CN109337981B - KCNJ4 gene related to lung adenocarcinoma and application thereof - Google Patents

Abstract

The invention discloses a KCNJ4 gene related to lung adenocarcinoma and application thereof. The invention firstly screens a KCNJ4 gene highly expressed in an A549 lung adenocarcinoma cell strain, then knocks out the expression of KCNJ4 by taking a KCNJ4 gene as a target in an RNA interference mode, and discovers that: the knock-out expression of KCNJ4 can inhibit the proliferation, invasion and migration capability of A549 cells. Therefore, the KCNJ4 gene can be used as a gene target for preventing or treating lung adenocarcinoma. Meanwhile, a human KCNJ4 gene is selected as a target gene sequence, the expression of the human KCNJ4 gene is efficiently inhibited in an RNA interference mode, the proliferation inhibitor of the human lung adenocarcinoma tumor cells is effectively inhibited, and a new idea and solution are provided for the prevention and treatment of the lung adenocarcinoma.

Description

KCNJ4 gene related to lung adenocarcinoma and application thereof

Technical Field

The invention relates to a gene related to lung adenocarcinoma and application thereof, belonging to the technical field of biology.

Background

Lung cancer is the cancer with the highest incidence and mortality worldwide. Lung adenocarcinoma (lung adenocarinoma) is a kind of lung cancer, and belongs to non-small cell carcinoma, and most of the adenocarcinoma originates from a small bronchus and is peripheral lung cancer. Early stages generally have no obvious clinical symptoms and are often found at chest X-ray examination. It is usually a slow growing mass, but sometimes early hematogenous metastases occur, manifested as a rounded or oval mass. Lymphatic metastasis occurs later. Adenocarcinoma, which differentiates well, is mainly composed of glandular structures, with glandular cavities or secretory mucosa, sometimes papillary structures. Adenocarcinoma with low differentiation degree has no glandular cavity structure, and cancer cells gather and are in a sheet shape or a cord shape. Adenocarcinoma cells are generally large, abundant in cytoplasm, contain secretory granules or myxoplasma, have large nuclei, and are rich in microvilli visible on the surface of the adenocarcinoma cells. Lung adenocarcinoma is common in women and tends to increase in men. Not much related to smoking, it grows mostly in the mucus glands of the marginal bronchi of the lung, and thus adenocarcinoma is most common in peripheral lung cancer. Adenocarcinoma accounts for approximately 25% of primary lung cancers. Adenocarcinoma tends to grow outside the tubes, but also spreads along the alveolar wall, often forming lumps of 2-4 cm in diameter at the lung margins. Adenocarcinoma is rich in blood vessels, so local infiltration and hematogenous metastasis are earlier than squamous carcinoma. It is easy to be transferred to liver, brain and bone, and is more likely to involve pleura, causing pleural effusion.

Lung adenocarcinoma is usually treated by surgery and radiotherapy, chemotherapy, targeted medicine, immunotherapy, etc. After the focus is removed by surgical treatment, the equipment irradiated in the cavity and in vitro is used as the postoperative adjuvant therapy to improve the success rate of the operation. Chemotherapy is a relatively important modality, both early and late, requiring combination with chemotherapy for increased survival, both surgery and radiation therapy. But the defects of weak treatment pertinence, reduction of the immunity of patients and low cure rate exist in both surgery, radiotherapy and chemotherapy. And the targeted therapy and immunotherapy can improve the effective rate of part of patients.

In addition, generally, the focus of early stage lung adenocarcinoma patients is small, tumors are limited in one lung lobe, proper treatment is timely carried out, canceration focus is eliminated, recurrence is prevented, radical cure can be achieved, namely the cure rate of early stage lung adenocarcinoma can reach 80-90%, but the early stage is usually difficult to find, the overall treatment effect of locally advanced patients is poor, the 5-year survival rate of IIIA stage patients is 15-23%, and the IIIB stage is only 6-7%. Therefore, the deep research on the gene mechanism related to the lung adenocarcinoma has important significance for the early diagnosis and the targeted therapy of the lung adenocarcinoma.

The gene KCNJ4 is the fourth gene of subfamily J forming potassium access channel, encodes inward rectification potassium channel protein, and may be related to the pathogenesis of some diseases such as refractory temporal lobe epilepsy. However, the expression of KCNJ4 in lung adenocarcinoma and its biological significance are not clear.

Disclosure of Invention

The invention firstly screens a KCNJ4 gene highly expressed in an A549 lung adenocarcinoma cell strain, and then screens an inhibitor for knocking out the expression of KCNJ4 in an RNA interference mode by taking a KCNJ4 gene as a target. After the expression of KCNJ4 is knocked out, the proliferation, invasion and migration capacity of A549 cells can be inhibited, so that the KCNJ4 gene can be used as a gene target for preventing or treating lung adenocarcinoma, and a preparation for preventing or treating lung adenocarcinoma is screened. The preparation for inhibiting the expression of KCNJ4 also provides a new idea and solution for preventing and treating lung adenocarcinoma.

The invention firstly provides application of a lung adenocarcinoma gene KCNJ4 as a gene target for preventing, treating or diagnosing lung adenocarcinoma.

The invention also provides a preparation for preventing or treating lung adenocarcinoma, which contains a quantitative inhibitor for inhibiting KCNJ4 gene.

Further, the inhibitor stably and specifically down-regulates the expression of KCNJ4 gene in an RNA interference mode, so that the proliferation of the human lung adenocarcinoma tumor cells is effectively inhibited.

Further, the inhibitor may be siRNA.

The construction method of the siRNA for inhibiting the KCNJ4 gene expression in the lung adenocarcinoma tumor cells comprises the following steps:

1) adopting a human A549 lung adenocarcinoma cell strain to carry out cell culture;

2) aiming at human KCNJ4 gene, designing siRNA1 and siRNA2 two interference sequences by using siRNA design tool, and synthesizing;

3) transfecting lung adenocarcinoma tumor cells by using the siRNA1 and the siRNA2 constructed in the step 2);

4) the KCNJ4 gene expression in lung adenocarcinoma tumor cells is detected by using a fluorescent quantitative real-time PCR technology, and siRNA construction is successful if KCNJ4 gene expression is inhibited or knocked out.

The sequence of the siRNA is shown as SEQ ID NO.1-2, the sequence shown as SEQ ID NO.1 is siRNA1, and the sequence shown as SEQ ID NO.2 issiRNA 2;

siRNA1 5’-CCGCUUCGUCAAGAAGAAC-3’(SEQ ID NO.1)；

siRNA2 5’-UGCAACGUGUACUUCGCCA-3’(SEQ ID NO.2)；

wherein, the siRNA1 has the best effect of inhibiting the expression quantity of the KCNJ4 gene and has the best capability of inhibiting the proliferation, invasion and migration of lung adenocarcinoma tumor cells.

The lung adenocarcinoma tumor cell is a human A549 lung adenocarcinoma cell strain.

The siRNA design tool adopts siDirect version 2.0siRNA design software.

Has the advantages that:

1. a KCNJ4 gene highly expressed in an A549 lung adenocarcinoma cell strain is screened, and the expression of KCNJ4 is knocked out, so that the proliferation, invasion and migration capacity of A549 cells can be inhibited. The KCNJ4 gene can be used as a gene target for preventing or treating lung adenocarcinoma, and a preparation for preventing or treating lung adenocarcinoma is screened.

2. The human KCNJ4 gene is taken as a target, a proper target gene sequence is selected, an inhibitor which can effectively inhibit the expression of the human KCNJ4 gene and effectively inhibit the proliferation of human lung adenocarcinoma tumor cells is designed in an RNA interference mode, and a new thought and a solution are provided for the prevention and treatment of lung adenocarcinoma.

Drawings

FIG. 1 shows the expression of KCNJ4 gene in different lung adenocarcinoma cell lines.

FIG. 2 Effect of siRNA1 and siRNA2 on the mRNA expression level of KCNL4 gene in A549 cells.

FIG. 3 Effect of siRNA1 and siRNA2 on protein expression levels of KCNL4 gene in A549 cells.

FIG. 4 Effect of KCNJ4 gene on A549 cell proliferation.

FIG. 5 influence of KCNJ4 gene on clonality of A549 cells.

Fig. 6 effect of KCNJ4 gene on invasion/migration of a549 cells.

FIG. 7 influence of KCNJ4 gene on the mechanism of phenotypic effect.

Detailed Description

Example 1 expression of KCNJ4 Gene in Lung adenocarcinoma cell lines

Materials and methods

1. Cell culture

Human lung cancer cell line A549 and normal cell line were purchased from ATCC. Using RPMI-1640 medium at 37 deg.C, 5% CO₂Performing conventional culture in the environment, wherein the serum concentration is 10%, the penicillin concentration is 100U/ml, and the streptomycin concentration is 0.1 mg/ml; after the cells entered logarithmic growth phase, wash 3 with PBSAnd secondly, digesting with pancreatin, adding the culture solution again to stop digestion after the cells become round, repeatedly blowing and beating to obtain single cell suspension, and planting the single cell suspension in a six-hole plate for subsequent experiments.

2. RNA extraction and fluorescent quantitative PCR detection

Respectively extracting total RNA of cells by using an RNA extraction kit, carrying out reverse transcription to form cDNA, and detecting the expression effect of KCNJ4 by real-time fluorescent quantitative PCR. The RNA primers used to detect RNA content were as follows:

F：5’-TAAACTTGGCCCTGCGTCTT-3’

R：5’-AGGTTGGCGAAGTACACGTT-3’

GAPDH-F:5’-GGAGCGAGATCCCTCCAAAAT-3’

GAPDH-R：5’–GGCTGTTGTCATACTTCTCATGG-3’

fluorescent quantitative PCR procedure: circulating at 95 deg.C for 5min and 95 deg.C for 30s for 40 times, at 60 deg.C for 45s and at 72 deg.C for 30 min; each group was provided with 3 duplicate wells, and the expression level of KCNJ4 was calculated by the 2-Delta Ct method. Repeated independently three times.

Second, statistical analysis

The experimental data were analyzed using SPSS22.0 statistical analysis software. The comparison of the two composition ratios of the grade data adopts chi-square test; the mean comparison between multiple samples was performed using one-way ANOVA analysis of variance.

Three, result in

Expression of KCNJ4 gene in lung adenocarcinoma cell line: QPCR detection shows that the KCNJ4 gene is highly expressed in A549 lung adenocarcinoma cell strains (as shown in figure 1).

Example 2 examination of KCNJ4 expression ability, tumor cell proliferation ability, cloning rate and invasion ability in tumor cells infected with siRNA interference sequence

Materials and methods

1 cell culture procedure as described in example 1.

2. Design of siRNA

Aiming at human KCNJ4 gene, 2 pairs of interference sequences are designed and synthesized by siDirect version 2.0siRNA design software.

3. RNA extraction and fluorescent quantitative PCR assay as described in example 1.

4. Transfection

Transfection was performed as described in the Lipofectamine2000 transfection kit. When the cells in the six-well plate grew to log phase, the fresh complete medium was replaced two hours before transfection. Dissolving liposome Lipofectamine 200010 ul in 250ul serum-free and antibiotic-free culture medium, gently mixing, and standing at room temperature for 5 min; 5ul siRNA was dissolved in 250ul serum-free and antibiotic-free medium. (KCNJ4si RNA 1: F: 5'-CCGCUUCGUCAAGAAGAAC-3' and si RNA 2: F: 5'-UGCAACGUGUACUUCGCCA-3'). Mixing the mixed solution of liposome and siRNA within 30min, gently mixing, and standing at room temperature for 30 min. Old media in 6-well plates was discarded and cells were washed 2 times with PBS. 500ul of the liposome and siRNA mixture was added to each well of cells, mixed gently and cultured in an incubator. After the cells were placed in an incubator and incubated for 6h, the complete culture medium was replaced. After 24h, the expression condition of the transferred siRNA can be observed or a subsequent experiment can be carried out.

5. Western blot experiment

After 48 hours of cell transfection, the six-well plate was placed on ice, protein was extracted by lysis with RIPA lysis buffer (protease inhibitor was added), the concentration was measured by BCA method, and the plate was heated at 95 ℃ for 5 min. Adding about 20 mu g of protein into each hole in a vertical electrophoresis tank, carrying out SDS polyacrylamide gel electrophoresis to separate protein samples, transferring the membrane to a PVDF membrane, sealing with 5% skimmed milk powder for 1h, incubating at 4 ℃ for one night, washing with TBST for 3X 5min, incubating at room temperature for 1h with secondary antibody, washing the membrane, and adding ECL for development. QUANTITY ONE software scans the gray scale values, GAPDH was used as an internal reference control, and the relative expression level of each protein was calculated for the target protein/internal reference. And (3) after the color development strip is scanned, detecting by using Bio ID software, and determining the density values of the target protein and beta-actin reference protein product strips. The optical density ratio of the target protein to the beta-actin protein is used as the relative content of the target protein.

6. CCK8 method for determining cell proliferation

Digesting and counting the cells after 24 hours of transfection, preparing cell suspension, taking 100ul of cell suspension, planting the cell suspension into a 96-well plate according to the standard of 1000 cells per well, conventionally culturing in a carbon dioxide incubator, detecting the cell activity once every 24 hours, adding 10ul of CCK8 reagent into each well before detection, incubating for 1.5 hours in the incubator at 37 ℃, detecting the OD value by using an enzyme-labeling instrument and using excitation light of 450nm, and drawing a proliferation curve.

7. Plate cloning experiment

Cells in log phase were trypsinized and blown into single cells, made into cell suspensions and counted. A60 mm dish containing 5mL of 37 ℃ pre-warmed medium was seeded at a gradient density of 1500 cells per dish and gently swirled to disperse the cells evenly. Control group added one thousandth of DMSO, experimental group added 10 uMPHEX. Culturing in a cell culture box with 37 deg.C and 5% CO2 and saturated humidity for 1-2 weeks. It was frequently observed that when macroscopic colonies appeared in the culture dish, the culture was terminated. The supernatant was discarded and carefully rinsed 2 times with PBS. Cells were fixed for 30 minutes by adding 5mL of 4% paraformaldehyde. Then the fixing solution is removed, an appropriate amount of 0.1% crystal violet staining solution is added for staining for 30 minutes, then the staining solution is slowly washed away by running water, and air drying is carried out. The clones were counted and compared for size and number of clone formations.

8. Transwell migration and invasion experiments

100ul of Matrigel (diluted by serum-free culture solution according to a ratio of 1: 6) which is melted overnight is added into an upper chamber of a transwell chamber of a 24-well plate, after being evenly shaken, the mixture is placed in a carbon dioxide incubator at 37 ℃ and is kept stand for 4 to 6 hours to form gel, then the culture solution is sucked dry, 500ul of serum-free culture solution is added into a lower chamber, and the mixture is kept stand for half an hour to hydrate a basement membrane. Cells after 24h of transfection were prepared in serum-free culture, 100ul of cell suspension (1X 105 cells) was added to the upper chamber and 500ul of complete medium was added to the lower chamber. After overnight, the chamber was removed and the residual cells in the upper chamber were wiped off with a cotton swab and fixed in 4% paraformaldehyde for 30min after washing with PBS. 0.1% crystal violet is stained for 20min, washed by PBS, and randomly selected 5 visual fields under a microscope for photographing, observation and counting. The migration procedure is similar to the invasion assay, the transwell chamber does not need to be plated with glue, and the cell number will be 5000. Second, statistical analysis

The experimental data were analyzed using SPSS22.0 statistical analysis software. The comparison of the two composition ratios of the grade data adopts chi-square test; the mean comparison between multiple samples was performed using one-way ANOVA analysis of variance.

Three, result in

1. KCNJ4 knockout efficiency test:

a knock-out vector carrying KCNJ4si RNA is transfected into a lung cancer cell line A549 cell with endogenous high expression of KCNJ4, and untransfected and transfected nonspecific sequences are used as controls (SCR). After transfection for 72h, total RNA and protein of cells are extracted, fluorescent quantitative PCR (as described in example 1) and Western blot detection are carried out, and as shown in figure 2, siRNA1 and siRNA2 can obviously reduce the expression level of RNA and protein of KCNJ4 in A549 cells, and the knockout efficiency reaches over 90%.

2. Experiment for detecting cell proliferation by CCK8 method

As shown in FIG. 3, after the expression of KCNJ4 is knocked out by the transfected siRNA, the proliferation of A549 cells can be inhibited.

3. Clone formation experiments

As shown in fig. 4, transfection siRNA knock-out of KCNJ4 significantly reduced the clonogenic rate of a549 cells.

4. Transwell migration and invasion experiments

The results of Transwell experiments (as shown in fig. 5) show that after the expression of KCNJ4 is knocked out by transfected siRNA, the invasion of A549 cells can be inhibited, the results are different remarkably (P <0.05), the migration capacity is reduced correspondingly, and the results are different remarkably (P < 0.05). Magnification: 100X.

5. Mechanism research verification of KCNJ4 on phenotype effect

As shown in FIG. 6, after the transfection siRNA knockdown the expression of KCNJ4, MAPK pathway is inhibited, and the expression level of phosphorylated MEK and phosphorylated ERK is reduced.

SEQUENCE LISTING

<110> thoracic hospital of Shandong province

<120> KCNJ4 gene related to lung adenocarcinoma and application thereof

<130> 0

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> RNA

<213> Artificial sequence

<400> 1

ccgcuucguc aagaagaac 19

<210> 2

<211> 19

<212> RNA

<213> Artificial sequence

<400> 2

ugcaacgugu acuucgcca 19

Claims (2)

1. Application of primers for detecting expression level of lung adenocarcinoma gene KCNJ4 in preparation of reagents for diagnosing lung adenocarcinoma.

2. The application of siRNA for inhibiting the expression of lung adenocarcinoma gene KCNJ4 in preparing a preparation for treating lung adenocarcinoma;

the siRNA comprises siRNA1 and siRNA2, wherein the siRNA1 is shown as a sequence in SEQ ID NO.1, and the siRNA2 is shown as a sequence in SEQ ID NO. 2.

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