CN113789381B

CN113789381B - Application of human CLDN10-AS1lncRNA in evaluating prognosis of lung adenocarcinoma patient and detection kit

Info

Publication number: CN113789381B
Application number: CN202111078017.5A
Authority: CN
Inventors: 赵小刚; 李培超; 田忠献; 郭嘉仲; 杨凌霄
Original assignee: Second Hospital of Shandong University
Current assignee: Second Hospital of Shandong University
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2022-05-17
Anticipated expiration: 2041-09-15
Also published as: CN113789381A

Abstract

The invention relates to an application of human CLDN10-AS1lncRNA in evaluating the prognosis of a patient with lung adenocarcinoma and a detection kit. The invention firstly proves that CLDN10-AS1lncRNA plays an important role in promoting cancer in the proliferation and migration of lung adenocarcinoma cells, and the up-regulation of the expression level of CLDN10-AS1lncRNA is closely related to the shortening of the overall survival period of lung adenocarcinoma patients. Therefore, the invention uses CLDN10-AS1lncRNA AS a biomarker of lung adenocarcinoma, and provides the application of CLDN10-AS1lncRNA in lung adenocarcinoma, in particular the application in preparing a lung adenocarcinoma prognosis evaluation product.

Description

Application of human CLDN10-AS1lncRNA in evaluating prognosis of lung adenocarcinoma patient and detection kit

Technical Field

The invention relates to an application of human CLDN10-AS1lncRNA in evaluating the prognosis of a patient with lung adenocarcinoma and a detection kit, belonging to the technical field of biomedicine.

Background

Lung cancer is increasing in incidence in our country and ranks first among cancer-related causes of death. Lung cancer can be classified into two main groups according to pathological types, namely small cell lung cancer and non-small cell lung cancer, and the non-small cell lung cancer accounts for about 85% of all lung cancer cases, including lung adenocarcinoma, lung squamous carcinoma and large cell carcinoma, wherein the lung adenocarcinoma is the most common pathological type in the non-small cell lung cancer, and accounts for about 40% of primary lung cancer. Among human malignancies, lung cancer has a relatively poor prognosis with a 5-year survival rate of less than 20%. At present, the invasion and metastasis mechanism of lung adenocarcinoma is not fully elucidated, the malignant biological behavior generating mechanism of lung adenocarcinoma is further explored, and appropriate intervention measures are given, so that the method has very important practical significance for improving the clinical diagnosis and treatment level of lung adenocarcinoma and improving the prognosis of patients, wherein the method is a problem which is urgently needed to be solved at present by defining new molecular markers related to the postoperative survival period of lung adenocarcinoma patients and applying the new molecular markers to the prognosis evaluation of lung adenocarcinoma.

The human genome is composed of 30 hundred million base pairs and can produce about 18 million transcripts, of which about 2 million transcripts are capable of encoding proteins and the remaining about 16 million transcripts are incapable of producing protein products, called non-coding RNAs (ncrnas), in which most ncrnas are more than 200 bases in length, called long-chain non-coding RNAs (incrnas). In recent years, with the development and popularization of sequencing technologies, more and more lncrnas have been detected and confirmed to participate in various physiological processes, such as myocyte differentiation, neuronal differentiation, synapse formation, re-editing of pluripotent stem cells, inflammatory regulation, and the occurrence and development of cancer. Among the lncrnas currently known to be associated with lung cancer, some have been shown to have cancer-driving effects, e.g., LINC00673, ANRIL, AGAP2-AS1, HOTTIP, SNHG 1; other lncRNA plays a role in inhibiting cancer genes, such AS GAS6-AS1, MEG3, LINC00961, SPRY4-IT1, and the like. The lncRNA plays an important role in the process of tumorigenesis and development by regulating and controlling tumor proliferation, invasion, apoptosis and drug resistance. At present, the research on the function and mechanism of lncRNA in lung adenocarcinoma is still far from enough, and further, a new lncRNA related to lung adenocarcinoma is found, and the function and the prognostic significance of the lncRNA in lung adenocarcinoma are clarified, so that the method has important significance for promoting the development of individualized clinical diagnosis and treatment strategies.

Polymerase Chain Reaction (PCR) is a technique for specifically amplifying a target DNA fragment in vitro under the action of DNA Polymerase by using the principle of base complementary pairing. The fluorescent quantitative PCR is to detect the PCR process in real time through a fluorescent signal embedded in a DNA double strand, and the copy number of target cDNA in a template has a linear relation with a Ct value in an exponential amplification stage, so that the quantitative analysis of the target RNA can be carried out. The upstream and downstream primer sequences for specifically recognizing the target fragment are important for accurate quantitative analysis of the target RNA.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an application of human CLDN10-AS1lncRNA in evaluating the prognosis of a patient with lung adenocarcinoma and a detection kit.

The technical scheme of the invention is as follows:

application of human CLDN10-AS1lncRNA in preparing lung adenocarcinoma prognosis evaluation products.

Preferably according to the invention, CLDN10-AS1lncRNA is a biomarker for prognostic assessment of lung adenocarcinoma in said use.

Preferably, the nucleotide sequence of CLDN10-AS1lncRNA is shown AS SEQ NO. 5.

Preferably, according to the invention, the product is used for assessing and predicting the overall survival of patients with lung adenocarcinoma.

Preferably, the lung adenocarcinoma prognosis evaluation product according to the present invention comprises a substance specifically recognizing CLDN10-AS1 incrna reverse transcription product cDNA.

Further preferably, the substance specifically recognizing CLDN10-AS1 incrna reverse transcription product cDNA is selected from a primer pair specifically amplifying CLDN10-AS1 incrna reverse transcription product cDNA.

Further preferably, the primer pair for specifically amplifying the cDNA of the reverse transcription product of CLDN10-AS1lncRNA is an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO. 2; or an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4.

According to a preferred embodiment of the present invention, the test sample of the lung adenocarcinoma prognosis evaluation product is selected from cells or tissues.

The application of a substance which specifically recognizes the cDNA of the reverse transcription product of CLDN10-AS1lncRNA in the lung adenocarcinoma prognosis evaluation product.

Preferably according to the invention, the substance specifically recognizing CLDN10-AS1 incrna reverse transcription product cDNA is selected from a primer pair specifically amplifying CLDN10-AS1 incrna reverse transcription product cDNA.

A prognostic evaluation kit for lung adenocarcinoma, comprising a substance specifically recognizing CLDN10-AS1 incrna reverse transcription product cDNA.

Preferably, the kit further comprises a detection reagent for real-time fluorescent quantitative PCR.

Has the advantages that:

1. the invention firstly proves that the expression of CLDN10-AS1lncRNA is up-regulated to promote the proliferation and migration of lung adenocarcinoma cells, and the up-regulated expression level of CLDN10-AS1lncRNA is obviously related to the shortening of the overall survival period of a lung adenocarcinoma patient. Therefore, the invention uses CLDN10-AS1lncRNA AS a biomarker of lung adenocarcinoma, and provides the application of CLDN10-AS1lncRNA in lung adenocarcinoma, in particular to the application in preparing a lung adenocarcinoma prognosis evaluation product.

2. The invention designs a primer pair for specifically amplifying CLDN10-AS1 incRNA, and can specifically and effectively detect the expression level of CLDN10-AS1 incRNA.

Drawings

FIG. 1 is a graph showing that the expression level of CLDN10-AS1lncRNA in 7 pairs of lung adenocarcinoma tissues and adjacent normal lung tissues is detected by an RNA sequencing technology, the expression level of CLDN10-AS1lncRNA in cancer tissues is calculated by taking the expression level of CLDN10-AS1lncRNA in adjacent normal lung tissues AS a standard, and Student's t is used for detecting and analyzing the statistical significance of the difference among the groups.

FIG. 2 is a graph showing that the kit of the present invention detects 10 expression levels of CLDN10-AS1lncRNA in lung adenocarcinoma tissue and adjacent normal lung tissue, AS shown in FIG. 2^-△△CTThe relative expression level of CLDN10-AS1 incrna in cancer tissues was calculated and a Student's t test was applied to analyze a statistically significant graph of differences between groups.

FIG. 3 is a graph showing that the kit of the present invention detects the expression level of CLDN10-AS1lncRNA in lung adenocarcinoma cell lines (A549, H1299, H1975) and normal bronchial epithelial cells (BEAS-2B) according to 2^-△△CTThe relative expression level of the lung adenocarcinoma cell line CLDN10-AS1lncRNA was calculated and a statistical significance map of differences between groups was analyzed using the Student's t test.

FIG. 4 is a line graph (left) and bar graph (right) showing the statistical significance of differences between groups analyzed using the Student's t test to examine the effect of stable knockdown of CLDN10-AS1 IncRNA on proliferation and migration of A549 cells using the CCK-8 assay and the Transwell assay.

FIG. 5 is a line graph (left) and bar graph (right) showing the statistical significance of differences between groups using the Student's t test to examine the effect of exogenous CLDN10-AS1 IncRNA expression on proliferation and migration of BEAS-2B cells using the CCK-8 assay and the Transwell assay.

FIG. 6 is a graph of the effect of expression levels of CLDN10-AS1 incRNA on the prognosis of patients with lung adenocarcinoma analyzed using the Kaplan-Meier Plotter database, comparing the prognostic difference between patients with high expression of CLDN10-AS1 incRNA and low expression of lung adenocarcinoma using the Kaplan-Meier method, and using Log-rank to test whether the difference between groups is statistically significant.

Detailed Description

The technical solution of the present invention is further described below with reference to the experimental examples, but the scope of the present invention is not limited thereto. The reagents and materials used in the examples are, unless otherwise specified, all of which are commonly commercially available products.

The example was approved by the ethical committee of medical science of the second hospital of Shandong university, and all cases received informed consent from the patients.

The normal human bronchial epithelial cell line BEAS-2B and the human lung adenocarcinoma cell lines A549, H1299 and H1975 were purchased from cell banks of the culture Collection of type members of the Chinese academy of sciences.

Example 1

The invention utilizes RNA-seq (Ribo-free) to detect the expression profiles of lncRNA in lung adenocarcinoma tissues and samples adjacent to normal lung tissues (7 cases respectively), and the bioinformatics analysis shows that the expression profiles of the lncRNA with up-regulated expression (Log2 Fold change >2 and p value <0.05) are 275 in total, and the expression profiles of the lncRNA with down-regulated expression (Log2 Fold change < -2 and p value <0.05) are 90 in total. Wherein CLDN10-AS1 IncRNA is the IncRNA with the highest upregulation fold in lung adenocarcinoma tissues. The expression level of CLDN10-AS1 incrna was significantly up-regulated in cancer tissue compared to adjacent normal lung tissue, and the results of the specific analysis are shown in fig. 1.

The specific implementation process is as follows:

(1) collecting a tissue sample: collecting 7 pairs of lung adenocarcinoma specimens removed by operation, wherein the tumor tissues are all taken from the central non-necrotic part, the adjacent normal tissues are taken from the area which is more than 5cm away from the tumor edge, and the specimens are quickly frozen by liquid nitrogen and preserved at-80 ℃. No radiotherapy and chemotherapy was performed on 7 patients before surgery, and the pathological result after surgery was confirmed to be lung adenocarcinoma. The above case collections were approved by the ethical committee of medical science of the second hospital of Shandong university and were informed consent from the patients;

(2) lncRNA sequencing: cracking the frozen and stored tissue of the liquid nitrogen by using a Trizol reagent, extracting RNA by using a phenol-chloroform-isopropanol method, evaluating the integrity of the extracted RNA by using agarose gel electrophoresis, detecting the concentration and the purity of the extracted RNA by using a NanoDrop2000, selecting an RNA sample with RIN >7 for library construction, and sequencing by using an RNA-seq (Ribo-free) technology of Beijing Nuo Poa biogenic bioinformatics science and technology ltd;

(3) bioinformatics analysis: analyzing the original data obtained by sequencing, sequentially performing quality evaluation, removing low-quality sequences, comparing with reference genome, screening lncRNA, performing differential expression analysis according to (Log)₂ Fold change>2，p value<0.05) or (Log)₂ Fold change<-2，p value<0.05) Standard screening differentially expressed lncRNA. The results show that CLDN10-AS1 incrna expression levels in lung adenocarcinoma tissue are significantly elevated compared to adjacent normal lung tissue.

Example 2

The method comprises the steps of collecting 10 samples of lung adenocarcinoma tissues and adjacent normal tissues after surgical resection, extracting total RNA, and detecting the expression level of CLDN10-AS1lncRNA in the samples by using a primer sequence of the cDNA of the reverse transcription product of the CLDN10-AS1lncRNA specifically identified by the invention to perform real-time fluorescence quantitative PCR, wherein the result shows that the expression level of the CLDN10-AS1lncRNA in the lung adenocarcinoma tissues is obviously increased compared with the adjacent normal tissues, and the specific analysis result is shown in figure 2.

The specific implementation process is as follows:

(1) collecting a tissue sample: 10 pairs of surgically excised lung adenocarcinoma specimens were collected, with tumor tissue all taken from the central non-necrotic area and adjacent normal lung tissue taken from a region greater than 5cm from the tumor margin, snap frozen in liquid nitrogen, and stored at-80 ℃. The pathological results of 15 patients after operation proved to be lung adenocarcinoma, and the patients did not receive radiotherapy and chemotherapy before the operation. The above case collections were approved by the ethical committee of medical science of the second hospital of Shandong university and were informed consent from the patients.

(2) Extracting total RNA of tissues: total RNA was extracted from adenocarcinoma lung tissue and adjacent normal lung tissue using RNA-Quick Purification Kit (RN 001), exactly as described in the specification, and the integrity of the extracted RNA was checked by agarose gel electrophoresis and the concentration and purity of the RNA was checked using NanoDrop 2000.

(3) RT-qPCR: removing genomic DNA by using an lnRcute lncRNA cDNA first strand synthesis kit (TIANGEN, KR202), and then carrying out reverse transcription to synthesize cDNA, specifically according to the steps of the specification, respectively carrying out fluorescent quantitative PCR by using an lnRcute lncRNA fluorescent quantitative detection kit (SYBR Green) (TIANGEN, FP402) and specific primers shown as SEQ NO.1 and SEQ NO.2, SEQ NO.3 and SEQ NO.4 in the invention, using GAPDH as an internal reference, and using 2^-ΔΔCTThe method calculates the relative expression level of lncRNA-CLDN10-AS1, and the result shows that the expression level of lncRNA-CLDN10-AS1 in the lung adenocarcinoma tissue is obviously increased compared with the adjacent normal lung tissue.

Wherein, the nucleotide sequence of the primer pair is as follows:

upstream primer # 1: 5'-CTGAGCATGTGGGTCCTTAT-3' (SEQ ID NO.1)

Downstream primer # 1: 5'-CTATACAGGGCAATACGGGG-3' (SEQ ID NO.2)

Upstream primer # 2: 5'-GAGCATGTGGGTCCTTATC-3' (SEQ ID NO.3)

Downstream primer # 2: 5'-CAGCTAGAGGAAGCAAAGAA-3' (SEQ ID NO.4)

Genomic DNA removal system: RNA (250 ng/. mu.L) 2. mu.L, 5 XgDNA Buffer 2. mu.L, RNase-Free ddH₂O6 μ L; reaction conditions are as follows: incubate at 42 ℃ for 3 minutes and place on ice until ready for use.

Reverse transcription PCR System: 10 XlnR RT Buffer 2. mu.L, lnR RT Enzyme Mix 1. mu.L, lnR-RT Primer Mix 2. mu.L, RNase-Free ddH₂O5 mu L, genome DNA removal system product 10 mu L; reaction conditions are as follows: incubate at 42 ℃ for 15 minutes, incubate at 95 ℃ for 3 minutes, and store at 4 ℃ for future use.

Real-time fluorescent quantitative PCR system (10 μ Ι _): the reverse transcription system product was 1. mu.L, 2 XlnR lncRNA PreMix 5. mu.L, primer diluent (1. mu.M) 3.8. mu.L, and 50 XROX Reference Dye 0.2. mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 20 seconds for 40 cycles; denaturation at 95 ℃ for 15 sec, incubation at 60 ℃ for 1 min, and denaturation at 95 ℃ for 15 sec.

The reagents are all from the first strand synthesis kit (TIANGEN, KR202) of the lnRCute lncRNA cDNA and the fluorescent quantitative detection kit (SYBR Green) (TIANGEN, FP402) of the lnRCute lncRNA.

Example 3

The expression levels of CLDN10-AS1lncRNA in normal bronchial epithelial cells (BEAS-2B) and 3 lung adenocarcinoma cell lines (A549, H1299 and H1975) were respectively detected by using the primer sequences of the invention shown in SEQ NO.1, SEQ NO.2, SEQ NO.3 and SEQ NO.4, which specifically recognize the cDNA of the CLDN10-AS1lncRNA reverse transcription product, and the results showed that the expression levels of CLDN10-AS1lncRNA in A549, H1299 and H1975 cells were significantly up-regulated compared to BEAS-2B cells, and the specific analysis results are shown in FIG. 3.

The specific implementation process is as follows:

(1) extracting total RNA of cells: total RNA was extracted from normal human airway epithelial cells (BEAS-2B) and 3 lung adenocarcinoma cells (A549, H1299, H1975) using the RNA-Quick Purification Kit (Shanghai Yiniao, RN001), the integrity of the extracted RNA was examined by RNA electrophoresis, and the concentration and purity of the RNA were examined using NanoDrop 2000.

(2) RT-qPCR: removing genomic DNA by using an lnRcute lncRNA cDNA first strand synthesis kit (TIANGEN, KR202), and then carrying out reverse transcription to synthesize cDNA, specifically according to the steps of the instruction, respectively carrying out fluorescent quantitative PCR by using specific primers shown in SEQ NO.1 and SEQ NO.2, SEQ NO.3 and SEQ NO.4 and an lnRcute lncRNA fluorescent quantitative detection kit (SYBR Green) (TIANGEN, FP402) in the invention, taking GAPDH as an internal reference, and using 2^-ΔΔCTThe method calculates the relative expression level of CLDN10-AS1lncRNA, and the result shows that the expression level of CLDN10-AS1lncRNA is remarkably up-regulated in 3 lung adenocarcinoma cell lines compared with BEAS-2B cells.

The PCR system was the same as in example 2.

Example 4

A549 cell strain with stably knocked-down expression of CLDN10-AS1lncRNA is constructed, CCK-8 experiment and Transwell experiment are applied to detect the influence of stable knocked-down of CLDN10-AS1lncRNA on proliferation and migration of A549 cells, and the result shows that the knocking-down expression level of CLDN10-AS1lncRNA obviously inhibits the proliferation and migration of A549 cells, and the specific result is shown in figure 4.

The specific implementation process is as follows:

(1) constructing a CLDN10-AS1lncRNA stable low-expression A549 cell strain: an RNAi Designer website is used for designing siRNA for specifically interfering expression of CLDN10-AS1lncRNA, further designing shRNA sequences, synthesizing shRNA oligo nucleic acid fragments, connecting the shRNA oligo nucleic acid fragments to an empty vector, transforming successfully constructed plasmids into competent cells, and sequentially carrying out bacterium shaking, plate coating, monoclonal selection and plasmid amplification. Plasmid was extracted using endotoxin-free plasmid medium extraction kit (kang century, CW2105), PMD2G, PSPAX2 plasmid and the above-mentioned plasmid-upgraded or empty vector plasmid were transfected into 293T cells using Lipo293 transfection reagent (Beyotime, C0521), the medium was collected after 48 hours, filtered using 0.45 μm filter membrane, 5 × PEG8000 solution was added to the filtrate, centrifuged after standing overnight at 4 ℃, the pellet was retained, and lentivirus was resuspended using serum-free medium. The obtained lentivirus is used for infecting A549 cells, and drug-resistant cells are screened by a culture medium containing puromycin (1 mu g/mL), so that a CLDN10-AS1lncRNA stable low-expression A549 cell strain (A549-sh-CLDN10-AS1) and a stable expression empty Vector cell strain (A549-Vector) can be obtained.

Wherein the nucleotide sequence of the shRNA is as follows:

shRNA: the target nucleotide sequence is: 5'-GCTGAGCATGTGGGTCCTTAT-3', respectively;

(2) the expression level of CLDN10-AS1 in A549-sh-CLDN10-AS1 and A549-Vector cells is verified: extracting the total RNA of the cells by using an RNA-Quick Purification Kit (Shanghai Yiniao, RN001), carrying out reverse transcription by using an lnRcute lncRNA cDNA first strand synthesis Kit (TIANGEN, KR202) to synthesize cDNA, carrying out fluorescence quantitative PCR by using an lnRcute lncRNA fluorescence quantitative detection Kit (TIANGEN, FP402) and a primer sequence which is shown AS SEQ NO.1 and SEQ NO.2, SEQ NO.3 and SEQ NO.4 and specifically recognizes the cDNA of the reverse transcription product of CLDN10-AS1lncRNA, using GAPDH AS an internal reference and using 2^-ΔΔCTThe method calculates the relative expression level of CLDN10-AS1 lncRNA;

(3) CCK-8 experiment: A549-sh-CLDN10-AS1 and A549-Vector cells were digested with trypsin, the cells were collected and cell suspensions were prepared, the cell concentration was adjusted to 20,000/mL of the medium, the above cell suspensions were added to a 96-well plate (100. mu.L/well), 6 wells were repeated, 10. mu.L of CCK-8 reagent (TargetMol, C0005) was added to each well at a designated time, the incubator was left at 37 ℃ for 1 hour, and the absorbance value at 450nm was measured using a microplate reader.

(4) Transwell experiment: the cells were digested with trypsin, collected and centrifuged, the cell pellet was resuspended with serum-free DMEM medium, the cell concentration was adjusted to 100,000/mL medium, a Transwell chamber (Corning, 3422) was placed in a 24-well plate (containing 600 μ L complete medium/well), 200 μ L of the cell suspension was aspirated and added to the chamber, incubated at 37 ℃ for 48 hours in an incubator, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, the cells were treated with methanol for 25 minutes, stained with 0.1% crystal violet for 30 minutes, unperforated cells on the inside of the chamber were scraped with a cotton swab, and the number of cells was recorded by observing the perforated cells under a microscope.

Example 5

The method comprises the steps of constructing a BEAS-2B cell strain with stable expression of exogenous CLDN10-AS1lncRNA, and detecting the influence of the expression of exogenous CLDN10-AS1lncRNA on the proliferation and migration of BEAS-2B cells by applying a CCK-8 experiment and a Transwell experiment, wherein the result shows that the proliferation and migration of the BEAS-2B cells are remarkably enhanced by the expression of exogenous CLDN10-AS1lncRNA, and the specific analysis result is shown in figure 5.

The specific implementation process is as follows:

(1) construction of a BEAS-2B cell line stably expressing exogenous CLDN10-AS1 lncRNA: CLDN10-AS1lncRNA plasmid and empty vector plasmid were synthesized by Biotech, Inc., of Beijing Okagaku. PMD2G, PSPAX2 and CLDN10-AS1lncRNA plasmids or empty vector plasmids were transfected into 293T cells using Lipo293 transfection reagent (Beyotime, C0521), the medium was collected after 48 hours, filtered using a 0.45 μm filter, 5 × PEG8000 solution was added to the filtrate, centrifuged after standing overnight at 4 ℃, the pellet was retained, and lentivirus was resuspended using DMEM medium without serum. The obtained lentivirus is used to infect BEAS-2B cells, and complete culture medium containing puromycin (1 mu g/mL) is used to screen drug-resistant cells, thus obtaining a BEAS-2B cell strain (BEAS-2B-CLDN10-AS1) and a stable expression empty Vector cell strain (BEAS-2B-Vector) which are stably expressed by exogenous CLDN10-AS1 lncRNA.

(2) Verifying expression levels of BEAS-2B-CLDN10-AS1 and CLDN10-AS1lncRNA in BEAS-2B-Vector cells: extracting the total RNA of the cells by using an RNA-Quick Purification Kit (Shanghai Yiniao, RN001), performing reverse transcription by using a lnRcute lncRNA cDNA first strand synthesis Kit (TIANGEN, KR202), performing fluorescence quantitative PCR by using an lnRcute lncRNA fluorescence quantitative detection Kit (TIANGEN, FP402) and a primer sequence which is shown AS SEQ NO.1 and SEQ NO.2, SEQ NO.3 and SEQ NO.4 and specifically recognizes the cDNA of the CLDN10-AS1lncRNA reverse transcription product, using GAPDH AS an internal reference, and using 2^-ΔΔCTThe method calculates the relative expression level of CLDN10-AS1 incrna.

(3) CCK-8 experiment: BEAS-2B-CLDN10-AS1 cells and BEAS-2B-Vector cells were digested with trypsin, cells were collected and cell suspensions were prepared, the cell concentration was adjusted to 15000 cells/mL of the medium, the above cell suspensions were added to a 96-well plate (100. mu.L/well), 6 wells were repeated, 10. mu.L of CCK-8 reagent (TargetMol, C0005) was added to each well at a designated time, incubated at 37 ℃ for 1 hour, and absorbance at 450nm was measured using a microplate reader.

(4) Transwell experiment: the above cells were digested and collected with trypsin, a cell suspension was prepared using serum-free DMEM medium, the cell concentration was adjusted to 100000 cells/mL medium, a Transwell chamber (Corning, 3422) was placed in a 24-well plate (containing 600 μ L of complete medium/well), 200 μ L of the above cell suspension was aspirated and added to the chamber, incubated in an incubator at 37 ℃ for 48 hours, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, the cells were treated with methanol for 25 minutes, stained with 0.1% crystal violet for 30 minutes, unperforated cells on the inside of the chamber were scraped with a cotton swab, and the number of penetrated cells was observed under a microscope and recorded.

Example 6

The Kaplan-Meier Plotter database is used for analyzing the prognosis difference between patients with high expression and low expression of CLDN10-AS1lncRNA, and the results show that the overall survival period of the patients with the lung adenocarcinoma is remarkably shortened by CLDN10-AS1lncRNA compared with the prognosis of the patients with the lung adenocarcinoma which is low expression of CLDN10-AS1lncRNA, and the specific analysis results are shown in FIG. 6.

The specific implementation process is as follows:

downloading prognosis information of lung adenocarcinoma patients and relative expression level of CLDN10-AS1 from a Kaplan-Meier Plotter website, analyzing the difference between high expression of CLDN10-AS1lncRNA and total Survival (over Survival) of low expression patients by using a Kaplan-Meier method, wherein the CLDN10-AS1lncRNA is highly expressed for 332 patients with moderate Survival and the median Survival is 95.07 months in total; CLDN10-AS1 incrna was underexpressed in 340 cases, median survival 110.27 months, and Log-rank test was used to analyze statistical significance of survival differences between groups.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

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Claims

1. An application of shRNA of CLDN10-AS1lncRNA in preparing a medicine for inhibiting proliferation and migration of lung adenocarcinoma cells is disclosed, wherein a targeting nucleotide sequence of the shRNA is AS follows: 5'-GCTGAGCATGTGGGTCCTTAT-3' are provided.

2. The use of claim 1, wherein the nucleotide sequence of CLDN10-AS1 incrna is represented by SEQ ID No. 5.