CN114632152B

CN114632152B - Application of lncRNA TC8260 as lung cancer treatment target

Info

Publication number: CN114632152B
Application number: CN202011479324.XA
Authority: CN
Inventors: 潘世扬; 徐娟; 顾春荣; 徐建; 金岳心子; 张世昌; 王芳
Original assignee: Jiangsu Kede Bio Medicine Technology Co ltd
Current assignee: Jiangsu Kede Bio Medicine Technology Co ltd
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2023-06-16
Anticipated expiration: 2040-12-16
Also published as: CN114632152A

Abstract

The invention discloses an application of lncRNA TC8260 as a lung cancer treatment target. The application of lncRNA TC8260 as a therapeutic target in preparing or screening medicaments for treating lung cancer and/or lung cancer metastasis. Application of lncRNA TC8260 or substances for promoting expression of lncRNA TC8260 in preparation of or screening of medicines for treating lung cancer and/or lung cancer metastasis. The expression level of HBE of lncRNA TC8260 in normal human bronchial epithelial cell strain is obviously higher than that of lung cancer cell strain. The expression level of lncRNA TC8260 in the serum of the lung cancer patient of the healthy control is obviously higher than that of the serum of the lung cancer patient. Downregulation of lncRNA TC8260 was found to promote SPC-A1 cell migration by cell scoring experiments. Therefore, lncRNA TC8260 can be used as lung cancer or lung cancer metastasis and can be applied to preparation of medicines for treating lung cancer and/or lung cancer metastasis.

Description

Application of lncRNA TC8260 as lung cancer treatment target

Technical Field

The invention belongs to the field of biological medicine, and relates to application of lncRNA TC8260 as a lung cancer treatment target.

Background

Lung cancer is one of the most common malignant tumors in humans, with both morbidity and mortality being the first worldwide. Epidemiological studies suggest that up to 1800 tens of thousands of patients with lung cancer in China will die from lung cancer by about 100 tens of thousands of people in China every year by 2025, which constitutes a serious threat to the life health of people. Lung cancer includes small cell lung cancer (Small cell lung cancer, SCLC) and Non-small cell lung cancer (Non-small cell lung cancer, NSCLC), with approximately 85% of new lung cancer cases being Non-small cell lung cancer, and lung adenocarcinoma being the most common subtype of Non-small cell lung cancer.

At present, the treatment of non-small cell lung cancer is mainly based on clinical stage of lung cancer, surgical excision is a main treatment means for I, II patients, and survival rates of IA, IB, IIA and IIB patients after operation for 5 years are respectively as follows: 77-92%, 68%, 60% and 53%; for those with lymph node metastasis, preoperative adjuvant chemotherapy or radiotherapy can be applied. For advanced patients, in addition to conventional radiotherapy and chemotherapy, corresponding personalized targeted therapies can be adopted according to the molecular typing of tumors. Although diagnosis and treatment of lung cancer have been advanced to some extent in recent years, the survival rate of lung cancer patients in 5 years is still low, about 15%, so that searching for new therapeutic targets and taking targeted personalized therapeutic measures are key to prolonging the survival time of patients and improving the therapeutic effect.

Tumor markers are a class of substances produced by tumor cells in tumorigenesis, development, infiltration, and metastasis. Traditional lung cancer markers, such as carcinoembryonic antigen (carcinoma embryonic antigen, CEA), cytokeratin 19 fragment (cytokeratin 19 fragment,CYFRA21-1), squamous cell carcinoma antigen (squamous cell carcinoma antigen, SCCA) and the like, have important application values for diagnosing and staging lung cancer, and monitoring the illness state and curative effect. However, the specificity and sensitivity of these individual indicators are not ideal, and the diagnosis of the tumor cannot be performed simply based on these indicators, and only a clue for further diagnosis can be provided. Therefore, in clinical practice, in order to improve the sensitivity of detection, a combination of multiple markers is usually adopted, but the combination of detection results in problems of reduced specificity, increased detection cost and the like, so that a new tumor marker needs to be searched. The subject group successfully prepares a monoclonal antibody NJ001 of specific anti-human lung adenocarcinoma SPC-A1 cells in the early stage. Monoclonal antibody NJ001 recognizes a specific protein molecule located in the plasma and on the cell membrane of lung adenocarcinoma cells, having a molecular weight of 70kD, which is subsequently named tumor-specific protein 70 by the subject group (Tumor specific protein, TSP70, also known as SP 70). Early studies demonstrated that SP70 is positively correlated with the poor prognosis of lung cancer patients, we also found that the use of NJ 001-blocked SP70 can significantly inhibit proliferation, invasion, metastasis of lung cancer cells and induce apoptosis, suggesting that SP 70-related molecular targets may have potential therapeutic value for lung cancer.

LncRNA (long non-coding RNA) is RNA with a transcript length of more than 200nt, which lacks an obvious open reading frame, is positioned in a cell nucleus or cytoplasm, does not participate in protein coding function, and regulates gene expression in multiple layers such as epigenetic, transcription and posttranscriptional in an RNA form, thereby participating in various physiological and pathological processes of an organism. A great deal of researches find that various lncRNA abnormal expression phenomena exist in different tumors including lung cancer, and the abnormal expression phenomena play an important role in the processes of proliferation, invasion, metastasis, apoptosis and the like of tumor cells. Studies show that lncRNA can play a role in oncogene and/or cancer suppressor gene to participate in tumor occurrence and development, and can also be used as a molecular target to predict tumor occurrence and development. However, there is little research on lncRNA as a target for lung cancer treatment, we used SPC-A1 cells after NJ 001-blocked SP70 as an experimental group, and SPC-A1 cells without NJ 001-blocked SP70 as a control group, and screening differentially expressed lncRNA TC8260, but no relevant literature for lung cancer treatment target has been reported.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and providing application of lncRNA TC8260 as a lung cancer treatment target.

Another object of the invention is to provide the application of lncRNA TC8260 as a lung cancer diagnosis target.

The aim of the invention can be achieved by the following technical scheme:

the application of the lncRNA TC8260 as a therapeutic target in preparing or screening medicaments for treating lung cancer and/or lung cancer metastasis is disclosed, and the cDNA sequence corresponding to the lncRNA TC8260 is shown as SEQ ID NO. 1.

Application of lncRNA TC8260 or substances for promoting expression of lncRNA TC8260 in preparation of or screening of medicines for treating lung cancer and/or lung cancer metastasis.

Application of lncRNA TC8260 as detection target in preparing lung cancer auxiliary diagnostic reagent.

The application of the reagent for detecting the expression level of the lncRNA TC8260 in preparing the lung cancer auxiliary diagnostic reagent.

In one preferred embodiment of the present invention, the reagent for detecting the expression level of lncRNA TC8260 is a specific primer for detecting lncRNA TC8260.

As a further preferred aspect of the invention, the specific primer for detecting the lncRNA TC8260 is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

The beneficial effects are that:

the early-stage experiment of the invention discovers that the NJ 001-blocked SP70 can obviously inhibit proliferation, invasion and metastasis of lung cancer cells and can induce apoptosis. Thus, the present invention uses SPC-A1 cells after NJ 001-blocked SP70 as an experimental group, and SPC-A1 cells without NJ 001-blocked SP70 as a control group, to screen differentially expressed LncRNA TC8260. The expression level of HBE of lncRNA TC8260 in normal human bronchial epithelial cell strain is significantly higher than that of lung cancer cell strain (SPC-A1, A549, H1299, H358, H460, H520) (P < 0.05). The expression level of lncRNA TC8260 was also significantly higher in healthy controls than in lung cancer patients serum (P < 0.05). Comparison of the ability of lncRNA TC8260 siRNA and corresponding control transfected cells to migrate was observed by cell scoring experiments, and down-regulating lncRNA TC8260 promoted SPC-A1 cell migration as shown in FIG. 6. The lncRNA TC8260 can be used as a therapeutic target spot to prepare or screen medicaments for treating lung cancer and/or lung cancer metastasis. The lncRNA TC8260 can also be used as a detection target spot to prepare an auxiliary diagnosis reagent for lung cancer.

Compared with the existing molecular therapeutic targets such as EGFR, ROS1, PD1 and the like, the lncRNA TC8260 can provide a new therapeutic strategy selection for advanced lung cancer patients which do not meet therapeutic selection standards, can also provide a new therapeutic thought for lung cancer patients with ineffective personalized targeted therapy, and is beneficial to the development of novel small-molecule drugs with potential therapeutic value.

Drawings

FIG. 1 shows cluster map of the differential expression of lncRNA of SPC-A1 and SPC-A1+NJ001.

Note that: SPC-A1+NJ001 is an SPC-A1 cell treated with NJ001 for 24 hours.

FIG. 2 shows the relative expression levels of lncRNA EN5775, lncRNA TC8260, and lncRNA TC9593 in SPC-A1+NJ001 and SPC-A1.

FIG. 3 shows the relative expression level of lncRNA TC8260 in lung cancer cell lines and HBE

Wherein 1 represents HBE,2 represents SPC-A1,3 represents A549,4 represents H1299,5 represents H358,6 represents H460, and 7 represents H520.

FIG. 4.LncRNA TC8260 expression is down-regulated in serum of lung cancer patients.

FIG. 5 interference efficiency of lncRNA TC8260 interference fragments

FIG. 6 scratch assay to detect the effect of lncRNA TC8260 siRNA on SPC-A1 cell migration ability

Detailed Description

Example 1

To screen for differentially expressed lncRNA, we used a core lncRNA microarray chip to obtain the lncRNA expression profile.

(1) Cell treatment: selecting SPC-A1 cells in logarithmic phase, digesting and collecting to obtain single cell suspension, and plating cell amount per well of 6-well cell culture plate by 2×10 ⁵ The culture supernatant was aspirated off overnight and the cells were washed once per well with 500. Mu.l of warm-bath Hank's solution. Adding a monoclonal antibody NJ001 solution into each hole of the SP70 closed group, wherein the final concentration is 300 ng/. Mu.l; 500 μl of culture medium was added to each well of the control group, and 3 parallel wells were placed in each group. After 36h of culture after intervention, the cell culture solution in the 6-well plate is discarded, 1ml of MZ lysate is directly added into the culture plate to lyse cells, the cells are blown by the MZ lysate for several times, and the cells are digested and lysed. After the cells are completely lysed, collecting cell suspension in an Eppendorf tube without RNase, and directly using the cell suspension for the next step of RNA extraction or placing the cell suspension in a refrigerator at the temperature of minus 80 ℃ for freezing and preserving after the cell suspension is uniformly mixed by shaking by a vortex oscillator.

(2) RNA extraction: cell suspension RNA of the above-mentioned MZ lysate was extracted using a miRcute miRNA extraction kit (Beijing Tiangen Biochemical technology Co., ltd.) according to the manual.

(3) RNA concentration and purity determination: the ratio of A260/A280 of the RNA sample is measured by a Nanodrop 2000 ultra-micro spectrophotometer (Sieimer's flight), and is 1.8-2.0, which indicates that the RNA has high purity and can be used for subsequent experiments.

(4) And (3) gene chip detection: the gene expression profile chip detection starts with the total RNA of a sample to be detected, after quality inspection, a single-stranded cDNA is obtained by reverse transcription synthesis of a strand, double-stranded cDNA is obtained by synthesis of double strands, and then biotin-labeled antisense RNA (aRNA) is obtained by in vitro transcription. The aRNA is purified by using magnetic beads, salt, enzyme and other impurities are removed, and the fragments are treated and hybridized with probes on a chip. And after washing and dyeing, scanning the image on the chip to obtain original data, and finally carrying out data analysis.

By detection on the lncRNA expression profiling chip, 83 lncRNAs were found to be expressed differentially between SPC-A1+NJ001 group and SPC-A1 group (FIG. 1).

Example 2

Expression levels of lncRNAEN5775, lncRNA TC8260 and lncRNATC9593 in SPC-A1 cells of blocked SP70 were detected by a real-time fluorescent quantitative PCR method, and SPC-A1 of unblocked SP70 was used as a control group.

(1) Reverse transcription reaction:

20 μl system: RNA (adjusted to 100. Mu.g-500. Mu.g) was mixed with 4. Mu.l of 5X PrimeScript RT Master Mix and made up to a total volume of 20. Mu.l with RNase-free water; reverse transcription conditions were 37℃for 15min,85℃for 5s and 4℃for infinity; preserving at-20 ℃ for standby.

(2) Real-time fluorescent quantitative PCR (Real-time PCR):

PCR primer: the first strand of cDNA is used as a template, and beta-actin is used as an internal reference to amplify lncRNA EN5775, lncRNA TC8260 and lncRNA TC9593.

The primers were dissolved with ddH2O to a final concentration of 100. Mu. Mol/L and stored at-20℃until use, working at a concentration of 10. Mu. Mol/ml. The primer sequences are shown in Table 1.

TABLE 1 PCR primer sequences

Reaction system (20 μl): 2X SYBR Premix Dimer Eraser. Mu.l, 0.8. Mu.l each of the upstream primer (10. Mu. Mol/ml) and the downstream primer (10. Mu. Mol/ml), 0.4. Mu.l each of ROX, ddH2O 6. Mu.l and 2. Mu.l each of the cDNA template.

Amplification procedure (two-step method): pre-denaturation at 95℃for 30s, denaturation at 95℃for 5s, annealing at 57℃for 30s, elongation at 72℃for 34s,40 cycles.

(3) Analysis of experimental results: beta-actin is used as an internal reference and a relative quantification method is adopted. Subtracting the Ct value of the corresponding beta-actin from the Ct value of each sample target gene to obtain a difference value, namely delta Ct=Ct target gene-Ct beta-actin, wherein the relative quantification of each sample is 2 ^-△Ct =2- (Ct gene of interest-Ct β -actin), and the two sets of samples were compared using unpaired t-test or rank-sum test. In the cell line, corresponding to the negative control group, the fold change of the target gene was 2- Δ C t method (ΔΔct= Δct target gene- Δct NC), and the paired t-test was used for comparison of the two groups of samples. Three duplicate wells were set and the assay was repeated 3 times per trial.

The results showed that lncRNA EN5775, lncRNA TC8260, lncRNA TC9593 were significantly elevated in SPC-A1+NJ001 group compared to SPC-A1 group (P < 0.05), consistent with the chip results (FIG. 2).

Example 3

(1) Cell culture: the basal culture mediums of SPC-A1, A549, H1299, H358, H460 and H520 are RPMI-1640, DMEM, RPMI-1640 and DMEM, DMEM, DMEM respectively, and are prepared into complete culture solutions containing 100U/ml penicillin, 100 mug/ml streptomycin and 10% fetal bovine serum before use; human bronchial epithelial cell line HBE was cultured in RPMI-1640 medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% fetal bovine serum, at 37℃under 5% CO2 and saturated humidity.

(2) RNA extraction: the cell RNA was extracted using a miRcute miRNA extraction and separation kit (Beijing Tian Gen) according to the instructions.

(4) RT-PCR: see example 2 for details.

The results showed that lncRNA TC8260 expressed significantly higher levels of HBE in normal human bronchial epithelial cell lines than in lung cancer cell lines (SPC-A1, a549, H1299, H358, H460, H520) (P < 0.05) (fig. 3).

Example 4

(1) Serum sample collection: the untreated lung cancer primary patients and healthy physical examination patients were collected and anticoagulated whole blood was centrifuged for 10min at 3000rmp, the supernatant serum was aspirated into RNase-free 1.5ml Eppendorf tubes, centrifuged for 10min at 12000rmp at 4℃and serum (note not to aspirate precipitation) was aspirated into fresh RNase-free 1.5ml Eppendorf tubes, and RNA was either frozen at-80℃or directly extracted.

(2) RNA extraction: RNA was extracted from 1ml of serum samples using the BioTeke extraction kit according to the instructions;

(4) Reverse transcription reaction:

20 μl system: mu.l of RNA was mixed with 4. Mu.l of 5X PrimeScript RT Master Mix and made up to a total volume of 20. Mu.l with RNase-free water; reverse transcription conditions were 37℃for 15min,85℃for 5s and 4℃for infinity; preserving at-20 ℃ for standby.

(5) Real-time fluorescent PCR: see example 2 for details.

The results showed that the expression level of lncRNA TC8260 in serum of healthy controls was significantly higher than that of lung cancer patients (P < 0.05) (fig. 4).

Example 5

(1) Dissolving siRNA: taking out the dry powder at-20deg.C, 12000rpm×2min; lightly opening the tube cover, adding DEPC water with corresponding volume according to instructions to prepare siRNA solution with final concentration of 2M, sub-packaging and freezing at-20 ℃ for later use.

(2) Cell culture: human lung adenocarcinoma SPC-A1 cells were cultured with RPMI-1640 medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% fetal bovine serum.

(3) Cell plating: collecting the supernatant which has good growth state and is centrifuged at 1000rmp for 5min; the cells were resuspended in RPMI-1640 medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% fetal bovine serum to maintain the cell density at (4.0-10.0). Times.10 ⁵ Per ml, inoculated into six-well culture plates, 2ml per well, 37 ℃, 5%Culturing under CO2 and saturated humidity for adherence for later use;

(4) Transfection: taking 1.5ml sterile EP tubes, adding 125 mu l RPMI-1640 basal medium and 7.5 mu l Lipofectamine3000 into each tube; another 1.5ml of sterile EP was obtained in the same amount, and 125. Mu.l of RPMI-1640 basal medium and 3.75. Mu.l of siRNA were added to each tube; standing at room temperature for 5min, and mixing the diluted Lipofectamine3000 and siRNA; standing at room temperature for 15min; sucking and discarding the RPMI-1640 complete culture solution in the 6-hole plate, washing 1 time by 1ml PBS, and sucking and discarding; adding mixed solution of Lipofectamine3000 and siRNA (250 μl) into each well, culturing at 37deg.C under 5% CO2 and saturated humidity; after 6 hours, changing into 2ml of RPMI-1640 complete culture solution for continuous culture for 48 hours; cells were collected and the transfection efficiency was measured by real-time fluorescent PCR.

(5) Real-time fluorescent PCR: as before.

The result shows that SPC-A1 cells with the transfection efficiency reaching the requirement are collected, RNA is extracted, and the interference efficiency is detected by qRT-PCR. The results show that the interference efficiency of the sh-TC8260-2 fragment in SPC-A1 cells reaches 50%, and the interference efficiency is better (FIG. 5). Thus, this interference fragment was selected for subsequent cell experiments.

Example 6

(1) Cell culture: human lung adenocarcinoma SPC-A1 cells were cultured with RPMI-1640 medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% fetal bovine serum.

(2) Cell plating: collecting the supernatant which has good growth state and is centrifuged at 1000rmp for 5min; the cells were resuspended in RPMI-1640 medium containing 100U/ml penicillin, 100. Mu.g/ml streptomycin and 10% fetal bovine serum to maintain the cell density at (4.0-10.0). Times.10 ⁵ Inoculating to six-hole culture plate, culturing at 37deg.C under 5% CO2 and saturated humidity for adherence, and standing;

(3) Transfection: taking 1.5ml sterile EP tubes, adding 125 mu l RPMI-1640 basal medium and 7.5 mu l Lipofectamine3000 into each tube; another 1.5ml of sterile EP was obtained in the same amount, and 125. Mu.l of RPMI-1640 basal medium and 3.75. Mu.l of siRNA were added to each tube; standing at room temperature for 5min, and mixing the diluted Lipofectamine3000 and siRNA; standing at room temperature for 15min; sucking and discarding the RPMI-1640 complete culture solution in the 6-hole plate, washing 1 time by 1ml PBS, and sucking and discarding; adding mixed solution of Lipofectamine3000 and siRNA (250 μl) into each well, culturing at 37deg.C under 5% CO2 and saturated humidity; after 6 hours, changing into 2ml of RPMI-1640 complete culture solution for continuous culture for 48 hours;

(4) Scoring: SPC-A1 cell migration was recorded by drawing "well" patterns on each well of a 6-well plate using a sterile 100-200. Mu.l yellow gun head and photographing with an Olympus optical microscope (Olympus Japan).

Comparison of the ability of lncRNA TC8260 siRNA and corresponding control transfected cells to migrate was observed by cell scoring experiments, and down-regulating lncRNA TC8260 promoted SPC-A1 cell migration as shown in FIG. 6.

Sequence listing

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Claims

1. The application of the reagent for detecting the expression quantity of the lncRNA TC8260 in preparing the lung cancer auxiliary diagnostic reagent is characterized in that the reagent for detecting the expression quantity of the lncRNA TC8260 is a specific primer for detecting the lncRNA TC8260, and the specific primer for detecting the lncRNA TC8260 is shown as SEQ ID NO. 4 and SEQ ID NO. 5.