Application of lncRNASEH1-AS1 as target in preparation of medicine for treating lung squamous cell carcinoma

Abstract

The invention discloses application of lncRNASEH1-AS1 as a target in preparation of a medicine for treating lung squamous cell carcinoma, and belongs to the technical field of biological medicine. The invention finds that the expression of the lncRNASEH1-AS1 in tissues, plasma and cell lines of a lung squamous cell carcinoma patient is higher than that of a control group of the lung squamous cell carcinoma patient. The lncRNASEH1-AS1 has clinical correlation with the tumor diameter of a lung squamous cell carcinoma patient. The results of CCK-8, clone formation, Transwell migration and invasion experiments show that after the lncRNASEH1-AS1 and the MCM7 are knocked down, the proliferation, migration and invasion ability of the lung squamous cell carcinoma cells is weakened, and after the lncRNASEH1-AS1 is over-expressed, the inhibition of the MCM7 on the proliferation, migration and invasion ability of the lung squamous cell carcinoma cells can be reversely knocked down. The result of a nude mouse subcutaneous tumor formation experiment shows that the tumor formation capability of the LUSC cells which stably knock down the lncRNASEH1-AS1 is weakened.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of lncRNASEH1-AS1 as a target in the preparation of a drug for treating lung squamous cell carcinoma. Background Art

[0002] Lung cancer (LC) is the second most common cancer in the world, accounting for about 11.4% of all diagnosed cancers. It is also the main cause of cancer death. Among the patients who die from various types of cancer each year, lung cancer patients account for as high as 18.0%. Lung cancer has become one of the important obstacles to improving life expectancy in every country. Lung cancer is divided into adenocarcinoma, squamous cell carcinoma, large cell carcinoma and other types according to the classification criteria released by the World Health Organization (WHO) in 2015. Further according to pathological characteristics, it is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC includes lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUSC usually originates from larger bronchi, so it is also called central lung cancer. LUSC is the most malignant subtype of NSCLC and one of the lung cancers with a higher rate of malignant transformation. It mainly occurs in elderly men and has a higher risk of metastasis and recurrence. The treatment effect and prognosis of LUSC patients are usually poor. Due to the continuous optimization of surgical methods and the emergence of effective therapeutic drugs, the mortality rate of lung cancer has declined in recent decades. Molecular targeted drug therapy and immunotherapy have achieved remarkable results in the treatment of lung adenocarcinoma, significantly prolonging the survival time of patients. However, the prognosis of LUSC patients is still not ideal. Therefore, screening and clarifying the molecular targets of lung squamous cell carcinoma and finding effective biomarkers for early diagnosis and treatment are of great significance to the survival and prognosis of LUSC patients.

[0003] Non-coding RNA (ncRNA) was once considered an insignificant substance because it could not perform the key task of encoding proteins, and therefore did not occupy an important position in the scientific community. In recent years, genome-wide association studies have made major breakthroughs in tumor samples, revealing many long non-coding RNAs (lncRNAs) that regulate key processes in cancer. However, the functions of most lncRNAs remain unexplained. A small number of studies have also confirmed that some lncRNAs are associated with precancerous lesions of LUSC and the malignant phenotype of LUSC. These findings have changed the previous understanding of lncRNAs and laid a solid theoretical foundation for exploring the pathogenesis of LUSC. Studying the functions and mechanisms of lncRNAs in LUSC will help find treatments for LUSC patients. lncRNASEH1-AS1 is the antisense lncRNA of lncRNASEH1, which is rarely reported in tumors. Summary of the invention

[0004] The purpose of the present invention is to provide an application of lncRNASEH1-AS1 as a target in the preparation of a drug for treating lung squamous cell carcinoma, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of lncRNASEH1-AS1 as a target in the preparation of a drug for treating lung squamous cell carcinoma.

[0007] The second technical solution of the present invention is the application of lncRNASEH1-AS1 in screening drugs for treating lung squamous cell carcinoma.

[0008] The third technical solution of the present invention is the use of lncRNASEH1-AS1 inhibitor in the preparation of drugs for treating squamous cell carcinoma of the lung.

[0009] A fourth technical solution of the present invention is a drug for treating lung squamous cell carcinoma, comprising the lncRNASEH1-AS1 inhibitor.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] The present invention found that the expression of lncRNASEH1-AS1 in the tissues, plasma and cell lines of lung squamous cell carcinoma patients was higher than that in the control group. There was a clinical correlation between lncRNASEH1-AS1 and the tumor diameter of lung squamous cell carcinoma patients. The results of CCK-8, clone formation, Transwell migration and invasion experiments showed that the proliferation, migration and invasion abilities of lung squamous cell carcinoma cells were weakened after knocking down lncRNASEH1-AS1 and MCM7, and overexpression of lncRNASEH1-AS1 could reverse the inhibition of MCM7 knocking down on the proliferation, migration and invasion abilities of lung squamous cell carcinoma cells. RNA Pull Down-MS and RIP experiments verified the interaction between lncRNASEH1-AS1 and MCM7 protein. The results of nude mouse subcutaneous tumor formation experiments suggested that the tumor formation ability of LUSC cells with stable knockdown of lncRNASEH1-AS1 was weakened. Therefore, lncRNASEH1-AS1 can be used as a target for the prevention and treatment of LUSC. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The expression level of lncRNASEH1-AS1 in LUSC cell lines and its proliferation, invasion and migration functions in cells. A. RT-qPCR detected the expression level of lncRNASEH1-AS1 in lung squamous cell carcinoma cell lines; B. RT-qPCR verified the knockdown efficiency of lncRNASEH1-AS1; C. CCK-8 method detected the effect of knockdown of lncRNASEH1-AS1 on cell viability; D. Clone formation experiment detected the effect of knockdown of lncRNASEH1-AS1 on cell proliferation ability; E. Transwell migration experiment detected the effect of knockdown of lncRNASEH1-AS1 on cell invasion ability (×100); F. Transwell invasion experiment detected the effect of knockdown of lncRNASEH1-AS1 on cell invasion ability (×100). *P<0.05, **P<0.01, ***P<0.001.

[0014] Figure 2RNAPull Down-MS combined with RIP experiment to screen and verify the interaction between lncRNASEH1-AS1 and MCM7 protein. A. Agarose gel electrophoresis to detect the sense and antisense chains of lncRNASEH1-AS1 amplified by PCR in vitro; B. Agarose gel electrophoresis to detect the in vitro transcribed fragments of the sense and antisense chains of lncRNASEH1-AS1; C. Agarose gel electrophoresis to detect RNAPull-down eluate; D. Partial mass spectrometry results; E. WB experiment proved the presence of MCM7 molecules in the precipitate; F. RT-qPCR experiment proved that the relative expression of the target gene was higher than that of the IgG group (Note: **P<0.01)

[0015] Figure 3 The proliferation, invasion and migration functions of MCM7 in LUSC cells. A. RT-qPCR detected the expression level of MCM7 in lung squamous cell carcinoma cell lines; B. RT-qPCR verified the knockdown efficiency of MCM7; C. CCK-8 method detected the effect of knockdown of MCM7 on cell viability; D. Cloning assay detected the effect of knockdown of MCM7 on cell proliferation ability; E. Transwell migration assay detected the effect of knockdown of MCM7 on cell invasion ability (×100); F. Transwell invasion assay detected the effect of knockdown of MCM7 on cell invasion ability (×100). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0016] Figure 4 Overexpression of lncRNASEH1-AS1 rescues the proliferation, migration and invasion of LUSC cells after knockdown of MCM7. A. Overexpression efficiency of lncRNASEH1-AS1; B. CCK-8 assay; C. Clone formation assay; D. Transwell migration assay (×100); E. Transwell invasion assay (×100).

[0017] Figure 5 The tumorigenicity of NCI-H520 cells with stable knockdown of lncRNASEH1-AS1 in nude mice. A. Stable cell line verification; B. Tumor gross image; C. Tumor volume. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0024] The embodiment of the present invention provides the use of lncRNASEH1-AS1 as a target in the preparation of a drug for treating lung squamous cell carcinoma.

[0025] The embodiments of the present invention also provide the use of lncRNASEH1-AS1 in screening drugs for treating lung squamous cell carcinoma.

[0026] The embodiments of the present invention also provide the use of lncRNASEH1-AS1 inhibitor in the preparation of a drug for treating lung squamous cell carcinoma.

[0027] In some specific embodiments, the lncRNASEH1-AS1 inhibitor is selected from at least one of a compound, a protein, a polypeptide, a polysaccharide, a glycoprotein, a glycopeptide or a nucleic acid.

[0028] The embodiment of the present invention also provides a drug for treating lung squamous cell carcinoma, comprising the lncRNASEH1-AS1 inhibitor.

[0029] Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) experiments were used to determine the differential expression of lncRNASEH1-AS1 in the plasma of lung squamous cell carcinoma patients and normal human plasma, lung squamous cell carcinoma cell lines and normal human lung epithelial cells. Fluorescence in situ hybridization was used to perform experiments on lung squamous cell carcinoma tissue chips to analyze the clinical relevance of lncRNASEH1-AS1. In vitro cultured lung squamous cell carcinoma cell lines NCI-H226 and NCI-H520 were divided into si-NC and si-RNASEH1-AS1 groups, si-NC and si-MCM7 groups, pcDNA3.1(+)-lncRNASEH1-AS1+si-MCM7, pcDNA3.1+si-MCM7 and pcDNA3.1+NC groups. CCK-8 assay, clone formation assay, Transwell migration and invasion assay were used to detect the proliferation, migration and invasion abilities of LUSC cells in the above groups. RNAPull Down-MS experiment screened out 97 proteins that may bind to lnc RNASEH1-AS1, and selected the downstream target protein MCM7. RIP experiment verified the binding of lnc RNASEH1-AS1 to MCM7. RT-qPCR experiment detected the difference in expression of MCM7 in lung squamous cell carcinoma cells and normal human lung epithelial cells. Nude mouse subcutaneous tumor formation experiment detected the effect of cells with stable knockdown of lncRNASEH1-AS1 on the growth of transplanted tumors.

[0030] The results showed that the expression of LncRNASEH1-AS1 in tissues, plasma and cell lines of lung squamous cell carcinoma patients was higher than that in the control group. LncRNASEH1-AS1 was clinically correlated with the tumor diameter of lung squamous cell carcinoma patients. The results of CCK-8, clone formation, Transwell migration and invasion experiments showed that the proliferation, migration and invasion abilities of lung squamous cell carcinoma cells were weakened after knocking down lncRNASEH1-AS1 and MCM7, and overexpression of lncRNASEH1-AS1 could reverse the inhibition of MCM7 knockdown on the proliferation, migration and invasion abilities of lung squamous cell carcinoma cells. RNA Pull Down-MS and RIP experiments verified the interaction between lncRNASEH1-AS1 and MCM7 proteins. The results of nude mouse subcutaneous tumor formation experiments showed that the tumorigenic ability of LUSC cells with stable knockdown of lncRNASEH1-AS1 was weakened.

[0031] Example 1

[0032] 1. Materials

[0033] 1 Clinical specimens: Plasma specimens were collected from Tangshan People's Hospital. 55 LUSC patients who visited Tangshan People's Hospital from 2022 to 2023 were collected and matched with 55 healthy subjects of similar age and gender. This experiment was approved and supervised by the Ethics Committee of Tangshan People's Hospital (approval number RMYY-LLKS-2021-103). The LUSC tissue chip containing 120 loci (HLugS120CS01) was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0034] 2 Cells and main reagents: Human lung squamous cell carcinoma cell lines (NCI-H226, NCI-H520, NCI-H1703, SK-MES-1) were purchased from Wuhan Pronocell Life Science Co., Ltd., human lung squamous cell carcinoma cell line KNS-62 was purchased from Zhejiang Meisen Cell Technology Co., Ltd., and human normal lung epithelial cells (BEAS-2B) were purchased from Beijing Zhongsheng Aobang Biotechnology Co., Ltd.; RPMI 1640 medium, MEM medium, DMEM medium, penicillin-streptomycin (PS) and 0.25% trypsin were purchased from Beijing Zhongsheng Aobang Biotechnology Co., Ltd.; fetal bovine serum (FBS), Matrigel matrix glue and opti-MEM medium were purchased from Corning, USA; phosphate buffered saline (Phosphate buffered saline) was purchased from Corning, USA. saline (PBS), cell freezing solution, puromycin and CCK-8 kit were purchased from Beijing Solaibao Technology Co., Ltd.; Lipofectamine 2000 and TRIzol were purchased from Invitrogen, USA; real-time quantitative polymerase chain reaction (RT-qPCR) kit was purchased from Takara, Japan; RIPA lysis buffer, BCA protein assay kit, gel preparation kit, polyvinylidene fluoride membrane, ECL chemiluminescence detection kit were purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.; glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) antibody and secondary goat anti-rabbit IgG antibody were purchased from Proteintech, USA; RNASEH1-AS1 fluorescent in situ hybridization probe, small interfering RNA (si-RNASEH1-AS1, si-MCM7) and its negative control (si-NC) were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.; RNASEH1-AS1, MCM7, GAPDH, U6 primers, lentiviral particles targeting silencing RNASEH1-AS1 (LV-sh RNASEH1-AS1) and its control (LV-shNC) were designed and synthesized by Beijing Xibei Hongcheng Biotechnology Co., Ltd.; 20 BALB / c-nu nude mice, 4 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd. [Animal License No.: SCXK(Jing)2023-SY-034].

[0035] 2. Experimental Methods

[0036] 1 Cell culture: Human LUSC cell lines (NCI-H226, NCI-H1703, NCI-H520) were cultured in RPMI-1640 medium, SK-MES-1 cells were cultured in MEM medium, KNS-62 cells were cultured in DMEM medium, and BEAS-2B cells were cultured in DMEM medium. All cells were cultured in a cell culture incubator (37°C, 5% CO2). When the cell confluence reached 85% to 90%, the subsequent steps were performed according to the experimental requirements.

[0037] 2 Fluorescence in situ hybridization probe was used to detect the expression level of lncRNASEH1-AS1 in LUSC tissues.

[0038] The fluorescent in situ hybridization probes were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and the screening experiments were performed according to the kit instructions.

[0039] 3 The downstream protein MCM7 of lncRNASEH1-AS1 was screened through RNA Pull Down-MS screening of interacting proteins.

[0040] The RNA Pull Down-MS screening experiment was performed according to the instructions of the RNA Pull Down-MS screening of interacting proteins experiment using NCI-H226 and NCI-H520 cells as materials.

[0041] 4. MCM7 was verified using RIP kit.

[0042] RNA binding protein immunoprecipitation (RIP) experiments were performed using the RNA Immunoprecipitation Kit, the experiment was carried out according to the instructions. The antibody used for RIP was purchased from Proteintech, USA. The immunoprecipitated lncRNASEH1-AS1 was detected by RT-qPCR, and the protein was detected by WB; the positive control was the total RNA in the Input, and the negative control was IgG.

[0043] 5. Cell transfection and grouping: Transfection was performed according to the instructions of the Lipofectamine 2000 kit.

[0044] To verify that lncRNA RNASEH1-AS1 participates in the development of LUSC by regulating MCM7, the experiment was divided into si-NC and si-RNASEH1-AS1 groups; si-NC and si-MCM7 groups; pcDNA3.1(+)-lncRNASEH1-AS1+si-MCM7, pcDNA3.1+si-MCM7 (knockdown expression) and pcDNA3.1+NC groups (overexpression).

[0045] 1) Digest and centrifuge the cells in the logarithmic growth phase, and evenly inoculate NCI-H226 and NCI-H520 cells in a six-well plate, place the plate in a cell culture incubator (37° C., 5% CO 2 ) and continue culturing. Transfection is performed when the cell density reaches 50% to 60%.

[0046] 2) Prepare transfection reagents: Solution A: 125 μL serum-free Opti-MEM + 5 μL Lipofectamine 2000, let stand at room temperature for 5 min; Solution B: 125 μL serum-free Opti-MEM + 5 μL si-RNASEH1-AS1 / si-NC / si-MCM7 / si-NC / pcDNA3.1(+)-lncRNASEH1-AS1+si-MCM7 / pcDNA3.1+si-MCM7 / pcDNA3.1+NC, let stand at room temperature for 5 min. Solution A + Solution B, let stand at room temperature for 15 min.

[0047] 3) 2 h before transfection, replace the old culture medium in the cell bottle with 5 mL of fresh serum-free culture medium, then evenly add the mixed transfection solution into the six-well plate and continue to culture in a cell culture incubator (37°C, 5% CO2). After 4 h of culture, replace the old serum-free culture medium in the bottle with 5 mL of fresh complete culture medium.

[0048] 6RNA extraction and RT-qPCR experiments

[0049] 6.1 RNA from each cell of the LUSC cell line was extracted using the Trizol method, and RT-qPCR experiments were performed using the reverse transcription and amplification kit from Takara, Japan. The primer sequences are shown in Table 1.

[0050] Table 1 Primer sequence list

[0051]

[0052] 6.2 Extraction of total RNA from plasma

[0053] The Trizol method was used to extract RNA from plasma. The plasma was obtained from 55 LUSC patients who were treated in Tangshan People's Hospital from 2022 to 2023 and matched with 55 healthy subjects of similar age and gender.

[0054] 6.3 RNA reverse transcription to cDNA

[0055] The total system in each tube is 10 μL, including: 1 μg sample RNA, 2 μL 5× gDNAEraser Buffer, 1 μL gDNA Eraser, and finally add RNase Free dH2O to 10 μL.

[0056] The reaction procedure is as follows: 42℃ for 2 minutes, immediately cool on ice for 2 minutes. Then add 1μL of PrimeScript RT Enzyme Mix I, 1μL of RT Primer Mix, 4μL of 5× PrimeScript Buffer 2, and 4μL of RNase Free dH2O to the above 10μL reaction solution. Gently mix the above mixture and centrifuge it instantly.

[0057] The reaction procedure is as follows: first stage: 37℃ for 15min; second stage: 85℃ for 5s, 4℃. Store at -20℃ for later use.

[0058] 6.4 RT-qPCR detection of RNA expression in transfected cells

[0059] The required reagents were melted at room temperature, and the following reagents were added to the 8-tube series in sequence: 10 μL of SYBR Premix ExTaqⅡ (2×), 2 μL of cDNA solution, 0.8 μL of Forward Primer (10 μM), 0.8 μL of Reverse Primer (10 μM), 0.4 μL of ROX Reference Dye II (50×), and 6 μL of ddH2O. The above mixture was gently mixed and centrifuged instantaneously to ensure that there were no bubbles in the 8-tube series. The reaction program was set as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, and 40 cycles. Each experiment was repeated 3 times independently, and the relative expression levels of lncRNASEH1-AS1 and MCM7 were calculated using the 2-ΔΔCT method with GAPDH as the internal reference. The detailed primer sequences are shown in Table 2.

[0060] Table 2 Real time-PCR primer sequences

[0061]

[0062] 7CCK-8 kit to detect cell activity

[0063] 1) After 36 hours of transfection, the cells were removed from the incubator, the old culture medium was discarded, and the cells were washed three times with PBS. Then, 0.25% trypsin solution was added for digestion. The digestion and centrifugation steps were the same as those for cell passaging. After centrifugation, the cells were resuspended in fresh complete culture medium and counted using a Corning counter. The cell density was adjusted to 2×10 4 The cell suspension was inoculated into a 96-well plate at 100 μL / well, with 3 replicate wells in each group.

[0064] 2) Place the 96-well plate in a cell culture incubator (37° C., 5% CO 2 ) and continue culturing.

[0065] 3) Fresh culture medium was replaced at 24 h, 48 h, 72 h and 96 h, and 10 μL of CCK-8 solution was added at the same time, and then the 96-well plate was placed in a cell culture incubator and incubated for another 2 h.

[0066] 4) Prepare a new 96-well plate, pipette 100 μL of solution into each well of each group into the new 96-well plate, measure the OD value at a wavelength of 450 nm using an ELISA reader and record the result.

[0067] 8. Clone formation assay to detect cell proliferation ability

[0068] After 36 h, resuspend the transfected NCI-H226 and NCI-H520 cells in complete medium to 2 × 10 4 2000 cells / mL, 2000 cells per well were seeded on a six-well plate, 3 replicates per group. The six-well plate was cultured in an incubator for about 13 days, and crystal violet staining was performed after the cells were clustered.

[0069] 1) 36 hours after cell transfection, cells were collected by digestion and centrifugation and counted, and the cell density was adjusted to 2×10 using complete medium. 4 The cell suspension was dripped into a 6-well plate containing 2 mL / well complete medium at a rate of 100 μL / well. The cells were shaken back and forth to mix thoroughly. Three replicate wells were set up for each group.

[0070] 2) Culture the shaken cells in an incubator (37°C, 5% CO2) for about 13 days, replace the culture medium with fresh culture medium every 3 days, observe the cell colony formation from time to time for about 1 week, and perform subsequent treatment when the cells grow to a suitable size.

[0071] 3) Remove the old culture medium, wash with PBS three times, add 1 mL of methanol solution to each well and fix at room temperature for 10 minutes, discard the methanol solution, add 1 mL of 0.1% crystal violet solution to each well, and continue for 10 minutes. After that, wash the 6-well plate with clean water, remove the excess crystal violet solution, place it in the room to dry naturally, and then take pictures and count.

[0072] 9Transwell assay to detect cell migration and invasion ability

[0073] 1) Migration experiment

[0074] (1) 36 h after transfection, digest and centrifuge the cells and count them. The steps are the same as the previous experimental steps. The cell density is adjusted to 6×10 in serum-free medium. 5 Pipette 100 μL of cell suspension and add it into the Transwell chamber.

[0075] (2) Add 650 μL of complete culture medium containing 25% fetal bovine serum to the lower chamber of the Transwell and culture in an incubator (37°C, 5% CO2) for 20 h. Discard the old culture medium in the lower chamber of the Transwell and add 700 μL of methanol solution to each lower chamber of the Transwell. Fix the cells at room temperature for 10 min.

[0076] (3) Discard the methanol solution, add 700 μL of 0.1% crystal violet solution to each lower chamber of the Transwell, stain for 10 min, carefully wash away the excess crystal violet solution with clean water, and then use a cotton swab to wipe off the cells that have not yet passed out of the upper chamber.

[0077] (4) After drying at room temperature, use a knife to gently remove the filter membrane of the small chamber, lay the filter membrane facing the lower chamber upward on a glass slide, seal the filter membrane with neutral gum and cover it with a cover glass. After the neutral gum is air-dried, observe and photograph it under an optical microscope, and calculate the experimental results.

[0078] 2) Invasion assay

[0079] (1) Matrigel was mixed with serum-free culture medium at a ratio of 1:8, 50 μL of the mixture was added to each upper chamber of the Transwell, and the cells were placed in an incubator (37°C, 5% CO2) for 24 h before the experiment.

[0080] (2) Cell digestion, centrifugation and counting were performed in the same manner as before. The cells were resuspended in serum-free medium to a concentration of 8 × 10 cells / mL and 100 μL of the cell suspension was added to each Transwell chamber containing Matrigel.

[0081] (3) Add 650 μL of complete culture medium containing 25% fetal bovine serum to the lower chamber of the Transwell and culture in an incubator (37° C., 5% CO 2 ) for 24 h.

[0082] (4) Terminate the culture after 24 hours, as in (2) to (4) of 1) migration assay.

[0083] 10Western blot experiment

[0084] To detect the expression of MCM7, 30 μg of protein sample was taken from each group for electrophoresis, the MCM7 was diluted to 1:1000 and incubated at 4°C overnight, the goat anti-rabbit IgG antibody (Sigma) was diluted to 1:10000 and incubated at room temperature for 1 h, and developed with BCA developer.

[0085] 11First, the cell line NCI-H520 with stable knockdown of lncRNASEH1-AS1 was constructed, and the expression level of lncRNASEH1-AS1 in NCI-H520 cells was detected by RT-qPCR experiment. Subsequently, 20 male nude mice aged 4 to 6 weeks were prepared and divided into two groups, named sh-NC group and sh-RNASEH1-AS1 group, with 10 mice in each group. Matrigel was prepared and melted overnight at 4°C. The cell line NCI-H520 with stable knockdown of lncRNASEH1-AS1 and Matrigel were mixed together to prepare a cell suspension. 3 million NCI-H520 cells without knockdown of lncRNASEH1-AS1 were inoculated in the sh-NC group, and 3 million NCI-H520 cells with stable knockdown of lncRNASEH1-AS1 were inoculated in the sh-RNASEH1-AS1 group. The injection volume of each nude mouse was 100 μL. At the beginning of the inoculation, wipe the needle insertion position with an alcohol cotton ball. The inoculation site is the armpit of the nude mouse. Each nude mouse is ear-tagged. The tumor growth and survival status of the nude mouse are observed at all times to ensure that the tumor volume does not exceed 2000mm 3 .

[0086] 12 Statistical analysis

[0087] GraphPad Prism8 software was used to perform statistical analysis on the experimental results. All experiments were repeated three times independently, and the data were expressed as mean ± standard deviation. The independent sample t test was used to compare two independent samples, the one-way analysis of variance was used to compare between groups, and the chi-square test was used to analyze clinical correlation. P < 0.05 indicated that the difference was statistically significant.

[0088] Experimental Results

[0089] 1LncRNA SEH1-AS1 is highly expressed in LUSC

[0090] RT-qPCR experiments found that the expression level of lncRNASEH1-AS1 in lung squamous cell carcinoma cell lines NCI-H226, NCI-H1703, NCI-H520, KNS-62, and SK-MES-1 was higher than that in BEAS-2B cells (Table 3, Figure 1Subsequently, lncRNASEH1-AS1 was knocked down in NCI-H226 and NCI-H520 cells to detect the effects of lncRNASEH1-AS1 on cell proliferation, migration and invasion. The results showed that after knocking down lncRNASEH1-AS1 in NCI-H226 and NCI-H520 cells, the expression level of lncRNASEH1-AS1 was reduced (Table 4, Figure 1 CCK-8 assay, clone formation assay, Transwell migration and invasion assay were used to detect the proliferation, migration and invasion abilities of LUSC cells in the above groups (Table 5, Figure 1 The above results indicate that lnc RNASEH1-AS1 is highly expressed in LUSC, and knocking down lnc RNASEH1-AS1 can inhibit the proliferation, migration and invasion of LUSC cells.

[0091] Table 3 Expression level of lncRNASEH1-AS1 in lung squamous cell carcinoma cell lines

[0092]

[0093] Table 4 Expression levels of lncRNASEH1-AS1 in lung squamous cell carcinoma cells after transfection

[0094]

[0095] Table 5 Effects of knocking down lncRNASEH1-AS1 on the activity, proliferation, migration and invasion of lung squamous cell carcinoma cells

[0096]

[0097] 2 Screening and verification of proteins binding to lncRNASEH1-AS1

[0098] The sense and antisense strands of lncRNASEH1-AS1 were amplified by PCR in vitro, and then detected by agarose gel electrophoresis. The results showed that the fragment size observed after electrophoresis gel was consistent with the target fragment size ( Figure 2 A). Then, T7 in vitro transcription kit was used Kit (Invitrogen) for transcription, followed by agarose gel electrophoresis, and it was observed that the size of the transcription product fragment was slightly larger than the target fragment and consistent with the expected position ( Figure 2 B). RNA pull-down experiments were performed using the sense strand and antisense strand to obtain the eluate, which was then tested by agarose gel electrophoresis ( Figure 2C). According to the results, in this experiment, the complex obtained by the LncRNASEH1-AS1 positive chain was used as the experimental group sample, and the sample obtained by the LncRNASEH1-AS1 antisense chain was used as the control group sample. The experimental group had a significant difference from the control group. No high-abundance protein appeared in the two groups of experimental samples, so the two groups of eluates were directly detected by mass spectrometry. From some mass spectrometry results, it can be seen that the score of minichromosome maintenance protein 7 (MCM7) is higher ( Figure 2 D), indicating that it is likely to bind to lncRNASEH1-AS1. MCM7 is a well-known cell proliferation marker, and studies have shown that MCM7 in tumor tissue is a potential marker for poor prognosis in NSCLC patients. Therefore, MCM7 was selected as a candidate molecule and its binding to lncRNASEH1-AS1 was verified by RIP experiment. According to the reagent instructions, MCM7 antibody and RNAImmunoprecipitation Kit (Jisai Bio) was used to detect the binding of lncRNASEH1-AS1 to MCM7 by RNA immunoprecipitation. IgG antibody was used as negative control. WB experiment detected the target protein. The negative control group was normal, the input group had clear bands, and the surface PCR amplification product could be used for the detection of this indicator. The target band of the IP group was clear, indicating that the target protein could bind to the RNA ( Figure 2 E). RT-qPCR analysis of purified RNA showed that the expression level of the target gene in the IP group was several times higher than that in the IgG group, and the difference was statistically significant ( Figure 2 Middle F).

[0099] 3MCM7 promotes LUSC cell proliferation, migration and invasion in vitro

[0100] RT-qPCR experiments found that the expression level of MCM7 in lung squamous cell carcinoma cells NCI-H226 and NCI-H520 was higher than that in BEAS-2B cells (Table 6, Figure 3 Subsequently, MCM7 was knocked down in NCI-H226 and NCI-H520 cells to detect the effects of MCM7 on cell proliferation, migration and invasion. The results showed that after knocking down MCM7 in NCI-H226 and NCI-H520 cells, the expression level of MCM7 was reduced (Table 7, Figure 3 CCK-8 assay, clone formation assay, Transwell migration and invasion assay were used to detect the proliferation, migration and invasion abilities of LUSC cells in the above groups (Table 8, Figure 3 The above results indicate that MCM7 is highly expressed in LUSC, and knockdown of MCM7 can inhibit the proliferation, migration and invasion of LUSC cells.

[0101] Table 6 MCM7 expression level in lung squamous cell carcinoma cells

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[0103] Table 7 MCM7 expression level in lung squamous cell carcinoma cells after transfection

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[0106] Table 8 Effects of knockdown of MCM7 on the activity, proliferation, migration and invasion of lung squamous cell carcinoma cells

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[0108] Effect of lncRNA SEH1-AS1 targeting MCM7 on LUSC cells

[0109] First, lncRNASEH1-AS1 was overexpressed in NCI-H226 and NCI-H520 cells and the efficiency was detected (Table 9, Figure 4 In Figure 1A), MCM7 was knocked down and lncRNASEH1-AS1 was overexpressed in NCI-H226 and NCI-H520 cells to detect the rescue effect of lncRNASEH1-AS1 on cell proliferation, invasion and migration. The experiment was divided into three groups: pcDNA3.1(+)-lncRNASEH1-AS1+si-MCM7, pcDNA3.1+si-MCM7 and pcDNA3.1+NC. CCK-8 assay, clone formation assay, Transwell migration and invasion assay were used to detect the proliferation, migration and invasion abilities of LUSC cells in the above groups (Table 10, Figure 4 Rescue experiments showed that overexpression of lncRNA SEH1-AS1 could rescue the effects of knockdown of MCM7 on the proliferation, migration and invasion of NCI-H226 and NCI-H520 cells.

[0110] Table 9 Expression levels of lncRNASEH1-AS1 in lung squamous cell carcinoma cells after overexpression

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[0113] Table 10 Downregulation of lncRNA SEH1-AS1 can reverse the effect of knockdown of MCM7 on the malignant phenotype of lung squamous cell carcinoma cells

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[0115] 5. In vivo verification of the function of lncRNASEH1-AS1 in a nude mouse subcutaneous tumor model

[0116] First, the cell line NCI-H520 with stable knockdown of lncRNASEH1-AS1 was constructed, and the expression level of lncRNASEH1-AS1 in NCI-H520 cells was detected by RT-qPCR experiment (Table 11, Figure 5 3 million cells were seeded in the armpits of two groups of 4-week-old nude mice. Through continuous measurement and observation, in vivo experiments confirmed that the tumor size of nude mice in the lncRNASEH1-AS1 knockdown group was significantly reduced (Table 12, Figure 5 BC). This suggests that the growth rate of LUSC cells with stable knockdown of lncRNASEH1-AS1 is attenuated in vivo.

[0117] Table 11 lncRNASEH1-AS1 expression levels in stable cell lines

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[0119] Table 12 Effect of knockdown of lncRNASEH1-AS1 on subcutaneous tumorigenesis of lung squamous cell carcinoma cells in nude mice

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[0121] Lung cancer has become an important obstacle to improving the life expectancy of all human beings. According to histopathological classification, lung cancer is divided into SCLC and NSCLC. About 85% of lung cancer patients are diagnosed with NSCLC after pathological diagnosis. The main subtypes of NSCLC are LUSC and LUAD. Patients with early lung cancer can undergo surgical resection of the tumor, while most patients with advanced lung cancer can only be treated with radiotherapy and chemotherapy. However, the side effects of radiotherapy and chemotherapy, as well as the acquired drug resistance of lung cancer patients, make the recovery prospects of patients not optimistic. Various targeted therapies and immunotherapies are increasingly being used in the treatment of NSCLC. Emerging treatment options have even played a significant role in the treatment of some advanced NSCLC patient groups. The average survival time of advanced LUAD patients has been significantly prolonged, and the 5-year survival rate has increased to 18%. However, compared with LUAD, the application effect of targeted therapy in LUSC patients is not obvious. The reason for this is that many scholars believe that it is closely related to the pathogenesis of LUSC. In the genomic testing of LUAD, more gene mutations encoding receptor tyrosine kinases were found, but in LUSC, most of them are functional loss of tumor suppressor genes such as PTEN. In recent years, a large number of cancer-related lncRNAs have been discovered through multi-center and repeatedly verified association analyses of a large number of tumor samples at the whole genome level. lncRNA was previously considered to be transcriptional junk, but recent studies have shown that some lncRNAs can not only encode small peptides and widely participate in regulating the life course of normal cells, but also play a key role in promoting the occurrence of malignant phenotypes of cancer, changing the gene network that maintains cell homeostasis, and promoting cancer resistance through various pathways. For example, lncRNA plays an important role in regulating the life process of cells such as chromatin remodeling and transcriptional activation and interference of genes. To date, a large amount of evidence has shown that the onset and progression of NSCLC are affected not only by protein-coding genes, but also by non-protein-coding genes. Therefore, in-depth research on the function of lncRNA in LUSC and its related molecular mechanisms has great scientific significance and practical application value, which will help to deeply understand the key processes such as the initiation, progression and metastasis of LUSC, help improve existing treatment options, and more accurately predict which patients may be sensitive or resistant to specific drugs, so as to develop personalized treatment plans for patients.

[0122] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. 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 claims of the present invention.

Claims

1. Application of lncRNASEH1-AS1 as a target in the preparation of drugs for the treatment of squamous cell lung carcinoma.

2. Application of lncRNASEH1-AS1 in screening drugs for the treatment of squamous cell lung carcinoma.

3. Application of lncRNASEH1-AS1 inhibitor in the preparation of drugs for the treatment of squamous cell lung carcinoma.

4. The use according to claim 3, characterized in that: The lncRNASEH1-AS1 inhibitor is selected from at least one of a compound, a protein, a polypeptide, a polysaccharide, a glycoprotein, a glycopeptide or a nucleic acid.

5. A drug for treating lung squamous cell carcinoma, characterized in that: Comprising the lncRNASEH1-AS1 inhibitor according to claim 3 or 4.

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