing: tip head, D-hanks sterilized at high temperature and high pressure; sterilizing a liquid transfer gun, a marker pen, a 15mL centrifuge tube and the like with alcohol, placing in a biological safety cabinet for ultraviolet irradiation for 30min, and ventilating for 10 min.

2) Plating and transfection: 25cm in the previous day ² And digesting the cells in a good state in the cell bottle, inoculating the cells into a 6-well plate, and transfecting ASO circSTX6 when the cell density is about 70% or transfecting an overexpression vector when the cell density is about 80-90%.

3) And after 12 hours of transfection, removing cell supernatant, washing for 3 times by D-hanks, adding 100 mu L of pancreatin into each hole of a 6-hole plate, blowing and transferring the cells to a 15mL centrifuge tube after the digested cells are round, centrifuging for 5min at 1000rpm, removing the supernatant, adding 1-2 mL culture medium, mixing uniformly, taking 10 mu L, adding into a cell counting plate, and counting the cells. Based on the cell count results, cells were diluted to 5000 cells/mL.

4) And adding the diluted cell suspension into a 96-well plate, adding 200 mu L of the cell suspension into each well, adding 200 mu L of Dank's buffer solution into the outermost circle of wells of the 96-well plate, and putting the wells into an incubator for continuous culture.

5) After the cells adhere to the wall for about 6-8 h, adding 20 mu L MTT into each hole, continuing culturing for 4h, carefully absorbing the culture supernatant in each hole, adding 200 mu L DMSO into each hole, placing on a shaking table, shaking for 10min, detecting the light absorption value of the DMSO sample adding hole on an enzyme linked immunosorbent detector by selecting the 490nm wavelength, and recording the result. Thereafter, absorbance measurements were performed daily at the same time points with MTT and DMSO, and statistical analysis was performed after 6 consecutive measurements.

Example 10: cell transwell invasion assay:

1) preparing matrigel: the BD Matrigel gel 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 diluting the gel are placed at-20 ℃ overnight, and therefore the Matrigel gel cannot be solidified too fast when the gel is laid in the next day of operation;

2) matrix glue dilution: BD Matrigel gel gum: adding 20 mu L of matrigel into 160 mu L of 1640 culture medium, blowing and mixing evenly;

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 cells after 24h of transfection, washing the cells for 2 times by using a serum-free medium, suspending the cells by using the serum-free medium, counting the cells, and adjusting the cell concentration to 2 ten thousand cells per 200 mu L;

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

6) adding 200 mu L of counted cell suspension into the upper chamber of the transwell, putting the 24-well plate back into the incubator at 37 ℃, and incubating for about 24-48 h according to the cell state and the cell invasion speed.

7) Taking out the 24-hole plate, washing twice with PBS or D-hanks, soaking and washing for 10min with 4% paraformaldehyde, and washing 3 times with 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 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 then photographed under an inverted microscope, optionally with 5 different fields of view, counted and statistically analyzed for significance of the differences using image J software.

In vitro silencing circSTX6 expression for inhibiting nasopharyngeal carcinoma invasion

To investigate whether silencing circSTX6 could affect nasopharyngeal carcinoma invasion, we performed Transwell cell matrigel invasion experiments in three cell lines, transiently transfected circSTX6 ASO and siNC into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circSTX 6. Transwell cell matrigel invasion experiments were performed in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, which are silent to circSTX6, and the results showed that the number of tumor cells observable under the Transwell cell surface in the ASO group was significantly less than that in the NC group, and the results of the three cell lines tended to be consistent. Random 5 photographs were taken and the number of cells was recorded, with significant differences between the two individual data in each cell line and statistical significance. The above results indicate that silencing the expression of circSTX6 in nasopharyngeal carcinoma cell lines can inhibit the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to invade in vitro (fig. 10).

In vitro overexpression of circSTX6 to promote invasion of nasopharyngeal carcinoma cells

We performed Transwell matrigel invasion experiments in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2 and observed the effect of silencing circSTX6 on cell invasion capacity. We also transiently transfected endotoxin-free plasmids pcDNA3.1(+) CircRNA Mini Vector and circSTX6 over-expression vectors into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 using Neofect, and continued culturing for 48 hours. The cells were collected and the expression level and cyclization efficiency of circSTX6 were determined by real-time fluorescent quantitative PCR. After confirming the good effect of overexpression of circSTX6 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 greater for the overexpressed plasmid group than for the unloaded group and that the trend of the results was consistent for the three cell lines. Random 3 photographs were taken and the number of cells was recorded, with significant differences between the two individual data in each cell line and statistical significance (see figure 10 for results). The above results show that overexpression of circSTX6 in nasopharyngeal carcinoma cell lines promotes the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to invade in vitro. The circular RNA circSTX6 was shown to promote the invasion of nasopharyngeal carcinoma cells by both forward and reverse directions (see FIG. 10 for results).

Example 11: cell scratch healing migration experiment

1) A cell illumination table: 1000 muL/10 muL Tip, D-Hank's sterilized at high temperature and high pressure, a ruler, a 1000 muL/10 muL pipette, 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 ASO 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-microliter gun head is perpendicular to the bottom of the 6-pore plate than a ruler to perform cross or # -shaped scratch quickly without inclination, and the force is consistent so as to ensure that the scratch width is consistent as much as possible;

4) the culture solution is discarded, and the culture solution is lightly washed for 3 times by D-hanks, so that the broken cells caused by scratches are washed away as far 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, and shooting the same position at intervals of 12h, and recording as 12 h;

8) and (5) taking the same position again at an interval of 24h until the scratch is healed, sorting all pictures and performing statistical analysis.

In vitro silencing of circSTX6 inhibits migration of nasopharyngeal carcinoma cells

ASO and siNC were transiently transfected into HONE1, HNE2 and CNE2 cell lines using Hiperfect reagent to silence the expression of circSTX 6. Scoring experiments were performed in nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE2, which are silent to circSTX6, to verify their effect on cell migration. Scratch healing experiments were confirmed at multiple time points in these cells: the migration ability of ASO group cells was significantly reduced compared to NC group. The scratch width difference is obvious and has statistical significance. The above results show that silencing the expression of circSTX6 in nasopharyngeal carcinoma cell lines can inhibit the ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 to migrate in vitro (fig. 11).

In vitro overexpression of circSTX6 to promote migration of nasopharyngeal carcinoma cells

The endotoxin-free plasmids pcDNA3.1(+) CircRNA Mini Vector and circSTX6 overexpression vectors were transiently transfected into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 using Neofect. After confirming the good effect of overexpression of circSTX6 overexpression plasmid, we performed cell scratch healing experiments on nasopharyngeal carcinoma cell lines HONE1, HNE2 and CNE 2. Scratch healing experiments were confirmed at multiple time points in these cells: the migration capacity of circSTX6 overexpressing plasmid cells was significantly enhanced relative to the unloaded pcdna3.1(+) CircRNA Mini Vector plasmid group. The width difference of the scratch is large and has statistical significance. The above results show that the expression of circSTX6 in the over-expressed nasopharyngeal carcinoma cell lines can promote the migration ability of nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 in vitro. As proved by forward and backward verification, circSTX6 can promote the migration of nasopharyngeal carcinoma cells (FIG. 11).

Example 12: in situ hybridization experiments

95 nasopharyngeal carcinoma clinical tissue samples are collected in the invention, NPE (paracarcinoma tissue) is 25, and NPC (nasopharyngeal carcinoma tissue) is 95. In situ hybridization kit (BOSTER, wuhan, china) was used to detect the expression of circSTX6 in paraffin-embedded nasopharyngeal carcinoma tissues and paracarcinoma tissues. The probe sequence (5'-TGATCTTTCATGTCCACTGGTCATATGAG-3') was designed to span the circSTX6 splice site (-30 nt). Staining density and depth were assessed using semi-quantitative scoring criteria. Double blind scoring is scored by two experienced pathologists. (1) Dyeing strength: 0, no dyeing; 1, light brown; 2, brown no background or dark brown with a light brown background (moderate positive); dark brown with no unspecified background (strong positive). (2) Fraction positive signals on a proportion basis account for total cell number: 0, no positive cells; 1, 0-25%; 2, the positive rate is 25-50%; 3, 50-70% positive rate; 4, the positive rate is 70-100%. The invention detects the expression of circSTX6 in nasopharyngeal carcinoma tissues and paracancer non-tumor tissues by using an in situ hybridization method, and finds that the circSTX6 is highly expressed in the nasopharyngeal carcinoma tissues (figure 12A). When the high-low expression of circSTX6 in NPE (para-carcinoma tissue) and NPC (nasopharyngeal carcinoma tissue) was analyzed simultaneously, the high expression was defined as a positive rate of circSTX6 of more than 25%, and the distribution of the high-low expression of circSTX6 in NPE and NPC was obtained (fig. 12B). In addition, a global survival (OS) curve was generated using Kaplan-Meier survival analysis and patients with higher circSTX6 levels in nasopharyngeal tissues were found to have significantly reduced global survival (FIG. 12C). When analyzing and counting the overall survival rate of nasopharyngeal carcinoma patients, the invention defines that the positive rate of the circSTX6 in the nasopharyngeal carcinoma is less than 50 percent as low expression of the circSTX6, and more than 50 percent as high expression of the circSTX 6. In the circSTX6 low expression group, 41 deaths and 22 survivors resulted in 35% overall survival in nasopharyngeal carcinoma patients. In the circSTX6 high expression group, 28 deaths, 4 survived, and the overall survival rate of nasopharyngeal carcinoma patients was 12.5% (fig. 12C).

Sequence listing

<110> university of Zhongnan

Application of <120> circSTX6 in preparation of nasopharyngeal carcinoma diagnosis and/or prognosis and treatment preparation, and diagnosis and treatment preparation

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 391

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 1

gacaugaaag aucagauguc aacuucaucu gugcaggcau uagcugaaag aaaaaauaga 60

caggcacugc ugggagacag uggcagccag aacuggagca cuggaacaac agauaaauau 120

gggcgucugg accgagagcu ccagagagcc aauucucauu ucauugagga gcagcaggca 180

cagcagcagu ugaucgugga acagcaggau gagcaguugg agcuggucuc uggcagcauc 240

ggggugcuga agaacauguc ccagcgcauc ggaggggagc uggaggaaca ggcaguuaug 300

uuggaagauu ucucucacga auuggagagc acucaguccc ggcuggacaa ugugaugaag 360

aaacuugcaa aaguaucuca uaugaccagu g 391

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggctggacaa tgtgatgaag 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agttctggct gccactgtct 20

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccatcgatga catgaaagat cagatgtcaa cttcat 36

<210> 5

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tccccgcggc actggtcata tgagatactt ttgcaag 37

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgatctttca tgtccactgg tcatatgag 29

<210> 7

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cauaugacca guggacauga tt 22

<210> 8

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ucauguccac uggucauaug tt 22

<210> 9

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gagaacggga uagcaucgac tt 22

<210> 10

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gucgaugcua ucccguucuc tt 22

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcaccaactg ggacgacatg 20

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtcaccggag tccatcacga t 21

Claims (10)

1. The application of circSTX6 in the preparation of nasopharyngeal carcinoma diagnosis and/or prognosis preparations, wherein the sequence of the circSTX6 is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein said nasopharyngeal carcinoma diagnosis and/or prognosis preparation comprises a reagent for detecting the expression level of circSTX6 by PCR or in situ hybridization.
3. 3. The use according to claim 1, wherein the reagent for detecting the expression level of circSTX6 by PCR or in situ hybridization comprises:

   circular RNA circSTX6 real-time quantitative PCR primer

   An upstream primer: 5'-GGCTGGACAATGTGATGAAG-3'

   A downstream primer: 5'-AGTTCTGGCTGCCACTGTCT-3', respectively;

   or amplifying circSTX6 full-length primer

   An upstream primer: 5'-CCATCGATGACATGAAAGATCAGATGTCAACTTCAT-3'

   A downstream primer: 5'-TCCCCGCGGCACTGGTCATATGAGATACTTTTGCAAG-3';

   circSTX6 in situ hybridization probe sequence: 5'-TGATCTTTCATGTCCACTGGTCATATGAG-3' are provided.
4. 4. A nasopharyngeal carcinoma diagnosis and/or prognosis preparation, characterized by comprising a reagent for detecting the expression level of circSTX6 by PCR or in situ hybridization, wherein the sequence of circSTX6 is shown in SEQ ID NO. 1.
5. 5. The diagnostic and/or prognostic preparation for nasopharyngeal carcinoma according to claim 4, characterized in that it comprises the primer or in situ hybridization probe according to claim 3.
6. 6. The application of a reagent for inhibiting the expression of circSTX6 in preparing a preparation for treating nasopharyngeal carcinoma, wherein the sequence of the circSTX6 is shown as SEQ ID NO. 1.
7. 7. The use according to claim 6, wherein the agent that inhibits the expression of circSTX6 comprises an antisense oligonucleotide.
8. 8. The use of claim 7, wherein said antisense oligonucleotide:

   sense strand (5'-3') CAUAUGACCAGUGGACAUGATT

   Antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT.
9. 9. A preparation for treating nasopharyngeal carcinoma, which is characterized by comprising an agent for inhibiting the expression of circSTX6, wherein the sequence of the circSTX6 is shown as SEQ ID NO. 1.
10. 10. The formulation for treating nasopharyngeal carcinoma according to claim 9, wherein said agent for inhibiting the expression of circSTX6 comprises an antisense oligonucleotide, preferably said antisense oligonucleotide:

    sense strand (5'-3') CAUAUGACCAGUGGACAUGATT

    Antisense strand (5'-3') UCAUGUCCACUGGUCAUAUGTT.

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