CN112980956B - Target for inhibiting lung cancer growth and diagnostic marker uc.336 and application thereof - Google Patents

Abstract

The invention discloses a target for inhibiting lung cancer growth, a diagnosis marker uc.336 and application thereof, discloses application of uc.336 in preparation of a lung cancer diagnosis marker, and also discloses application of a uc.336 expression promoter in preparation of a medicament for inhibiting lung cancer growth. According to the invention, lung cancer cells A549 and HCC827 are taken as models, the in-vitro malignant proliferation capacity of the lung cancer cells A549 and HCC827 can be obviously inhibited by over-expression of uc.336, animal experiments show that the in-vivo proliferation capacity of the lung cancer cells A549 can be obviously inhibited by over-expression of uc.336, and the uc.336 can be taken as a potential treatment target for inhibiting lung cancer proliferation.

Description

Target for inhibiting lung cancer growth and diagnostic marker uc.336 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to discovery of a new tumor marker and a new target for inhibiting lung cancer growth and application thereof.

Background

According to the data of the cancer analysis in 2018, the incidence rate of lung cancer accounts for 11.6% of all cancers, the mortality rate accounts for 18.4% of all cancers, and the incidence rate and the mortality rate of the lung cancer are at the top of all large cancers. The data of the incidence of cancer in China in 2015 show that 78.7 ten thousand cases (accounting for 20.03%) and 63.1 ten thousand cases (accounting for 26.99%) of new lung cancer in China are respectively the first cases of malignant tumors in China. Although the targeted therapy of lung cancer is developed nationwide, the five-year survival rate of lung cancer is still very low, only about 15%. Therefore, the search for more accurate and specific diagnosis markers and effective tumor proliferation inhibition therapeutic targets and the improvement of the diagnosis and treatment effects of lung cancer patients are the problems to be urgently solved in the current clinical practice of the industry.

Before the discovery of non-coding RNAs, it has been thought that biological behavior of organisms at the molecular level is achieved by protein-protein interactions. Later studies found that the encoded gene in the human genome was only 3% of the human genome. While 75% of the genomic sequence is transcribed into RNA, nearly 74% of the transcripts are Non-coding RNA (ncRNA), which were originally thought to be transcription "noise" of gene expression and do not have any biological function per se. However, as research progresses, more and more research in recent years has revealed that non-coding RNA plays an extremely important role in the life process.

Super conserved regions (UCRs), also known as ultraconserved elements (UCEs), are highly conserved regions in the human genome with 100% similarity in orthologous regions of distantly related mammalian species (e.g., human, mouse, rat). A total of 481 UCRs in the human genome, found by Bejerano and coworkers in 2004, were 100% identical in the human, mouse and rat genomes, did not contain insertions or deletions, and were greater than 200bp in length. Transcribed super conserved regions (T-UCRs) are a long-chain non-coding RNA transcribed from 481 UCRs, also called super conserved genes (uc).

T-UCR is mainly regulated in cancer by 2 ways: and (3) interacting with the microRNA, and performing epigenetic modification. And obvious antisense complementarity exists between some T-UCRs and the base sequences of the microRNAs. And forming T-UCR according to the principle of complementary pairing of at least 6 base sequences at the 5' end: the microRNAs are paired, so that endogenous competition is formed, and the expression amount of the target gene protein is changed. Other partial studies have found that T-UCR is closely related to methylation of CpG islands. In recent years, there have been studies showing that malignant tumors are associated with changes in T-UCR expression, and T-UCR expression profiles can be used to distinguish different cancer types, suggesting that they may be novel relevant tumor biomarkers. However, a plurality of T-UCR molecules which have important effects but unknown functions are not researched and reported, so that the identification and mechanism elucidation of new T-UCR molecular targets are beneficial to comprehensively understanding the biological functions of the T-UCR and promoting the research and development of new markers and medicines for efficiently diagnosing and treating lung cancer.

uc.336 found high conservation in 2004 by Bejerano et al, and until now, no research reports the function and molecular mechanism of uc.336 in lung cancer development.

Currently available targets for inhibiting lung cancer include EGFR, ALK gene rearrangement, ROS1, HER2, MET, KRAS, and the like. New medicines aiming at the targets are also endless, and have better curative effect when being put into clinical use successively, but the occurrence of the drug resistance of the targeted medicines not only limits the treatment effect, but also further increases the treatment difficulty; in addition, the occurrence probability of the mutant target is low, the population capable of receiving targeted therapy is limited, and the application of targeted therapy is limited to a certain extent, so that the search for a new effective therapeutic target is still very important.

Disclosure of Invention

The invention aims to provide uc.336 as a target and a diagnostic marker for inhibiting the growth of lung cancer and application thereof.

In order to solve the technical problem, the invention provides application of uc.336 in preparation of a lung cancer diagnosis marker.

The invention also provides the application of the uc.336 expression promoter in the preparation of the medicine for preventing/diagnosing lung cancer; inhibit the proliferation of lung cancer cell in vivo.

The uc.336 expression promoter is an over-expression uc.336 plasmid.

The invention also provides a composition for preventing or/and treating lung cancer, which comprises the following components in part by weight: uc.336 expression promoter; a pharmaceutically acceptable carrier.

The invention also provides a reagent for detecting uc.336 expression, wherein the reagent for detecting uc.336 expression comprises a reagent based on a fluorescent quantitative PCR quantitative detection method, and the reagent for the fluorescent quantitative PCR quantitative detection method comprises a pair of specific primers: 5'-AGGCATTCACTCGGAAGCA-3' for F (upstream primer); r (downstream primer) 5'-ATTACAACAGCCATTACAGCCG-3'.

According to researches, the uc.336 gene is a long-chain non-coding RNA transcribed from a super-conservative region, has absolute conservation in human, rats and mice, and is remarkably low-expressed in lung cancer tissues, so that the uc.336 gene is suggested to be possibly used as a therapeutic target and a diagnostic marker for inhibiting the growth of lung cancer.

The invention aims to show that uc.336 can be applied as a diagnostic marker for lung cancer growth and a therapeutic target.

The technical scheme adopted by the invention is as follows: by carrying out high-throughput sequencing on 5 pairs of matched lung cancer clinical samples and normal tissues, T-UCR-uc.336 with obvious expression difference is screened out by utilizing the high-throughput sequencing, and the expression difference in lung cancer tissues and lung cancer cell lines is verified by qPCR.

According to the invention, after uc.336 overexpression vectors are constructed and stable cell strains of A549 cells and HCC827 cells are established, the malignant proliferation function of the cells in lung cancer cells is researched through in-vitro experiment soft agar.

The invention adopts a subcutaneous injection mode to establish a nude mouse ectopic transplantation tumor model and observe the growth condition of A549 cells under the nude mouse skin. The research finds that uc.336 remarkably inhibits the in vivo proliferation capacity of the lung cancer cell A549 cell.

The real-time fluorescent quantitative PCR technology is used for detecting the expression of uc.336 in lung cancer clinical tissues, and the expression shows a remarkable down-regulation trend. It can be seen that the detection of uc.336 expression is closely related to the development of lung cancer. The invention is expected to provide a new target and corresponding measures for inhibiting lung cancer proliferation.

According to the invention, the experiment technologies such as Q-PCR and the like in the high-throughput sequencing result show that uc.336 in the cancer tissue has a remarkable down-regulation trend in the transcription level compared with the paracancer normal tissue, and the uc.336 can be used as a lung cancer diagnosis marker. Meanwhile, the lung cancer cells A549 and HCC827 are further taken as models, the in-vitro malignant proliferation capacity of the lung cancer cells A549 and HCC827 can be remarkably inhibited by over-expression of uc.336, and animal experiments show that the in-vivo proliferation capacity of the lung cancer cells A549 can be remarkably inhibited by over-expression of uc.336, which indicates that uc.336 can be used as a potential treatment target for inhibiting lung cancer proliferation.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows that uc.336 is low expressed in lung cancer tissues and lung cancer cells;

in the context of figure 1 of the drawings,

a, detecting the relative expression quantity condition of uc.336 in 60 pairs of lung cancer tissues by using a uc.336 specific primer; of these, 60 of the lung cancer tissues showed overexpression in 6 pairs.

B, detecting the relative expression quantity of uc.336 in normal lung bronchial epithelial cells and lung cancer cell lines by using a uc.336 specific primer;

FIG. 2 shows that uc.336 can significantly inhibit the in vitro malignant proliferation capacity of lung cancer cells A549 and HCC 827;

in the context of figure 2, it is shown,

A. b is the overexpression efficiency identified by Q-PCR after the uc.336 is overexpressed in A549 and HCC827 cells;

C. d, verifying the influence of uc.336 on the in-vitro malignant proliferation capacity of the lung cancer cells through a soft agar experiment;

fig. 3 shows that uc.336 can remarkably inhibit the in vivo proliferation capacity of lung cancer cell A549; (ii) a

In the context of figure 3, it is shown,

a is the obtained tumor body weight;

b, establishing a nude mouse ectopic transplantation tumor model by adopting a subcutaneous injection mode, and observing the growth condition of A549 cells compared with control cells under the nude mouse skin after uc.336 is over-expressed;

c is the uc.336 expression in both groups of tumours detected by qPCR.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

examples 1, 60 paired clinical Lung cancer tissues and expression of uc.336 in Lung cancer cells

The RT-QPCR technology is utilized to detect the expression level of uc.336 in a clinical lung cancer tissue sample, the result is shown in figure 1A, the uc.336 is obviously low expressed in the clinical lung cancer tissue sample, and the specific implementation steps are 1), 2) and 4); similarly, uc.336 was also significantly low expressed in lung cancer cell lines by RT-QPCR experiments, with the following steps 3), 4):

1) tissue sample

Clinical tissue samples of lung cancer that have been diagnosed and surgically resected are provided by the first hospital affiliated with the university of medical Wenzhou, and sample collection and utilization have been approved by the ethical committee of the university of medical Wenzhou, and are strictly collected and utilized according to relevant regulations and procedures. Tumor tissue was taken from each specimen, and normal tissue was excised approximately 3cm from the periphery of the tumor as a control. Each sample was cryopreserved in liquid nitrogen after tissue ex vivo.

2) Tissue total RNA extraction

a. The lung cancer clinical samples were taken out of the ultra-low temperature refrigerator, about 30-50mg of each sample was put into a 1.5mL enzyme-removed EP tube, 1000. mu.L Trizol was added and mixed well, and the tissue was cut into pieces and sufficiently crushed with a tissue crusher.

b. Adding 200 μ L chloroform, shaking vigorously for 1min, and standing on ice for 5 min. The centrifuge was precooled to 4 ℃ in advance. Centrifuge at 4 ℃ at 12000g for 15 min.

c. The supernatant was aspirated with 200. mu.L of the enzyme removal tip and transferred to a new EP tube at about 400. mu.L. An equal volume of 400-600. mu.L of precooled isopropanol was added, mixed by inversion and allowed to stand on ice for 10 min. Centrifugation was carried out at 4 ℃ at 12000g for 10min, and the supernatant was discarded.

d. Preparing 75% alcohol with DEPC water, adding 1ml of prepared 75% alcohol into the above precipitate, blowing and beating the precipitate, centrifuging at 4 deg.C, 7500g, 5min, discarding supernatant, and repeating the steps.

e. Discarding the supernatant, then performing air separation for 5min, sucking the residual supernatant by a small enzyme-removing gun head, and leaving white sediment at the bottom. And opening the cover and airing, and adding enzyme-removing water after the white precipitate at the bottom is transparent. Dissolving at 58 deg.C for 10min, and determining RNA concentration.

RT-QPCR experiments were performed according to step 4).

3) Extraction of total RNA of lung cancer cell line

a. Cell about 1X10⁷After the medium was discarded, 1000. mu.L of Trizol blown cells were added and collected in a 1.5mL de-enzymed Ep tube.

b. Adding 200 μ L chloroform, shaking vigorously for 1min, and standing on ice for 5 min. The centrifuge was precooled to 4 ℃ in advance. Centrifuge at 4 ℃ at 12000g for 15 min.

c. The supernatant was aspirated with 200. mu.L of the enzyme removal tip and transferred to a new EP tube at about 400. mu.L. Adding 400-equivalent of precooled isopropanol of 600 mu L, reversing and mixing evenly, and standing for 10min on ice. Centrifugation was carried out at 4 ℃ at 12000g for 10min, and the supernatant was discarded.

d. Preparing 75% alcohol with DEPC water, adding 1ml of prepared 75% alcohol into the above precipitate, blowing and beating the precipitate, centrifuging at 4 deg.C, 7500g, 5min, discarding supernatant, and repeating the steps.

e. Discarding the supernatant, then performing air separation for 5min, sucking the residual supernatant by a small enzyme-removing gun head, and leaving white sediment at the bottom. And opening the cover and airing, and adding enzyme-removing water after the white precipitate at the bottom is transparent. And dissolving at 58 ℃ for 10min, and determining the RNA concentration.

RT-QPCR experiments were performed according to the procedure 4).

4)、RT-QPCR

After the completion of the extraction and determination of the RNA concentration according to step 2), 3), GoScript (available from Promega, USA) was used^TMReverse Transcription is carried out by a Reverse Transcription System Reverse Transcription kit, and the Reverse Transcription reaction System is as follows according to the instruction of a reagent:

the first step is as follows: 5 μ L System

Shaking, mixing, spotting, placing in PCR instrument, reacting at 70 deg.C for 5min, standing on ice for 5 min; then, the second step of operation is carried out:

the second step is that: 15 μ L system

After the system is mixed uniformly, the mixed solution is dripped, 15 mu L of the mixed solution system is added into the 5 mu L system in the first step, after the mixed solution is mixed uniformly, the mixed solution is dripped, and reverse transcription is carried out according to the following procedures:

a, annealing: 5min at 25 DEG C

b, extension: 42 ℃ for 1h

c, inactivation: 15min at 70 DEG C

Temporarily stored at-80 deg.C at 4 deg.C for a long time.

After obtaining cDNA from the desired cells, PCR was carried out according to GoTaq qPCR kit (Promega, USA) to prepare a PCR reaction system (10. mu.L) as follows (on ice):

PCR Forward Primer:5'-AGGCATTCACTCGGAAGCA-3'；

PCR Reverse Primer:5'-ATTACAACAGCCATTACAGCCG-3'。

and (3) fully and uniformly mixing the reaction systems, adding the mixture into a 384-well plate, setting 3 multiple wells for each sample, centrifuging at 3000rpm for 3min to uniformly mix the reagents, placing the reagents at the bottom of the wells, and placing the reagents in a Q6 fluorescent quantitative PCR instrument for detection. Q-PCR reaction conditions:

the results obtained are depicted in FIG. 1A: uc.336 expression is relatively down-regulated in lung cancer tissues; likewise, uc.336 was significantly under-expressed in lung cancer cell lines, as shown in fig. 1B. Therefore, uc.336 can be used as a diagnostic marker for lung cancer.

Example 2

uc.336 remarkably inhibits the in vitro malignant proliferation capacity of lung cancer cells

1) A549 cells and HCC827 cells are selected, pCDH-CMV-MCS-EF1-GFP-pruo plasmid vectors are adopted to construct uc.336 overexpression vectors, uc.336 is overexpressed in the A549 cells and the HCC827 cells, and stable transfected cells A549 uc.336, HCC827-uc.336 and control A549-Vector, HCC827-Vector and Q-PCR experiments verify overexpression efficiency, as shown in FIGS. 2A and B.

From FIGS. 2A-2B, it can be seen that: stable transgenic cell lines overexpressing uc.336 were successfully constructed in A549 and HCC827 cells.

uc.336 over-expression vector can be constructed by Oncokaceae company according to conventional mode, the used vector is pCDH-CMV-MCS-EF1-GFP-pruo, and the enzyme cutting site is NheI-NotI. The over-expression plasmid has a full length of 8429 bp.

The method for stably transfecting the cells A549 uc.336 and HCC827-uc.336 comprises the following steps:

(1) lentiviral packaging

a. Inoculating a proper amount of 293T cells into a 6-well plate, and inoculating for about 12h until the density reaches about 80% for transfection;

b. changing the culture solution 0.5-1h in advance, and incubating in an incubator with 2mL of free-DMEM culture medium per well;

c. transfection system (amount of 1 well): solution A: adding 2 μ g of the objective plasmid (uc.336 overexpression plasmid or control plasmid), 1.2 μ g of pMD2.G plasmid, 1.2 μ g of psPAS X2 plasmid into 50 μ L of free-DMEM medium, and mixing; and B, liquid B: 6 uL of Polyjet^TMAdd 50. mu.L of free-DMEM and mix well. Adding solution B into solution A, mixing, and standing for 15 min;

d. then adding the transfection compound into the hole, uniformly mixing while adding the cross shape, changing the culture solution into a complete culture medium after the transfection is carried out for 12 hours in an incubator, and continuously culturing for 48-72 hours;

e.48h, collecting the supernatant in a centrifuge tube, centrifuging at 3000rpm for 30min, filtering the supernatant with a 0.45 μm filter to obtain a filtrate, i.e. the virus supernatant, and subpackaging in EP tubes with 1mL per tube and storing at-80 ℃;

(2) selection of stably transformed cell lines

a. Inoculating cells into a 6-well plate, infecting viruses when the cell density reaches 50% -60%, replacing a fresh culture medium with 1mL of the culture medium in each well, then adding 1mL of virus supernatant, uniformly mixing, culturing in an incubator for 24h, observing fluorescence expression under a mirror, and further detecting the transfection efficiency of the cells;

b. changing the solution after infecting the virus for 12-24 h, continuously culturing, and carrying out passage after the cells grow full;

c. after passage, adding a culture medium containing puromycin antibiotics with a proper concentration for screening when the attachment is adhered;

d. fresh medium with antibiotics was changed on average for 2-3 days and antibiotic concentrations were increased continuously until the cells grew out of clonal population, and finally A549 cells were maintained at a final concentration of 5. mu.g/mL puromycin antibiotic for 4 days and HCC827 cells were maintained at a final concentration of 7. mu.g/mL puromycin antibiotic for 4 days. The average time is about 12 to 15 days for drug screening;

e. after the cloned cells are digested, blowing and uniformly mixing, paving the mixture in a six-hole plate again, supplementing to 2mL of fresh culture medium (containing no antibiotics), and placing the culture medium in an incubator with 37 ℃ and 5% carbon dioxide for continuous culture;

f. when the density of the six-hole plate reaches about 90%, one third of the six-hole plate is transferred to a 10cm culture dish, and when the density reaches 90%, the six-hole plate is used for identification, and after total RNA is extracted, the RT-QPCR test identifies the over-expression efficiency, as shown in FIGS. 2A and 2B. After the identification is correct, the cells in the 10cm culture dish are frozen one by one in three dishes for seed preservation, and the rest part is reserved for subsequent experiments.

2) Tumor cells a549 uc.336, HCC827-uc.336 were evaluated using a soft agar colony formation (soft agar) assay for their anchorage-independent malignant proliferative capacity compared to control a549-Vector, HCC827-Vector, as shown in fig. 2C, D.

The method comprises the following specific steps: 1.2ml of 1.25% agarose solution (sterilized at high temperature) and 1.8ml of prepared culture medium (medium, filtration sterilization) are put into a 15ml centrifuge tube according to each hole, and are lightly blown and uniformly mixed, and then are added into the holes of a 6-hole plate, so that air bubbles are prevented from being blown and uniformly paved, and the air bubbles are prevented from being generated. After standing for at least 2h, paving the upper layer glue according to the following system:

first, 1.25% agarose gel and medium are mixed evenly and put into a 42 ℃ water bath for preheating, then 0.25% pancreatin is used for digesting cells in logarithmic phase, the cells are blown into single cell suspension by a culture medium, after counting, the cells with corresponding suspension volume are added into the agarose gel and the medium with corresponding cell number of 1.25%, and then the mixture is plated. Standing for 1-2 hours, sealing the 6-hole plate with a sealing film, then placing the 6-hole plate into a cell incubator with 37 ℃ and 5% carbon dioxide for continuous culture, beginning to observe the growth state of the clone after about 20 days, taking a picture by using a microscope 5-fold mirror when the clone grows to a proper size, counting, and calculating the formation rate of the cell colony number.

The results obtained were: over-expression of uc.336 can obviously inhibit the in vitro malignant proliferation capacity of the lung cancer cells; therefore, uc.336 can be used as a new therapeutic target for inhibiting the proliferation of lung cancer.

Example 3

Over-expression of uc.336, and remarkable inhibition of in vivo proliferation capacity of lung cancer cells

1) Animal feeding

BALB/C-nu female nude mice, week age 3-4 weeks, weight 15 + -0.5 g, experimental animals purchased from the experimental animals center of Wenzhou university of medical science, and raised in the SPF grade experimental area of the experimental animals center of Wenzhou university of medical science. The animal experiments performed have been approved by the ethical committee of experimental animals at the university of medical science in wenzhou and the experimental procedures comply with the ethical requirements of the ethical committee on animals.

2) Subcutaneous injection

0.25% pancreatin digested A549-Vector cells, A549-uc.336 cells in logarithmic growth phase; terminating digestion by using a culture medium, collecting cells of all culture dishes into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, then discarding a supernatant culture medium, washing the cells once by using PBS (phosphate buffer solution) for heavy suspension, centrifuging at 1500rpm for 5min again, then discarding the PBS, adding 1ml of PBS for heavy suspension, diluting the cells according to a certain proportion, filling a pool for counting, and calculating the required cell amount. Each nude mouse was injected subcutaneously with 100. mu.L of cell suspension containing 250 ten thousand cells. The subcutaneous injection part of the nude mice is wiped and disinfected by medical iodophor, the cells are fully and uniformly mixed before inoculation, 100 mu L of cell suspension is absorbed by a 1ml sterile insulin syringe and is uniformly injected at the subcutaneous position of the right back of the mice, and 6 nude mice are injected in each group.

3) Determination of photographs

PBS was fully absorbed by nude mice for about 1 week; when the nude mice are inoculated with tumor cells and grow for about 4 weeks, the nude mice are killed after being anesthetized with 0.5% sodium pentobarbital, tumor bodies are dissected and taken out, photographed (fig. 3A) and weighed (fig. 3B), and RNA is extracted from tumor tissues to carry out RT-QPCR to detect the expression of uc.336 (fig. 3C).

The results obtained were: over-expression of uc.336 can obviously inhibit the in vivo proliferation capacity of lung cancer cells; therefore, uc.336 can be used as a new therapeutic target for inhibiting the lung cancer proliferation.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

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Claims (2)

1. The application of uc.336 expression promoter in the preparation of the medicine for inhibiting the growth of lung cancer cells is characterized in that: the uc.336 expression promoter is an overexpression plasmid of uc.336.
2. 2. Use according to claim 1, characterized in that: inhibit the proliferation of lung cancer cell in vivo.

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