CN116350788A - Application of inhibitor of lncRNA EBLN3P functional expression in preparation of medicines - Google Patents

Abstract

The invention discloses application of an inhibitor of lncRNAEBLN3P functional expression in preparation of medicines. Relates to the technical field of biological medicine. The nucleotide sequence of the lncRNAEBLN3P is shown as SEQ ID No.1. Related applications are also provided. The invention discovers that lncRNAEBLN3P with low expression after heavy ion irradiation inhibits the expression of a cancer related gene TNPO1 in tumor cells, thereby leading to increased apoptosis. Meanwhile, the lncRNAEBLN3P can be used as a new biological molecular target, and the expression of the lncRNA is inhibited in the ordinary photon radiotherapy process, so that the apoptosis is promoted, and especially for some hypoxic tumor tissues, the apoptosis level of tumor cells can be effectively improved, the tumor cure rate is increased, and the prognosis of patients is improved.

Description

Application of inhibitor of lncRNA EBLN3P functional expression in preparation of medicines

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an inhibitor of lncRNA EBLN3P functional expression in preparation of medicines.

Background

Currently, cancer is the first killer to threaten human life. Due to the limitations of the current technology level, the treatment approaches for cancer remain relatively limited. These include traditional surgery, photon radiation therapy, chemotherapy, and targeted gene therapy developed in the last decade. Although the cure rate of a few of these cancers is high, the prognosis of most cancers is still less than ideal and the cure rate is also relatively low.

In face of these problems scientists and clinicians are continually exploring more effective ways of treatment. The rapid development of radiation therapy has greatly improved the cure rate of malignant tumors in recent decades. In particular heavy ion radiotherapy, due to its excellent physical properties (i.e. "bragg peak"), tumor cells can be killed as much as possible during the treatment while surrounding normal tissues are protected to the greatest extent.

The occurrence of heavy ion radiotherapy obviously improves the cure rate of some malignant tumors and improves the prognosis of patients.

However, the cost of heavy ion radiotherapy is quite high, as the cost of manufacturing heavy ion accelerators is very expensive.

Thus, most patients still need to rely on traditional three major treatment regimens.

In clinical practice, common photon radiotherapy (such as X-rays, gamma rays and the like) has the problem of poor effect in treating hypoxic tumors. The reason is that the continuous proliferation of tumor cells consumes oxygen to form a hypoxic microenvironment, and the killing effect of the common photon radiotherapy mainly depends on the reaction of oxygen and free radicals, so that the killing effect on the tumor cells in the hypoxic microenvironment is weaker.

In contrast, heavy ion radiotherapy can directly deposit energy and produce biological effect, has smaller dependence on oxygen and has obvious killing effect on tumor cells in a hypoxia microenvironment.

Based on the above, if the molecular mechanism of heavy ion radiotherapy can be explored, the difference between the heavy ion radiotherapy and photon radiotherapy is revealed, and some biological molecular targets are found, it is possible to add adjuvant therapy for these targets in conventional photon radiotherapy, so as to improve the therapeutic effect and tumor cure rate of the conventional therapeutic method.

Therefore, whether to provide lncRNA EBLN3P for preparing auxiliary drugs for radiation therapy of hypoxic tumors is a problem to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the present invention provides the use of inhibitors of lncRNA EBLN3P functional expression in the manufacture of a medicament. Relates to the promotion of the apoptosis of hypoxic tumor cells after knocking down long-chain non-coding EBLN 3P. Provides a potential therapeutic target for the problem of low killing rate of the hypoxic tumor in the common photon radiotherapy.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the application of an inhibitor of the functional expression of the lncRNA EBLN3P in the preparation of medicaments is provided, wherein the nucleotide sequence of the lncRNA EBLN3P is shown as SEQ ID No.1.

Preferably: the medicine is an auxiliary medicine for treating the radiation therapy of the hypoxic tumor and selectively induces apoptosis of tumor cells.

Preferably: the inhibitor is siRNA or miRNA.

Preferably: the sequence of siRNA is:

si-LNC EBLN3P sense:5'-ACAGAAAGCACCAAACAGGGA-3', SEQ ID No.2;

si-LNC EBLN3P anti: 5'-CCUGUUUGGUGCUUUCUGUUU-3', SEQ ID No.3.

Preferably: the miRNA is miR-144-3P, and the common sequence of miR-144-3P combined with wild type lncRNA EBLN3P and wild type TNPO1 is as follows: 5'-UACAGUAUAGAUGAUGUACU-3' is shown as SEQ ID No.4.

Preferably: the hypoxic tumors include lung cancer.

Preferably: the radiotherapy method is X-ray or carbon ion irradiation.

Compared with the prior art, the invention discloses the application of the inhibitor for the functional expression of the lncRNA EBLN3P in preparing medicines, and the beneficial effects are that:

in hypoxic tumor cells, both lncRNA EBLN3P and TNPO1 were significantly down-regulated after heavy ion irradiation compared to X-ray irradiation. Bioinformatics analysis showed that there was a ceRNA regulatory relationship between lncRNA EBLN3P and TNPO 1.

Further experiments show that the low-expression lncRNA EBLN3P after heavy ion irradiation inhibits the expression of the cancer related gene TNPO1 in tumor cells, thereby leading to increased apoptosis. The molecular mechanism of the lncRNA EBLN3P in the heavy ion radiotherapy of the hypoxic tumor is discovered for the first time, and the reason that the cure rate of the tumor after the heavy ion radiotherapy is higher is explained to a certain extent. Meanwhile, the lncRNA EBLN3P can be used as a new biological molecular target, and the expression of the lncRNA is inhibited in the ordinary photon radiotherapy process, so that the apoptosis is promoted, and especially for some hypoxic tumor tissues, the apoptosis level of tumor cells can be effectively improved, the tumor cure rate is increased, and the prognosis of patients is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the expression of TNPO1 in lung cancer cell regulated by lncRNA EBLN 3P.

FIG. 2 is a graph showing the results of the flow cytometry provided by the present invention, wherein the left 4 graphs show the actual image of the flow cytometer, and the right 2 graphs show the quantized images.

FIG. 3 is a diagram showing a cell proliferation control experiment provided by the invention.

FIG. 4 is a diagram showing a control experiment of the luciferase reporter gene provided by the invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses application of an inhibitor of lncRNA EBLN3P functional expression in preparation of medicines.

Example 1

Cell culture

The stabilized lung cancer cell line a549 (purchased from the Shanghai national cell bank) was selected and the morphology of the cells was checked before delivery to the next generation cells to ensure good growth. Cells were seeded in a cell incubator containing 5% carbon dioxide using RPMI-1640 medium at 37 ℃. When the cell density reached about 80%, the cells were rinsed 2-3 times with sterile PBS solution. 0.25% pancreatin solution was added to the cells and allowed to stand for 1-2 minutes to separate the cells from the culture dish. It is checked whether the cells are completely isolated to avoid affecting the success rate of the next cell culture. Complete medium of 10% fetal bovine serum was added, the cells were resuspended, and the health of the cells was examined using microscopy. The resuspended cells were aliquoted into new dishes and expanded by addition of RPMI-1640 medium until the cell density reached the desired level.

Cell hypoxia culture: cell hypoxia culture was performed using a ratio of 5% carbon dioxide, 94% nitrogen and 1% oxygen at 37 ℃.

Cell transfection

Cells were transfected with LNC EBLN3P small interfering RNA (si-LNC EBLN 3P), negative control small interfering RNA (si-NC), LNC EBLN3P over-expression plasmid (pcDNA3.1-LNC EBLN 3P), empty vector (pcDNA3.1). All siRNAs and plasmids were synthesized by Sangon Biotech (Shanghai, china) and the sequences used to prepare the siRNAs and plasmids were as follows:

si-NC sense：5′-AGGUGUUGGUUAAUUGGUUUA-3′

si-NC antisense：5′-AACACCUACAAACUAUACCUA-3′

si-LNC EBLN3P sense:5'-ACAGAAAGCACCAAACAGGGA-3', SEQ ID No.2;

si-LNC EBLN3P anti: 5'-CCUGUUUGGUGCUUUCUGUUU-3', SEQ ID No.3.

The specific experimental steps are as follows: a549 cells were seeded in 6-well plates and cultured to 60% confluency. The cells were replaced with fresh medium without antibiotics and without serum. After the liquid exchange, the cells are put back into the incubator, and the transfection reagent starts to be prepared. Each transfection system was prepared by mixing A, B tubes in equal volume in 50. Mu.l to A, B tubes of opti-MEM, the A tube contained no plasmid, the B tube contained different plasmids, and the B tube was divided into B1, B2, B3, etc. according to the plasmids. 2.5. Mu.l of lipo3000 reagent was added to the A tube, 2.5. Mu.l of P3000 reagent was added to the B tube, and 1. Mu.g of the different plasmids were added to the different B tubes. Gently blow the liquid in the pipe A, take out and add the liquid into the pipe B, gently blow and mix the liquid evenly. After mixing, the mixture was left at room temperature for 10 to 15 minutes. Then taking out the cells from the incubator, marking each hole according to different components, dripping the transfection reagent into different cell holes, and gently shaking and mixing. And (5) placing the culture medium back into the incubator for further culture.

Radiation treatment

The experimental components are divided into two groups, and the experimental whole is divided into two parts: carbon ions and X-rays were irradiated in Japan in the early stage, and 2Gy X-rays and 2Gy carbon ions were irradiated, respectively, while the control group was not irradiated, and the results were subjected to a belief analysis to screen out differentially expressed lncRNA.

Carbon ion irradiation was carried out on the kilo-leaf Heavy Ion Medical Accelerator (HIMAC) of the national institute of radioscience. In this experiment, cultured a549 cells were divided into a control group and an experimental group, each group containing about 1×108 cells. In the carbon ion irradiation experiments, the Linear Energy Transfer (LET) of C290 at the plateau entrance was 13.3keV/μm, while the LET in the extended Bragg peak was 80keV/μm.

The subsequent experimental steps did not involve carbon ion irradiation, and lnRNA-EBLN3P with significantly reduced expression was screened under carbon ion irradiation, and then lnRNA was used for subsequent X-ray experiments to investigate the different effects of the lnRNA under X-rays.

For X-ray related experiments, an electric energy-activated X-ray irradiator (model RAD SOURCE, RS-2000) was used, with a voltage of 225kVp and a dose rate of 1.12Gy/min.

Extraction of RNA

Cells from which the medium was washed were placed on ice and washed 3 times with PBS. 1ml of trizol was added to each sample for cell lysis, and lysis was performed on ice for 5 to 10 minutes. The lysate was transferred to 1.5ml centrifuge tubes and each sample was transferred to 1 centrifuge tube. 200 μl of chloroform was added to each centrifuge tube and added below trizol. The vortex shaker was thoroughly shaken to thoroughly mix the chloroform and trizol. Centrifuge at 12000rpm for 15min at 4 ℃. After centrifugation, the cells will be separated into 3 layers, the lowest layer being trizol, the middle layer being protein and cell debris, the uppermost clear liquid being the chloroform layer in which RNA has been solubilized. In a new set of tubes, 500. Mu.l of isopropanol was added each, the upper RNA solution in the original tube was transferred over, 200. Mu.l each time, as much as possible the chloroform layer was transferred, but millions of layers were avoided from transferring the middle or lower trizol layer. The tube was inverted and mixed well and allowed to stand at room temperature for 10 minutes. Centrifuge at 12000rpm for 10min at 4deg.C, note the consistent orientation of the centrifuge tubes. The concentration was 70% using analytically pure alcohol plus DEPC water. After centrifugation was completed, the bottom of the tube was precipitated as RNA, and the upper isopropanol layer was carefully poured off. 1ml of 70% ethanol was added to each tube in order to wash off the isopropanol. Centrifugation was performed at 7500rpm for 5 minutes at 4℃to check RNA precipitation and carefully pour off the ethanol from the upper layer. Centrifugation at 7500rpm at 4℃for 5min was performed with the centrifuge tube in the same direction, and secondary centrifugation was performed to better separate RNA from ethanol, to better precipitate RNA, and to discard the supernatant with a 10. Mu.l tip to avoid aspiration of RNA precipitate. All centrifuge tubes were uncapped until ethanol evaporated, and the RNA became clear, but excessive drying of RNA was avoided. According to the RNA amount, a proper amount of DEPC water is added for dissolution. The RNA sample should be stored in a refrigerator at-80 ℃ to avoid degradation if not used.

qRT-PCR

Reverse transcription of total RNA was performed using PrimeScript kit, 5X PrimeScriptRTM asterMix L was added, appropriate amount of RNA was added, and RNaseF reedH was supplemented ₂ O to 10L system; the reverse transcription is carried out for 15min at 37 ℃, 5s at 85 ℃ and 4 ℃; diluting cDNA obtained by reverse transcription by 5 times to be used as a template of quantitative PCR;

related primer sequences:

primer sequence for lncRNA EBLN3P transcript fluorescent quantitative PCR experiment

Forward：5′-TACGCGTTTTGGTCCCTGTT-3′

Reverse：5′-GCCACTTGGCTCAAAAGACTG-3′

GAPDH：

Forward：5′-AGCCACATCGCTCAGACAC-3′

Reverse：5′-GCCCAATACGACCAAATCC-3′

TNPO1：

Forward：5′-GTCTTAACAGAGTTAGAACTTGGG-3′

Reverse：5′-CTTCTGGGAGTATCTTGAAAGAG-3′

The primer was diluted to a concentration of 2M; after denaturation at 95℃for 10min, using PrimeScript kit and according to the instructions formulation and set-up procedure, 40 cycles were started, cycles 95℃15s,60℃30s,72℃30s; and calculating the expression change times of the target lncRNA transcripts between the experimental group and the control group according to the Ct value of the fluorescent quantitative PCR experiment, and plotting according to the results of three independent experiments.

LncRNA EBLN3P sequence (siRNA binding sites in bold and miRNA binding sites in underlined): GTGTTGTCCCGGAAGTGCCTTCTCTCCTCCCGGGTCTGCGCGGACGCGGCCTCCTTACCTCATTTGTCCTCGCCCCTCCCCGTCCCTCTACGCGTTTTGGTCCCTGTTTGGTGCTTTCTGTTTGCAGCTACGGCAGTGAGTATGTATGTGACGGACCCCGAGTCACCCGCGGCCTGGGACCCCTGCCTACCCTCCGTCTCGCCAGCCGAGCTGTGGAACTAGCGCGTGCCCCCTCGCCGACCTCGGCGTCTCCGGTCCGCCCCTCACTTGTGGTGGGGCGCAGCTCCTGGTCCCTCAGCTGCGCGCCGCCCCACGCGGCCGGGCTGCGGGTCTAGGGGGTCCGCATCTCCCTGGCTTTCCAAGGGCTAAGGTCGT

GATTCTAGGGCGGCTGGGCGTCCAGGGCCTCGGTGGGGGTGGCGTGTCTGCCCTTTTTATCTCCC

CGCAAGGCCCCCAGTCTTCTAGGGAAGCCAGTCAGTGAAGCGCGGAGGTCCGGGCGCGCCGAGA

GAGAGTCCAGTCTTTGAGGACCGAGTAGTCCTGGGCCACCTCCCGCCTCTGCTGTCAGAAGCAGC

AGCTGCCGCCGTGGAATCCAAAATTTCGGGAGCTGTGACCCTTTCCTCATGTAAAACGAGTAGTC

TTGGACGATCTGGGCATAGGAACCAATCAGAAACAATCGCTTCAGCAATCAAGACCATTGTTCAT

CATGGAGGAACCCATGGATACCTCTGAGCCTCTATCTGCATTACCATTCACTGGGCAGCAGTCTT

TTGAGCCAAGTGGCAAATTTGGACAGTATCCATCGATGCAGATGAACCACATCCAGGCACTGGG

GAAGTGGAGGACATAGAACAGCTCAATCAGTGTTTGATCCAACACTTCCATCTCATTAAGACAAG

TTTGATTTTTCTTTGCTTTTTATTTCATGGAATACATGAGAATCTCTTAACTGTTGGAGTTTCCAAG

GAGGCATACCTCATGACTTCAGTTAATGGAAAGAACAAAACTAAAATGCTGTATGGCCAAAGCC

ACAAAGGGAAGGATCCACAATTTACATTCTTGGAGCTATCATCTGTACTGTACTGTTGTGATCTA

CTGATTGGCATTGGCATAGTAGTAGGGGTCAAGTGACAGAATCCGTGCCAGCAGTCTCCAGGTTC

AGAAGCAATTCAAGACCCTGATGATAGCTCTCCAGCAACCAACACATGGTGATTGTGCCAACTTG

TTGCTCAGTTATAATGCAGGGCCAGTGATTGGTTTAAGTGAAGACCATGGTTGAGATCATTTGTC

TTTGGTCTAATAGAATTTGAGTCTAGTAGAATTTGAGTCTCCAGGGAAAGAGCTACTTGACCAAA

TTAAACTAGTAGCAGGTAGAGCATGAATGACAGCATATTATACCATCAAGATGTTCTTAGAGCAG

TGTATGGATGGATCGATTGTACTGCCATCAGTTGTGACTGACGTTGTATTCAAGGAGAAAGAGAA

ACTTGTTTAGAAAGCACTTTGAAAGTTTTTTGAGTACGGGGGTGCCCTGTATCACCCCGTTATGGT

TGAACTTTCTCCTTCAAAATTACCAGACTTGGCAGCAGTGGCAAATTATTGGGCTAAAAGACTTA

ATCAGACATATTCTGGGTTCAAGGCTCCTAATATAATACCTGGTGCAAACATTATACTTCCACTC

ATTCAGATGGTTGCATCCTGCCAGGCATCCAGTGGGACTGGGAATATGGACACTTGAACATTAAA

CATCCTGAAGAATTTTGGAATGACAGGTTACAAGTGAACATAATCAGTTCTCTATATTAAAAAAA

AAAAAAGATTTGGGCATAGATTGGGCTCACATAATCTTTAGAGAGATTTTTTCATTTTTGTGAAA

GTACAGAAATTCTTAACTGCTTATGAAATGCTGATTGTTAAACAGCATCCACAGCTATTTTGTGTT

GTTTCCCTGACCCCACCCTGAAGAAAAGAAAAATTATGGCATATTGAAAACAGCAGTATGATGT

AAGAGAAAAGATCACAAATTCCTTGAGGGTGGGTCTTTTCCATACTCATAAGCCTATTTATAATA

TTCAGAGTAATTTATTGACACATATTAATATTCCCTCCTATCCCATTAATTGCCAAATCATCAAAC

ATTTATTGAGCACCTACTCTGTGTAGGGTGTAAGCAGTACTGGGGACTTACAGTGATAAAAGACA

TAGTTTTAGCCTTAAGTGATGTACTGTTTCACTGAAGAGATGGAATATAAGAAGCCAAGTAATTT

ATGGAATTCTCTTCTTTGTTTTTCTTTTTTTTTTTTGGCAGTGGAGTCTTGCTCTTTAAATAGAGCAAGGCTAGTCTGGAACTCCTGGGCTCATGTGGTCCTCCCGCCTTAGCTTTCAGTGTAGCTGGGACTACAGGTGTGAACACAGCTTGGAAATCTCTTAACCATGGGAGTTAAGTCTCAAAATTCTGGTGATACAAGTGGTTGAAACTTAAAACTGTATTTAAAAAATAGGATTCGTGAATTTGAGATAGTTCATAAGTCTGCAAAAGGCTGTATAAATACATATTTTACATTTACTATTATTAATTTTGTAGTAAATTTGAGTACAGCACTCTCTTTATCTGTGGAAACTTCAGACTCTCCCCTATTACTTTAATTTCAGTGAGACATTATTAAATATAAGTGGGCTTACACATTTGTTTTGCTTTACTGACAAATAATACACAACTTGGAGGCTTTTTTTTCCTTTCTATTCTTCCTCTAAATGTTCAACACTTTTCTGATTTTGTGATTTGAGGTTGTTTAATAGCTTCCTGAGGCTCCATTGAGACCGTATATACGTGACACTTAACAGTCTAGCCTTCCTCGGTACATATAGATATATGATGGTGGCTTTGCCTGTAGTAAATTCATGCCAAAACATAGGCTTTCAGTGCCTATTACATATGGCTTTCAGCTCTCTCTACTGAGGGATGTAGGAGTTTATTTCTGAGGTCTGAGCCTCTTTTCCTTTACTTCCTTTACTCTTTCCTAAGCCTTCTTTATAAAAACTATGCATGTTCTATTGTTTTCCTTTTTGATTCCCTTTCTTTTATTATCCCCAGTAGGAGTGACTTGTAATTCTCATATGTTAGAAAGGCAGATCTCCTGGTTGAAGAAAAGATCCACCCAAGCAAGTCAGCATGTTTAATAATTTTTGAGGGGGATCTCAAATGTGGGAAGGATTGTTATATAAGACAACCAAATGATGACATGAGACAATAAATGCTATAGGAATTATGGAGGAATAATTAGCTATTTATTTTCTTGGTTAGGGAAGAGATATTATTAGTTGTAGAAGTAATTACTAACTTCTACATTTTTTATTGTGGAAATCAAAAATATATATATGAAAATAAAATGTTATAATTGACTTCAGTGTCCCATAAACCAGCTTCAACAATTACCAAATTGTGACCAATCTTTACACACATGCACAGGTGTCCCTCAGTATCTGTGGGGCATTGGTTCTAGGACCACTTATGGATACCAACATCTATGGATGCTCAAGTCCCTGATATAAAATGGTGGACTATTTGCATATAACCTGTGTACATCCCGTATTATTTAAATCATCCCTAGATCACTTATAATACGTAATACAATGTAAATGCCATGTAAATAACTGTTATACTGTATTAAGGAATAACAACAAGAAAAATGTACATGTTCAGTACAGACGCAATTTTTTTTGTGTGTGGAATATTTTCATTCCAAGGTCAGTTGAACCCATGGACATAGGAGGCTGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATACAGACACACATATTTCTGAAATGTAAATATTCTCTTCTTAAAAAAATTATTATCACAGCTAAACAAATTACCAGTAATTCTTTTATCCTCATATACCCGGTGTTCAGATTTTCTAGATTGGCTCCTAATTTTTTTACAGATTATTTGAATCTGATTCAATTCATGTACTGTAATGTTTGATAACTTAAGTACCCTTTATAGGTTCTCTTTTACCTCTTCTTTATTAAATTCCTTGTAATTTGTTGTACTAAATAGATTGTCTTCTAGAATTTCCTGTAGTCTGAATTATGTAGTATTGTTTCACATGTTCCAGTGTCCTCTTATTTCCTGTGAGTTGGTAGTTAGATCTAGAAGCTTGATTAAATTCAGATTTTCTCTCTTTAGATCATCAACTTTAGATCATCAACTTGGATCATTTGTTTCATTTTGCTTTTGATATGTTGTTTTTTAGAATTACCTCTTAAAATTTTGATTTAATTTTATAATCATG

TAAAATGTTTATAAATTTCCAAATTCAGATCAGCAAAACACAATAAAATCTATTCAGAGAAGGCAAACCTTCAAAAAAA as shown in SEQ ID No.1.

Example 2

Western immunoblotting

Total protein was extracted from cells using radioimmunoassay lysates (Beyotime, shanghai, china) and protein concentration of each sample was determined using BCA protein assay kit (Beyotime, china). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes (allington high-earth albesom, usa). Chemiluminescent signals were detected using an enhanced chemiluminescent detection kit (Millipore, burlington, MA, USA) and analyzed using a polychromatic fluorescent chemiluminescent imaging analysis system (Alpha Innotech, san Leandro, CA, USA).

Anti-transporter-1 antibodies (catalog number: 38700;Cell Signaling Technology,Danvers,MA, U.S.) and glyceraldehyde-3-phosphate dehydrogenase (catalog number: 5174;Cell Signaling Technology). mir-144-3P:

miR10000436-1-5，hsa-miR-144-3p mimic,5nmol

miR20000436-1-5，hsa-miR-144-3p inhibitor,5nmol

miR1N0000001-1-5，micrON mimic NC#22。

experimental results: referring to FIG. 1 (experimental group: pcDNA3.1-LNC EBLN3P, LNC EBLN3P insert cleavage site: bstX I; control group: pcDNA3.1; FIG. 1B, experimental group: si-LNC EBLN3P, control group: si-NC), lncRNA EBLN3P positively regulates expression of lung cancer cell TNPO1

(A, C) overexpression of lncRNA EBLN3P increased TNPO1 mRNA and protein levels in A549 cells.

(B, D) downregulation of the lncRNA EBLN3P gene reduced TNPO1 mRNA and protein levels in A549 cells.

a-D represents the average ± SD (n=3). * P <0.01, p <0.001.

Example 3

Apoptosis assay

After over-expression or knocking down lncRNA EBLN3P in a549 cells, culturing for 24h, collecting all cells including cells in supernatant, washing twice with sterile PBS, digesting adherent cells with 0.25% pancreatin for 1-2min, stopping digestion with RPMI-1640 complete medium containing 10% fetal bovine serum, recovering digested cell suspension in the same centrifuge tube as supernatant, centrifuging at 1000 rpm for 5min at room temperature, discarding supernatant, and removing anexinv-Alexa from fomis company, and collecting the supernatant

647 binding buffer (1 Xbinding buffer) in detection kit for apoptosis resuspended cells at 200L/test followed by addition of Annexin V dye at 5L/test at 10

PI dye was added in the amount of L/test, and after incubation for 10-15min in the dark, apoptotic cells were detected on a BD company flow cytometer.

The results shown in fig. 2 indicate that: overexpression of lncRNA EBLN3P reduces apoptosis rate in cells, while knockdown of EBLN3P increases apoptosis rate in cells. * P <0.01, p <0.001.

Example 4

Cell proliferation assay

A549 cells overexpressing EBLN3P were cultured according to 1×10 ⁴ Is planted in 96-well plates, and the proliferation level of the cells is measured every 24 hours. The cell proliferation test adopts the CCK-8 method, the original old culture medium is discarded before the test, the complete culture medium containing 10% of CCK-8 reagent is prepared, the complete culture medium is added into cells to be detected in a 96-well plate according to the dosage of 100L/hole, then the cells are incubated for 2 hours at 37 ℃ in a dark place, the absorbance is measured at 450nm on a multifunctional enzyme labeling instrument after the incubation is finished, the value of the absorbance (OD value) is positively related to the cell proliferation level, and the larger the OD value is, the more the cell proliferation is represented.

The results shown in fig. 3 indicate that:

(A) After the lncRNA EBLN3P is overexpressed using the plasmid, the amount of intracellular lncRNA EBLN3P expression;

(B) Over-expressing lncRNA EBLN3P can enhance cell proliferation;

(C) After downregulating lncRNA EBLN3P using siRNA, intracellular lncRNA EBLN3P expression levels;

(D) Down-regulating lncRNA EBLN3P can inhibit cell proliferation and colony formation, and promote apoptosis.

**p<0.01，***p<0.001

Example 5

Luciferase reporter assay

A549 cells were seeded on 6-well plates and co-transfected with luciferase reporter plasmids lncRNA EBLN3P 3'utr wild-type (WT) or 3' utr Mutant (MUT) and miR-144-3P mock or negative control (miR-NC). 48 hours after transfection, cells were treated according to the instructions of the double luciferase reporter assay kit (beyotidme, shanghai, china) and firefly and Renilla luciferase activities were measured using a multifunctional microplate reader (BioTek Instruments).

All vectors were synthesized by RiboBio.

See fig. 4:

(A) The interaction site of lncRNA EBLN3P with miR-144-3P is indicated. After co-transfection of lncRNA EBLN3P wild-type (WT) or Mutant (MUT) luciferase reporter plasmids and miR-144-3P mimic or negative control (miR-NC), the relative luciferase activity of a549 cells was detected.

Conclusion: there is an interaction site between the lncRNA EBLN3P and miR-144-3P, and the miR-144-3P can regulate the intracellular level of the lncRNA EBLN 3P.

(B) The interaction of miR-144-3p and TNPO1 is shown; after co-transfection of TNPO1 wild-type (WT) or Mutant (MUT) luciferase reporter plasmid and miR-144-3p mimic or negative control (miR-NC), A549 cells were assayed for relative luciferase activity.

The common sequence of miR-144-3P and the combination of wild type lncRNA EBLN3P and wild type TNPO1 is as follows: 5'-UACAGUAUAGAUGAUGUACU-3', SEQ ID No.4.

Conclusion: the miR-144-3p has an interaction site with TNPO1, and the miR-144-3p can regulate the expression level of TNPO 1.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. Use of an inhibitor of the functional expression of lncRNAEBLN3P in the preparation of a medicament, wherein the nucleotide sequence of lncRNAEBLN3P is shown as SEQ ID No.1.

2. The use of claim 1, wherein the medicament is an adjunct to radiation therapy for treating hypoxic tumors, and selectively induces apoptosis in tumor cells.

3. The use of claim 2, wherein the inhibitor is an siRNA or miRNA.

4. The use of claim 3, wherein the siRNA has the sequence:

si-LNCEBLN3Psense:5'-ACAGAAAGCACCAAACAGGGA-3' as shown in SEQ ID No.2;

si-LNCEBLN3Pantisense:5'-CCUGUUUGGUGCUUUCUGUUU-3', SEQ ID No.3.

5. The use of claim 4, wherein the miRNA is miR-144-3P and the common sequence for miR-144-3P binding to wild-type lncrrnaebln 3P and wild-type TNPO1 is: 5'-UACAGUAUAGAUGAUGUACU-3' is shown as SEQ ID No.4.

6. The use according to claim 5, wherein the hypoxic tumor comprises lung cancer.

7. The use according to any one of claims 1 to 6, wherein the method of radiotherapy is X-ray or carbon ion irradiation.

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