CN113476618B

CN113476618B - Application of miR-199a-3p in preparation of medicine for treating nasopharyngeal carcinoma

Info

Publication number: CN113476618B
Application number: CN202110772224.4A
Authority: CN
Inventors: 罗海清; 李祥勇; 胡文佳; 任静; 戴秋芹; 陈燃; 张敬通; 黄语萌; 许云仕
Original assignee: Guangdong Medical University; Affiliated Hospital of Guangdong Medical University
Current assignee: Guangdong Medical University; Affiliated Hospital of Guangdong Medical University
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2023-03-31
Anticipated expiration: 2041-07-08
Also published as: CN113476618A

Abstract

The invention discloses application of miR-199a-3p in preparation of a medicine for treating nasopharyngeal carcinoma, and belongs to the technical field of biological medicines. The application of miR-199a-3p in preparation of the medicine for treating nasopharyngeal carcinoma disclosed by the invention discusses the action mechanism and clinical diagnosis significance of miR-199a-3p in occurrence and development of nasopharyngeal carcinoma through in vivo and in vitro experiments and clinical sample analysis, and provides a new scientific basis for molecular targeted therapy of nasopharyngeal carcinoma.

Description

Application of miR-199a-3p in preparation of medicine for treating nasopharyngeal carcinoma

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of miR-199a-3p in preparation of a medicine for treating nasopharyngeal carcinoma.

Background

Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the Nasopharyngeal mucosa. At present, radiotherapy is preferred to be used for treating nasopharyngeal carcinoma, surgery treatment and chemotherapy are used as auxiliary treatment methods, but the disease has high malignancy degree, is difficult to diagnose in early stage, is easy to generate lymph node metastasis, and has limitation, so that the recurrence rate of a patient is high, and the prognosis is poor. Recurrent metastasis of nasopharyngeal carcinoma is the leading cause of death in patients. However, the distant metastasis mechanism of nasopharyngeal carcinoma is extremely complex and not completely understood so far, so that the molecular mechanism of NPC invasion and metastasis is actively discussed, and a valuable molecular treatment target is searched, so that the method has very important practical significance for the treatment of nasopharyngeal carcinoma.

miRNA is a non-coding small RNA molecule widely expressed in animals and plants, is approximately composed of about 18-24 nucleotides, has the characteristics of high conservation and high stable inheritance in the evolution process, mainly degrades mRNA of a target gene by combining with 3' UTR of mRNA of the target gene or leads the translation process of the mRNA of the target gene to be inhibited, and carries out negative regulation and control on the target gene at the post-transcriptional level so as to participate in biological processes of regulating and controlling differentiation, proliferation, apoptosis, invasion and transfer, autophagy and the like. miR-199a is a non-coding small molecular RNA which is obtained from rat cell clone for the first time by German scientist Lagos-Quintena and the like in 2003, and two mature miRNAs are obtained after Dicer cracking: miR-199a-5p and miR-199a-3p. Research shows that the two mature bodies of miR-199a have abnormal expression in various tumor cells and participate in regulation of life activities such as tumor cell proliferation and metabolism, invasion and metastasis, angiogenesis, apoptosis and the like. However, the regulation and mechanism research of miR-199a-3p in nasopharyngeal carcinoma have not been reported yet.

Therefore, the application of miR-199a-3p in the preparation of medicines for treating nasopharyngeal carcinoma is a problem to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides an application of miR-199a-3p in preparation of a medicine for treating nasopharyngeal carcinoma, wherein miR-199a-3p inhibits invasion and metastasis of nasopharyngeal carcinoma by regulating SCD1 expression.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of miR-199a-3p in preparing a medicine for treating nasopharyngeal carcinoma is characterized in that the nucleotide sequence of miR-199a-3p is as follows:

5’-ACAGUAGUCUGCACAUUGGUUA-3’；SEQ ID NO.1。

further, the miR-199a-3p can inhibit nasopharyngeal carcinoma invasion and metastasis by regulating SCD1 expression.

Further, the application of SCD1 in preparing a medicine for treating nasopharyngeal carcinoma is disclosed, wherein the SCD1 is a target gene of miR-199a-3p.

According to the technical scheme, compared with the prior art, the application of miR-199a-3p in preparation of the medicine for treating nasopharyngeal carcinoma is provided, the action mechanism and the clinical diagnosis significance of miR-199a-3p in occurrence and development of nasopharyngeal carcinoma are discussed through in vivo and in vitro experiments and clinical sample analysis, and a new scientific basis is provided for molecular targeted therapy of nasopharyngeal carcinoma.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing the expression of miR-199a-3p in a public chip database; GSE32960 chip data: the total number of samples was 330 independent samples, N (Normal) was Normal nasopharyngeal epithelial tissue (18 cases), T (Tumor) was nasopharyngeal carcinoma tissue (312 cases);

FIG. 2 is a drawing showing the expression of miR-199a-3p in a public chip database; GSE36682 chip data: total number of samples was 68 independent samples, N was nasopharyngeal tissue (6 cases), T (Tumor) was nasopharyngeal carcinoma tissue (62 cases), P <0.05;

FIG. 3 is a diagram showing the RT-qPCR detection of miR-199a-3p expression in nasopharyngeal carcinoma tissue and normal nasopharyngeal carcinoma tissue according to the present invention; wherein NT (n = 8) is 8 cases of inflamed tissues of nasopharynx; NPC (n = 19) was 19 nasopharyngeal carcinoma tissues; * P <0.05;

FIG. 4 is a diagram showing relative expression of miR-199a-3p in a nasopharyngeal carcinoma cell line detected by RT-qPCR of the invention; * P <0.05;

FIG. 5 is a diagram showing the expression of SCD1 of the present invention in nasopharyngeal carcinoma tumor tissues and paracarcinoma normal tissues;

FIG. 6 is a graph of immunohistochemical scoring of SCD1 of the present invention in 82 pairs of clinical tissues;

FIG. 7 is a graph showing the expression of SCD1 according to the present invention in tumor tissues of different clinical stages;

FIG. 8 is a graph showing immunohistochemical scores of SCD1 of the present invention in tissues of different tumor grade; * P <0.05;

FIG. 9 is a graph showing the efficiency of miR-199a-3p mimic transfection of CNE-2 and 5-8F cells in the invention; * P <0.01, at least three replicates;

FIG. 10 is a graph showing the efficiency of miR-199a-3p inhibitor in transfecting CNE-2 and 5-8F cells; * P <0.01, at least three replicates;

FIG. 11 is a drawing showing that miR-199a-3p is over-expressed, and a scratch test detects wound healing conditions of 5-8F and CNE-2 cells at 0h, 24h, 48h and 72h (100 x);

FIG. 12 is a graph showing migration (200X) of various groups of cells detected by the miR-199a-3p overexpression and Trans-well chamber migration assay of the present invention;

FIG. 13 is a graph showing the detection of invasion (200X) of each group of cells in the miR-199a-3p overexpression and Trans-well chamber invasion assay of the present invention;

in fig. 11-13, control is blank control and no treatment; NC: transfecting a micm negative control with a final concentration of 50nM; mimic: miR-199a-3p imic is transfected, and the final concentration is 50nM;

FIG. 14 is a drawing showing that miR-199a-3p is inhibited and expressed, and wound healing conditions (100 x) of 5-8F and CNE-2 cells at 0h, 24h, 48h and 72h are detected by a scratch experiment;

FIG. 15 is a graph showing that miR-199a-3p is inhibited and a transwell chamber migration experiment is used for detecting the migration condition of each group of cells (200X);

FIG. 16 is a drawing showing that the miR-199a-3p is inhibited and the invasion condition of each group of cells is detected by a transwell chamber invasion experiment (200X) according to the invention;

in fig. 14-16, control is blank control, no treatment; NC: transfection inhibitor negative control, final concentration 100nM; inhibitor: transfecting miR-199a-3p inhibitor with the final concentration of 100nM;

FIG. 17 is a Westernblot assay of expression of 5-8F cell-associated proteins in each treatment group in accordance with the present invention;

FIG. 18 is a diagram showing the expression of CNE-2 cell-associated protein in each treatment group detected by Westernblot in accordance with the present invention;

FIG. 19 shows RT-qPCR detection of 5-8F cell-associated gene expression in each treatment group; * P <0.05,. P <0.01;

FIG. 20 is the RT-qPCR assay of CNE-2 cell-associated gene expression at P <0.01 for each treatment group according to the invention;

FIG. 21 is a drawing showing the binding site of miR-199a-3p and SCD13' UTR and the mutation site of the inserted sequence in the mutant plasmid;

FIG. 22 is a diagram showing the detection of luciferase activity in each group by the dual luciferase activity detection kit of the present invention; SCD1-WT/NC: SCD1 wild type/miR-199 a-3p mic NC (Negative control); SCD1-WT/miR-199a-3p: SCD1 wild type/miR-199 a-3p imic; SCD1-Mut/NC: SCD1 mutant/miR-199 a-3p mic NC (Negative control); SCD1-Mut/miR-199a-3p: SCD1 mutant/miR-199 a-3p imic; * P <0.05;

FIG. 23 is a diagram showing expression conditions of SCD1 and PTEN proteins after transfection of miR-199a-3p mimic/inhibitor by 5-8F and CNE-2 cells in a Western blot experiment according to the invention; control: blank control; NC: negative control; mimic: miR-199a-3p mimic; inhibitor: miR-199a-3p inhibitor;

FIG. 24 is a diagram showing the expression change of SCD1 gene after miR-199a-3p mimic transfection detected by RT-qPCR experiment of the invention; NC: miR-199a-3p micnc (Negative control); * P <0.05;

FIG. 25 is a graph showing the effect of the present GC assay on the ratio of miR-199a-3p to C16:0/C16:1 and C18:0/C18:1 of nasopharyngeal carcinoma cells 5-8F; NC: NC on the left side of the graph is negative control of mimic, NC on the right side of the graph is negative control of inhibitor; * P <0.05;

FIG. 26 is a graph showing that the influence of miR-199a-3p on the ratio of C16:0/C16:1 and C18:0/C18:1 of nasopharyngeal carcinoma cells CNE-2 is detected by GC assay according to the present invention; NC: NC on the left side of the graph is negative control of mimic, and NC on the right side of the graph is negative control of inhibitor; * P <0.05;

FIG. 27 is a graph showing that the CCK-8 method of the present invention detects the cytotoxicity of oleic acid OA at various concentrations for 5-8F under 24 hours; NC: blank control; BSA: solvent control;

FIG. 28 is a graph showing that the CCK-8 method of the present invention detects the cytotoxicity of oleic acid OA at various concentrations for 5-8F under the condition of 48 hours; NC: blank control; BSA: solvent control; * P <0.05;

FIG. 29 is a graph showing the change in invasion-migration ability (200X) of 5-8F cells in OA (80. Mu.M) according to the Trans-well assay of the present invention;

FIG. 30 is a schematic diagram showing the Trans-well assay of the present invention detecting the change in invasion and migration ability of CNE-2 cells by OA (80. Mu.M) (200X);

FIG. 31 is a diagram showing a Western blot experiment for detecting expression changes of each related protein under the treatment of miR-199a-3p mimic and OA (80 mu M) transfected by 5-8F and CNE-2 cells or the combined treatment of mimic and OA;

FIG. 32 is a diagram showing the Western blot experiment for detecting the expression of SCD1, PTEN, p-Akt, t-Akt, E-cadherin, N-cadherin, MMP2 and MMP9 proteins in 5-8F and CNE-2 cells overexpressing SCD1;

wherein, the first lanes of 5-8F and CNE-2 are both transfected with empty vector without SCD1 gene; the second lane is the transfected miR-199a-3p mimic; the third column, lanes, is the miR-199a-3p micc transfection and SCD1 overexpression plasmid vector;

FIG. 33 is a Western blot experiment for detecting the expression of p-Akt, t-Akt, E-cadherin, N-cadherin and MMP2 proteins after a PI3K inhibitor LY294002 treats 5-8F and CNE-2 cells;

FIG. 34 is a drawing showing the effect of miR-199a-3p of the invention on the in vivo growth of 5-8F cell xenograft tumors; wherein, A: representative images of nude mice inoculated with the transplantation tumor of each treatment group; b: the change curve of the volume of the transplanted tumor of each treatment group of nude mice; c: representative graphs of the transplanted tumors for each treatment group; d: difference in weight of tumor tissue for each treatment group; * P <0.05,n =5;

FIG. 35 is a drawing showing the effect of miR-199a-3p of the invention on the in vivo growth of CNE-2 cell xenograft tumors; wherein, A: representative images of nude mice inoculated with the transplantation tumor of each treatment group; b: change curve of the transplanted tumor volume of each treatment group of nude mice; c: representative graphs of the transplanted tumors for each treatment group; d: difference in weight of tumor tissue for each treatment group; * P <0.05,n =5;

FIG. 36 is a graph showing the effect of miR-199a-3p of the present invention on body weight of nude mice;

FIG. 37 is a diagram showing the RT-qPCR detection of CNE-2 cell xenograft tumor tissue-associated gene expression in accordance with the present invention; detecting relative expression levels of miR-199a-3P and SCD1 in the agomiR-NC group and the agomiR-199a-3P group, # P <0.05, n =5;

FIG. 38 is a photograph of H & E staining (200X magnification to 400X) of CNE-2 cell xenograft tumor tissues of the invention;

FIG. 39 is a diagram showing the detection of CNE-2 cell xenograft tumor tissue-associated protein expression by immunohistochemical method according to the present invention; in tumor tissues of nude mice, the expression levels of each protein of the agomiR-NC group and the agomiR-199a-3p group were examined, A: SCD1; b: PTEN; c: e-cadherin; d: MMP2; (200X magnification to 400X).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 expression of miR-199a-3p in nasopharyngeal carcinoma cell lines and clinical tissues

1) miR-199a-3p obviously has low expression in nasopharyngeal carcinoma gene chip of public database

After collating the microRNA chip data of two groups of nasopharyngeal carcinoma tissues in the public database of National Center of Biotechnology Information (NCBI), making a scatter diagram and further performing statistical analysis, the expression content of miR-199a-3p in the nasopharyngeal carcinoma tissues is found to be significantly lower than that of normal nasopharyngeal epithelial tissues and nasopharyngeal inflammation tissues (FIG. 1-FIG. 2).

2) miR-199a-3p is low expressed in clinical nasopharyngeal carcinoma tissues

8 nasopharyngeal inflammation tissues and 19 nasopharyngeal cancer tissues are collected, the expression level of miR-199a-3p is detected by using an RT-qPCR experiment, the U6 gene is used as an internal reference, the relative expression quantity of the miR-199a-3p gene in each tissue is calculated by using a delta Ct method, and the result is shown in figure 3. The results in FIG. 3 show that the expression level of miR-199a-3p in nasopharyngeal carcinoma tissue samples is lower than that of miR-199a-3p in nasopharyngeal inflammation tissues, which is consistent with the results of public databases.

3) miR-199a-3p is low expressed in nasopharyngeal carcinoma cells

RT-qPCR experiments are adopted to detect the expression level of miR-199a-3p in an immortalized nasopharyngeal epithelial cell line NP69-SV40T and nasopharyngeal cancer cell lines CNE-1, CNE-2, HONE1, 5-8F and 6-10B, the result takes U6 gene as internal reference and NP69 as reference, and the relative expression of miR-199a-3p in each cell line is calculated by a delta-delta Ct method, and the result is shown in figure 4.

FIG. 4 shows that compared with NP69, miR-199a-3p is significantly low expressed in nasopharyngeal carcinoma cells HONE1, CNE-2, 6-10B and 5-8F; the expression content of miR-199a-3p is NP69>5-8F >6-10B >HONE1>CNE-1 >. In view of the fact that 5-8F and CNE-2 are less differentiated (i.e., more malignant) and the relative expression of 5-8F and CNE-2in nasopharyngeal carcinoma cell lines is the most different, the present invention selects 5-8F and CNE-2 cell lines for subsequent experiments.

Wherein, the RT-qPCR test method in 2) and 3) is as follows:

(1) Total RNA extraction of tissues or cells: strictly according to the experimental steps in the mircute miRNA extraction and separation kit (DP 501).

(2) And (3) RT reaction: according to the instructions of RT reaction kit (Takara), RT reaction solution was prepared, and PCR program was set for reverse transcription to obtain cDNA.

(3) RT-qPCR: according to SYBRqPCR purchased from Takara: according to the specification of the SYBRPremixExTaq kit, RT-qPCR reaction solution is prepared, and the expression of the gene is detected by using an ABI7500qPCR instrument.

After the reaction was completed, the data were analyzed by SDS software (v 1.4) provided in the system. The analytical method used for the relative quantification experiment is the comparative Ct value method. The Ct value represents the threshold cycle, which is the number of cycles that the fluorescence signal intensity goes through when it exceeds the set threshold intensity. Because there is a linear correspondence between the Ct value and the logarithm of the initial copy number of the template in the reaction tube, according to the formula:

ΔΔCt＝(CtmiR-199a-3p-CtU6) _{sample 1} -(CtmiR-199a-3p-CtU6) _{Sample 2}

Calculating the expression level 2 of CNE-1, CNE-2, HONE1, 5-8F and 6-10B cell strains relative to NP69 cell strain miR-199a-3p ^-△△Ct (ii) a Calculating the expression quantity 2 of other clinical tissues relative to the clinical nasopharynx inflammatory tissue miR-199a-3p ^-△△Ct 。

4) Targetscan predicted SCD1 to be the target gene of miR-199a-3p

According to the prediction result of Targetscan of the miRNA target gene prediction website, SCD1 is predicted to be one of target genes of miR-199a-3p, and a seed region of miR-199a-3p is contained in a 3' UTR region of the SCD1 gene.

5) SCD1 is highly expressed in clinical nasopharyngeal carcinoma tissues and is related to clinical staging

Collecting 82 pieces of clinical pathological tissue slices of a pathologist department in a hospital in the central Jiangmen city, nasopharyngeal carcinoma tissues (TT) and paracancer normal tissues (ANT) respectively, and performing immunohistochemical staining analysis; as a result, the expression of SCD1 protein is localized in nucleus and cytoplasm of nasopharyngeal carcinoma tissue cells, and is yellowish brown, and the expression level of SCD1 protein is obviously increased in nasopharyngeal carcinoma tissues relative to paracancerous tissues, and the difference is statistically significant (FIG. 5). The samples were read for SCD1 expression using immunohistochemical scoring criteria and found that SCD1 scores were concentrated in the para-carcinoma tissues at 0-4 points and tumor tissue scores were distributed between 2-12 points (FIG. 6). Further studies found that SCD1 expression increased with increasing clinical stage of the patient's tumor, with statistical differences (fig. 7-8).

The results suggest that miR-199a-3p functions as a cancer suppressor gene in nasopharyngeal carcinoma. In addition, the Targetscan software predicts that the target gene of miR-199a-3p contains a fatty acid metabolism-related gene SCD1. The clinical pathological section immunohistochemical experiment shows that SCD1 is obviously and highly expressed in nasopharyngeal carcinoma tissue and is related to clinical pathological characteristics, which indicates that the fatty acid metabolism related to SCD1 is obviously enhanced in nasopharyngeal carcinoma. We hypothesize that the occurrence and development of nasopharyngeal carcinoma are likely related to the low expression of miR-199a-3p and the high expression of SCD1; the invention further develops the correlation research of miR-199a-3p and nasopharyngeal carcinoma.

Example 2 Effect of miR-199a-3p on nasopharyngeal carcinoma cell invasion, migration and EMT and its mechanisms

Nasopharyngeal carcinoma cells 5-8F and CNE-2 are taken as research objects, and the influence on the invasion and migration capacity of tumor cells and the change of fatty acid metabolism after miR-199a-3p mimic/inhibitor transfects the nasopharyngeal carcinoma cells are observed through a lipo3000 mediated transfection method; finally, the targeting effect of the miR-199a-3p and SCD1 is verified through a dual-luciferase reporter gene experiment, and the action mechanism of the miR-199a-3p for inhibiting the invasion and migration of the nasopharyngeal carcinoma cells is disclosed by further carrying out detection and verification through a western blot experiment, an immunohistochemical experiment and a gas chromatography method.

1) Transfection efficiency of miR-199a-3p mimic/inhibitor for transfecting nasopharyngeal carcinoma cells

(1) Cell plating: before transfection, inoculating an appropriate number of cells into a 6-well or 96-well culture dish until the cells grow to a fusion degree of 30% -50%;

(2) Preparing a transfection solution: miR-199a-3p mimic/inhibitor/negative control (available from Ruibo, guangzhou, 5 nmol) + 250. Mu.l RNase-free water at a final concentration of 20 pmol/. Mu.l, i.e., 20. Mu. Mol/l.

(1) Diluting miR-199a-3p mimic/inhibitor/Negative control: diluting miR-199a-3p mim/inhibitor/Negative control with Opti-MEM serum-free medium, mixing gently, and incubating at room temperature for 5min (miR-199 a-3p mim cell transfection final concentration is 50nmol/L, miR-199a-3p inhibitor cell transfection final concentration is 100 nmol/L);

(2) dilution Lipo3000: diluting the Lipo3000 solution with Opti-MEM serum-free medium to a final concentration of 0.1875% (v/v), gently mixing and incubating at room temperature for 5min;

(3) gently mixing the above (1) and (2), and incubating at room temperature for 15min.

(3) Cell transfection: discarding a cell primary culture medium to be transfected, adding a 1640 complete culture medium, adding the prepared transfection solution, culturing for 48h in an incubator at 37 ℃, and detecting the change of the expression level of miR-199a-3p by adopting an RT-qPCR method.

The relative expression of miR-199a-3p under the transfection conditions was calculated using the Delta-Delta Ct method with the U6 gene as the internal reference and the 5-8F and CNE-2 negative control groups as the controls, and the results are shown in FIG. 9 and FIG. 10.

The results in FIG. 9 show that the expression level of miR-199a-3p in cells of a transfection group of CNE-2 and 5-8F cells is remarkably increased under the conditions that the miR-199a-3p mimic transfection concentration is 50nmol/L and the transfection time is 48 h. The results in FIG. 10 show that miR-199a-3p inhibitor with the concentration of 100nmol/L transfects 5-8F and CNE-2, and the expression quantity of miR-199a-3p is only 0.24 times and 0.14 times of that of a negative control group after 48 hours of action. The difference was statistically significant compared to the control group (P < 0.01).

2) Over-expression miR-199a-3p for inhibiting migration invasion capacity of nasopharyngeal carcinoma cells

A. And observing the change of the wound healing capacity of the transfected nasopharyngeal carcinoma cells by miR-199a-3p mimic by adopting a scratch experiment.

(1) Drawing a horizontal line mark on the back of the 6-hole plate by using a marker pen before inoculating the cells on the 6-hole culture plate (facilitating the positioning in the same visual field when photographing at different time points);

(2) Appropriate number of cells were seeded in 6-well plates, each set of cells was controlled, NC, mic 3, each set was established with 3 replicate wells, and transfection was performed separately. When transfected cells grow to be fused in a single layer, the old culture medium is discarded, and 20 mu l of tip heads are used for scratching the single-layer cells in an I shape with the help of a ruler;

(3) Carefully cleaning with PBS for three times, adding serum-free RP1640 culture medium, continuously culturing, observing wound healing degree after scratching for 0h, 24h, 48h and 72h, and taking pictures with microscope under 100 times visual field (eyepiece 10 x, objective 10 x);

(4) And analyzing the result according to the collected picture data.

The migration of the cells was observed by an inverted microscope at each time point of 0h, 24h, 48h and 72h and photographed, and the results are shown in FIG. 11. The results in FIG. 11 show that, over time, the 5-8F and CNE-2NC groups of cells have stronger healing capacity than the miR-199a-3p mimic group of cells at each time point.

B. Transwell cell invasion (invasion) experiment

(1) Coating of basement membrane (on ice operation): (1) placing Matrigel (Matrigel) in a refrigerator at 4 ℃ overnight to slowly melt the Matrigel into liquid, and placing an EP tube and a gun head for experiments at-20 ℃ for cold storage for later use; (2) the Matrigel was diluted with pre-cooled RP1640 according to a ratio of 8 (RP 1640: matrigel); (3) the transwell chamber was placed on a 24-well plate with tweezers, and 80. Mu.l of the diluted gel was pipetted into the 24-well plate upper chamber (transwell chamber) to avoid air bubbles during the experimental procedure; (4) the transwell cell was incubated at 37 ℃ for at least 4h to form a solid artificial basement membrane.

(2) Hydration of basement membrane: the residual liquid in the plate was aspirated, and 50. Mu.l of serum-free medium containing 10g/L BSA was added to each well at 37 ℃ for 30min.

(3) Preparing a cell suspension: after 24h of transfection treatment, each group of cells are washed once by PBS, cultured for 12h by a medium without serum, digested and collected, washed twice by PBS, resuspended by RP1640 without serum, and diluted to have the cell density of 2 multiplied by 10 ⁵ Perml, aspirate cell suspension 200. Mu.l into transwell chamber, lift the upper chamber with forceps, add 600. Mu.l of medium containing 10% FBS into the lower 24-well plate, gently replace the upper chamber into the 24-well plate, and take care to avoid air bubbles. Culturing in an incubator at 37 ℃ for 24h.

(4) Fixing: the transwell chamber was removed from the 24-well plate, the upper chamber medium was removed, the upper chamber cells were wiped off with a cotton swab carefully rotating, and washed 2 times with PBS. The chamber was fixed in 4% paraformaldehyde for 30min.

(5) Dyeing: the transwell chamber was removed from the 24-well plate, 600. Mu.l of 0.1% crystal violet was added to the lower chamber to avoid bubbles between the bottom of the upper chamber and the staining solution, incubated at 37 ℃ for 30min, and washed 2-3 times with PBS.

(6) And (4) observation and counting: the number of cells passing through the membrane as a whole was observed under a high power field, and several fields were randomly picked up for photographing under a 200 power field (eyepiece 10 ×, objective 20 ×), and 3 to 5 fields were randomly selected for counting and recording.

C. Transwell cell migration (migration) experiments: the other experimental procedures were the same as in the transwell chamber invasion experiment without Matrigel.

the results of the transwell chamber experiments show that the number of transmembrane cells in the upper chamber of the transwell after miR-199a-3p mimic transfection of nasopharyngeal carcinoma cells is obviously less in the migration experiment (FIG. 12) and the invasion experiment (FIG. 13) than in the blank control group and the NC group. The difference is statistically significant.

3) Inhibiting migration and invasion capacity of miR-199a-3p expression reversible nasopharyngeal carcinoma cells

And (3) observing the change of the wound healing capacity of the transfected nasopharyngeal carcinoma cells by using a scratch experiment, wherein the experiment operation is the same as that in the step 2) A. The migration of the cells was observed by an inverted microscope at each time point of 0h, 24h, 48h and 72h and photographed, and the results are shown in FIG. 14. The results in FIG. 14 show that the 5-8F and CNE-2inhibitor groups had stronger healing capacity than the NC group cells at each time point, and the difference became more and more obvious with the passage of time. In addition, the results of the transwell chamber experiments show that the number of transmembrane cells in the upper chamber of the transwell after miR-199a-3p inhibitor transfects nasopharyngeal carcinoma cells in the migration experiment (FIG. 15) and the invasion experiment (FIG. 16) is obviously less than that in the blank control group and the NC group, and the difference is statistically significant.

4) miR-199a-3p regulates expression of nasopharyngeal carcinoma cell migration invasion related gene and protein

The EMT characterization protein is E-cadherin and N-cadherin; and (3) transferring related proteins MMP2 and MMP9.

In order to clarify the action mechanism of miR-199a-3p for inhibiting nasopharyngeal carcinoma cell migration and invasion capacity, a western blot experiment is adopted to detect the expression change of cell migration and invasion related proteins before and after nasopharyngeal carcinoma cell transfection. As a result, compared with the control group and the NC group, the expression levels of EMT (epithelial-mesenchymal transition) and metastasis related proteins N-cadherin, MMP2 and MMP9 of the miR-199a-3p imic transfected 5-8F cells are reduced, and the expression level of E-cadherin protein is increased; meanwhile, when the expression of miR-199a-3p is reduced, the expression of N-cadherin, MMP2 and MMP9 is increased, and the expression of E-cadherin is reduced (FIG. 17); similar results were obtained in miR-199a-3p imic transfected CNE-2 cells (FIG. 18). RT-qPCR experiments are utilized to detect the expression change of related genes, and the results show that after miR-199a-3p imic is transfected, the mRNA expression levels of the related genes of 5-8F and CNE-2 cells are different, and the difference has statistical significance (figures 19-20).

5) Expression of target gene SCD1 regulated by miR-199a-3p

Early-stage experiment results show that miR-199a-3p can inhibit invasion and migration of nasopharyngeal carcinoma cells and EMT (acute respiratory syndrome) effect. In order to disclose a molecular mechanism of miR-199a-3p for inhibiting nasopharyngeal carcinoma cell migration and invasion, a bioinformatics method is adopted to predict a target gene of miR-199a-3p, a gene SCD1 which has higher score and is related to fatty acid metabolism is screened, and a conserved binding site ACUACUG (SEQ ID NO. 2) between miR-199a-3p and SCD13' UTR is predicted, as shown in figure 21.

To further verify that miR-199a-3p can be directly bound to SCD13' UTR, dual luciferase vectors human-SCD-wt and human-SCD-mut were constructed. The insertion sequences of the wild type and mutant plasmids are shown in FIG. 21.

The construction method of the dual-luciferase vectors human-SCD-wt and human-SCD-mut comprises the following steps: construction of wt/mut-SCD13' UTR plasmid, PCR amplification primers based on SCD1 (human) 3' UTR sequence information (Gene accession No.: NM-005063.5), PCR amplification of SCD1 gene 3' UTR sequence using 293T genomic DNA as a template, cloning to pmiR-RB-REPORT ^TM In a dual-luciferase report vector (purchased from Rumbo biology, guangzhou), the report fluorescence of the used vector is Renilla luciferase gene hRluc, and the corrected fluorescence is firefly luciferase gene hLuc (serving as an internal reference gene);

wherein the primer sequence of the amplification primer is as follows:

h-SCD-3UTR-F(8)：

5’-GGCGCTCGAGGTCCCTCAGGTTCCTTTTTC-3’；SEQ ID NO.3；Xho I；

h-SCD-3UTR-R(651)：

5’-AATGCGGCCGCGGGCTTGAAGTCCTCATTAGG-3’；SEQ ID NO.4；Not I。

designing a mutation primer to mutate a target sequence ACTACTACTG (SEQ ID NO. 5) (67-73) into TGATAC (SEQ ID NO. 6) on the basis of a wild type (wt) vector by utilizing a PCR mutation method to construct a mutant type (mut) vector;

wherein, the primer sequence of the mutation primer is as follows:

h-SCD-3UTR-MUT-F：

5’-GTTTATTATGATGACAATAATGCTACCAGGATG-3’；SEQ ID NO.7；

h-SCD-3UTR-MUT-R：

5’-GCATTATTGTCATCATAATAAACAGACATTAAA-3’；SEQ ID NO.8。

inoculating an appropriate amount of A549 cells into a 24-well plate, adding 5% CO ₂ And culturing in an incubator at 37 ℃, and performing cotransfection when the cell fusion degree reaches 50-70 percent, wherein the cotransfection comprises 4 groups: SCD1-WT/NC, SCD1-WT/miR-199a-3p, SCD1-Mut/NC, SCD1-Mut/miR-199a-3p. After 48h of cotransfection, the original culture medium was discarded, washed 2 times with PBS and blotted dry. Adding 100 μ l of 1 XPBL lysate into each well, placing on a shaking table, shaking for 15min at room temperature, fully cracking, absorbing 20 μ l into a black 96-well plate, preparing 3 composite wells for each sample, adding 100 μ l of fluorescein reaction reagent LAR II into each well, mixing gently for 3-4 times by a gun, detecting on a machine for 2sec, delaying for 10sec, recording the activity of firefly luciferase (hLuc), taking out the 96-well plate, adding 100 μ l into each well, and performing light shaking for 15min

And mixing gently with gun for 3-4 times, and detecting on machine to obtain renninase activity (hRluc). The relative activity of luciferase was taken as the ratio of renilla luciferase activity to firefly luciferase.

The result shows that the relative luciferase activity of the cell group co-transfected by the miR-199a-3p mimic and the wild-type plasmid is obviously lower than that of the cell group co-transfected by the miR-199a-3p mimic and the mutant plasmid, and the difference has statistical significance (figure 22). The result shows that SCD1 is a direct target gene of miR-199a-3p, and miR-199a-3p can perform biological function regulation through SCD1.

The result of Western Blot experiment shows that the expression level of SCD1 in the cell group transfected with miR-199a-3p imic is reduced, the protein expression level of SCD1 is increased after miR-199a-3p inhibitor is transfected, and RT-qPCR experiment also shows similar expression change, which indicates that the expression change of SCD1 is targeted and inhibited by miR-199a-3p at the transcription level to influence the protein expression level (fig. 23-24). In addition, PTEN protein molecule expression change is also found to be opposite to SCD1, and is probably related to downstream signal pathway molecule regulation and is needed to be further verified subsequently.

6) miR-199a-3p inhibits invasion and migration of nasopharyngeal carcinoma cells through regulation and control of SCD1 on fatty acid metabolism change

(1) In order to understand the relationship between miR-199a-3p and fatty acid metabolism, the relative content change of the fatty acid after miR-199a-3p mimic/inhibitor transfection of 5-8F and CNE-2 cells is analyzed by a gas chromatography experiment. Since SCD1 is stearoyl-coa desaturase 1, also known as a delta-9-fatty acid desaturase, the relative amounts of the four fatty acids C16:0, C16:1, C18:0, and C18:1 are of interest.

The experimental procedure for gas chromatography was as follows:

(1) 5-8F and CNE-2 cells were plated in 6cm dishes and transfected with miR-199a-3p imic/inhibitor for 48h, and the cells were collected in a clean EP tube for use.

(2) And (3) extraction: a. preparing chloroform: methanol =2 extract in a 50ml EP tube for use; b. after numbering each sample, adding 4ml of extract liquor, fully and uniformly mixing cells, and transferring the cells into a methyl esterification tube; c. vortexed for 30sec; shaking overnight; d. shaking overnight, adding 1ml PBS, vortexing for 30sec, and centrifuging at 3000r/m for 5min; e. and sucking the lower layer liquid into a new EP pipe, and drying by nitrogen.

(3) Methyl esterification: a. adding 1.5ml of BF3 and 1.5ml of n-hexane into a sample, fully and uniformly mixing, transferring into a methyl esterification pipe, and numbering; b. vortexed for 30sec; c. heating in a 100 ℃ dry heating instrument for 1h, vortexing once every 20min, checking whether a cover of the methyl esterification pipe is screwed (whether a page is greatly reduced within 5min of heating at the beginning, if yes, the page is not screwed), taking out the methyl esterification pipe and cooling to normal temperature if not screwed, complementing normal hexane to the original liquid level height, or replacing a new methyl esterification pipe and reheating; d. cooling at room temperature for 5min; e. adding 1ml PBS, covering the cover tightly, whirling for 30sec, and centrifuging for 5min at 3000 r/m; f. transferring the supernatant to a clean EP tube, and drying by nitrogen; g. adding 100 μ l n-hexane to dissolve white powder completely, transferring to a new sample loading chamber, numbering, and storing in a 4 deg.C refrigerator or immediately testing on a machine.

(4) Detecting on a gas chromatograph: corresponding solvent control wells (n-hexane), standard wells and sample wells were set in the GC instrument. If the number of samples is large, the number of solvent control wells and standard wells should be increased between about 6 assays to increase the accuracy of the assay.

As a result, compared with the control group and the NC group, after miR-199a-3p mimic transfection, the ratio of C16:0: c16:1 and C18:0: the C18:1 ratio increased, in contrast, after miR-199a-3p inhibitor transfection, C16:0: c16:1 and C18:0: the C18:1 ratio decreased (fig. 25 and 26). The results demonstrate that miR-199a-3p can regulate fatty acid metabolism of nasopharyngeal carcinoma cells by targeting SCD1. Three independent replicates of this experiment were performed, and the differences were statistically significant.

(2) The role of stearoyl-CoA desaturase SCD1 is to convert saturated fatty acids into unsaturated fatty acids, of which C18:1 Oleic Acid (OA) is one of its important products. Subsequent experiments were performed to select appropriate drug concentrations to act on cells in order to understand the toxic effects of OA on nasopharyngeal carcinoma cells.

The CCK-8 method is used for detecting the toxic effect of OA on nasopharyngeal carcinoma cells, and the specific operation is as follows:

(1) cells in the logarithmic growth phase were collected by digestion and counted by resuspension. The total cell demand was calculated from experimental grouping and the duplicate well requirements, 5000 cells were seeded per well in 96-well plates, 100. Mu.l complete medium per well. 37 ℃,5% of CO ₂ The culture was conditioned overnight.

(2) Transfecting the cells, and after 8 hours of transfection treatment, respectively treating 5-8F cells with OA under a concentration gradient of 20-640 mu M;

(3) after OA treatment for 24h or 48h, the original culture medium is discarded, CCK-8 reaction reagent is added, 90. Mu.l of complete culture medium and 10. Mu.l of CCK-8 reagent are added into each well of a 96-well plate, incubation is carried out at 37 ℃ for 2h, 100. Mu.l of PBS is added into a blank hole circle around to prevent culture evaporation, and the absorbance value at 450nm is measured by an enzyme-linked immunosorbent assay.

The toxic effect of OA on nasopharyngeal carcinoma cells was found to be slight, and the change in absorbance of the cells only started to be statistically significant when the cells were exposed to 320. Mu.M-640. Mu.M for 48 hours (FIGS. 27 and 28), so that the cells were treated under the condition that OA (80. Mu.M) was exposed for 24 hours.

(3) 5-8F and CNE-2 cells were treated with C18:1 or C18:1 in combination with miR-199a-3pmimic, and transwell chamber migration and invasion experiments were performed to see if the migration invasion capacity of the cells changed. The experimental results show that the number of cell penetrating cells is increased after the cells are treated by OA (80 mu M), and the number of cell penetrating cells is correspondingly reduced after the cells are treated by OA in combination with miR-199a-3pmimic, which indicates that the OA can promote the migration and invasion of nasopharyngeal carcinoma cells, and the miR-199a-3pmimic can reverse the cancer promotion effect of the OA (FIG. 29 and FIG. 30).

(4) To further understand whether OA modulates invasive migration of nasopharyngeal carcinoma cells at the protein level, western blot experiments were performed. Experimental results show that after cells are treated by OA, the protein expression of SCD1, p-Akt, N-cadherin, MMP2 and MMP9 is increased, the protein expression of PTEN and E-cadherin is reduced, and the protein expression change can be correspondingly reversed after the treatment of miR-199a-3p mim (figure 31). The result shows that OA can reversely over-express the invasion migration inhibition effect caused by miR-199a-3p to a certain extent, and the action of OA can be related to the expression regulation of cancer suppressor PTEN.

(5) SCD1 can reverse the influence of miR-199a-3p on nasopharyngeal carcinoma cell migration invasion and EMT through PI3K-Akt signal pathway

In order to further verify that miR-199a-3p plays a role in inhibiting invasion and migration of nasopharyngeal carcinoma cells by virtue of targeted regulation of expression of SCD1, plasmids which over-express SCD1 are transfected into 5-8F and CNE-2 cells, the expression level of exogenous SCD1 of the cells is improved, and further the expression change of cell transfer related proteins is observed.

(1) Preparation of vector for overexpression of SCD1 plasmid

Amplifying the SCD1 segment by using a primer to construct an overexpression SCD1 plasmid vector;

wherein the primer sequences are as follows:

SCD(41369-1)-P1：

5’-CGAGCTCAAGCTTCGAATTCCGCCACCATGCCGGCCCACTTGCTGCAGG-3’；SEQ ID NO.9；

SCD(41369-1)-P2：

5’-TGGTGGCGACCGGTGGATCCCGGCCACTCTTGTAGTTTCCATCTC-3’；SEQ ID NO.10。

(2) cell plating: an appropriate number of cells were seeded into 6-well plates and ready for cell transfection when the cells were grown to a confluence of 50% -80%.

(3) Preparing a transfection solution: tube A, 125. Mu.l of Opti-MEM + 2.5. Mu.g of SCD1 overexpression plasmid + 5. Mu.l of P3000TM reagent, gently mixing, and incubating at room temperature for 5min; tube B125. Mu.l Opti-MEM + 3.75. Mu.l

3000 reagent, gently mixing, and incubating at room temperature for 5min; mix and mix the tubes and incubate at room temperature for 15min.

(4) Cell transfection: discarding the original culture medium of the cells to be transfected, adding 1.75ml 1640 complete medium, adding the prepared transfection solution thereto, shaking the culture solution in a culture dish uniformly, 5% CO ₂ Culturing in an incubator for 48h, and carrying out Western blot experiment.

Western blot results show that after 5-8F cells are transfected with SCD1 overexpression plasmids for 48 hours, compared with transfected unloaded plasmid groups, the expression of SCD1, p-Akt, N-cadherin, MMP2 and MMP9 proteins is increased, and the expression of PTEN and E-cadherin proteins is reduced; meanwhile, the SCD1 overexpression can inhibit miR-199a-3p from causing the reduction of SCD1, p-Akt, N-cadherin, MMP2 and MMP9 protein expression of 5-8F cells and the increase of PTEN and E-cadherin protein expression. In the same way, consistent experimental results were obtained in CNE-2 cells (FIG. 32). The result shows that the transfection SCD1 overexpression plasmid can reverse the inhibition effect of miR-199a-3p on the invasion and migration of nasopharyngeal carcinoma cells, and the suggestion that miR-199a-3p can play the inhibition effect on the invasion and migration of nasopharyngeal carcinoma cells and EMT through the targeted regulation and control of SCD1 expression.

Meanwhile, in order to disclose a molecular mechanism of SCD1 for regulating invasion and migration of nasopharyngeal carcinoma cells, and combine all the experimental results presented above, SCD1 may influence the invasion and migration of nasopharyngeal carcinoma and EMT by influencing PI3K-Akt pathway; whether this signal pathway has a direct effect is reversely verified by the PI3K inhibitor LY 294002. LY294002 was dissolved in DMSO and acted on cells at a final concentration of 20. Mu.M, and the experimental results showed that, after addition of the LY294002 inhibitor, the expression of the p-Akt molecule decreased and the expression of downstream molecules changed, the PI3K-Akt pathway was inhibited, and that the LY 294002-associated miR-199a-3p inhibitor-treated group restored to some extent the inhibition of the signaling pathway compared with the LY294002 inhibitor-treated group alone (FIG. 33).

Example 3 Effect of miR-199a-3p on the in vivo tumorigenic ability of nasopharyngeal carcinoma cells

1) miR-199a-3p can remarkably inhibit in-vivo tumorigenesis capability of nasopharyngeal carcinoma cells

In order to further verify the inhibition effect of miR-199a-3p overexpression on nasopharyngeal carcinoma invasion and metastasis, a human nasopharyngeal carcinoma nude mouse transplantation tumor model is established by respectively adopting human nasopharyngeal carcinoma 5-8F and CNE-2 cells. The influence of miR-199a-3p on nasopharyngeal carcinoma is observed under in vivo conditions.

(1) The method comprises the steps of establishing an animal model by adopting cultured nasopharyngeal carcinoma cell allotransplantation, culturing a large number of human nasopharyngeal carcinoma 5-8F and CNE-2 cells, performing conventional digestion and collection on each group of cells, gently blowing and beating by using a pipetting gun, fully and uniformly mixing, and calculating the number of living cells, wherein the number of the living cells is more than 95 percent, and the cells can be used for subsequent experiments. The medium was removed by centrifugation at 1000rpm for 5min, washed once by addition of serum-free 1640 medium and centrifuged to remove the supernatant. Adding appropriate amount of PBS to resuspend cells, and counting cells to make cell suspension density 2X 10 ⁷ And (3) mixing the cell suspension uniformly, subpackaging the cell suspension in 100 mu l/tube PCR, marking, placing on ice, and inoculating as soon as possible within 30 minutes.

(2) And selecting 4-week-old nude mice, breeding for one week, and then marking the numbers of the ear nails for later use. Taking the waist of a nude mouse as an inoculation part, sucking 100 mu l of the uniformly subpackaged cell suspension by using a 1ml syringe after local disinfection, injecting the cell suspension into the subcutaneous tissue of the waist of the nude mouse stably and slowly, slightly pressing the needle insertion part for a plurality of seconds, and then pulling out the needle head, wherein the whole process complies with the aseptic operation principle. On the sixth day after inoculation, the nude mice were observed to have obvious tumor tissue masses at the inoculated parts. The experimental process keeps the feed, drinking water and growth environment of experimental animals normal, the life quality of the experimental animals is observed every 2 days, and the weight and the tumor volume of the nude mice are measured and recorded. The long diameter (a) and the short diameter (b) of the tumor tissue were measured with a vernier caliper, and the tumor volume formula was V = ab ² 2, unit is mm ³ 。

(3) Intratumoral injection of agomir-199a-3p and agomir-NC was initiated 10 days after subcutaneous tumorigenesis of tumor cells. The experiments were divided into 4 groups, namely, 5-8F agomiR-NC group, 5-8F agomiR-199a-3p group, CNE-2agomiR-NC group, and CNE-2agomiR-199a-3p group. SPF grade 5 BALB/c-nu mice per group. The fractions agomir-199a-3p and agomir-NC were dissolved in a sterile worktop once every 4 days, with a first injection of 1nmol, a second injection of 1.5nmol, a third injection of 1.5nmol, and a fourth injection of 2nmol per tumor. And (3) injecting chloral hydrate into the abdominal cavity of the nude mice to kill the nude mice after the 26 th day of subcutaneous tumorigenesis, taking out tumor tissues, and respectively drawing a weight change curve and a tumor volume growth curve of the nude mice.

The results show that both of the cell-transplanted tumors in the mice of the agomiR-199a-3 p-treated group grew more slowly and tumor volumes were significantly smaller with statistical significance compared to the agomiR-NC control group over time from day 6 (fig. 34 and fig. 35). In addition, the body weights of the nude mice of the treatment groups were not significantly different (FIG. 36), but the tumor weights of the agomiR-NC group were significantly greater than those of the agomiR R-199a-3p group, suggesting that miR-199a-3p can effectively inhibit the in vivo tumorigenic ability of nasopharyngeal carcinoma cells.

2) miR-199a-3p can inhibit expression of metastasis related protein in nude mouse tumor nasopharyngeal carcinoma transplanted tumor tissue

Injecting chloral hydrate into abdominal cavity to kill nude mice after subcutaneous tumorigenicity day 26, taking out tumor tissue, storing one part of the tumor tissue in a refrigerator at-80 ℃ for extracting tissue RNA to perform RT-qPCR experiment, fixing the other part of the tumor tissue with 4% paraformaldehyde for 24h, and performing conventional dehydration, paraffin embedding and slicing to perform HE staining and immunohistochemical staining to detect expression change of various related proteins.

RT-qPCR experiments are carried out on total RNA of tissue extracted from CNE-2 cell xenograft tumor tissues, gene expression levels of miR-199a-3p and SCD1 are detected, and as a result, the Agomir-NC group and the agomiR-199a-3p group are found to have higher SCD1 expression level and relatively lower miR-199a-3p expression level (FIG. 37). In order to explore whether the molecular expression of the xenograft tumor under the in vivo experimental condition has expression difference after injecting the agomiR-NC and the agomiR-199a-3p in the tumor, the expression of related protein is detected by paraffin-embedded immunohistochemical staining. HE staining results are shown in fig. 38. Immunohistochemistry results show (FIG. 39), that SCD1 protein is generally expressed in tumor tissues, but the protein expression intensity in the agriR-199 a-3p group is lower than that in the agrimir-NC group, and MMP2 protein expression also has similar results. However, the expression of E-cadherin and PTEN proteins was higher in the agromiR-199 a-3p group, where E-cadherin localization was very apparent in the cell membrane. This is consistent with the results of the experiments in the cell experiments.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Guangdong medical university Hospital affiliated to Guangdong medical university

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> RNA

<213> Artificial Sequence

<400> 1

acaguagucu gcacauuggu ua 22

<210> 2

<211> 7

<212> RNA

<213> Artificial Sequence

<400> 2

acuacug 7

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 3

ggcgctcgag gtccctcagg ttcctttttc 30

<210> 4

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 4

aatgcggccg cgggcttgaa gtcctcatta gg 32

<210> 5

<211> 7

<212> DNA

<213> Artificial Sequence

<400> 5

actactg 7

<210> 6

<211> 7

<212> DNA

<213> Artificial Sequence

<400> 6

tgatgac 7

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 7

gtttattatg atgacaataa tgctaccagg atg 33

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 8

gcattattgt catcataata aacagacatt aaa 33

<210> 9

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 9

cgagctcaag cttcgaattc cgccaccatg ccggcccact tgctgcagg 49

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 10

tggtggcgac cggtggatcc cggccactct tgtagtttcc atctc 45

Claims

The application of miR-199a-3p in the preparation of medicines for treating nasopharyngeal carcinoma is characterized in that the miR-199a-3p can inhibit the invasion and metastasis of the nasopharyngeal carcinoma by regulating the expression of SCD1; SCD1 is a target gene of the miR-199a-3 p;

the nucleotide sequence of the miR-199a-3p is shown in SEQ ID NO. 1:

5’-ACAGUAGUCUGCACAUUGGUUA-3’。