Application of long noncoding RNA LINC01635 in the preparation of anti-lung cancer drugs

Abstract

The present invention discloses an application of a long non-coding RNA LINC01635 in the preparation of an anti-lung cancer drug. It relates to the sequence of LINC10635 and a target sequence for downregulating the expression of LINC01635. The present invention first discovered a long non-coding RNA, LINC01635, which has never been reported. Experimental verification found that it is highly expressed in lung cancer tissues and lung cancer cell lines. Downregulating the expression of LINC01635 in lung cancer cells can effectively reduce the proliferation and migration of lung cancer. The long non-coding RNA can provide a new target for the diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-lung cancer, and in particular to application of long-chain non-coding RNA LINC01635 in the preparation of anti-lung cancer drugs. Background Art

[0002] Lung cancer is one of the most common malignant tumors and the leading cause of cancer-related deaths worldwide. Among lung cancer patients, non-small cell lung cancer (NSCLC) accounts for approximately 85%. Despite the development of a variety of therapies over the past few decades, the 5-year survival rate of NSCLC patients remains low. This situation of non-small cell lung cancer is due to difficulties in early diagnosis, lack of targeted therapy, drug resistance, and easy recurrence. Therefore, the discovery of new biomarkers for early diagnosis and treatment is crucial for the clinical treatment of NSCLC.

[0003] Bioinformatics analysis of the human genome shows that less than 2% of the genome sequence is protein-coding genes, while more than 70% is transcribed into RNA, i.e., non-coding RNA. Long non-coding RNA (lncRNA) is an important category of non-coding RNA, and the length of lncRNA is more than 200 nucleotides. It is reported that lncRNA plays an important role in various tumors through multiple mechanisms. Through comparative analysis of microarray and next-generation sequencing data of NSCLC and normal tissues, thousands of lncRNAs are differentially expressed in NSCLC samples. To date, although some studies have shown that several lncRNAs can promote or inhibit the progression of NSCLC, the discovery of new oncogenic lncRNAs is crucial for the diagnosis and treatment of NSCLC. Summary of the invention

[0004] The present invention provides application of long-chain non-coding RNA LINC01635 in preparing anti-lung cancer drugs.

[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows: The present invention is the use of long-chain non-coding RNA LINC01635 in the preparation of anti-lung cancer drugs, and the long-chain non-coding RNA contains three transcripts, the nucleotide sequence of the first transcript is shown in SEQID No.1, the nucleotide sequence of the second transcript is shown in SEQID No.2, and the nucleotide sequence of the third transcript is shown in SEQID No.3.

[0006] Furthermore, small interfering RNA (siRNA) was designed to downregulate the three transcripts of LINC01635, and its nucleotide sequence was: 5'-GGAGUUUUGGAUACAUUCU-3'.

[0007] Beneficial effects: The present invention discovered for the first time a long non-coding RNA, LINC01635, which has never been reported before. Experimental verification found that it is highly expressed in lung cancer tissues and lung cancer cells. Down-regulating the expression of LINC01635 in lung cancer cells can effectively reduce the proliferation and migration of lung cancer cells. This long non-coding RNA provides a new target for the diagnosis and treatment of lung cancer. The present invention also found that LINC01635 is distributed in the nucleus and cytoplasm of lung cancer cells. Further studies have found that it can directly bind to miR-455-5p, thereby regulating the expression of a series of downstream tumor-related genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The present invention inhibits the proliferation and migration of lung cancer cells by knocking down LINC01635 through siRNA.

[0009] Figure 2 The lung cancer tissue samples and lung cancer cell lines of the present invention were verified to have upregulated the expression of LINC01635 and contained 3 different transcripts.

[0010] Figure 3 The present invention verifies that knocking down LINC01635 reduces the proliferation and metastasis of lung cancer in the zebrafish lung cancer transplant tumor model.

[0011] Figure 4 The present invention localizes LINC01635 in lung cancer cells and regulates the expression of genes related to lung cancer proliferation and metastasis by binding to miR-455-5p. DETAILED DESCRIPTION

[0012] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings, but the present invention can be implemented in different forms and is not limited to the embodiments described in the text. On the contrary, these embodiments are provided to make the content disclosed in the present invention more thorough and comprehensive.

[0013] Reagents used in the examples:

[0014] Isopropanol, chloroform, glycerol, anhydrous ethanol, methanol, β-mercaptoethanol, DEPC water, crystal violet, agarose, LB medium, etc. were purchased from Nanjing Saihongrui Biotechnology Co., Ltd.;

[0015] Nucleic acid loading buffer, ampicillin, TAE buffer solution, DNA molecule marker, gel recovery kit, plasmid extraction kit, one-step ligation kit, DH5α competent cells, etc. were purchased from Tiangen Biotechnology Company.

[0016] Fetal bovine serum, 1640 culture medium, DMEM culture medium, opti-MEM reduced serum culture medium, phosphate-buffered saline, Lipo2000 transfection reagent, trypsin, penicillin-streptomycin solution, Trizol, HBSS buffer solution, DiI fluorescent dye, low-melting point agarose, RNA later, nucleocytoplasmic separation kit, etc. were purchased from Thermo Fisher Scientific.

[0017] PCR kits, reverse transcription kits, quantitative PCR kits, etc. were purchased from Bio-Rad Biotechnology Co., Ltd.

[0018] CCK-8 kit, Transwell, 6-well plate (transfection), 24-well plate (transwell), 96-well plate (CCK8), culture flask, culture dish, 50 ml centrifuge tube, 15 ml centrifuge tube (cell culture), 1.5 ml centrifuge tube (transfection), 5 ml centrifuge tube, 0.2 ml PCR tube, etc. were purchased from

[0019] PCR kits, reverse transcription kits, qPCR kits, etc. were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0020] The specific method used in the embodiment:

[0021] Detection of LINC01635 expression level in lung cancer clinical tissue samples

[0022] Surgical specimens of lung cancer patients were dissected to separate lung cancer tissue and adjacent tissue, placed in RNA later solution, and stored at -20 degrees. After a certain number of samples were collected, the tissues were cut into small pieces the size of rice grains, ground with liquid nitrogen, and then Trizol was added to extract total RNA from the tissues. cDNA was obtained using a reverse transcription kit with random primers, and the expression levels of LINC01635 in lung cancer tissue and adjacent tissue were compared using qPCR.

[0023] Detection of LINC01635 expression level in lung cancer cell lines

[0024] Trizol was used to extract total RNA from different lung cancer cell lines and normal lung epithelial cells, and cDNA was obtained using a reverse transcription kit with random primers. The expression levels of LINC01635 in lung cancer cell lines and normal lung epithelial cells were compared by qPCR.

[0025] Detection of LINC01635 transcripts

[0026] The cDNA of A549 lung cancer cells was obtained by random primers using a reverse transcription kit. Primers for amplifying a longer fragment of LINC01635 were designed using the cDNA clone BC039533 reported in ENSEMBL and NCBI. Specific bands were amplified by PCR and cloned into the pMD19-T vector. Different transcripts expressed by LINC01635 were sequenced and analyzed.

[0027] Detection of cell proliferation and migration after knockdown of LINC01635 in lung cancer cells

[0028] According to the cloned LINC01635 transcripts, the public sequence was selected from the universal biological design and synthesis of siRNA and control siRNA. The designed siRNA was transfected into the lung cancer cell line using Lipofectamine 2000, and the knockdown efficiency was detected by qPCR 24 hours after transfection. After siRNA transfection, 2000 lung cancer cells were plated in a 96-well plate, and 10uL of CCK-8 reagent was added to each well. After 2 hours, the reading was read at OD 450nm by a microplate reader, and then every 24 hours for 96 hours, and then the cell proliferation level was analyzed. After siRNA transfection, the lung cancer cells were resuspended in serum-free culture medium, and the cell migration experiment was carried out using an 8-um pore size Transwell chamber. The resuspended cells were plated on the upper layer, and the culture medium containing 10% FBS was added to the lower layer. After 24 hours, methanol was used to fix for 30 minutes, and then 0.1% crystal violet was used for staining for 20 minutes. The surface of the upper layer was cleaned with a cotton swab, and then the migrated cells in the lower layer were imaged by an inverted microscope, and the migration level of the knockdown combination control group was compared.

[0029] Detection of LINC01635 localization in lung cancer cells

[0030] The cultured lung cancer A549 cells were collected, and the total RNA in the cytoplasm and nucleus was extracted using the PARIS kit. The cDNA in the cytoplasm and nucleus was synthesized using a reverse transcription kit, and the localization of LINC01635 in the cytoplasm and nucleus was analyzed by qPCR.

[0031] In vitro experiments verified the binding relationship between LINC01635 and miR-455-5p

[0032] The first transcript (the longest) of LINC01635 was cloned into a dual-luciferase expression vector to construct a reporter plasmid. The binding site of miR-455-5p on LINC01635 was predicted by mutation using a point mutation PCR kit. The plasmid and miR-455-5p mimic, the mutant plasmid and miR-455-5p mimic plasmid and control miRNA, and the mutant plasmid and control miRNA were co-transfected into HEK293T cells. The readings of firefly luciferin and Renilla luciferin were detected by a dual luciferin reporter kit and a microplate reader, respectively, to analyze the binding relationship between LINC01635 and miR-455-5p.

[0033] Detection of cell proliferation and metastasis after knockdown of LINC01635 in zebrafish transplant tumor model

[0034] Transgenic Tg(fli1a:EGFP) zebrafish larvae with labeled endothelial cells were used for cell transplantation. 24 hours after siRNA transfection, lung cancer cells were collected and stained with a fluorescent dye (CM-DiI) for injection, and the stained cells were examined under a fluorescence microscope before injection. Approximately 300-400 CM-DiI-labeled cells were transplanted into the perivitelline space of zebrafish larvae 48 hours after fertilization using a Picosprizer III microinjector. One day after injection, larvae with similar size of transplanted tumor cell clusters were selected and cultured in a 34°C light incubator until the end of the experiment. 96 hours after injection, zebrafish larvae were fixed with 1.2% low-melting-point gel, and the abdomen and trunk were imaged using an Olympus MVX10 stereo microscope or Fluoview3000 confocal microscope (20X water objective). The image resolution was 1600×1200 (MVX10) or 1024×1024 pixels (Fluoview 3000). The levels of lung cancer cell proliferation and migration were calculated by counting the fluorescence values ​​of the abdomen and trunk separately.

[0035] Example 1

[0036] Expression and transcript analysis of LINC01635 in lung cancer

[0037] In this example, total RNA was extracted from lung cancer tissues of 14 patients and paracancerous tissues of 15 patients, and the expression levels of LINC01635 in lung cancer tissues and paracancerous tissues were compared by qPCR. It was found that the expression of LINC01635 in lung cancer tissues was significantly higher than that in paracancerous tissues ( Figure 1A); Total RNA was extracted from different lung cancer cell lines and normal lung epithelial cells, and the expression of LINC01635 in lung cancer cell lines and normal lung epithelial cells was compared by qPCR. It was found that the expression of LINC01635 in lung cancer cell lines was significantly higher than that in normal lung epithelial cells ( Figure 1 B); Using the cDNA clone LINC01635 of A549 lung cancer cell line, we found that two specific bands could be amplified ( Figure 1 C), predicting that LINC01635 contains multiple transcripts; to further understand the specific sequences of different bands, we cloned and sequenced different bands separately and found that LINC01635 contains three transcripts, of which the largest transcript (transcript 1) is 1008bps (see Seq No.1), the second transcript (transcript 2) is 973bps (see Seq No.2), and the smallest transcript (transcript 3) is 904bps (see Seq No.3). The exon-intron structures of these three transcripts are shown in Figure 1 D.

[0038] Example 2

[0039] Cell experiments verified that downregulation of LINC01635 reduced the proliferation and migration of lung cancer cells

[0040] In this example, two lung cancer cell lines, A549 and H1975, were selected for functional studies. First, siRNAs targeting three transcripts were designed to interfere with the expression of LINC01635. A specific interference target was found at the N-terminus of the three transcripts. The interference efficiency of siRNA was further tested by qPCR. It was found that the interference efficiency of siRNA in A549 and H1975 was 70.7% and 53.5%, respectively (see Figure 2 A and B); After transfection with siRNA, we used CCK-8 kit to detect the OD 450 value at 2 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours after transfection, and found that compared with the control group, transfection with siRNA significantly reduced the proliferation of two lung cancer cell lines A549 and H1975 (see Figure 2 C and D); Transwell assay was used to investigate cell migration ability. After cells were transfected with siRNA, the migration ability of the two lung cancer cell lines was significantly reduced compared with the control group (see Figure 2 E and F).

[0041] Example 3

[0042] LINC01635 regulates the expression of tumor-related genes by binding to miR-455-5p

[0043] In this example, the mechanism of LINC01635 was further studied. First, the nuclear and cytoplasmic localization of LINC01635 was determined. Total RNA was extracted from the cytoplasm and nucleus of lung cancer cells, and qPCR was used to detect that LINC01635 was distributed in both the nucleus and cytoplasm (see Figure 3 A), its localization in the cytoplasm suggests that it may play a role by binding to miRNA; secondly, through online analysis of miRDB and LncBase databases, it was found that LINC01635 contains multiple possible miRNA binding sites. Cross-comparison revealed 9 common possible miRNA binding sites, from which the highest-scoring possible binding sites for miR-455-5p were selected for further study (see Figure 3 B); The longest LINC01635 transcript was cloned and cloned into a dual-luciferase reporter vector, and the possible binding sites of miR-455-5p were mutated using a point mutation PCR kit to construct a mutant plasmid (see Figure 3 C), co-transfected with miR-455-5p mimic and wild-type reporter plasmid, miR-455-5p mimic and mutant reporter plasmid, miRNA control and wild-type reporter plasmid, miRNA control and mutant reporter plasmid, and detected the readings of firefly luciferin and Renilla luciferin, respectively. The analysis results showed that miR-455-5p could directly bind to LINC01635 and down-regulated the level of the reporter gene containing LINC01635 wild type (see Figure 3 D).

[0044] Example 4

[0045] In vivo experiments verified that downregulation of LINC10635 could inhibit the proliferation and migration of lung cancer cells.

[0046] In this example, the potential research of LINC01635 as a drug target in living animals was further studied. After lung cancer cells were transfected with control and LINC01635 siRNA, 300-400 lung cancer cells were transplanted into the yolk space of zebrafish larvae 2 days after fertilization by microinjection, and cultured in a 34-degree light incubator. After 24 hours of transplantation, zebrafish larvae in the control and experimental groups with similar numbers of transplanted cells were selected for further culture, and the abdomen and tail of the zebrafish larvae were imaged by stereo fluorescence microscopy or confocal microscopy 96 hours after transplantation, and the fluorescence signal areas of the abdomen and tail were counted to calculate the level of lung cancer cell proliferation and migration. The results showed that compared with the control group, the use of siRNA to knock down LINC01635 can also detect a significant decrease in the proliferation and migration of the two lung cancer cells tested in the zebrafish transplant tumor model (see Figure AL).

[0047] The invention discloses an application of a long non-coding RNA LINC01635 in the preparation of an anti-lung cancer drug, wherein the long non-coding RNA contains three transcripts, the nucleotide sequence of the transcripts is shown in SEQID No.1, the nucleotide sequence of the transcripts is shown in SEQID No.2, and the nucleotide sequence of the transcripts is shown in SEQID No.3.

[0048] siRNA targeting three transcripts was designed to downregulate the expression vector of LINC01635, and the nucleotide sequence of the expression vector was: 5'-GGAGUUUUGGAUACAUUCU-3'.

[0049] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents. Sequence Listing <110> Lianshui County People's Hospital Nanjing Xinjia Pharmaceutical Technology Co., Ltd. <120> Application of long noncoding RNA LINC01635 in the preparation of anti-lung cancer drugs <130> 2021 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1008 <212> DNA <213> Artificial sequence (nucleotide sequence of the first transcript) <400> 1 ttaccgtgcg gagttttgga tacattctta gcagcttttt cgctccggag aagttcttta 60 tctttctgag ccgcagtttg cccaactgca aaatggaaag agatgaagca ggaattaaga 120 tactctagga gctcgcccac agctgattca gggccccatt ctgaggttac acccttaagc 180 ctttgcacca ctcaaaggaa ggaaaccaag agagaggggt ggctggctgc tgtttggaga 240 cagtggtcct tgtattggcg ccccgcggaa tcactccaga tgagtcagtg gatacagtct 300 ataggtcccg cctcattgta cgcaaacagc agtgggcata tggccgtggg gggttgatac 360 agcctgtccg gggtgggact ctgtgtgtat gggaggctaa atctcagcag gacttttcca 420 gttgctacca gactacgcac tgtcgcctga tggttaagaa ctgaagcttt agccgtgcgc 480 ggtggctcac gcctgtaatc ccagcacttt aggaggcaga ggcgtgtgga tcgcctgagg 540 tcaggagttc gagaccagct tgaccaacaa gctggcgtat ccgaatgggc tgagctaccg 600 gattaagagg gacaccccaa agcccccatt gctgggttat tgctccagag ccaatgttct 660 tggggaaagg aagatatgcc ctttgtcaac attgccactg ctggtctgta aactcctaga 720 cggccagctg gtggttcaca aaccaggact ccttgctctg gccctacccc tacctaccag 780 aatgaccgtg aacccttccc cactcactcc tacaaccagg tttccatctc ctctctcagc 840 ttaggtttcc ctaactgtaa aatagaaggg ttggactagg ttaaggactt cctgctattt 900 ctctctccca cactctaagg ttccttagga atgcttcaga aaacagcagg ggttggggca 960 agaatgccac ttgagtccca gagcaactcc aatttcatag gcacataa 1008 <210> 2 <211> 973 <212> DNA <213> Artificial Sequence (Nucleotide Sequence of the Second Transcript) <400> 2 ttaccgtgcg gagttttgga tacattctta gcagcttttt cgctccggag aagttcttta 60 tctttctgag ccgcagtttg cccaactgca aaatggaaag agatgaagca ggaattaaga 120 tactctagga gctcgcccac agctgattca gggccccatt ctgaggttac acccttaagc 180 ctttgcacca ctcaaaggaa ggaaaccaag agagaggggt ggctggctgc tgtttggaga 240 cagtggtcct tgtattggcg ccccgcggaa tcactccaga tgagtcagtg gatacagtct 300 ataggtcccg cctcattgta cgcaaacagc agtgggcata tggccgtggg gggttgatac 360 agcctgtccg gggtgggact ctgtgtgtat gggaggctaa atctcagcag gacttttcca 420 gttgctacca gactacgcac tgtcgcctga tggttaagaa ctgaagcttt agccgtgcgc 480 ggtggctcac gcctgtaatc ccagcacttt aggaggcaga ggcgtgtgga tcacctgagc 540 tggcgtatcc gaatgggctg agctaccgga ttaagaggga caccccaaag cccccattgc 600 tggttattgc tccagagcca atgttcttgg ggaaaggaag atatgccctt tgtcaacatt 660 gccactgctg gtctgtaaac tcctagacgg ccagctggtg gttcacaaac caggactcct 720 tgctctggcc ctacccctac ctaccagaat gaccgtgaac ccttccccac tcactcctac 780 aaccaggttt ccatctcctc tctcagctta ggtttcccta actgtaaaat aaaagggttg 840 gactaggtta aggacttcct gctatttctc tctcccacac tctaaggttc cttaggaatg 900 cttcagaaaa cagcaggggt tggggcaaga atgccacctg ctccagagca actccaattt 960 cataggcaca taa 973 <210> 3 <211> 904 <212> DNA <213> Artificial Sequence (Nucleotide Sequence of the Third Transcript) <400> 3 ttaccgtgcg gagttttgga tacattctta gcagcttttt cgctccggag aagttcttta 60 tctttctgag ccgcagtttg cccaactgca aaatggaaag agatgaagca ggaattaaga 120 tactctagga gctcgcccac agctgattca gggccccatt ctgaggttac acccttaagc 180 240 cagtggtcct tgtattggcg ccccgcggaa tcactccaga tgagtcagtg gatacagtct 300 ataggtcccg cctcattttg ctaccagact acgcactgtc gcctgatggt taagaactga 360 agctttggcc gtgcgcggtg gctcacgcct gtaatcccag cactttagga ggcagaggcg 420 tgtggatcac ctgaggtcag gagttcgaga ccagcttgac caacaagctg gcgtatccga 480 atgggctgag ttaccggatt aagagggaca ccccaaagcc cccattgctg ggttattgct 540 ccagagccaa tgttcttggg gaaggaa tatgcccttt gtcaacattg ccactgctgg 600 tctgtaaact cctagacggc cagctggtgg ttcacaaacc aggactcctt gctctggccc 660 tacccctacc taccagaatg accgtgaacc cttccccact cactcctaca accaggtttc 720 catctcctct ctcagcttag gtctccctaa ctgtaaaata aaagggttgg actaggttaa 780 ggacttcctg ctatttctct ctcccacact ctaaggttcc ttaggaatgc ttcagaaaac 840 agcaggggtt ggggcaagaa tgccacttga gtcccagagc aactccaatt tcataggcac 900 ataa 904

Claims

1. Use of an inhibitor of long non-coding RNA LINC01635 in the preparation of an anti-lung cancer drug, characterized in that: The long non-coding RNA contains three transcripts, the nucleotide sequence of the first transcript is shown in SEQID No.1, the nucleotide sequence of the second transcript is shown in SEQID No.2, and the nucleotide sequence of the third transcript is shown in SEQID No.3; the inhibitor is a small interfering RNA, which is used to downregulate the expression of all transcripts of LINC01635, and its nucleotide sequence is: 5'-GGAGUUUUGGAUACAUUCU-3'.

2. The use according to claim 1, wherein the inhibitor can reduce the expression level of LINC01635 in lung cancer cells, and the anti-lung cancer effect is to inhibit the proliferation and migration of lung cancer.

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