Application of piRNA as target spot in preparation of medicine for treating lung cancer or lung injury caused by tobacco exposure

Abstract

The invention relates to application of piRNA as a target spot in preparation of a medicine for treating lung cancer or lung injury caused by tobacco exposure, and belongs to the technical field of biomedicine. The invention provides application of piR-2850767 and / or piR-157618 as a target spot in preparation of a medicine for treating lung cancer or lung injury caused by tobacco exposure, and particularly relates to application of an expression inhibitor of piR-2850767 and / or an overexpression reagent of piR-157618 in preparation of a medicine for treating lung cancer or lung injury caused by tobacco exposure. The inhibitor and the overexpression preparation can cause growth inhibition of lung cancer representative cells A549 and cause apoptosis of the lung cancer representative cells A549, and can recover cell cycle arrest, cell apoptosis, cell proliferation and motor ability decline of lung epithelial cells caused by tobacco exposure as a preventive drug for lung injury caused by tobacco exposure.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and specifically to the use of piRNA as a target in the preparation of drugs for treating lung cancer or lung damage caused by tobacco exposure. Background Art

[0002] PIWI-interacting RNA (piRNA) is a non-coding small RNA with a length of 23-31 nucleotides. As a new epigenetic indicator, it can play an epigenetic regulatory role by silencing transposons, regulating DNA methylation modification, and controlling mRNA transcription and translation, affecting the expression level of genes, and thus participating in the occurrence and development of various diseases. Studies have shown that the abnormal expression of multiple piRNAs in the plasma and cancer tissues of lung cancer patients may involve multiple signal transduction pathways, such as inflammation, apoptosis, and cell cycle; some specific piRNAs are abnormally expressed in lung cancer, and perturbations of these specific piRNAs can affect cell malignant phenotypes such as proliferation, metastasis, and invasion. These research results suggest that piRNA is expected to become a biomarker for early diagnosis of lung cancer and a new therapeutic target. Summary of the invention

[0003] The present invention provides the use of piRNA as a target in the preparation of a drug for treating lung cancer or lung damage caused by tobacco exposure, and discloses the use of an expression inhibitor of piR-2850767 and / or an overexpression agent of piR-157618 in the preparation of a drug for treating lung cancer or lung damage caused by tobacco exposure. The expression inhibitor and / or overexpression agent are used to inhibit the growth and apoptosis of A549 cells in lung cancer, and can be used as a preventive drug for lung damage caused by tobacco exposure, and can restore cell cycle arrest, apoptosis, cell proliferation and decreased motility of lung epithelial cells caused by tobacco exposure.

[0004] According to the purpose of the present invention, there is provided the use of piR-2850767 and / or piR-157618 as targets in the preparation of drugs for treating lung cancer or lung damage caused by tobacco exposure, the nucleotide sequence of the piR-2850767 being shown as SEQ ID NO 1, and the nucleotide sequence of the piR-157618 being shown as SEQ ID NO 4.

[0005] Preferably, the expression inhibitor of piR-2850767 and / or the overexpression agent of piR-157618 are used in the preparation of a drug for treating lung cancer or lung damage caused by tobacco exposure.

[0006] Preferably, the nucleotide sequence of the expression inhibitor is shown as SEQ ID NO 3, and the nucleotide sequence of the overexpression agent is shown as SEQ ID NO 5 and SEQ ID NO 6.

[0007] Preferably, the expression inhibitor and / or the overexpression agent is used to inhibit the growth and apoptosis of A549 cells in lung cancer.

[0008] Preferably, the lung cancer is non-small cell lung cancer.

[0009] Preferably, the expression inhibitor and / or the overexpression agent is used to inhibit cell cycle arrest, cell apoptosis, decreased cell proliferation ability and decreased cell motility ability of lung epithelial cells induced by tobacco exposure.

[0010] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0011] (1) The present invention discloses that the expression levels of piR-2850767 and piR-157618 in lung epithelial cells after tobacco exposure are significantly changed, the expression of piR-2850767 increases with the increase of tobacco exposure dose, and the expression level of piR-157618 decreases with the increase of tobacco exposure dose; in addition, the present invention also finds that inhibiting the expression of piR-2850767 or increasing the expression of piR-157618 can significantly restore the cell cycle arrest, apoptosis, cell proliferation and decreased motility of lung epithelial cells caused by tobacco exposure, and can cause growth inhibition and apoptosis of A549, a representative cell of lung cancer. Therefore, the application of piR-2850767 and piR-157618 as lung cancer treatment agents or drugs has important value.

[0012] (2) There were significant statistical differences in the expression of piR-2850767 and piR-157618 in the plasma of patients with non-small cell lung cancer and healthy controls. The expression of piR-2850767 in the plasma of patients with lung cancer was higher, while that of piR-157618 was lower. However, the expression of piR-2850767 in the plasma of patients with lung cancer after surgery was significantly reduced, while that of piR-157618 was significantly increased. Compared with adjacent adjacent tissues, piR-2850767 was significantly more highly expressed in cancer tissues, while piR-2850767 was significantly less expressed. Using piR-2850767 and piR-157618 as therapeutic targets for lung cancer to develop related therapeutic agents or drugs has important significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Changes in the cellular expression levels of candidate piRNAs in lung epithelial cells after tobacco exposure.

[0014] Figure 2 This is the effect of piR-2850767Inhibitor on the expression level of piR-2850767 in lung cells and the effect of piR-157618Mimic on the expression level of piR-157618 in lung cells.

[0015] Figure 3 Inhibition of piR-2850767 expression or overexpression of piR-157618 inhibits A549 cell growth.

[0016] Figure 4 To inhibit piR-2850767 expression or piR-157618 overexpression to cause apoptosis of A549 cells.

[0017] Figure 5 To inhibit piR-2850767 expression or overexpression of piR-157618 to affect a series of cellular phenotypic changes caused by tobacco exposure. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0019] In the present invention, the nucleotide for down-regulating the expression of piR-2850767 is used as an expression inhibitor, which is a nucleotide for targeted down-regulation of the expression of piR-2850767, and the effective sequence is the reverse complementary sequence of the original sequence of piR-2850767.

[0020] SEQ ID NO.1 (piR-2850767 sequence):

[0021] 5'-GCUGG GAAGG CCCGG CGGGG AAGGG G-3'

[0022] SEQ ID NO.2 (DNA sequence corresponding to piR-2850767 sequence):

[0023] 5'-gctgg gaagg cccgg cgggg aaggg g-3'

[0024] SEQ ID NO.3 (complementary sequence of SEQ ID NO.1):

[0025] 5'-CCCCUUCCCCGCCGGGCCUUCCCAGC-3'

[0026] piR-2850767Inhibitor competitively binds to the mature piR-2850767 sequence (SEQ ID NO.1) through chemically modified RNA single-stranded nucleotides (SEQ ID NO.3), thereby weakening the gene transcription efficiency of endogenous piR-2850767 in tissues / cells and downregulating the expression of piR-2850767. Specifically, piR-2850767Inhibitor is an inhibitor designed specifically for the expression of piR-2850767 in tissues / cells. It is synthesized by chemical synthesis and can specifically target and downregulate the expression level of piR-2850767.

[0027] In the present invention, the nucleotide for up-regulating the expression of piR-157618 is used as an over-expression reagent (Mimic), which is a nucleotide for up-regulating the expression of piR-157618 in a targeted manner, and the effective sequence is a double-stranded RNA identical to the original sequence.

[0028] SEQ ID NO.4 (piR-157618 sequence):

[0029] 5'-AGUUG GUCUG AGUGU UGUGG GUUAU UGUUA AG-3'

[0030] SEQ ID NO.5 (DNA sequence corresponding to piR-157618 sequence):

[0031] 5'-agttg gtctg agtgt tgtgg gttat tgtta ag-3'

[0032] SEQ ID NO.6 (the complementary sequence of SEQ ID NO.4):

[0033] 5'-UAACAAUAACCCACAACACUCAGACCAACUUU-3'

[0034] Mimics are usually designed as double-stranded structures, so the effective sequence of piR-157618Mimic is a double-stranded RNA identical to the original sequence. The mechanism of action of piR-157618Mimic is to synthesize piR-157618Mimic through chemical synthesis, mimicking endogenous mature piR-157618 in cells, to specifically target and upregulate the expression level of piR-157618.

[0035] The following are specific embodiments

[0036] Example 1: Tobacco exposure causes increased expression of piR-2850767 and decreased expression of piR-157618 in lung epithelial cells

[0037] All primer pairs of piR-2850767, piR-157618 and U6 (this gene sequence is a non-coding small RNA with a length of about 100 nucleotides, which is stably expressed in a variety of tissues and cells and does not involve the regulatory pathway of small RNA. It is often used as a housekeeping gene for the detection of non-coding RNA expression levels in cells, blood, and tissues) used in this example were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] The reverse transcription primer sequence of piR-2850767 used in the reverse transcription experiment was 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCTT-3' (SEQ ID NO.7), and the reverse transcription primer sequence of piR-157618 was

[0039] 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCT TAAC-3' (SEQ ID NO. 8), and the reverse transcription primer sequence of U6 is 5'-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO. 9).

[0040] The upstream primer sequence of piR-2850767 used in the real-time fluorescence quantitative PCR experiment was 5'-GCTGGGAAGGCCCGGCGGGG-3' (SEQ ID NO.10), the upstream primer sequence of piR-157618 was 5'-AGTTGGTCTGAGTGTTGTGGGTTATT-3' (SEQ ID NO.11), and the downstream primer sequences of piR-2850767 and piR-157618 were both 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQ ID NO.12). The upstream primer sequence of U6 was 5'-CTCGCTTCGGCAGCACA-3' (SEQ ID NO.13), and the downstream primer sequence of U6 was 5'-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO.14).

[0041] 1. Experimental Methods

[0042] (1) Cell culture: The lung epithelial cell lines BEAS-2B and 16HBE were used as cell models. T25 culture flasks were used and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The culture medium was MEM (containing NEAA) medium with 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin antibody).

[0043] (2) Cell counting: When the cells are in the logarithmic growth phase, the original culture medium is discarded, 1 mL of PBS is added to wash three times, 1 mL of trypsin is added and the T25 is placed in the incubator for incubation for 2 minutes. Gently shake until a large area of ​​cells fall off. Add 3 mL of culture medium to terminate cell digestion, and use a sterile Pasteur pipette to blow the cells to mix well. Take 10 μL and count in a cell counting plate.

[0044] (3) Cell plating and infection treatment: 1×10 5 Cells were inoculated in a 6-well plate, and the cells were evenly distributed in the wells, and then the 6-well plate was placed in a constant temperature incubator for culture. When the cell density reached 80% (24 hours), the original culture medium was discarded, and 2 mL of serum-free and double-antibody culture medium containing 0.25‰, 0.5‰, and 1‰ CSE (tobacco smoke agglutinating fluid) was added for poisoning treatment, and then the 6-well plate was placed in a constant temperature incubator for culture.

[0045] (4) Total RNA extraction: After CSE treatment for 24 hours and 48 hours, the culture medium was discarded, 1 mL of PBS was added to each well for washing three times, and then 1 mL of Trizol was added and placed in a refrigerator at 4°C for 15 minutes to promote complete cell lysis. After blowing the cells, the cells were transferred to a new 1.5 mL EP tube, and 200 μL of chloroform was added respectively. After vigorous shaking and extraction, the tube was placed at room temperature for 15 minutes, centrifuged at 12,000 rpm for 15 minutes, and 500 μL of the upper colorless aqueous phase (containing RNA) was drawn into a new EP tube 5 times (100 μL each time) and the same volume of isopropanol was added. After inversion and mixing, the tube was placed at room temperature for 15 minutes, and centrifuged at 412,000 rpm for 10 minutes. The supernatant was discarded and 1 mL of 80% ethanol was added for washing, and the tube was centrifuged at 4°C and 12,000 rpm for 8 minutes. This step was repeated 3 times. Discard the supernatant and dry for 20 minutes, add 40 μL of RNase-Free ddH2O to dissolve RNA, and let it stand at room temperature for 20 minutes to allow the RNA to fully dissolve.

[0046] (5) Reverse transcription: 1) Removal of genomic DNA: Take 200 μL of eight tubes without nuclease, prepare 20 μL of reaction system according to the following table, centrifuge and set the reaction program (incubate at 42°C for 2 min).

[0047] RNase-Free ddH2O 6μL

[0048] 5×gDNA Wiper Mix 4μL

[0049] >100ng / uL RNA Template 10μL

[0050] 2) First-strand cDNA synthesis: In the above 200 μL nuclease-free eight-tube, prepare 40 μL reverse transcription reaction system according to the table below. After centrifugation, set the reaction program (preheat at 25°C for 5 min, incubate at 50°C for 15 min, and incubate at 80°C for 5 min to inactivate reverse transcriptase). The synthesized cDNA can be stored at -20°C for use.

[0051]

[0052] The stem-loop primer (reverse transcription primer) is a nucleotide sequence specifically designed for different small RNAs. The reverse transcription primer sequence of piR-2850767 is 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCC CCTT-3' (SEQ ID NO.7), and the reverse transcription primer sequence of piR-157618 is

[0053] 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCT TAAC-3' (SEQ ID NO. 8), and the reverse transcription primer sequence of U6 is 5'-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO. 9).

[0054] (6) Real-time fluorescence quantitative PCR technology to detect the relative expression of piR-2850767 and piR-157618: Prepare 10 μL of qPCR reaction system according to the table below, vortex and mix, add to the 386-well plate, seal with sealing film and centrifuge. Then place the 386-well plate in the ABIQ7 real-time fluorescence quantitative PCR instrument and react according to the following procedure: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, denaturation and extension for 40 cycles, and then melt curve reaction.

[0055]

[0056]

[0057] The Specific Primer is the upstream primer, which is a nucleotide sequence specifically designed for different small RNAs. The upstream primer sequence of piR-2850767 is

[0058] 5'-GCTGGGAAGGCCCGGCGGGG-3' (SEQ ID NO.10), the upstream primer sequence of piR-157618 is 5'-AGTTGGTCTGAGTGTTGTGGGTTATT-3' (SEQ ID NO.11). In addition, mQ primer R is a universal downstream primer for small RNA reverse transcription products, and its sequence is 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQID NO.12), which is used as the downstream primer of piR-2850767 and piR-157618. The upstream primer sequence of U6 is 5'-CTCGCTTCGGCAGCACA-3' (SEQ ID NO.13), and the downstream primer sequence is 5'-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO.14).

[0059] 2. Experimental results

[0060] like Figure 1 As shown in the figure, CSE was exposed for 24 hours ( Figure 1 Middle a) and 48 hours ( Figure 1 After exposure to tobacco, the expression of piR-2850767 in lung epithelial cells increased, while the expression of piR-157618 decreased. The candidate piRNAs screened by the research team in the early stage, among which piR-2850767 and piR-157618 are potential non-small cell lung cancer specific diagnostic markers. Combined with the above, it is suggested that piR-2850767 and piR-157618 have the potential to be used as targets for the preparation of preventive drugs for lung damage caused by tobacco exposure.

[0061] Example 2: Verification of piR-2850767Inhibitor Silencing Efficiency and piR-157618Mimic Overexpression Efficiency

[0062] 1. Experimental Methods

[0063] The nucleotide sequences of the piR-2850767 expression inhibitor and piR-157618 overexpression reagent used in this example were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The inhibitor control used the company's Inhibitor negative control, i.e., the Inhibitor NC group, with the sequence 5'-CAGUACUUUUUGUGUAGUACAA-3' (SEQ ID NO.15), and the overexpression reagent control used the company's Mimic negative control, i.e., the Mimic NC group 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.16) and its complementary sequence 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.17).

[0064] (1) Using lung epithelial cell lines BEAS-2B and 16HBE and lung cancer cell line A549 as cell models, the experimental methods of cell culture and cell counting were the same as those in Example 1.

[0065] (2) Cell plating and transfection: 1×10 per well 5 Inoculate cells in a 6-well plate and ensure that the cells are evenly distributed in the wells, then place the 6-well plate in a constant temperature incubator for culture. Transfection is performed when the cell density reaches 40%. Add 250μL RNase-Free ddH2O water to the reagent bottle containing 5nmol of the interference sequence to obtain a 20μmol / L interference sequence stock solution. Dilute 5μL of 20μmol / L interference sequence with 245μL serum-free and double antibody culture medium, mix gently, and incubate at room temperature for 5min. Dilute 5μL Lipofectamine with 245μL serum-free and double antibody culture medium TM 3000, gently mix and incubate at room temperature for 5 minutes. Gently mix the two mixtures, incubate at room temperature for 20 minutes, add 1500μL of serum-free and dual antibody culture medium, so that the final concentration of the interference sequence is 50nmol / L. Aspirate the original culture medium, add 2mL of transfection mixture to each well, and shake gently. After culturing the 6-well plate in an incubator for 6 hours, aspirate the culture medium containing the interference sequence, replace it with fresh complete culture medium containing serum and dual antibodies, and culture it for another 48 hours. The cell transfection groups include Inhibitor NC group, Mimic NC group, piR-2850767Inhibitor group, and piR-157618Mimic group.

[0066] (3) The experimental methods for total RNA extraction, reverse transcription and qPCR detection of the relative expression of piR-2850767 and piR-157618 were the same as those in Example 1. The results were expressed as 2^-ΔΔCT, and the final treatment groups were compared with the NC group.

[0067] 2. Experimental results

[0068] like Figure 2 As shown, after 48 hours of cell transfection, the expression of piR-2850767 ( Figure 2 middle a) and piR-157618 expression levels ( Figure 2 There was no significant change in the middle (Fig. b), but the expression level of piR-2850767 in the piR-2850767Inhibitor group cells decreased, and all three cell types dropped below 50% ( Figure 2 (a), the expression level of piR-157618 in the piR-157618 Mimic group cells was significantly increased, and the three types of cells were increased by more than 3 times that of the control group ( Figure 2 The results suggest that piR-2850767Inhibitor can effectively down-regulate the expression of piR-2850767 in normal lung epithelial cells and lung cancer cells, and piR-157618Mimic can effectively up-regulate the expression of piR-157618 in normal lung epithelial cells and lung cancer cells.

[0069] Example 3: piR-2850767 expression inhibitor or piR-157618 overexpression reagent can inhibit cell growth and induce apoptosis in lung cancer cell line A549

[0070] 1. Experimental Methods

[0071] (1) Lung cancer cell line A549 was used as a cell model, and the experimental methods of cell culture and cell counting were the same as those in Example 1.

[0072] (2) Cell viability assay: 3000 cells were seeded per well in a 96-well plate, and the cells were evenly distributed in the wells. The 96-well plate was then placed in a constant temperature incubator for culture. When the cell density reached 40% (18 hours), the original culture medium was discarded, and 200 μL of serum-free and dual antibody culture medium containing the interference sequence (transfection mixture) was added and gently shaken. After culturing the 96-well plate in an incubator for 6 hours, the culture medium containing the interference sequence was discarded, and fresh complete culture medium containing serum and dual antibodies was replaced, and then cultured for another 24 hours and 48 hours. The cell transfection groups included Inhibitor NC group, Mimic NC group, piR-2850767Inhibitor group, and piR-157618Mimic group. After 24 hours and 48 hours of transfection, the original culture medium was discarded, and 110 μL of newly prepared complete culture medium containing 10 μL of CCK-8 reagent was quickly added. After incubation in a constant temperature incubator for 40 minutes, the absorbance value was detected in a microplate reader to evaluate cell viability, and the detection wavelength was 450 nm. The results were calculated as cell proliferation rate (%) = (experimental group OD-blank group OD) / (control group OD-blank group OD), and finally the transfected groups were compared with the NC group.

[0073] (3) Hoechst / PI apoptosis staining experiment: 0.3×10 5 Cells were inoculated in a 12-well plate, and the cells were ensured to be evenly distributed in the wells, and then the 12-well plate was placed in a constant temperature incubator for culture. When the cell density reached 40% (18 hours), the original culture medium was aspirated and 600 μL of serum-free and dual-antibody culture medium containing interference sequences (transfection mixture) was added and gently shaken. After culturing the 12-well plate in an incubator for 6 hours, the culture medium containing the interference sequence was aspirated and replaced with fresh complete culture medium containing serum and dual antibodies, and then cultured for 24 hours and 48 hours. The cell transfection groups included Inhibitor NC group, Mimic NC group, piR-2850767Inhibitor group, and piR-157618Mimic group. After 48 hours of transfection, Hoechst / PI apoptosis staining experiment was performed. After preheating the anti-dual antibody-free culture medium at 37°C, prepare the Hoechst / PI working solution. Add 5μL Hochest33342 staining solution (100×) and 5μL PI staining solution (100×) to every 500μL anti-dual antibody-free culture medium to form the staining working solution. Take a 12-well plate, discard the original culture medium, wash with an appropriate amount of PBS, add 500μL of staining working solution to each well, incubate at room temperature and avoid light for 15 minutes, wash once with an appropriate amount of PBS, and then add 1mL PBS to each well to prevent the cells from drying up. Then use an inverted fluorescence microscope to take pictures to detect cell apoptosis.

[0074] 2. Experimental results

[0075] The results of CCK-8 assay are as follows Figure 3 The results showed that the cell viability of the Inhibitor NC group and the Mimic NC group was above 97%; compared with the Inhibitor NC group, the 24-hour cell viability of the piR-2850767Inhibitor group was 74.81%±3.19%, and the 48-hour cell viability was 67.93%±1.64%; compared with the Mimic NC group, the 48-hour cell viability of the piR-157618Mimic group was 76.94%±3.67%, and the 48-hour cell viability was 64.28%±3.26%. The results of the Hoechst / PI apoptosis staining experiment are shown in Figure 2. Figure 4 As shown, 48 hours after transfection, compared with the control group (Inhibitor NC / Mimic NC), apoptosis was detected in A549 cells transfected with piR-2850767Inhibitor or piR-157618Mimic (the cells showed bright blue after Hoechst staining, which was an early apoptosis phenomenon - nuclear condensation, and the cells showed red after PI staining, which was a late apoptosis phenomenon). The results suggest that the piR-2850767 expression inhibitor or piR-157618 overexpression reagent can cause growth inhibition and apoptosis of A549, a representative lung cancer cell, indicating that piR-2850767 and piR-157618 can be used as targets to prepare drugs for the treatment of lung cancer, and have certain application prospects.

[0076] Example 4: piR-2850767 expression inhibitor or piR-157618 overexpression agent restores tobacco exposure-induced cell cycle arrest, apoptosis, cell proliferation and decreased motility of lung epithelial cells

[0077] 1. Experimental Methods

[0078] (1) The experimental methods of cell culture, cell counting, cell plating and transfection treatment are the same as those in Example 2.

[0079] (2) Cell poisoning: 24 hours after cell transfection, the original culture medium was discarded, and 2 mL of serum-free and double-antibody culture medium containing 0.5‰ CSE was added for poisoning, and then the 6-well plate was placed in a constant temperature incubator for culture. After 24 hours of CSE treatment, cell cycle (PI staining flow cytometry) and cell apoptosis (PI staining flow cytometry) detection, plate cloning experiment, scratch experiment, and transwell experiment were performed.

[0080] (3) Cell cycle detection: Cell pretreatment was performed according to the instructions of the "PI staining flow cytometry detection kit", PI red fluorescence was detected by flow cytometer, and cell cycle analysis was performed using FlowJo software. The results were the percentage (%) of cells in the G1 phase, S phase, and G2 / M phase of the total cell number.

[0081] (4) Cell apoptosis detection: Cell pretreatment was performed according to the instructions of the “ANNEXIN V-FITC / PI Apoptosis Detection Kit”. FITC green fluorescence and PI red fluorescence were detected by flow cytometry. FlowJo software was used for cell apoptosis analysis. The number of early apoptotic and late apoptotic cells was summed up as the number of apoptotic cells. Finally, all treatment groups were compared with the NC group.

[0082] (5) Plate cloning experiment: After 24 hours of CSE treatment, the original culture medium was discarded, 1 mL of PBS was added to wash three times, 0.5 mL of trypsin was added and the 6-well plate was placed in an incubator for incubation for 2 minutes. Gently shake to see large-scale cell detachment, add 1 mL of culture medium to stop cell digestion, and use a sterile Pasteur pipette to blow the cells and mix them evenly. After centrifugation, discard the supernatant, add 2 mL of complete culture medium containing serum and double antibody to resuspend the cells, and take 10 microliters to count in a cell counting plate. 400 cells were inoculated in each well of a 6-well plate, and the cells were evenly distributed in the wells. Then the 6-well plate was placed in a constant temperature incubator for 14 days. When the clones visible to the naked eye appeared in the culture dish, that is, the number of cells in most single clones was greater than 50, the culture was terminated, and the medium was changed every 3 days to observe the cell status. Then the original culture medium was discarded, 1 mL of PBS was added to wash once, 1 mL of 4% paraformaldehyde was added to fix for 30 minutes, 1 mL of PBS was added to wash once, and 1 mL of crystal violet staining solution was added to stain the cells for 20 minutes. Finally, add 1 mL of PBS to wash several times, dry, and take pictures of the entire six-well plate and each well. Use ImageJ software to count clones, and count the number of clones with more than 50 cells. Finally, calculate the clone formation rate. The result is clone formation rate = (number of clones / number of inoculated cells) × 100%, and the final treatment group is compared with the NC group.

[0083] (6) Scratch test: Before cell plating, a marker pen was used to draw parallel lines evenly on the back of the 6-well plate with a ruler. The spacing between each horizontal line was 0.5 cm, and 5 horizontal lines were drawn in each well. 24 hours after cell transfection (cell density 100%), a ruler was used to draw a cell scratch with a 20ul pipette tip perpendicular to the well plate and the line, so that the scratch intersected with the marked line. Then 1mL PBS was added for washing three times, the cells were poisoned, and photos were taken with a microscope. As a control for 0 hours, the 6-well plate was placed in a constant temperature incubator for culture. Subsequently, the cells were removed at 6, 12, and 24 hours, the scratch width was observed under a microscope, and photos were taken with a microscope. The pre-made marks must be used as fixed observation points for timed photography. ImageJ software was used to measure the pixels of the scratch area to quantitatively compare the speed of cell migration. Finally, the treated groups were compared with the NC group.

[0084] (7) Transwell experiment: 750 μL of complete culture medium containing serum and dual antibodies was added to the lower chamber of a 12-chamber / 24-well plate. The plates were divided into Inhibitor NC group, Mimic NC group, Inhibitor NC + 0.5‰ CSE-treated group, Mimic NC + 0.5‰ CSE-treated group, piR-2850767Inhibitor group, piR-157618Mimic group, piR-2850767Inhibitor + 0.5‰ CSE-treated group, and piR-157618Mimic + 0.5‰ CSE-treated group. 24 hours after cell transfection, the original culture medium was discarded, 1mL PBS was added to wash three times, 0.5mL pancreatin was added and the 6-well plate was placed in the incubator for incubation for 2 minutes. Gently shake to see that the cells fell off in a large area, 1mL culture medium was added to stop cell digestion, and the cells were blown and mixed with a sterile Pasteur pipette, and the supernatant was discarded after centrifugation, and 2mL serum-free and double-antibody culture medium was added to resuspend the cells, and 10 microliters were taken to count in a cell counting plate. 200 cells per well were inoculated in the upper chamber of the 12-chamber / 24-well plate (no more than 200μL), and it was ensured that there were no bubbles between the upper and lower chambers, and then the 24-well plate was placed in a constant temperature incubator for 24 hours. The original culture medium of the upper and lower chambers was discarded, 1mL PBS was added to the upper chamber to wash once, 1mL 4% paraformaldehyde was added to fix for 30min, 1mL PBS was added to wash once, 0.3mL crystal violet stain was added, and the cells were stained for 20min, 1mL PBS was added to wash several times, and dried, and each upper chamber was photographed under a microscope. 3-5 fields of view were randomly selected, and the number of cells that migrated to the lower side of the membrane was counted and counted using ImageJ software. The results were used to calculate the percentage of cell migration (%), and the treatment groups were finally compared with the NC group.

[0085] 2. Experimental results

[0086] like Figure 5As shown in the figure, the results of in vitro functional validation of piR-2850767 and piR-157618 in two lung epithelial cell lines suggest that, compared with the control group, inhibition of piR-2850767 expression or overexpression of piR-157618 can restore the lung epithelial cell cycle arrest caused by tobacco exposure ( Figure 5 a), cell apoptosis ( Figure 5 b), decreased proliferation ( Figure 5 (Figure c), decreased plane movement ability ( Figure 5 Middle d), decreased longitudinal movement ability ( Figure 5 e). Therefore, the piR-2850767 expression inhibitor or the piR-157618 overexpression agent can be used to prepare a preventive drug for lung damage caused by tobacco exposure. Based on the results of all the above examples, it is suggested that the use of piR-2850767 and piR-157618 as targets in the preparation of drugs for treating lung cancer or lung damage caused by tobacco exposure has important value.

[0087] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of piR-2850767 and / or piR-157618 as targets in the preparation of drugs for treating lung cancer or lung damage caused by tobacco exposure, the nucleotide sequence of piR-2850767 is shown in SEQ ID NO 1, and the nucleotide sequence of piR-157618 is shown in SEQ ID NO 4.

2. The use according to claim 1, characterized in that Use of the piR-2850767 expression inhibitor and / or the piR-157618 overexpression reagent in the preparation of a drug for treating lung cancer or lung damage caused by tobacco exposure.

3. The use according to claim 1, characterized in that The nucleotide sequence of the expression inhibitor is shown in SEQ ID NO 3, and the nucleotide sequence of the overexpression agent is shown in SEQ ID NO 5 and SEQ ID NO 6.

4. The use according to claim 1, characterized in that The expression inhibitor and / or the overexpression agent is used to inhibit the growth and apoptosis of A549 cells in lung cancer.

5. The use according to claim 1, characterized in that The lung cancer is non-small cell lung cancer.

6. The use according to claim 1, characterized in that The expression inhibitor and / or the overexpression agent is used to inhibit cell cycle arrest, cell apoptosis, decreased cell proliferation ability and decreased cell motility ability of lung epithelial cells caused by tobacco exposure.

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