Application of piRNA as a target in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure.

Abstract

This invention relates to the application of piRNA as a target in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure, belonging to the field of biomedical technology. Specifically, this invention provides the application of piR-2850767 and / or piR-157618 as targets in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure, specifically the application of piR-2850767 expression inhibitors and / or piR-157618 overexpression agents in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure. The inhibitors and overexpression agents of this invention can induce growth inhibition and apoptosis in lung cancer representative cells A549, and as preventative drugs for lung injury caused by tobacco exposure, can restore cell cycle arrest, apoptosis, cell proliferation, and decreased motility in lung epithelial cells induced by tobacco exposure.

Description

Technical Field

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

[0002] PIWI-interacting RNAs (piRNAs) are small, non-coding RNAs of 23-31 nucleotides in length. As novel epigenetic markers, they exert epigenetic regulatory functions by influencing gene expression levels through transposon silencing, regulating DNA methylation, and controlling mRNA transcription and translation, thereby participating in the development and progression of various diseases. Previous studies have shown that the abnormal expression of various piRNAs in the plasma and cancer tissues of lung cancer patients may involve multiple signal transduction pathways, such as inflammation, apoptosis, and cell cycle. Certain specific piRNAs are abnormally expressed in lung cancer, and perturbation of these specific piRNAs can affect malignant cellular phenotypes such as proliferation, metastasis, and invasion. These findings suggest that piRNAs hold promise as biomarkers for early diagnosis of lung cancer and novel therapeutic targets. Summary of the Invention

[0003] This invention provides the application of piRNA as a target in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure. It discloses the application of an expression inhibitor of piR-2850767 and / or an overexpression agent of piR-157618 in the preparation of drugs for treating lung cancer or lung injury 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 injury caused by tobacco exposure, restoring cell cycle arrest, apoptosis, cell proliferation, and decreased motility of lung epithelial cells caused by tobacco exposure.

[0004] According to the present invention, the use of piR-2850767 and / or piR-157618 as targets in the preparation of medicaments for treating lung cancer or lung injury caused by tobacco exposure is provided, wherein the nucleotide sequence of piR-2850767 is shown in SEQ ID NO1 and the nucleotide sequence of piR-157618 is shown in SEQ ID NO4.

[0005] Preferably, the use of the expression inhibitor of piR-2850767 and / or the overexpression agent of piR-157618 in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure.

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

[0007] Preferably, the expression inhibitor and / or the overexpression reagent are 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 reagent are used to inhibit cell cycle arrest, apoptosis, decreased cell proliferation, and decreased cell motility in lung epithelial cells induced by tobacco exposure.

[0010] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0011] (1) This invention discloses that the expression levels of piR-2850767 and piR-157618 are significantly altered in lung epithelial cells after tobacco exposure. The expression of piR-2850767 increases with increasing tobacco exposure dose, while the expression level of piR-157618 decreases with increasing tobacco exposure dose. Furthermore, this invention also finds that inhibiting piR-2850767 expression or increasing piR-157618 expression can significantly restore the cell cycle arrest, apoptosis, cell proliferation, and motility reduction induced by tobacco exposure in lung epithelial cells, and can induce growth inhibition and apoptosis in A549, a representative lung cancer cell. Therefore, piR-2850767 and piR-157618 have significant value as therapeutic agents or drugs for lung cancer.

[0012] (2) The expression of piR-2850767 and piR-157618 in the plasma of non-small cell lung cancer patients and healthy controls showed statistically significant differences. Lung cancer patients had higher plasma piR-2850767 expression levels and lower piR-157618 expression levels. However, after surgery, the plasma piR-2850767 expression level in lung cancer patients was significantly reduced, while the plasma piR-157618 expression level was significantly increased. Compared with adjacent normal tissues, piR-2850767 was significantly highly expressed in cancerous tissues, while its expression level was significantly low. Developing therapeutic agents or drugs targeting piR-2850767 and piR-157618 for lung cancer treatment is of great significance and has promising application prospects. Attached Figure Description

[0013] Figure 1 This study investigated the changes in the cellular expression levels of candidate piRNAs in lung epithelial cells following tobacco exposure.

[0014] Figure 2 The effects of piR-2850767 Inhibitor on the expression level of piR-2850767 in lung cells and the effects of piR-157618 Mimic on the expression level of piR-157618 in lung cells were investigated.

[0015] Figure 3 To inhibit the expression of piR-2850767 or the overexpression of piR-157618 to suppress the growth of A549 cells.

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

[0017] Figure 5 Inhibition of piR-2850767 expression or overexpression of piR-157618 affects a series of cellular phenotypic changes induced by tobacco exposure. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0019] In this invention, the nucleotide that downregulates the expression of piR-2850767 is used as an expression inhibitor. It is a nucleotide that targets and downregulates the expression of piR-2850767, and the effective sequence is the reverse complementary sequence of the original piR-2850767 sequence.

[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):

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

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

[0025] 5'-CCCCUUCCCCGCCGGGCCUUCCCAGC-3'

[0026] The piR-2850767 inhibitor competitively binds to the mature piR-2850767 sequence (SEQ ID NO.1) via a chemically modified RNA single-stranded nucleotide (SEQ ID NO.3), thereby weakening the transcriptional efficiency of endogenous piR-2850767 in tissues / cells and downregulating piR-2850767 expression. Specifically, the piR-2850767 inhibitor is a chemically synthesized inhibitor specifically designed to target and downregulate piR-2850767 expression in tissues / cells. It specifically targets and downregulates piR-2850767 expression levels.

[0027] In this invention, the nucleotide that upregulates piR-157618 expression is used as an overexpression reagent (Mimic). It is a nucleotide that targets and upregulates piR-157618 expression, and the effective sequence is a double-stranded RNA with the same sequence as 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):

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

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

[0033] 5'-UAACAAUAACCCACAACACUCAGACCAACUUU-3'

[0034] Mimics are typically designed as double-stranded structures; therefore, the effective sequence of piR-157618Mimic is a double-stranded RNA identical to the original sequence. The mechanism of action of piR-157618Mimic involves chemically synthesizing piR-157618Mimic to mimic the endogenous maturation of piR-157618 in cells, thereby specifically targeting and upregulating the expression level of piR-157618.

[0035] The following are specific embodiments.

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

[0037] In this embodiment, all primer pairs for piR-2850767, piR-157618, and U6 (this gene sequence is a non-coding small RNA of approximately 100 nucleotides in length, stably expressed in various tissues and cells, and does not involve small RNA regulatory pathways; it is often used as a housekeeping gene for experiments to detect the expression level of non-coding RNA in cells, blood, and tissues) were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

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

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

[0040] The upstream primer sequence for piR-2850767 used in the real-time quantitative PCR experiment was 5'-GCTGGGAAGGCCCGGCGGGG-3' (SEQ ID NO.10), the upstream primer sequence for piR-157618 was 5'-AGTTGGTCTGAGTGTTGTGGGTTATT-3' (SEQ ID NO.11), and the downstream primer sequence for both piR-2850767 and piR-157618 was 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQ ID NO.12). The upstream primer sequence for U6 was 5'-CTCGCTTCGGCAGCACA-3' (SEQ ID NO.13), and the downstream primer sequence for 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. The cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was MEM (containing NEAA) medium with 10% fetal bovine serum and 1% penicillin-streptomycin antibody added.

[0043] (2) Cell counting: When the cells are in the logarithmic growth phase, the original culture medium is aspirated, 1 mL of PBS is added to wash three times, 1 mL of trypsin is added, and T25 is placed in an incubator for 2 minutes. The cells can be seen to detach in large areas when gently shaken. 3 mL of culture medium is added to stop cell digestion, and the cells are blown and mixed with a sterile Pasteur pipette. 10 μL is taken and counted in a cell counting chamber.

[0044] (3) Cell plating and treatment: 1×10⁶ cells per well 5 One cell line was seeded into each well of a 6-well plate, ensuring even distribution of cells within the wells. The 6-well plates were then incubated in a constant temperature incubator. When the cell density reached 80% (24 hours), the original culture medium was aspirated, and 2 mL of serum-free and antibiotic-containing medium containing 0.25‰, 0.5‰, and 1‰ CSE (tobacco smoke agglutination solution) was added for treatment. The 6-well plates were then incubated in a constant temperature incubator.

[0045] (4) Total RNA extraction: After treatment with CSE for 24 and 48 hours, the culture medium was discarded, and each well was washed three times with 1 mL of PBS. Then, 1 mL of Trizol was added, and the cells were incubated at 4°C for 15 min to promote complete cell lysis. After pipetting the cells, they were transferred to new 1.5 mL EP tubes, and 200 μL of chloroform was added to each tube. After vigorous vortexing and extraction, the cells were incubated at room temperature for 15 min, centrifuged at 12,000 rpm for 15 min, and 500 μL of the colorless aqueous phase (containing RNA) was aspirated five times (100 μL each time) to new EP tubes. An equal volume of isopropanol was added, and the mixture was inverted and mixed. The cells were incubated at room temperature for 15 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and the cells were washed with 1 mL of 80% ethanol. The cells were then centrifuged at 4°C and 12,000 rpm for 8 min. This step was repeated three times. Discard the supernatant and dry for 20 min. Add 40 μL of RNase-Free ddH2O to dissolve the RNA. Let stand at room temperature for 20 min to allow the RNA to dissolve completely.

[0046] (5) Reverse transcription: 1) Genomic DNA removal: Take 200 μL of nuclease-free eight-tube strips, prepare 20 μL of reaction system according to the table below, centrifuge and set the reaction program (incubate at 42℃ for 2 min).

[0047] RNase-Free ddH2O 6μL

[0048] 5×gDNA Wiper Mix 4μL

[0049] >100 ng / uL RNA Template 10 μL

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

[0051]

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

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

[0054] (6) Real-time quantitative PCR was used to detect the relative expression levels of piR-2850767 and piR-157618: 10 μL of qPCR reaction system was prepared according to the table below, vortexed and mixed, and then added to a 386-well plate. The plate was sealed with sealing film and centrifuged. The 386-well plate was then placed in an ABIQ7 real-time quantitative PCR instrument and the reaction was performed according to the following program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 sec, 60℃ annealing for 30 sec, denaturation and extension for 40 cycles, followed by melting curve reaction.

[0055]

[0056]

[0057] The Specific Primer, or upstream primer, is a nucleotide sequence specifically designed for different small RNAs. The upstream primer sequence for piR-2850767 is as follows:

[0058] The upstream primer sequence for piR-157618 is 5'-GCTGGGAAGGCCCGGCGGG-3' (SEQ ID NO.10), and the downstream primer sequence for piR-157618 is 5'-AGTTGGTCTGAGTGTTGTGGGTTATT-3' (SEQ ID NO.11). Additionally, mQ primer R is a universal downstream primer for small RNA reverse transcription products, with the sequence 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQ ID NO.12), which serves as the downstream primer for piR-2850767 and piR-157618. The upstream primer sequence for 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, CSE was exposed to the drug for 24 hours ( Figure 1 (Figure a) and 48 hours ( Figure 1 After [Figure b], the expression of piR-2850767 in lung epithelial cells increased, while the expression of piR-157618 decreased. Among the candidate piRNAs screened in previous studies, piR-2850767 and piR-157618 are potential specific diagnostic biomarkers for non-small cell lung cancer. Based on the above, piR-2850767 and piR-157618 have the potential to be targets for the development of preventative drugs against lung damage caused by tobacco exposure.

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

[0062] 1. Experimental Methods

[0063] The nucleotide sequences of the piR-2850767 expression inhibitor and piR-157618 overexpression reagent used in this embodiment 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'-CAGUACUUUUGUGUAGUACAA-3' (SEQ ID NO.15). The overexpression reagent control used the company's Mimic negative control, i.e., the Mimic NC group, with the sequence 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.16) and its complementary sequence 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.17).

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

[0065] (2) Cell seeding and transfection: 1 × 10⁶ cells per well 5 Cells were seeded into 6-well plates, ensuring uniform distribution within the wells, and then the plates were incubated in a constant temperature incubator. Transfection was performed when the cell density reached 40%. 250 μL of N-methylmethionine-free ddH2O was added to a reagent bottle containing 5 nmol of the interfering sequence to obtain a 20 μmol / L stock solution of the interfering sequence. 5 μL of the 20 μmol / L interfering sequence was diluted with 245 μL of serum-free and antibiotic-free medium, gently mixed, and incubated at room temperature for 5 min. 5 μL of Lipofectamine was diluted with 245 μL of serum-free and antibiotic-free medium. TM 3000, gently mix and incubate at room temperature for 5 min. Gently mix the two mixtures and incubate at room temperature for 20 min. Add 1500 μL of serum-free and antibiotic-free medium to bring the final concentration of the interfering sequence to 50 nmol / L. Discard the original medium, add 2 mL of transfection mixture to each well, and gently mix. Incubate the 6-well plate in an incubator for 6 hours, then discard the medium containing the interfering sequence, replace with fresh complete medium containing serum and antibiotics, and incubate for another 48 hours. Cell transfection groups included 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 levels of piR-2850767 and piR-157618 were the same as in Example 1. The results are 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 culture following transfection, the piR-2850767 cells in the Inhibitor NC group and the Mimic NC group... Figure 2 Figure a) and piR-157618 expression levels ( Figure 2 (Figure b) There was no significant change. The expression level of piR-2850767 in the piR-2850767 Inhibitor group was reduced, and the expression level of piR-2850767 in all three cell types decreased to below 50%. Figure 2 (Figure a) In the piR-157618Mimic group, the expression level of piR-157618 was significantly increased, and the level in all three cell types was more than three times that of the control group. Figure 2 (Figure a). The results suggest that piR-2850767 Inhibitor can effectively target and downregulate the expression of piR-2850767 in normal lung epithelial cells and lung cancer cells, while piR-157618 Mimic can effectively target and upregulate the expression of piR-157618 in normal lung epithelial cells and lung cancer cells.

[0069] Example 3: piR-2850767 expression inhibitors or piR-157618 overexpression agents can inhibit the growth of lung cancer cell line A549 and induce its apoptosis.

[0070] 1. Experimental Methods

[0071] (1) The lung cancer cell line A549 was used as a cell model. The experimental methods for cell culture and cell counting were the same as in Example 1.

[0072] (2) Cell viability assay: 3000 cells per well were seeded into 96-well plates, ensuring uniform cell distribution. The plates were then incubated in a constant temperature incubator. When the cell density reached 40% (18 hours), the original culture medium was discarded, and 200 μL of serum-free and antibiotic-free medium containing the interfering sequence (transfection mixture) was added, and the mixture was gently shaken. After incubating the 96-well plates for 6 hours, the medium containing the interfering sequence was discarded, and fresh complete culture medium containing serum and antibiotics was added. The plates were then incubated for another 24 and 48 hours. Cell transfection groups included the Inhibitor NC group, the Mimic NC group, the piR-2850767Inhibitor group, and the piR-157618Mimic group. 24 and 48 hours after transfection, the original culture medium was discarded, and 110 μL of freshly prepared complete culture medium containing 10 μL of CCK-8 reagent was quickly added. The cells were incubated in a constant temperature incubator for 40 minutes, and then the absorbance was measured using a microplate reader at a wavelength of 450 nm to assess cell viability. The cell proliferation rate (%) was calculated as (experimental group OD - blank group OD) / (control group OD - blank group OD). Finally, all transfected groups were compared with the NC group.

[0073] (3) Hoechst / PI apoptosis staining assay: 0.3 × 10⁻⁶ per well 5 Cells were seeded into 12-well plates, ensuring uniform distribution within the wells, and then the plates were incubated in a constant temperature incubator. When the cell density reached 40% (18 hours), the original culture medium was discarded, and 600 μL of serum-free and antibiotic-containing medium (transfection mixture) containing the interfering sequence was added, followed by gentle mixing. The plates were incubated for 6 hours, then the medium containing the interfering sequence was discarded, and fresh complete culture medium containing serum and antibiotics was added. The plates were then incubated for another 24 and 48 hours. Cell transfection groups included the Inhibitor NC group, the Mimic NC group, the piR-2850767Inhibitor group, and the piR-157618Mimic group. Forty-eight hours after transfection, Hoechst / PI apoptosis staining was performed. After preheating the antibiotic-free culture medium to 37℃, prepare Hoechst / PI working solution. Add 5μL of Hoechst 33342 staining solution (100×) and 5μL of PI staining solution (100×) to each 500μL of antibiotic-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 in the dark for 15 minutes, wash once with an appropriate amount of PBS, and then add 1mL of PBS to each well to prevent cell drying. Subsequently, use an inverted fluorescence microscope to photograph and detect cell apoptosis.

[0074] 2. Experimental Results

[0075] The results of the CCK-8 experiment are as follows: Figure 3 The results showed that the cell viability of both 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-2850767 Inhibitor 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-157618 Mimic group was 76.94% ± 3.67%, and the 48-hour cell viability was 64.28% ± 3.26%. Hoechst / PI apoptosis staining results are as follows... 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 appeared bright blue after Hoechst staining, indicating early apoptosis—nuclear condensation; the cells appeared red after PI staining, indicating late apoptosis). The results suggest that piR-2850767 expression inhibitors or piR-157618 overexpression agents can inhibit the growth of A549 lung cancer representative cells and induce apoptosis, indicating that piR-2850767 and piR-157618, as targets, can be used 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 reagent restored tobacco exposure-induced cell cycle arrest, apoptosis, cell proliferation, and decreased motility in lung epithelial cells.

[0077] 1. Experimental Methods

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

[0079] (2) Cell treatment: 24 hours after cell transfection, the original culture medium was discarded, and 2 mL of serum-free and antibiotic-free culture medium containing 0.5‰ CSE was added for treatment. The 6-well plates were then placed in an incubator. After 24 hours of CSE treatment, cell cycle (PI staining flow cytometry) and apoptosis (PI staining flow cytometry) assays, plate colony assay, scratch assay, and transwell assay 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 G1 phase, S phase, and G2 / M phase to the total number of cells (%).

[0081] (4) 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. Apoptosis analysis was performed using FlowJo software. The total number of cells with early and late apoptosis was the number of apoptotic cells. Finally, the treatment groups were compared with the NC group.

[0082] (5) Plate cloning assay: After 24 hours of CSE treatment, the original culture medium was discarded, and the cells were washed three times with 1 mL of PBS. 0.5 mL of trypsin was added, and the 6-well plate was incubated for 2 minutes. Gentle shaking revealed large areas of cell detachment. 1 mL of culture medium was added to stop cell digestion, and the cells were dispersed and mixed using a sterile Pasteur pipette. After centrifugation, the supernatant was discarded, and the cells were resuspended in 2 mL of complete culture medium containing serum and antibiotics. 10 μL of the resuspended cells were counted in a cell counting chamber. 400 cells were seeded per well in a 6-well plate, ensuring even distribution. The 6-well plate was then incubated for 14 days. Culture was terminated when visible clones appeared in the culture dish, meaning the majority of individual clones contained more than 50 cells. The medium was changed and cell status observed every 3 days. The original culture medium was then discarded, and the cells were washed once with 1 mL of PBS. 1 mL of 4% paraformaldehyde was added for fixation for 30 minutes, followed by another wash with 1 mL of PBS. 1 mL of crystal violet staining solution was added, and the cells were stained for 20 minutes. Finally, wash several times with 1 mL of PBS, air dry, and photograph the entire six-well plate and each well. Use ImageJ software to count clones, counting those with more than 50 cells. Calculate the colony formation rate. The result is colony formation rate = (number of clones / number of seeded cells) × 100%. All treatment groups were compared with the NC group.

[0083] (6) Scratch assay: Before cell seeding, parallel lines were evenly drawn on the back of the 6-well plate using a marker and a ruler, with a spacing of 0.5 cm between each horizontal line, and 5 horizontal lines were drawn in each well. 24 hours after cell transfection (100% cell density), cell scratches were created using a 20 μL pipette tip perpendicular to the well plate and the lines, with the scratches intersecting the marker lines. Cells were then washed three times with 1 mL PBS for cell infection treatment and photographed under a microscope as a 0-hour control. The 6-well plate was then incubated in a constant temperature incubator. Cells were harvested at 6, 12, and 24 hours, and the scratch width was observed and photographed under a microscope. Pre-marked observation points were used for timed photography. ImageJ software was used to measure the pixel density of the scratched area to compare cell migration speed. Finally, the treated group was compared with the NC group.

[0084] (7) Transwell assay: 750 μL of complete culture medium containing serum and double antibodies was added to the lower chamber of a 12-well / 24-well plate. The plates were divided into four groups: Inhibitor NC group, Mimic NC group, Inhibitor NC + 0.5‰ CSE exposure group, Mimic NC + 0.5‰ CSE exposure group, piR-2850767Inhibitor group, piR-157618Mimic group, piR-2850767Inhibitor + 0.5‰ CSE exposure group, and piR-157618Mimic + 0.5‰ CSE exposure group. 24 hours after cell transfection, the original culture medium was aspirated, and the cells were washed three times with 1 mL of PBS. 0.5 mL of trypsin was added, and the 6-well plate was incubated for 2 minutes. Gentle shaking revealed large-scale cell detachment. 1 mL of culture medium was added to stop cell digestion, and the cells were dispersed and mixed using a sterile Pasteur pipette. After centrifugation, the supernatant was discarded, and the cells were resuspended in 2 mL of serum-free and antibiotic-containing medium. 10 μL of the resuspended cells were counted in a cell counting chamber. 200 cells per well were seeded into the upper chamber of a 12 / 24-well plate (not exceeding 200 μL), ensuring no air bubbles between the upper and lower chambers. The 24-well plate was then incubated for 24 hours. The original culture medium in both chambers was aspirated. The upper chamber was washed once with 1 mL of PBS, fixed with 1 mL of 4% paraformaldehyde for 30 min, washed once with 1 mL of PBS, stained with 0.3 mL of crystal violet solution for 20 min, washed several times with 1 mL of PBS, and air-dried. Each upper chamber was photographed under a microscope. Three to five fields of view were randomly selected, and the number of cells that migrated to the submembrane side was counted using ImageJ software. The results were calculated as the percentage of cell migration (%), and all treatment groups were compared with the NC group.

[0085] 2. Experimental Results

[0086] like Figure 5As shown, in vitro functional validation results of piR-2850767 and piR-157618 in two lung epithelial cell lines indicated that, compared with the control group, inhibiting piR-2850767 expression or overexpressing piR-157618 could restore tobacco exposure-induced lung epithelial cell cycle arrest. Figure 5 Figure a), apoptosis ( Figure 5 Figure b), reduced proliferation ( Figure 5 (Figure c) Decreased planar motion ability ( Figure 5 (Figure d) Decreased longitudinal motor ability ( Figure 5 (See Figure e). Therefore, piR-2850767 expression inhibitors or piR-157618 overexpression reagents can be used to prepare preventive drugs for lung injury caused by tobacco exposure. Based on the results of all the above examples, it is suggested that piR-2850767 and piR-157618 have significant value as targets in the preparation of drugs for treating lung cancer or lung injury caused by tobacco exposure.

[0087] Those skilled in the art will readily understand that the above description is merely 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 within the scope of protection of the present invention.

Claims

1. The use of an expression inhibitor of piR-2850767 or an overexpression reagent of piR-157618 in the preparation of a drug for treating lung cancer or lung injury caused by tobacco exposure, wherein the nucleotide sequence of piR-2850767 is shown in SEQ ID NO 1, the nucleotide sequence of piR-157618 is shown in SEQ ID NO 4; the nucleotide sequence of the expression inhibitor is shown in SEQ ID NO 3, and the nucleotide sequence of the overexpression reagent is a double-stranded RNA formed by SEQ ID NO 4 and SEQ ID NO 6.

2. The application as described in claim 1, characterized in that, The expression inhibitor and / or the overexpression reagent are used to inhibit the growth and apoptosis of A549 cells in lung cancer.

3. The application as described in claim 1, characterized in that, The lung cancer in question is non-small cell lung cancer.

4. The application as described in claim 1, characterized in that, The expression inhibitor and / or the overexpression reagent are used to inhibit cell cycle arrest, apoptosis, decreased cell proliferation, and decreased cell motility in lung epithelial cells caused by tobacco exposure.

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