CN111979319A - Biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation - Google Patents
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- CN111979319A CN111979319A CN202010781168.6A CN202010781168A CN111979319A CN 111979319 A CN111979319 A CN 111979319A CN 202010781168 A CN202010781168 A CN 202010781168A CN 111979319 A CN111979319 A CN 111979319A
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Abstract
The invention discloses a biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation. In the present invention, we demonstrated that circCIT is highly expressed in nasopharyngeal carcinoma cell lines compared to normal nasopharyngeal epithelial cell NP 69. The detection result of a clinical tissue sample shows that the expression of circCIT is up-regulated in nasopharyngeal carcinoma tissues and is positively correlated with stages. In vitro cell function experiments show that circCIT promotes proliferation, migration and invasion of cells and promotes mesenchymal transformation of cell epithelium. Therefore, the circCIT can be used as a biomarker to be applied to preparation of nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation reagents, kits or detection devices; can also be used as a target point to be applied to screening medicines for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis. The reagent for inhibiting the circCIT expression can be used for preparing medicaments for nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation.
Background
Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy, with the onset characterized by geographical accumulation, usually occurring in the south of china, especially the guangdong and guangxi. Nasopharyngeal carcinoma is characterized by high malignancy, high metastasis rate, early metastasis and early lack of distinct specific symptoms. Most nasopharyngeal carcinoma patients are not diagnosed until the middle and late stages, which significantly reduces the 5-year survival rate. Therefore, early diagnosis and treatment are effective ways to improve the quality of life of patients with nasopharyngeal carcinoma. The key factors affecting prognosis in relapse and metastasis, and at present, no specific marker can predict the prognosis of NPC patients. Therefore, development of a marker having high sensitivity and specificity is urgently required.
The etiology of nasopharyngeal carcinoma is not clear, and the medical field considers that the nasopharyngeal carcinoma is closely related to the following factors: genetic factors, dietary factors, environmental factors and EB virus infection. Because the nasopharynx position is hidden, the examination is not easy, and simultaneously, the early symptoms of the nasopharyngeal carcinoma are relatively complex and lack of characteristics, so the nasopharyngeal carcinoma can be easily ignored by people, and the diagnosis and treatment are delayed. Therefore, the early screening and diagnosis of nasopharyngeal carcinoma, especially the body fluid examination with low traumatic property and convenient material acquisition, has important significance for treating and improving the survival rate of patients.
Unlike conventional linear RNA, circular RNA (circrna) exists in vivo in a closed loop structure, is stably expressed in various body fluids, and exhibits high tissue and cell specificity. In recent years, with the rapid development of high-throughput sequencing and bioinformatics, circRNA has been demonstrated to be highly expressed in various diseases, especially tumors. It has been reported that circRNA specifically expressed in tissues plays an important role in the development and progression of tumors such as gastric cancer, liver cancer, breast cancer, colon cancer and prostate cancer. These findings suggest that circRNA may be a novel diagnostic biomarker and therapeutic target for cancer, and have important clinical value for early diagnosis of tumors, improvement of disease conditions and quality of life of patients. Although circRNA has been extensively studied in tumors, NPC is rarely studied. Current research is focused on radiotherapy of nasopharyngeal carcinoma. Therefore, the influence of circRNA expression on nasopharyngeal carcinoma and pathogenesis thereof is further deeply researched, and a preliminary clinical basis is provided for treatment of the nasopharyngeal carcinoma.
Disclosure of Invention
The invention provides a biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation and application thereof. In the invention, the circCIT is highly expressed in nasopharyngeal carcinoma tissues and cells, and can promote the migration, invasion and tumor metastasis of nasopharyngeal carcinoma cells.
The technical scheme of the invention is as follows:
the application of the circCIT as a biomarker in the preparation of reagents, kits or detection devices for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation.
The application of the product for detecting the circCIT molecule in nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation.
A nasopharyngeal carcinoma detection kit is characterized by comprising a detection object specifically bound with circCIT.
The detector preferably comprises at least one of a primer pair and a probe which specifically binds to circCIT.
The sequences of the primer pairs are as follows: 5'-AAGGTCTTTGCTGCCATCCT-3' (SEQ ID NO.1) and 5'-CGGGCTTGTGCTTCGAGATA-3' (SEQ ID NO. 2).
The circCIT is used as a target point to be applied to screening of drugs for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
The circCIT is used as a treatment target to be applied to the preparation of the medicine for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
The application of the reagent for inhibiting or interfering the circCIT expression in the preparation of the medicine for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
The invention has the beneficial effects that:
circular RNA (circRNA), a subset of non-coding RNA, plays an important role in regulating gene expression in eukaryotes. However, their role in nasopharyngeal carcinoma is poorly understood. In the present invention, we demonstrated that circCIT is highly expressed in nasopharyngeal carcinoma cell lines compared to normal nasopharyngeal epithelial cell NP 69. The detection result of a clinical tissue sample shows that the expression of circCIT is up-regulated in nasopharyngeal carcinoma tissues and is positively correlated with stages. In vitro cell function experiments show that circCIT promotes proliferation, migration and invasion of cells, and promotes Epithelial Mesenchymal Transition (EMT) of cells. Therefore, the circCIT can be used as a biomarker to be applied to preparation of nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation reagents, kits or detection devices; can also be used as a target point to be applied to screening medicines for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis. The reagent for inhibiting or interfering the circCIT expression can be used for preparing the medicine for nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
Drawings
FIG. 1 is a graph showing the up-regulation of the expression level of circCIT in nasopharyngeal carcinoma tissues according to the present invention.
FIG. 2 is a schematic diagram of the first generation sequencing validation head-to-tail cleavage of circCIT of the present invention.
FIG. 3 is a schematic representation of amplification of circCIT of the invention in cDNA and gDNA.
FIG. 4 is a schematic diagram showing the detection of the expression level of circCIT of the present invention after RNase R digestion.
FIG. 5 is a schematic diagram showing that the expression of circCIT of the nasopharyngeal carcinoma cell line of the present invention is significantly higher than that of immortalized nasopharyngeal carcinoma epithelial cell NP-69.
FIG. 6 relationship between circCIT and clinical staging of the invention
FIG. 7 shows the design of two interfering RNAs for circCIT in accordance with the present invention.
FIG. 8 is a schematic representation of the interference of circCIT with one of the proliferative, colony forming effects of 5-8F and 6-10B cells of the invention.
FIG. 9 is a second schematic of the present invention interfering with the proliferative, colony forming effects of circCIT on 5-8F and 6-10B cells.
FIG. 10 is a graph showing the effect of the invention on migration invasion of 5-8F and 6-10B cells by interfering with circCIT.
FIG. 11 is a second schematic diagram illustrating the effect of interfering with circCIT on migration and invasion of 5-8F and 6-10B cells according to the present invention.
FIG. 12 is a schematic diagram showing the effect of the interference circCIT of the present invention on EMT key proteins.
Detailed Description
For the convenience of understanding the technical scheme of the invention, the technical scheme of the invention is described in detail in a specific use mode as follows:
the invention is further described below with reference to the accompanying drawings.
The following examples relate to materials and methods
Patient sample
All 40 nasopharyngeal carcinoma samples come from the radiotherapy department of tumor hospital in Jiangsu province, and all the pathologies are clearly diagnosed as nasopharyngeal carcinoma. According to the declaration of helsinki, each patient signed a written informed consent and was approved by the ethical review board of the tumor hospital, Jiangsu province. The nasopharyngeal carcinoma staging standard is UICC eighth edition staging. Collecting clinical data: inclusion criteria were: firstly, no treatment has been received before; ② nasopharyngeal carcinoma is confirmed by pathology; (iii) no other tumor history; fourthly, the clinical pathological data are complete; age greater than 18 years old. Exclusion criteria: patients who have received other treatments before treatment; ② patients with non-primary nasopharyngeal carcinoma; patients with other serious diseases affecting radiotherapy; patients who have incomplete clinical data and cannot be followed.
Cell lines
Six human nasopharyngeal carcinoma cell lines (CNE1, CNE2, 6-10B, SUNE1,5-8F and c666) and a permanent nasopharyngeal epithelial cell line (NP69) were obtained from the clinical tumor research center of tumor hospital in Jiangsu province (Nanjing, Jiangsu, China). Human NPC cell lines were cultured in RPMI-1640 medium (Corning, Manassas, Va., USA) supplemented with 10% calf serum (Gibco, Grand Island, USA) at 37 ℃ in a humid atmosphere of 5% CO 2. NP-69 was propagated in keratinocyte/serum-free medium (Invitrogen) containing bovine pituitary extract (BD Biosciences, san Diego, Calif., USA) and grown in saturated CO2 at 37 ℃.
Agarose gel electrophoresis experiment
20ml of TBE buffer is taken and added with water to 200ml to prepare 1 XTBE dilution buffer for later use. The appropriate agarose was weighed, placed in a conical flask, and added to about 70ml of TBE dilution buffer, heated in a microwave oven and observed while heating. The heater was removed and shaken during the heating period to mix the agarose particles thoroughly. Preparing a rubber plate: and cooling the melted agar to 50-60 ℃, putting the cooled agar into a glue making mold (a sample comb is inserted in advance), pulling out the comb after the glue is completely solidified, taking out the glue block, and putting the glue block into an electrophoresis tank with TBE electrophoresis buffer solution. Mu.l of DNA sample was mixed with 2. mu.l of the loading solution and carefully added to the sample well. And closing the electrophoresis tank cover and switching on a power supply. The control voltage is kept at 100V, and the current is above 40 mA. The electrophoresis direction is from negative electrode to positive electrode, and when the bromophenol blue band moves to about 2cm from the front edge of the gel, the electrophoresis is stopped. The electrophoresis gel plate is firstly placed under an ultraviolet lamp for observation. The presence of DNA showed an orange-red fluorescent band discernible to the naked eye. When observing, the user needs to wear the safety glasses.
Sanger sequencing
Designing a reverse primer of the circRNA cross-shearing site by Sharpu Ribo Biotechnology Co., Ltd, carrying out Sanger sequencing on a PCR amplified product by Scopy Biotechnology Co., Ltd, comparing sequences by Jellyfish software, confirming that a sequencing result is matched with a back-splice junction sequence, and verifying qRT-PCR primer specificity.
Nuclear mass separation experiment
To prepare (2X lysine/Binding solution), 415. mu.l of amphiphobic ethanol was added. Washsolution 2/3 Add 64. mu.l of absolute ethanol. (iii) Fractional Buffer and Distruppotionbuffer on ice. Fourthly, heating 200 mu l of eluent to 95 ℃ by using an RNA-free EP tube. The digested cells were centrifuged at low speed to pellet and the supernatant was aspirated, the cell pellet was washed once with PBS and placed on ice. Add 300. mu.l of precooled Fractional Buffer to each tube, flick the tube to loosen the cells, and gently swirl or blow to resuspend the cells. Incubating on ice for 5-10min, centrifuging at 4 deg.C and 500g for 1-5 min. The supernatant was aspirated and placed in a new RNA-free tube, and labeled with cytoplasm. The precipitate was observed, and if the precipitate was loosened by sucking the supernatant in the step (5), the precipitate was gently flicked by adding a Fractional Buffer into an EP tube, and then centrifuged at 500g for 1min at 4 ℃. If there is no loose, add the same volume of the Fractional Buffer of the Distruption Buffer, vigorously vortex or blow to lyse the cell nucleus. To each of the cytoplasm and nucleus was added 300. mu.l of 2 × lysine/Bindingsolution. Add 300. mu.l of absolute ethanol to each tube and gently blow it to mix well. The mixture was added to a fresh EP tube with a filter cartridge, not more than 700. mu.l each time, centrifuged at 12000g for 1min and the filtrate discarded. 700 μ l Wash Solution 1 was washed 1 time and centrifuged at 12000g for 15s and the filtrate discarded. Mu.l Wash solution 2/3 was washed 2 times and centrifuged at 12000g for 15s and the filtrate discarded. Centrifugation was continued for 10-30s to remove residual Wash Solution 2/3. Mu.l of preheated Elation Solution was added to the filter column and centrifuged at 12000g for 30 s. The elution was repeated once and approximately 100. mu.l of sample was collected. And (3) detecting the concentration and purity of the RNA by using the NanoDrop, recording data, carrying out a reverse transcription experiment in the next step, and storing the residual RNA in a refrigerator at the temperature of-80 ℃.
Cell transfection
The nasopharyngeal carcinoma cell strains are respectively transfected and the subsequent biological functions are explored. The specific cell transfection procedure is as follows: collecting when nasopharyngeal carcinoma cells grow to 80-90%, inoculating into 6-well plate, and culturing at 37 deg.C with 5% CO2Culturing for 24 h; diluting the transfection product: the transfectants were removed from the freezer and diluted with predominantly Opti-MEM at 20 nM. Mixing, and incubating at room temperature for 5 min; dilution Lipo 3000: diluting Lipo 3000 with Opti-MEM culture solution by 50 times, quantifying to 250 μ L, mixing well, and incubating at room temperature for 5 min; changing the culture solution in the 6-well plate to a serum-free culture medium, respectively adding Lipo 2000 and the transfectants, and culturing for 4-6h at 37 ℃; and pouring out serum-free culture solution, washing with PBS for 2 times, adding fresh culture solution, and culturing at 37 ℃ for 48h, wherein the transfection is successful.
Cell viability assay and colony formation assay
The growth curve of stably transfected cells was studied using a cell counting kit-8 (Beyotime, China). NPC cells were seeded in 96-well plates at a density of 1.5 × 103 cells per well in triplicate. The absorbance was recorded at 490nm after 24,48 and 72 hours by using an ELX800 spectrophotometric plate reader (Bio-Tek, Winooski, VT, USA). In colony formation experiments, stably transfected cells were seeded at a rate of 500 cells per well in 6-well plates and cultured in media for 7-12 days. The colonies were observed by crystal violet staining. Colonies were counted with ImageJ and the count was >50 cells.
Invasion and migration testing
Migration capability: digesting the nasopharyngeal carcinoma cells after transfection, centrifuging and discarding the culture solution, and adding 0.2% BSA serum-free culture medium for resuspension. The cell density needs to be maintained at 5X 105Per mL; 200 u L cell suspension is accurately measured, then it is added to the Transwell chamber, accurately measured 600 u L containing 10% FBS medium, then it is added to the lower chamber, at 37 degrees C temperature conditions, placed in the CO2Culturing for 48h in an incubator; the transwell chamber was removed, fixed with methanol, stained with crystal violet, and cells were observed at random in five fields under a 400-fold microscope to calculate the average cell number.
Invasion capacity: the upper compartment face of the bottom membrane of the Transwell cell was coated with 50. mu.L of 50mg/L Matrigel 1: 4 coating with diluent. After digestion of transfected nasopharyngeal carcinoma cells, centrifugation, the culture medium was discarded, carefully washed with PBS, which was done 2 times, and 0.2% BSA in serum-free medium was added to the resuspension. The cell density needs to be maintained to 5X 105Per mL; 200. mu.L of cell suspension was accurately measured and then added to a Transwell chamber, and 600. mu.L of medium containing 10% FBS was accurately measured and then added to the lower chamber at 37 ℃ and CO2Culturing for 48h in an incubator; the Transwell chamber was removed, fixed with methanol, stained with crystal violet, and cells were randomly observed in five fields of view under a 400-fold microscope and counted.
Wound healing assay
Before inoculating cells, marking on the back of a 12-hole plate by using marker stroke horizontal lines; after digestion of the cells, they are seeded in 12-well plates, preferably with the bottom of the plate; the same width was maintained using a 200 μ L tip scratch. However, the difficulty of ensuring consistent width of the scratch is relatively large, and the experimental result is influenced to a certain extent; sucking out cell culture solution, washing with PBS (phosphate buffer solution), wherein the operation needs to be carried out for three times in total to ensure that no cell fragments are generated; and replacing the culture medium with a serum-free culture medium, transferring the culture medium into an incubator to continue culturing, observing cell migration, and simultaneously taking pictures, wherein the corresponding operation nodes are 0h and 24 h.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Liquid nitrogen was extracted from the tissue samples and transferred to a mortar for grinding. After completion based on 100mg tissue samples: TRIzol was added at a rate of 1mL, homogenized, and allowed to stand at room temperature. After 5min, it was transferred to a new EP tube and centrifuged at 4 ℃ based on 12,000rpm, which is a total time consumption of 10 min. The supernatant was transferred to a new EP tube and chloroform was added in the ratio of 1mL TRIzol to 0.2mL chloroform. Mixing well, and standing for 3 min. The centrifugation treatment was performed at 4 ℃ based on 12,000rpm, and the time consumption of this step was 15min in total. Collecting the upper layer water solution, adding isopropanol (ratio of 1: 1) therein, mixing well, and storing at room temperature for 30 min. The centrifugation treatment was performed at 4 ℃ based on 12,000rpm, and the time consumption of this step was 15min in total. The precipitate was collected, washed with 75% ethanol and added to an EP tube, and the ethanol solution was pre-cooled at 4 ℃. The pellet was washed with 1mL TRIzol corresponding to a ratio of 1mL 75% ethanol. Then, the centrifugation treatment was performed at 4 ℃ based on 5,000rpm, and the time consumption of this step was 5min in total. Discarding the supernatant, and standing at room temperature for 2-3 min. Then 20-30. mu.L RNase-Free ddH was further added2O was added thereto, and RNA was dissolved and stored at-20 ℃. A circCIT back primer designed by RiboBio (Guangzhou, China) and a miR-133a-5p specific RT primer or IGFBP3 random primer (Promega) were used for reverse transcription qRT-PCR. qRT-PCR was performed in an ABI7500 real-time PCR instrument (Applied Bio-systems). Specific back primers 5'-AAGGTCTTTGCTGCCATCCT-3' (SEQ ID NO.1) and 5'-CGGGCTTGTGCTTCGAGATA-3' (SEQ ID NO.2) for circCIT, 5 'CACCATTGGCAATGAGCGGTTC-3' and 5'-AGGTCTTTGCGGATGTCCACGT-3' for β -actin; the 2- Δ Δ Ct method was used to calculate the fold change in circCIT expression.
Protein sample extraction
A number of 1.5ml EP tubes of RNA-free were prepared and precooled to 4 ℃ by a high-speed centrifuge. Preparation of lysate, phosphatase inhibitor: PMSF: RIPA ═ 1: 1: 100. discard the medium and wash 3 times with ice PBS. The prepared lysate was added in an amount of 300. mu.l per dish (200. mu.l per well in a six-well plate) and the cells were scraped off using a cell scraper (ice-on procedure). The lysate was aspirated into an EP tube, placed in a precooled high-speed centrifuge at 13200rpm/min, and centrifuged for 30 min. After centrifugation, the supernatant was transferred to a new EP tube, labeled, and stored in a-80 ℃ refrigerator.
Protein pretreatment
The concentrations of the samples from each group were adjusted to agreement (lowest concentration, fixed volume 65ul) with RIPA based on the protein concentration determined. For each 65ul protein sample, 25ul Buffer (NuPAGE, LDSample Buffer4x), 10ul Reducing (NuPAGE Reducing Agent 10X) was added. Boiling protein, performing metal bath at 70 deg.C for 10min, fully denaturing, and storing at-80 deg.C.
Preparation of precast rubber
10% of separation glue and 5% of concentrated glue are prepared according to the following system
The cleaned and dried electrophoresis plate is inserted into a frame to be clamped tightly, and glue is poured after leakage detection.
Adding 10% prepared separation gel, adding TEMED, shaking up immediately, and pouring gel into the tank. And (3) slowly adding double-distilled water to seal the colloid when the colloid surface reaches a proper height, keeping the colloid surface as horizontal as possible, placing the colloid surface in a fume hood for about 30min, and when the water and the colloid present a remarkable boundary, saying that the gelatin is solidified. The water in the gel upper layer was poured off and then the water was removed by suction with filter paper. Preparing 5% concentrated gelatin solution according to the system, sequentially adding the reagents into a beaker, and fully and uniformly mixing; and (3) slowly injecting the prepared concentrated glue into the upper layer of the separation glue from left to right by using a liquid-moving gun, slightly inserting the comb teeth, gently moving to avoid generating bubbles, and supplementing the extruded glue. Standing at normal temperature for about 15 min. After the concentrated gel is condensed, the comb is pulled out slightly and uniformly, and the gel is placed into a prepared electrophoresis tank to be clamped for sample loading.
Sample loading and electrophoresis
Protein loading: the total amount of loading in each well was 15ul, and the deficient fraction was filled with protein loading buffer (1X). 5ul of pre-dyeing indicator Marker is added in the middle for estimating the position of a target strip, so that the film shearing is facilitated. Buffer tailing is added to holes on two sides. And (3) running electrophoresis at a constant voltage of 160V, observing the position of the marker, evaluating the position of the needed protein, and stopping the electrophoresis after the target protein is completely run out, wherein the whole process is about 1 h.
Rotary film
Preparing: taking out the prepared membrane transferring liquid, pouring the membrane transferring liquid into a tray, and putting the experimental appliance into the tray in advance to soak for about 20 min. The gel block of the desired protein of interest is cut out with a light motion to prevent crumbling of the gel. Put into a tray with a transfer buffer. The PVDF membrane (a little larger than the rubber block) is cut out, soaked in methanol for about 30s for activation, and then moved to a tray. Spreading a film-rotating clamping plate, sequentially spreading sponge, filter paper, a glue block, a PVDF film, the filter paper and the sponge on the film-rotating clamping plate according to the sequence of a black glue film and a white film, rolling bubbles by using a glass rod, and closing and clamping. The fixed splint is arranged in the film-transferring groove, two stages of power supplies (paying attention to distinguishing the cathode and the anode) are connected, the film-transferring buffer solution is poured, an ice bag is filled around the film-transferring buffer solution, and the current of 300mA is set to flow for about 2 hours.
Sealing of
And preparing membrane washing solution (TBST one package powder, 1L deionized water and 2ml TWEEN) in advance. And (5) refrigerating the membrane washing solution at 4 ℃, and washing the membrane for 3 times, each time for 3-5min, after the membrane transferring is finished. The PVDF membrane is put into 10ml of sealing liquid and sealed for 2 hours at normal temperature on a shaking table. After blocking, the membrane was washed 3 times with TBST for 10min each.
Immune response
Applying a first antibody: the cut antibody strips were placed in a prepared primary antibody (DNMT1,1: 2000; beta-actin, 1:1000), sealed with plastic envelope and labeled, and wrapped in moist cotton cloth in a refrigerator at 4 ℃ overnight. Applying a second antibody: after the completion, the antibody-coated strips were removed and washed with TBST for 3 times, 5-10min each time. And diluting the secondary antibody with a sealing solution according to the proportion of 1:5000, then preparing the secondary antibody, putting the antibody strip into the secondary antibody diluent, and incubating for 2 hours on a shaking table.
ECL chemiluminescence method development
And (4) turning on the power supply of the imager in advance, and preheating the imager for about 30 min. Taking out the solution A and the solution B in the ECL kit from dark at-4 ℃, and preparing and mixing the solution A and the solution B uniformly according to the proportion of 1:1 for later use. Uniformly covering the prepared ECL mixed solution on the PVDF membrane, adjusting the dosage of the mixed solution according to the size to optimally uniformly cover the surface, putting the membrane into an exposure platform by using tweezers, and setting the program and the exposure time. And selecting proper time according to specific conditions during exposure, and increasing the exposure time to make the strip become thick, otherwise, making the strip become thin. The results were observed, named tag and saved.
Statistical analysis
The above experiments were repeated 3 more times. Results were analyzed using graphpad7.0 and spss23.0 statistical software, with the data measured as means ± standard deviation, using t-test to compare differences between two groups, and One-way ANOVA to compare differences between groups. Correlation analysis was evaluated using the Person's correlation coefficient. Assuming that the test level is judged as α ═ 0.05, when P <0.05 is considered statistically significant, P values <0.05 (. x.), P values < 0.01 (. x.) and P values < 0.001 (. x.).
Example 1
Expression of circCIT in nasopharyngeal carcinoma tissues and cells
Compared with normal tissues, the expression level of the circCIT in nasopharyngeal carcinoma tissues is up-regulated (figure 1, P <0.05), and the expression of the circCIT is positively correlated with the clinical pathological stage of the nasopharyngeal carcinoma (figure 6). Similarly, circCIT expression in nasopharyngeal carcinoma cell lines was significantly lower than that of immortalized nasopharyngeal carcinoma epithelial cell NP-69 (FIG. 5). Sanger sequencing further confirmed head-to-tail splicing (fig. 2). However, head-to-tail splicing may be not only trans-splicing, but also the result of genomic rearrangements. To distinguish these two possibilities, forward primers for circCITmRNA were designed with the following sequences: forward Primer, 5' CCGATTGGGACCCCCAGATTA-. cDNA and gDNA were extracted from 5-8F cells and detected by nucleic acid electrophoresis. The results showed that circCIT was detected only in cDNA and not in gDNA (fig. 3). Further RNase R digestion experiments were used to confirm the stability of circCIT. As expected, the linear transcript of the host gene CIT was degraded by RNase R, whereas the circular transcript of circCIT was resistant to RNase R treatment (FIG. 4). These data confirm the presence of circCIT and that circCIT may play a tumor promoting role in nasopharyngeal carcinoma.
In vitro regulation of proliferation, invasion and migration of nasopharyngeal carcinoma cells by circCIT
In order to explore the in vitro biological function of circCIT, 5-8F, 6-10B cell lines with higher expression level are selected for subsequent functional experiments. siRNA can stably knock down the expression of circCIT in most cells, we designed 2 si, sequences as follows: Si-circCIT # 1: ACAACATTCGTAACTGTTTCA are provided. Si-circCIT # 2: CAACATTCGTAACTGTTTCAT are provided. Knock down efficiency is shown in fig. 7. Knocking down circCIT expression compared to control group significantly inhibited nasopharyngeal carcinoma cell proliferation, invasion and migration in vitro (fig. 8-11). Further analyzing Epithelial Mesenchymal Transition (EMT) related protein highly related to tumor metastasis. Western blot analysis showed that the expression level of E-cadherin was significantly increased in circCIT cells with down-regulated expression, while the expression level of N-cadherin, vimentin was significantly decreased (FIG. 12).
In the present invention, we detected that circCIT is significantly upregulated in nasopharyngeal carcinoma tissues and several nasopharyngeal carcinoma cell lines, suggesting that circCIT is likely to be a potential cancer-related gene in nasopharyngeal carcinoma. Further we explored the function of circCIT in vitro. The fact that the knockdown of the circCIT inhibits the growth, invasion and migration of nasopharyngeal carcinoma cells in vitro and in vivo and the process migration of EMT shows that the circCIT has the effect of promoting the development of cancers in the nasopharyngeal carcinoma.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
- Application of CircCIT as a biomarker in preparation of reagents, kits or detection devices for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation.
- 2. Application of the reagent for detecting the expression of the CircCIT in preparing a nasopharyngeal carcinoma metastasis diagnosis reagent and/or a prognosis evaluation reagent.
- 3. A nasopharyngeal carcinoma detection kit is characterized by comprising a detection object specifically bound with CircCIT.
- 4. The nasopharyngeal carcinoma detection kit of claim 3, wherein said detector comprises at least one of a primer pair, a probe that specifically binds to CircCIT.
- 5. The nasopharyngeal carcinoma detection kit of claim 4, wherein said primer pair has the sequence shown in SEQ ID No.1 and SEQ ID No. 2.
- Application of the CircCIT as a target point in screening of drugs for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
- Application of the CircCIT as a treatment target in preparation of a medicine for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
- 8. The application of the reagent for inhibiting or interfering the expression of the CircCIT in the preparation of the medicine for treating nasopharyngeal carcinoma or nasopharyngeal carcinoma metastasis.
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