CN115948548A

CN115948548A - circRNA related to development and development of non-small cell lung cancer and application thereof

Info

Publication number: CN115948548A
Application number: CN202211100552.0A
Authority: CN
Inventors: 王友亮; 林艳丽; 崔雨萌; 吴晓洁; 李响
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-09-09
Filing date: 2022-09-09
Publication date: 2023-04-11

Abstract

The invention discloses circRNA related to the occurrence and development of non-small cell lung cancer and application thereof. The invention provides an application of a substance for detecting the expression of circular RNA circ _0035796 in preparing a product with any function of 1) diagnosing or assisting in diagnosing non-small cell lung cancer; 2) Screening or screening-assisted screening for non-small cell lung cancer; the nucleotide sequence of the circular RNA circ _0035796 is 21 th to 1163 rd position of the sequence 1. The invention discovers that the expression quantity of the circRNA in the cancer tissue of a lung cancer patient is obviously up-regulated compared with the expression quantity of the circRNA in the tissue beside the cancer. An overexpression or knockout experiment is carried out on circ _0035796, and the experiment proves that the polypeptide can effectively promote the proliferation, invasion and metastasis of lung cancer and is closely related to the occurrence and development of the lung cancer. The circ _0035796 is expected to become a novel biomarker for early screening and diagnosis of lung cancer and a new target point for accurate diagnosis and treatment.

Description

circRNA related to development and development of non-small cell lung cancer and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to circRNA related to development of non-small cell lung cancer and application thereof.

Background

Lung cancer is one of malignant tumors with high incidence, high malignancy, poor drug treatment effect, high mortality and poor prognosis worldwide. The development and progression of lung cancer is a multi-stage and multi-step complex process of genetic alteration of proto-oncogenes and tumor suppressor genes, and besides, epigenetic alterations are also widely present in the development and progression of tumors. The genome of tumor cells is essentially genetically altered, for example, by DNA methylation, histone modification, regulation of non-coding RNA, and upregulation of certain epigenetic factors. Non-coding RNA, one of the apparent regulatory effects, has been of great interest in current tumor studies, circular RNA (circRNA), an RNA in a closed circular structure, produced primarily from pre-mRNA by "reverse splicing" (an alternative RNA splicing), and located mostly in the cytoplasm and only a few in the nucleus. Compared with linear RNA, the covalent ring structure of the circRNA is not easy to be degraded by exonuclease, and the structural stability is higher.

In recent years, with the rapid development of new-generation sequencing technology and bioinformatics, it is found that circRNA plays an important role in the occurrence and development of tumors, and the circRNA widely participates in the proliferation, apoptosis, differentiation, invasion, migration, angiogenesis and the like of tumor cells and has close relation with the occurrence, development and prognosis of various tumors. In particular, the circRNA can show differential expression in various malignant tumors and normal tissues and participate in the occurrence and development processes of a series of tumors including lung cancer. The major biological functions of circRNA include acting as a miRNA sponge, interacting with RNA binding proteins, and as a protein translation template, among others. The circRNA molecule plays a role of miRNA sponge in cells, so that the inhibition effect of miRNA on target genes is relieved, and the expression level of the target genes is improved, and the effect is called a competitive endogenous RNA mechanism (cerRNA).

Disclosure of Invention

An object of the present invention is to provide a use of a substance for detecting the expression of circular RNA circ _ 0035796.

The invention provides an application of a substance for detecting circular RNA circ _0035796 expression in preparing a product with any one of the following functions:

a1 Diagnosis or assisted diagnosis of non-small cell lung cancer;

a2 Differential diagnosis or differentiation of benign nodules of the lung from non-small cell lung cancer;

a3 Screening or assisted screening for non-small cell lung cancer;

a4 Diagnosing or aiding in diagnosing whether the test patient is a non-small cell lung cancer patient;

a5 Screening or assisting in screening whether the patient to be tested is a non-small cell lung cancer patient;

a6 To diagnose or aid in diagnosing whether the test sample is derived from non-small cell lung cancer tissue;

a7 Screening or assisting in screening whether the patient to be tested is derived from non-small cell lung cancer tissue;

the nucleotide sequence of the circular RNA circ _0035796 is 21 th to 1163 rd position of the sequence 1.

In the application, the substance for detecting the expression of the circular RNA circ _0035796 comprises a probe specifically binding to circ _0035796 or a primer specifically amplifying circ _ 0035796.

In the embodiment of the invention, the primer for specifically amplifying circ _0035796 is a primer pair consisting of a sequence 2 and a sequence 3.

The sample to be tested in the above application is derived from plasma or tumor tissue.

It is another object of the invention to provide a product.

The product provided by the invention comprises a substance for detecting the expression of circular RNA circ _ 0035796;

the product has any one of the following functions:

a1 Diagnosis or assisted diagnosis of non-small cell lung cancer;

a3 Screening or screening aid for non-small cell lung cancer;

a5 Screening or screening the patient to be tested for non-small cell lung cancer;

a6 ) diagnosing or aiding in diagnosing whether the test sample is derived from non-small cell lung cancer tissue;

a7 Screening or aiding in screening whether the test patient is derived from non-small cell lung cancer tissue.

In the above, the subject to be screened or diagnosed is a lung tumor patient;

in the above, the patient to be tested is a suspected non-small cell lung cancer patient; the sample to be detected is derived from a sample of a suspected non-small cell lung cancer patient;

the application of the above circular RNA circ _0035796 as a marker in the development or design of products for diagnosis, auxiliary diagnosis, screening or auxiliary screening of non-small cell lung cancer is also the protection scope of the present invention.

The application of the substance for inhibiting the expression of the circular RNA circ _0035796 in any one of the following is also within the protection scope of the invention:

b1 For treating non-small cell lung cancer;

b2 Inhibit proliferation of non-small cell lung cancer cells;

b3 Inhibit migration of non-small cell lung cancer cells;

b4 Inhibit non-small cell lung cancer cell invasion;

b5 Inhibit the formation of tumors in vivo by non-small cell lung cancer cells.

It is yet another object of the invention to provide such another product.

The product provided by the invention is a substance for inhibiting the expression of circular RNA circ _ 0035796;

the product has any one of the following functions:

b1 For treating non-small cell lung cancer;

b2 Inhibit proliferation of non-small cell lung cancer cells;

b3 Inhibit migration of non-small cell lung cancer cells;

b4 Inhibit non-small cell lung cancer cell invasion;

b5 Inhibit the formation of tumors in vivo by non-small cell lung cancer cells.

The substance for inhibiting the expression of the circular RNA circ _0035796 is a substance for interfering or silencing the expression of circ _ 0035796.

The substance for interfering or silencing the expression of circ _0035796 is specifically a DNA molecule (sequence 4 in the embodiment of the invention) for interfering the expression of circ _0035796 or a vector for expressing the DNA molecule.

The application of the circular RNA circ _0035796 in preparing an animal model or a cell model for screening the medicine for treating the non-small cell lung cancer is also within the protection scope of the invention.

The invention discovers that circ _0035796 is up-regulated in the plasma of a patient with non-small cell lung cancer, and the sensitivity and the specificity of the invention for diagnosing the non-small cell lung cancer are higher. The expression level of the circRNA in the cancer tissue of the patient with the non-small cell lung cancer is obviously up-regulated compared with that in the tissue beside the cancer. The circ _0035796 is subjected to overexpression or knockout experiment, and the experiment proves that the polypeptide can effectively promote the proliferation, invasion and metastasis of the non-small cell lung cancer, and is closely related to the generation and development of the lung cancer. The circ _0035796 is expected to become a novel biomarker for early screening and diagnosis of the non-small cell lung cancer and a new target point for accurate diagnosis and treatment.

Drawings

FIG. 1 is a heat map of the expression of circ _0035796 in the plasma of benign patients, non-small cell lung adenocarcinoma patients and non-small cell lung squamous carcinoma patients.

FIG. 2 shows the expression of circ _0035796 in the plasma of different patients.

FIG. 3 is a ROC curve showing the relative expression of circ _0035796 in plasma.

FIG. 4 shows the expression of circ _0035796 in tumor and paracancerous tissues of a patient with non-small cell lung cancer.

FIG. 5 shows the circ _0035796 overexpression vector and siRNA expression in NCI-H3255 cells.

FIG. 6 shows the results of growth curves for circ0035796 high expressing cells and circ0035796 knock-out cells using CCK8 assay.

FIG. 7 shows the proliferation assay results of circ0035796 high expressing cells and circ0035796 knock-out cells using edu.

FIG. 8 is a graph showing the results of migration experiments for circ0035796 high expressing cells and circ0035796 knock-out cells.

FIG. 9 is a graph showing the results of invasion experiments in circ0035796 high expressing cells and circ0035796 knock out cells.

FIG. 10 is a graph showing the results of in vivo inhibition of tumor cell proliferation by circ0035796 high expressing cells and circ0035796 knock out cells.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Blood sample sources in the following examples: the subject group cooperates with the thoracic surgery of the general hospital of the people's liberation military in China, all non-small cell lung cancer patients and benign tubercle lung patients admitted to the hospital to be treated are selected as research objects, and the patients are fully informed and signed with informed consent before being admitted to the hospital to be treated. Then, a preoperative blood sample is collected and separated into three parts of blood plasma, PBMC and RBC, and the three parts are subpackaged into an RNase-free EP tube for freezing storage. The detailed medical record data of the patient including sex, age, smoking amount, smoking age, infectious virus infection, cancer history and other information is recorded and maintained during specimen collection.

The criteria for sample inclusion and grouping in the following examples were:

according to the clinical data of the patients, all blood samples of patients with the past cancer history or infectious virus infection (such as infectious hepatitis, syphilis, HIV and the like) are removed. Differential expression gene screening and detection were carried out using plasma specimens of patients with non-small cell lung adenocarcinoma and non-small cell lung squamous carcinoma who had not undergone any surgery, radiotherapy, chemotherapy or drug treatment (non-small cell lung carcinoma NSCLC) and plasma specimens of patients with BENIGN nodules of the lung (BENIGN, including alveolar epithelial hyperplasia, chronic granuloma, fibroplasia, polyp, etc.) as subjects (see table 1).

Table 1 is a summary of clinical data of patients

Note: the P value is calculated from the Chi-square test or Fisher's exact test for categorical variables and the T test or Wilcoxon rank-sum test for continuous variables.

Some of the experimental methods in the examples below are as follows:

1. separation of plasma

10mL of venous blood before fasting operation of a patient is collected in a blood collecting pipe containing EDTA as an anticoagulant, and separation operation is carried out within 2 hours after blood collection.

1) Freshly collected whole blood was centrifuged at 400 Xg for 15min at room temperature;

2) After the centrifugation is finished, the upper layer yellow clear liquid is plasma and is transferred into a 1.5mL centrifuge tube, and the plasma is frozen and stored at the temperature of 80 ℃ below zero for standby.

2. Extraction of plasma Total RNA

The miRNeasy Serum/Plasma Kit (QIAGEN, when used, buff RWT and Buff RPE were prepared by adding the specified absolute ethanol according to the instruction;

1) Taking 250 μ L of plasma separated from blood;

2) Centrifuging at 16000 Xg for 10min at 4 deg.C;

3) Carefully sucking 200. Mu.L of the upper layer plasma into a 1.5mL centrifuge tube;

4) Adding 1mL of QIAzol lysate, uniformly blowing, and standing at room temperature for 5 minutes;

5) Adding chloroform with the original volume (200 mu L) of the plasma sample, tightly covering, and uniformly mixing by vortex oscillation for 15 seconds;

6) Standing for 2-3 minutes at room temperature;

7) Centrifuging at 12000 Xg for 15min at 4 ℃;

8) After centrifugation the solution in the tube clearly separated into 3 layers and carefully pipetted the upper layer of about 600. Mu.L of aqueous phase into a new centrifuge tube, taking care not to pipette proteins in the middle layer. Adding 1.5 of absolute ethyl alcohol into the upper layer water phase (900 mu L) which is sucked out, fully blowing, beating and uniformly mixing;

9) Transferring the sample into a purification column sleeved with a collecting pipe, transferring at most 700 mu L of sample each time, covering tightly, centrifuging at room temperature 8000 Xg for 15s, and removing the filtrate in the collecting pipe;

10 Step 9) is repeated until all the sample is added to the purification column;

11 Add 700. Mu.L of Buff RWT to the spin column, cover tightly and centrifuge at 8000 Xg for 15 seconds at room temperature, discard the filtrate in the collection tube;

12 Add 500. Mu.L of Buff RPE to the spin column, cover tightly and centrifuge at 8000 Xg for 15 seconds at room temperature, discard the filtrate in the collection tube;

13 Adding 500 μ L of 80% absolute ethanol, covering tightly, centrifuging at room temperature of 8000 Xg for 2 min, and removing the collection tube and the filtrate in the tube;

14 Replacing the collection tube, opening the purification cover, centrifuging at high speed (about 15000 rpm) for 5 minutes at room temperature to dry the membrane on the purification column, and discarding the filtrate in the tube and the collection tube;

15 Changing the 1.5ml recovery tube, carefully adding 30. Mu.L of RNase-free-water to the center of the purification column membrane, standing at room temperature for 1 minute, and centrifuging at high speed (about 15000 rpm) for 1 minute to elute the RNA into the recovery tube.

3. Reverse transcription of plasma total RNA

1) Preparing 2 × Reverse Transcription mixed solution (Table 2, all components from Kit High capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Co., ltd., product number 4374967), and keeping on ice;

table 2 shows the reverse transcription reaction system

2) After preparing the reverse transcription mixed solution, adding the mixed solution into 8 connecting tubes respectively, and then adding 15 mu L of RNA samples respectively to prepare a reaction system of 30 mu L. Slightly blowing, uniformly mixing, tightly covering, and centrifuging for a short time;

3) Placed in a 96-well PCR instrument and set the reaction conditions as follows (table 3):

table 3 shows the reverse transcription PCR conditions

Example 1, circ _0035796 high expression in plasma and lung cancer tissues of lung cancer patients

1. Discovery of circular RNA circ _0035796

Deep sequencing of the whole transcriptome in plasma from non-small cell lung cancer (lung adenocarcinoma patients and lung squamous carcinoma patients) and benign sarcoidosis of the lungs using a second generation high throughput sequencing technique found that circular RNA circ _0035796 (hsa _ circ _ 0035796) was the circRNA with the highest fold up (fig. 1). The nucleotide sequence of the circular RNA circ _0035796 is 21 st to 1163 rd position of the sequence 1.

2. Expression of circular RNA circ _0035796 in plasma and lung cancer tissues of non-small cell lung cancer patients

1. Primer design primers were designed for real-time fluorescent quantitative PCR reactions based on the gene sequences of circ _0035796 and GAPDH, the specific primer names and sequences are given in table 4:

table 4 shows the sequence information of the primers used in the PCR reaction

2. qRT-PCR reaction system and conditions

Extracting RNA in blood plasma to be detected, carrying out reverse transcription to obtain cDNA as a template, and carrying out qRT-PCR by using the following system and program:

adding appropriate amount of RNase-free-water according to the primer synthesis instruction to prepare 10 μ M primer solution, using THUNDERBIRD from TOYOBO ^TM SYBR qPCR MiThe x Without ROX kit is prepared into a reaction system according to the instruction (table 5):

TABLE 5 qRT-PCR reaction systems

Adding the prepared qRT-PCR reaction system shown in Table 5 into a 96-well PCR plate, adding 18 mu L of the qRT-PCR reaction system shown in Table 5 into each reaction well, adding 2 mu L of cDNA into the reaction system to prepare 20 mu L of reaction system, and placing the reaction system into a real-time fluorescent quantitative PCR instrument for reaction, wherein the PCR reaction conditions are as follows (Table 6):

table 6 shows the reaction conditions of qRT-PCR

Each sample was plated with 3 duplicate wells using GAPDH as the reference gene. And after the qPCR reaction is finished, taking the average Ct value of three duplicate wells of the target gene in each sample as a final result. The calculation formula of the relative expression quantity of each target gene is 2^ -delta Ct, wherein the delta Ct = Ct (target gene) -Ct (reference gene).

The expression of circ _0035796 in the plasma of 48 non-small cell lung adenocarcinoma patients (labeled lung adenocarcinoma patients in the figure), 17 non-small cell lung squamous carcinoma patients (labeled lung squamous carcinoma patients in the figure) and 17 benign nodule in the lung patients (labeled normal patients in the figure) was detected by qRT-PCR.

As shown in FIG. 2, the circRNA was expressed in plasma of non-small cell lung cancer patients at a high level (P < 0.01) compared to normal patients.

Respective ROC curves were plotted in GraphPad Prim 6 based on the relative expression of circ _ 0035796.

The results are shown in fig. 3, the possibility of the circRNA as the non-small cell lung cancer diagnosis molecular marker is evaluated through the AUC value (area under the curve), the larger the AUC value, the higher the sensitivity and specificity of the gene as the non-small cell lung cancer diagnosis molecular marker, and it can be seen that the AUC value of the circRNA is 0.7790 (P = 0.0014-0.01), which indicates that the circRNA can be used as the non-small cell lung cancer diagnosis molecular marker and has higher sensitivity and specificity as the non-small cell lung cancer diagnosis.

The relative expression level of the lung cancer tissues and the paracancerous tissues circ _0035796 of 10 non-small cell lung cancer patients (8 non-small cell lung adenocarcinoma patients and 2 non-small cell lung adenocarcinoma patients) was detected by qRT-PCR. The template is cDNA obtained by reverse transcription of RNA of a tissue to be detected.

As shown in FIG. 4, the relative expression level of circ _0035796 in lung cancer tissue is significantly higher than that in paracarcinoma tissue (P < 0.05).

Therefore, circ _0035796 can be used as a molecular marker for diagnosing or screening non-small cell lung cancer; specifically, whether the patient to be detected is lung cancer is determined or screened by detecting the expression level of circ _0035796 in blood or tumor tissue of the patient to be detected;

if the circ _0035796 expression level in the blood of the patient to be detected is higher than the circ _0035796 expression level in the blood of the patient to be detected, the patient to be detected is or is selected as the patient with non-small cell lung cancer;

if the circ _0035796 expression level in the tumor tissue to be detected is higher than the circ _0035796 expression level in the tissue beside the cancer, the tumor tissue to be detected is derived or candidate derived from the patient with the non-small cell lung cancer.

The patient to be detected or the tumor to be detected is a suspected non-small cell lung cancer patient.

Example 2 use of circ _0035796 in the treatment of Lung cancer

1. construction of circ _0035796 overexpression plasmids or cells and knockdown plasmids or cells

1. construction of circ _0035796 overexpression plasmids and knockdown plasmids

Overexpression vector pLC5-circ _0035796: replacing DNA molecules between EcoR I enzyme cutting sites and BamH I enzyme cutting sites of a circRNA overexpression vector pLC5-ciR (Guangzhou Gisela Biotechnology Ltd.) with DNA molecules (sequence 1) containing a circ _0035796 linear sequence to obtain a vector;

the nucleotide sequence of the DNA molecule containing the circ _0035796 linear sequence is sequence 1, wherein the 3 rd to 8 th positions of the sequence 1 are EcoRI enzyme cutting sites, the 9 th to 18 th positions are forward cyclization mediation sequences, the 19 th to 20 th positions are AG receptors, the 1164 th to 1165 th positions are GT donors, the 1166 th to 1175 th positions are reverse cyclization mediation sequences, and the 1176 th to 1181 th positions are BamHI enzyme cutting sites.

The silent expression vector pLKO.1-circ _0035796 is a vector obtained by replacing a DNA molecule (sequence 4, sequence GCATCACTATCAGATATCA (joint of circular RNA, positions 1151-1163 and 21-26 of sequence 1) which interferes with the AgeI and EcoRI cleavage sites of plasmid pLKO.1 (Addgene) with a DNA molecule which interferes with the expression of circ _ 0035796.

2. construction of circ _0035796 overexpressing and knockdown cells

The over-expression vector pLC5-circ _0035796, the empty vector pLC5-ciR, the silent expression vector pLKO.1-circ _0035796 and the empty vector pLKO.1 were transfected into lung cancer cell NCI-H3255 (Shanghai Cheng Biotech Co., ltd., QC 473) respectively to obtain pLC5-circ _0035796 cell, pLC5-ciR cell, pLKO.1-circ _0035796 cell and pLKO.1 cell.

RNA was extracted from the cells of pLC5-circ _0035796, pLC5-ciR, pLKO.1-circ _0035796 and pLKO.1, and reverse transcribed according to the above method to obtain cDNA as a template, followed by the above RT-PCR amplification.

Results of expression levels of circ _0035796 in pLKO.1-circ _0035796 cells (noted as knockdown) and pLKO.1 cells (noted as control) are shown in the right panel of FIG. 5, and it can be seen that the expression level of circ _ 3532 in pLKO.1-circ _0035796 cells was significantly reduced (P < 0.01) compared to pLKO.1 cells, indicating that cells with silent circ _0035796 expression were obtained.

The expression levels of the cells pLC5-circ _0035796 (shown as overexpression) and pLC5-ciR (shown as control) are shown in the left panel of FIG. 5. It can be seen that the expression level of circ _0035796 in pLC5-circ _0035796 cells is up-regulated (P < 0.01) compared to that in the empty vector, indicating that cells overexpressing circ _0035796 are obtained.

2. circ _0035796 is associated with proliferation potency of non-small cell lung cancer cells

The proliferation capacity of the lung cancer cells is detected by adopting a CCK-8 experiment and an EdU experiment.

1. CCK-8 assay for cell proliferation

The thus-obtained pLC5-circ _0035796 cells, pLC5-ciR cells, pLKO.1-circ _0035796 cells and pLKO.1 cells were counted using a cell counter plate from Bio-Rad, and cell suspensions (density of about 2X 10) were prepared ⁴ mL), seeded into 96-well plates at 100 μ L per well, 3 wells per group were set and added after cells attached. 10. Mu.L of CCK-8 solution (Cell Counting Kit-8, APExBIO K1018) was added to each well, and the mixture was incubated at 37 ℃ for 120min in a Cell incubator, followed by measurement of optical density OD (450 nm) using a microplate reader, after which each group of cells cultured for 24, 48, 72, and 96 hours was subjected to optical density measurement using the same method, and the values were recorded and a growth curve was plotted.

As a result, as shown in FIG. 6, the right panel is the silent group, and the left panel is the overexpression group, it can be seen that, in the CCK-8 experiment, after 96h of culture, the optical density value of the transgenic pLC5-circ _0035796 cell (shown as overexpression in the figure) is obviously increased at 450nm (P <0.01, left panel in FIG. 6) compared with the transgenic pLC5-ciR cell (shown as control in the figure) in the overexpression group; in the silent group, pLKO.1-circ _0035796 cells (shown as knockdown) had significantly lower optical density values at 450nm (P <0.01, right panel in FIG. 6) compared to pLKO.1 cells (shown as control).

2. EdU test

The pLC5-circ _0035796 cells, pLC5-ciR cells, pLKO.1-circ _0035796 cells and pLKO.1 cells were EdU labeled according to the BeyoClick Edu-594 cell proliferation assay kit (Biyunyuan, cat # C0078L), fixed with 4% paraformaldehyde for 15min, washed twice with PBS for 5min, permeabilized with 1 Triton-X100 at room temperature for 10min, removed from the liquid permeation, washed twice with PBS for 5 min. 1mL of dyeing solution is prepared according to the following formula: 860 μ L-Click Reaction Buffer,40 μ L-CuSO4,2 μ L-Azide 594, 100 μ L-Click Additive Solution. The cells were stained with the freshly prepared staining solution for 30min in the dark and washed twice with PBS for 5min each. Cells were stained with DNA using a fixative DAPI (Mounting medium with DAPI, abcam, cat # ab 104139) in the dark, and photographed under a fluorescent microscope.

The results are shown in fig. 7, the left panel is an overexpression panel, the right panel is a knock-out panel, 5 visual fields are respectively collected, in the overexpression panel, the positive cell rate of the transgenic lc5-circ _0035796 cell (marked as overexpression in the figure) is 51.64%, the positive cell rate of the transgenic lc5-ciR cell (marked as control in the figure) is 28.15%, the number of positive cells is remarkably increased, and the cell proliferation activity is remarkably increased (P <0.01, left panel of fig. 7); in the knockout group, the positive cell rate in the pLKO.1-circ _0035796 cell (designated as knockdown in the figure) was 8.93%, the positive cell rate in the pLKO.1 cell group (designated as control in the figure) was 32.39%, the number of positive cells was significantly decreased, and the cell proliferation activity was significantly decreased (P <0.01, right panel in FIG. 7).

The results show that the non-small cell lung cancer cell proliferation can be inhibited by silencing the expression of circ _ 0035796.

3. circ _0035796 is associated with migration and invasion capacity of non-small cell lung cancer cells

And detecting the migration and invasion capacity of the non-small cell lung cancer cells by adopting a cell scratch experiment and a Transwell cell migration and invasion experiment.

1. Cell scratch test

Cells were seeded on streaked 6-well cell culture plates the day before transfection, preferably with a cell density of 70% to 80% the next day; transfection experiments were performed in which pLC5-ciR and overexpression vector pLC5-circ _0035796 were transfected with NCI-H3255 (Shanghai Cheng Biotech Co., ltd., product number QC 473)/BEAS-2B cells (Shanghai Ganning Biotech Co., ltd., CM-H365), pLKO.1 and silencing expression vector pLKO.1-circ _0035796 were transfected with NCI-H3255/A549 cells (ATCC, product number CCL-185), respectively.

After the liquid is changed within 4-6h of transfection, continuously culturing the cells until the culture holes are full of cells; aligning the scribed parallel lines with a pipette tip, and paying attention to uniform force; removing the culture solution, and washing the crushed cells with PBS; adding DMEM culture medium containing 1% fetal calf serum; immediately take a picture under the microscope, this time point was noted as 0 hour, and the corresponding observed position was recorded with a marker pen. After photographing, the cells were returned to the incubator to continue culturing, and the cells were photographed at the same position every 12 hours, and the experiment was repeated three times.

The results are shown in FIG. 8, the right panel is a knock-out panel, the left panel is an overexpression panel, and it can be seen that in the cell scratch experiment, the relative mobility of the cells transfected with pLC5-ciR (noted as control in the figure) in the overexpression panel is 41%, the relative mobility of the cells transfected with pLC5-circ _0035796 (noted as overexpression in the figure) is 70.2%, and the relative mobility of the cells transfected with pLC5-circ _0035796 is increased by 1.71 times (P <0.01, left panel in FIG. 8) compared with the cells transfected with pLC 5-ciR); pLKO.1-circ _0035796 transfected cells (noted as knockouts) in the knockout group had a relative mobility of 68.9% and pLKO.1-circ _0035796 transfected cells (noted as knockouts) had a relative mobility of 40.3%, with pLKO.1-circ _0035796 transfected cells having a 1.73-fold decrease in relative mobility (P <0.01, FIG. 8) compared to pLKO.1 transfected cells.

The results show that silencing the expression of circ _0035796 can inhibit the migration of lung cancer cells.

2. Transwell cell migration experiment and Matrigel invasion experiment

A. Transwell cell migration experiment

The pLC5-ciR and over-expression vector pLC5-circ _0035796 were transfected into NCI-H3255/BEAS-2B cells, respectively, and pLKO.1 and silencing expression vector pLKO.1-circ _0035796 were transfected into NCI-H3255/A549 cells, respectively.

2.1 matrigel Panels

The cells were diluted with Matrigel 1:8 from BD, which was determined based on the amount of mmp produced by the cells, coated on the upper surface of the bottom membrane of the Transwell chamber, and allowed to stand at 37 ℃ for 2 hours to polymerize Matrigel into a gel. The base membrane is hydrated prior to use.

2.2 preparation of cell suspensions

The cells were digested, the digestion was stopped, the medium was discarded by centrifugation, (1-2 times with PBS), and resuspended in serum-free medium. Adjusting cell density to 5X 10 ⁵ /ml。

2.3 seeding of cells

(1) 100. Mu.l of the cell suspension was taken and added to a Transwell chamber.

(2) The lower chamber of the 24-well plate is typically filled with 600. Mu.l of medium containing 20% serum, and no cells are produced.

(3) Culturing cells by conventional culture for 10-48h

2.4 statistics of results

The Transwell chamber was removed, the medium from the wells was discarded, washed 2 times with PBS, fixed with 90% ethanol for 30 minutes, and the chamber was appropriately air-dried.

0.1% crystal violet for 30min, gently wipe off the upper non-migrated cells with a cotton swab, and wash 3 times with PBS.

Cells were observed in five fields immediately under 400-fold microscope and counted.

B. Matrigel invasion assay

1) Preparation of an invasion cell: the Matrigel gel (Matrigel Matrix base membrane, corning, cat No. 354234) solidifies very quickly at ambient temperature, so it was taken out from-20 ℃ and placed at 4 ℃ to melt, and the whole process was put on ice for operation. After dilution with serum-free medium (1:5-1:8), 400. Mu.L of the resulting solution was applied to a Transwell membrane, and a Transwell chamber (Transwell programmable supports 6.5mm insert,24well plate, cat # 00721069) was placed in a 6-well plate and left to stand at 37 ℃ for 3 hours for solidification.

2) Inoculating cells: the experimental cells (pLC 5-circ _0035796 transfected cells, pLC5-ciR transfected cells, pLKO.1-circ _0035796 transfected cells and pLKO.1 transfected cells) were digested and centrifuged, and then resuspended in serum-free medium. 1mL of tumor cell suspension was added to the chamber at a cell count of 3X 10 ⁵ -6×10 ⁵ (the appropriate number of cells is selected depending on the invasive potential of the tumor cells). The cell was filled with 3mL of medium containing 1.5% fetal bovine serum.

3) Culturing the cells: culturing for 24-48h (according to tumor cell invasion ability).

4) Fixing and dyeing: the Transwell chamber was removed, washed once with PBS, the cells on the upper layer of the microporous membrane were wiped off with a cotton swab, fixed with 1% crystal violet-formaldehyde solution and stained for 20-30 min, the staining solution was aspirated, then the residual staining solution was slowly washed off with tap water, and air dried.

5) Microscopic examination: photographs were taken under an inverted microscope, 5 fields were counted per sample, and statistical analysis was performed after counting.

As a result, as shown in FIG. 9, the upper panel is an overexpression panel, and the lower panel is a knock-out panel, it can be seen that in the Transwell cell migration experiment, cells transfected with pLC5-circ _0035796 (denoted as overexpression in the figure) and pLC5-ciR (denoted as control in the figure) were photographed at 10hr and counted, and the number of cells transfected with pLC5-circ _0035796 was significantly increased by 10.9 times (P <0.01, FIG. 9) compared to cells transfected with pLC 5-ciR; in invasion experiments, cells transfected with pLC5-circ _0035796 (designated as overexpression) and pLC5-ciR (designated as control) were photographed at 16hr and counted, and the number of migrating cells transfected with pLC5-circ _0035796 was significantly increased by 8-fold (P <0.01, FIG. 9) compared to cells transfected with pLC 5-ciR; the results show that after circ _0035796 is over-expressed, the migration and invasion of lung cancer cells can be enhanced; in the Transwell chamber migration experiment, pLKO.1-circ _0035796 transfected cells (shown as knockdown) and pLKO.1 transfected cells (shown as control) were photographed and counted at 16hr in the knockdown group, and the number of migrated cells was significantly reduced by 6-fold compared to pLKO.1 transfected cells (P <0.01, FIG. 9), and in the invasion experiment, photographed and counted at 22hr, and pLKO.1-circ _0035796 transfected cells were significantly reduced and pLKO.1-circ _0035796 transfected cells were significantly reduced by 12.5-fold compared to pLKO.1 transfected cells (P <0.01, FIG. 9).

The results show that silencing the expression of circ _0035796 can inhibit migration or invasion of non-small cell lung cancer cells.

4. cir _0035796 is associated with in vivo tumorigenesis and metastasis

In order to explore the influence of circ _0035796 on tumor growth and metastasis in vivo, NCI-H3255 cells stably overexpressed by circ _0035796 and corresponding control cells are respectively inoculated to the subcutaneous tissues of female nude mice, and a nude mouse transplanted tumor growth and metastasis model is successfully constructed.

The method comprises the following specific steps:

1) Control cells used in conventional culture (pLC 5-ciR cells and pLKO.1 cells) or transfected with cell lines carrying stable overexpression of circ-0035796 (pLC 5-circ _0035796 cells) and with knockdown of circ _0035796 (pLKO.1-circ _0035796 cells) were expanded in sufficient numbers to prepare for seeding.

2) Digesting cells with pancreatin, and centrifugally collectingAfter cell counting, cells were resuspended in PBS. The inoculation amount is generally 100 mu L/tube, which contains 4X 10 ⁶ And (4) tumor cells. Cells to be injected were placed on ice.

3) Nude mice to be injected were briefly comatosed with ether and the cell suspension was injected subcutaneously into the axilla using a 1mL syringe.

4) The experimental nude mice were kept for about one month, tumor changes were observed at regular time, tumor volume was measured with a vernier caliper and recorded, live imaging of the small animals was performed before tumor body was taken out, 15mg/ml fluorescein substrate (Xenolight D-luciferase potassium salt, perkinElmer, cat # 12799) solution was prepared with PBS, the mice were injected at 10 μ L/g, and pictures were taken by an imaging system (Living image4.5, perkinElmer) after 10-15 minutes of intraperitoneal injection.

5) And taking out tumor bodies, measuring the weight, recording the numerical value, and performing statistical analysis.

The results are shown in FIG. 10, in which the overexpression group is mice injected with cells of pLC5-circ _0035796, the knockdown group is mice injected with cells of pLKO.1-circ _0035796, and the overexpression control group and the knockdown control group are mice injected with cells of pLC5-ciR and pLKO.1, respectively; it can be seen that the tumor volume and mass of circ _0035796 over-expressed group was significantly higher than that of over-expressed control group (P <0.01, fig. 10) and that of in-vivo tumor volume and mass of knock-down group was significantly lower than that of knock-down control group (P <0.01, fig. 10) compared to the over-expressed control group.

The tumor growth curve shows that the tumor growth speed of circ _0035796 in the overexpression group is obviously faster than that of the overexpression control group (P <0.01, FIG. 10), while the tumor growth speed of the knockdown group in vivo is obviously slower than that of the knockdown control group (P <0.01, FIG. 10).

The fluorescence intensity of circ _0035796 over-expressed group was significantly enhanced compared to the control group (P <0.01, fig. 10) and the fluorescence intensity of the knockdown group was significantly reduced compared to the control group (P <0.01, fig. 10) as shown by small animal in vivo imaging (PE).

The results show that the exogenous high expression of circ _0035796 can obviously enhance the in vivo tumor formation of the non-small cell lung cancer cell, and the low expression can also obviously inhibit the in vivo tumor formation of the non-small cell lung cancer cell.

Claims

1. The application of the substance for detecting the expression of the circular RNA circ _0035796 in preparing the product with any one of the following functions:

a1 Diagnosis or assisted diagnosis of non-small cell lung cancer;

a2 ) differentially diagnosing or differentiating benign nodules from non-small cell lung cancer of the lung;

a3 Screening or assisted screening for non-small cell lung cancer;

a7 Screening or screening the patient to be tested for non-small cell lung cancer tissue;

the nucleotide sequence of the circular RNA circ _0035796 is 21-1163 th position of the sequence 1.

2. Use according to claim 1, characterized in that:

the substance for detecting the expression of the circular RNA circ _0035796 comprises a probe which is specifically bound to circ _0035796 or a primer which is specifically amplified to circ _ 0035796.

3. A product comprising a substance that detects the expression of circular RNA circ _ 0035796;

the product has any one of the following functions:

a1 Diagnosis or assisted diagnosis of non-small cell lung cancer;

a3 Screening or screening aid for non-small cell lung cancer;

4. The application of the circular RNA circ _0035796 as a marker in developing or designing a product for diagnosing, assisting in diagnosing, screening or assisting in screening non-small cell lung cancer; the nucleotide sequence of the circular RNA circ _0035796 is 21 th to 1163 rd position of the sequence 1.

5. Use of a substance that inhibits the expression of circular RNA circ _0035796 in any of:

b1 For treating non-small cell lung cancer;

b2 Inhibit proliferation of non-small cell lung cancer cells;

b3 Inhibit migration of non-small cell lung cancer cells;

b4 Inhibit non-small cell lung cancer cell invasion;

b5 Inhibit the formation of tumors in vivo by non-small cell lung cancer cells.

6. Use according to claim 5, characterized in that: the substance for inhibiting the expression of the circular RNA circ _0035796 is a substance for interfering or silencing the expression of circ _ 0035796.

7. A product comprising a substance of claim 5 or 6 which inhibits the expression of circular RNA circ _ 0035796;

the product has any one of the following functions:

b1 For treating non-small cell lung cancer;

b2 Inhibit proliferation of non-small cell lung cancer cells;

b3 Inhibit migration of non-small cell lung cancer cells;

b4 Inhibit non-small cell lung cancer cell invasion;

b5 Inhibit the formation of tumors in vivo by non-small cell lung cancer cells.

8. The application of circular RNA circ _0035796 in preparing animal models or cell models for screening drugs for treating non-small cell lung cancer.