One kind KCNJ4 gene related with adenocarcinoma of lung and its applicationTechnical field
The present invention relates to a kind of gene related with adenocarcinoma of lung and its applications, are to belong to field of biotechnology.
Background technique
Lung cancer is global incidence and the highest cancer of the death rate.Adenocarcinoma of lung (lung adenocarcinoma) is lung cancerOne kind, belong to non-small cell carcinoma, most gland cancer originate from lesser bronchus, are peripheral type carcinoma of lung.It is typically no in early days brightAobvious clinical symptoms are often found in chest X-ray.Performance is round or oval lump, and general growth is slower, butSometimes hematogenous metastasis occurs for early stage.Lymph Node Metastasis then occurs later.The gland cancer of had better differentiation is mainly by gland structure groupAt with lumen of gland or secretion mucous membrane, sometimes in mamilla.The low gland cancer of differentiation degree can be without lumen of gland structure, cancer cell collectionIt is poly-, in the form of sheets or strand.Adenocarcinoma cell is generally large, and endochylema is abundant, and containing secretory granules or mucocyst, karyon is larger, and cancer is thinThe visible microvillus abundant of cellular surface.Adenocarcinoma of lung women is common, also has the tendency that increasing in male.It is little with Smoking,It is grown in the muccus gland of margo border of the lung bronchium, therefore most commonly seen with gland cancer in peripheral type carcinoma of lung more.Gland cancer accounts for about primaryThe 25% of property lung cancer.Gland cancer tends to pipe outgrowth, but also follows steep that wall sprawling, often forms the swollen of 2~4cm of diameter in margo border of the lung portionBlock.Gland cancer richness blood vessel, therefore local infiltration and hematogenous metastasis are compared with squamous carcinoma morning.It easily is transferred to liver, brain and bone, is easier to involve pleura and drawPlay pleural effusion.
Adenocarcinoma of lung usually selects operative treatment and radiotherapy, chemotherapy, targeted drug treatment and immunization therapyDeng.Intracavitary, extracorporeal irradiation equipment is used after operative treatment removal of lesions as postoperative adjuvant treatment improve operation atPower.Chemotherapy is a kind of more important mode, either early stage or advanced stage, no matter performs the operation or radiotherapy all needsSurvival rate could be improved in conjunction with chemotherapy.But no matter perform the operation, radiotherapy or chemotherapy all exist treatment specific aim it is not strong, reduceThe not high disadvantage of patient's immunity, cure rate.And the effective percentage of some patientss can be improved in targeted therapy and immunization therapy.
In addition, general early stage patients with lung adenocarcinoma lesion is smaller, tumour is confined in a lobe of the lung, carries out appropriate control in timeIt treats, removes canceration lesion, prevent from recurring, can get the cure rate that radical cure is early stage adenocarcinoma of lung can reach 80-90%, but usuallyEarly stage is difficult to find, and the overall treatment effect of the patient of Locally Advanced is poor, and 5 years survival rates of III A phase patient are 15-23%, III B phase was only 6-7%.Therefore, adenocarcinoma of lung related gene mechanism, early diagnosis and target for adenocarcinoma of lung are furtherd investigateThere is important meaning to treatment.
Gene KCNJ4 is No. four gene for constituting the subfamily J of potassium access way, encodes inward rectifyimg potassium channel eggIt is white, it may be related with causing a disease for some diseases such as intractable temporal epilepsy.However, expression of the KCNJ4 in adenocarcinoma of lung and itsBiological significance is unclear.
Summary of the invention
The present invention screens a kind of KCNJ4 gene highly expressed in A549 lung adenocarcinoma cell line first, then with KCNJ4Gene is target, filters out a kind of inhibitor of expression that KCNJ4 is knocked out in a manner of RNA interference.Knock out the expression of KCNJ4Afterwards, it is able to suppress proliferation, invasion and the transfer ability of A549 cell, therefore KCNJ4 gene can be used as prevention or treatment lungThe preparation of the gene target of gland cancer, screening prevention or treatment adenocarcinoma of lung.And the preparation of this expression for inhibiting KCNJ4 is also lung glandThe prevention and treatment of cancer provide new approaches and solution.
Present invention firstly provides adenocarcinoma of lung gene KCNJ4 as the gene target for preventing, treating or diagnosing adenocarcinoma of lungApplication in point.
The present invention also provides a kind of preventions or the preparation for the treatment of adenocarcinoma of lung, the reagent to contain quantitative inhibition KCNJ4 baseThe inhibitor of cause.
Further, the inhibitor expression of lowering KCNJ4 gene stable and specific with RNA conflicting mode, fromAnd effectively inhibit the proliferation of human lung adenocarcinoma tumour cell.
Further, the inhibitor can be siRNA.
The construction method for inhibiting the siRNA of KCNJ4 gene expression in adenocarcinoma of lung tumour cell, includes the following steps:
1) cell culture is carried out using people A549 lung adenocarcinoma cell line；
2) it is directed to the KCNJ4 gene of the mankind, designs two kinds of interference sequences of siRNA1 and siRNA2 using siRNA design toolColumn, and synthesized；
3) adenocarcinoma of lung tumour cell is transfected using siRNA1 and siRNA2 constructed in step 2)；
4) the KCNJ4 gene expression in adenocarcinoma of lung tumour cell is detected using fluorescent quantitation real time pcr,KCNJ4 gene expression is suppressed or knocks out then siRNA and constructs successfully.
The sequence of the siRNA is as shown in SEQ ID NO.1-2, and the sequence as shown in SEQ ID NO.1 is siRNA1, such asSequence shown in SEQ ID NO.2 is siRNA2；
siRNA1 5'-CCGCUUCGUCAAGAAGAAC-3'(SEQ ID NO.1)；
siRNA2 5'-UGCAACGUGUACUUCGCCA-3'(SEQ ID NO.2)；
Wherein siRNA1 inhibits the effect of KCNJ4 gene expression amount best, inhibits proliferation, the invasion of adenocarcinoma of lung tumour cellIt is best with transfer ability.
The adenocarcinoma of lung tumour cell behaviour A549 lung adenocarcinoma cell line.
Above-mentioned siRNA design tool uses siDirect version 2.0siRNA design software.
The utility model has the advantages that
1, a kind of KCNJ4 gene highly expressed in A549 lung adenocarcinoma cell line is screened, after the expression for knocking out KCNJ4,It is able to suppress proliferation, invasion and the transfer ability of A549 cell.KCNJ4 gene can be used as prevention or treat the base of adenocarcinoma of lungBecause of target spot, the preparation of screening prevention or treatment adenocarcinoma of lung.
2, it using people KCNJ4 gene as target, by choosing suitable target-gene sequence, is designed in a manner of RNA interferenceWith the efficient expression for inhibiting people KCNJ4 gene, and the inhibitor of the proliferation of human lung adenocarcinoma tumour cell is effectively inhibited, is lungThe prevention and treatment of gland cancer provide new approaches and solution.
Detailed description of the invention
Expression of Fig. 1 KCNJ4 gene in different lung adenocarcinoma cell lines.
Influence of Fig. 2 siRNA1 and siRNA2 to the mRNA expression of KCNL4 gene in A549 cell.
Influence of Fig. 3 siRNA1 and siRNA2 to the protein expression level of KCNL4 gene in A549 cell.
The influence of Fig. 4 KCNJ4 gene pairs A549 cell Proliferation.
The influence of the cloning efficiency of Fig. 5 KCNJ4 gene pairs A549 cell.
Invasion/migration influence of Fig. 6 KCNJ4 gene pairs A549 cell.
The influence of the mechanism of Fig. 7 KCNJ4 gene pairs phenotype effect.
Specific embodiment
Expression of the embodiment 1KCNJ4 gene in lung adenocarcinoma cell line
One, material and method
1, cell culture
Human lung carcinoma cell line A549 and normal cell strain are purchased from ATCC.Using RPMI-1640 culture solution in 37 DEG C, 5%CO2Routine culture under environment, serum-concentration 10%, penicillin concn 100U/ml, streptomysin 0.1mg/ml；To cellWhen into logarithmic growth phase, is cleaned 3 times using PBS, then digested with pancreatin, after cell rounding, rejoin culture solutionDigestion is terminated, piping and druming is single cell suspension repeatedly, and plantation carries out subsequent experimental into six orifice plates.
2, RNA extracting and fluorescence quantitative PCR detection
Cell total rna is extracted respectively using RNA extraction agent box, after reverse transcription forms cDNA, real-time fluorescence quantitative PCR inspectionSurvey the expression effect of KCNJ4.It is as follows to detect RNA primer used in rna content:
F:5 '-TAAACTTGGCCCTGCGTCTT-3 '
R:5 '-AGGTTGGCGAAGTACACGTT-3 '
GAPDH-F:5’-GGAGCGAGATCCCTCCAAAAT-3’
GAPDH-R:5 '-GGCTGTTGTCATACTTCTCATGG-3 '
Quantitative fluorescent PCR program: 95 DEG C of 5min, 95 DEG C of 30s are recycled 40 times, 60 DEG C of 45s, 72 DEG C of 30min；Every group sets 3Multiple holes, 2- △ △ Ct method calculate the expression quantity of KCNJ4.Independently in triplicate.
Two, it statisticallys analyze
Experimental data is analyzed using SPSS22.0 statistical analysis software.The comparison of two composition ratios of ranked data is adoptedUse Chi-square Test；Mean value between multisample compares using single factor test ANOVA variance analysis.
Three, result
Expression of the KCNJ4 gene in lung adenocarcinoma cell line: QPCR detection discovery, KCNJ4 gene is in A549 lung glandHigh expression (such as Fig. 1) in cancer cell line.
Embodiment 2 detects the increasing of the ability to express, tumour cell of KCNJ4 in the tumour cell for infecting siRNA interference sequenceGrow ability, cloning efficiency and infection ability
One, materials and methods
1 cell culture step as described in example 1 above.
2, siRNA is designed
For the KCNJ4 gene of the mankind, 2 pairs of interference are designed using siDirect version 2.0siRNA design softwareSequence is simultaneously synthesized.
3, RNA extracting as described in Example 1 and fluorescence quantitative PCR detection.
4, it transfects
Illustrate to be transfected according to Lipofectamine2000 transfection reagent box.It is long to logarithm to the cell in six orifice platesWhen the phase, in the complete culture solution that transfection replacement in first two hours is fresh.Liposome Lipofectamine2000 10ul is dissolved in 250ulIn serum-free antibiotic-free culture medium, 5min is stored at room temperature after mixing gently；5ul siRNA is dissolved in 250ul serum-free nonreactiveIn raw element culture medium.(KCNJ4si RNA1:F:5 '-CCGCUUCGUCAAGAAGAAC-3 ' and si RNA2:F:5 '-UGCAACGUGUACUUCGCCA-3').The mixed liquor of Coliposomes and siRNA in 30min, is stored at room temperature after mixing gently30min.The old culture medium in 6 orifice plates is discarded, PBS is cleaned cell 2 times.Every hole is added in 500ul liposome and siRNA mixed liquorIt in cell, mixes gently, puts in incubator and cultivate.After cell is put into incubator incubation 6h, complete culture solution is replaced.After for 24 hoursIt can observe and be transferred to siRNA expression or progress subsequent experimental.
5, Western blot is tested
After cell transfecting 48 hours, six orifice plates are placed on ice, RIPA lysate (adding protease inhibitors) cracking is added to mentionAlbumen is taken, BCA method measures concentration, 95 DEG C of heating 5min.About 20 μ g albumen, SDS polypropylene is added in Vertial electrophorestic tank in every holeProtein sample, transferring film to pvdf membrane is separated by electrophoresis in acrylamide gel, and 5% skimmed milk power closes 1h, 4 DEG C of primary antibody overnight incubations, TBST3 × 5min is washed, secondary antibody is incubated at room temperature 1h, washes after film plus ECL develops.QUANTITY ONE software scans gray value is with GAPDHInternal reference control, destination protein/internal reference calculate the relative expression quantity of each albumen.It is examined after colour developing Scan with Bio ID softwareIt surveys, measures the density value of destination protein and β-actin internal reference protein product band.With the light of destination protein and β-actin albumenThe relative amount of density ratio albumen as a purpose.
6, CCK8 method measures cell proliferative conditions
Cell dissociation after transfecting 24 hours is counted, cell suspension is prepared, takes 100ul cell suspension, with every hole 1000The standard species of a cell is into 96 orifice plates, routine culture in carbon dioxide incubator, every detecting a cell viability for 24 hours, examinesEvery hole adds 10ul CCK8 reagent before surveying, and is incubated for 1.5h in 37 DEG C of incubators, excites light detection OD value using microplate reader 450nm,Draw growth curve.
7, plate clone is tested
It by the cells trypsinised of logarithmic phase and blows and beats into individual cells, cell suspension is made and counts.With everyIt in the 60mm ware of Graded Density inoculation 37 DEG C of pre-temperature culture solutions containing 5mL of 1500 cells of ware, and gently rotates, makes cell pointIt dissipates uniform.Control group adds one thousandth DMSO, and experimental group adds 10uMPHEX.Set the cell culture of 37 DEG C of 5%CO2 and saturated humidityIt is cultivated 1-2 weeks in case.Often observation terminates culture when occurring macroscopic clone in culture dish.Liquid is discarded supernatant, is usedPBS carefully embathes 2 times.The fixed cell 5mL of 4% paraformaldehyde is added to fix 30 minutes.Then fixer is removed, adds appropriate 0.1%Violet staining liquid contaminates 30 minutes, then slowly washes away dyeing liquor with flowing water, is air-dried.Clone is counted, and compares clone's shapeAt size and number.
8, Transwell migration and Matrigel
The Matrigel matrigel 100ul melted overnight in advance (serum-free medium is diluted by 1:6) is added to 24 holesIn the upper chamber of the cell transwell of plate, shakes even be placed in 37 DEG C of carbon dioxide incubators and stand 4-6 hours plastics, it is rear to inhaleDry culture solution in lower room plus 500ul serum-free medium, stands half an hour aquation basilar memebrane.Cell after transfection for 24 hours is usedFree serum culture prepares cell suspension, and 100ul cell suspension (1 × 105) is added in upper chamber, and it is complete that 500ul is added in lower roomFull nutrient solution.After overnight, cell is taken out, cotton swab wipes cell remaining in upper chamber, and after PBS cleaning, 4% paraformaldehyde is fixed30min.After 0.1% violet staining 20min, PBS cleaning, 5 visuals field are randomly selected under microscope, observation of taking pictures counts.It movesIt moves experimental procedure and is similar to Matrigel, the cell transwell need not carry out paving glue processing, and cell number will be 5000.Two,Statistical analysis
Experimental data is analyzed using SPSS22.0 statistical analysis software.The comparison of two composition ratios of ranked data is adoptedUse Chi-square Test；Mean value between multisample compares using single factor test ANOVA variance analysis.
Three, result
1, KCNJ4 knocks out Efficiency testing:
The knockout carrier for carrying KCNJ4si RNA is transfected to the lung cancer cell line A549 cell of the high expressing K CNJ4 of endogenous,(SCR) is compareed with untransfected and the nonspecific sequence of transfection.Cell total rna and albumen are extracted after transfecting 72h, carries out fluorescenceQuantitative PCR (as described in example 1 above) and Western blot detection, as shown in Fig. 2, siRNA1 and siRNA2 can significantly dropThe RNA and protein expression level of KCNJ4 in low A549 cell knocks out efficiency up to 90% or more.
2, the experiment of CCK8 method detection cell Proliferation
As shown in figure 3, being able to suppress the proliferation of A549 cell after the expression of transfection siRNA knockout KCNJ4.
3, colony formation
As shown in figure 4, transfection siRNA, which knocks out KCNJ4, significantly reduces the cloning efficiency of A549 cell.
4, Transwell migration and Matrigel
Transwell is the experimental results showed that (such as Fig. 5) is able to suppress A549 after transfection siRNA knocks out the expression of KCNJ4The invasion of cell, result difference is significant (P < 0.05), and transfer ability also reduces accordingly, and result difference is significant (P < 0.05).It putsBig multiple: 100X.
5, KCNJ4 verifies the Mechanism Study that phenotype acts on
As shown in fig. 6, MAPK access is suppressed, MEK, the phosphorus of phosphorylation after the expression of transfection siRNA knockout KCNJ4The ERK expression quantity of acidification also declines therewith.
SEQUENCE LISTING
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