CN104293796A - CACNA1E mutant gene as well as detection method and application thereof - Google Patents

Abstract

The invention provides a method for detecting a CACNA1 mutant gene. The method is a whole genome depth sequencing method or a capillary electrophoresis sequencing method. The invention also provides a CACNA1 mutant gene. Further, the invention provides a kit for detecting the CACNA1 mutant gene and application of the kit in prevention, detection and/or treatment on lung cancers. The invention also provides the mutant gene and application of the detection method in prevention, detection and/or treatment on lung cancers. Based on whole genome sequencing and specificity polymerase chain reaction (PCR), gene CACNA1E mutation sites are found in lung-cancer patient samples; a theoretical basis on molecular classification, therapeutic targeting and the like is provided for clinical treatment and medication by detecting the mutation, and a new concept is provided for preventing and treating cancer.

Description

CACNA1E mutator gene and detection method thereof and application

Technical field

The invention belongs to oncobiology field, be specifically related to a kind of detection method and mutator gene thereof of CACNA1E mutator gene.Further, the test kit of detection said mutation gene and the application of this test kit is related to.In addition, the invention still further relates to the application in the prevention of lung cancer, detection and/or treatment of detection method, gene and/or test kit.

Background technology

Oncogene is the gene that a class can bring out cancer.The generation of cancer is often to the unconventionality expression of oncogene and activate relevant.In tumor tissues sample, the general high expression level of associated oncogene, causes its activity greatly to strengthen after these oncogene mutation simultaneously.For EGF-R ELISA (EGFR), in lung cancer tumor sample, the high expression level of EGFR result in PI3K-AKT signal path abnormal activation, thus result in cellular abnormality hyperplasia.In conjunction with analysis of clinical, EGFR sudden change causes patient to produce susceptibility to medicine (Gefitinib etc.), and the sudden change as occurred in EGFR the 21st exon (L858R) and the 19th exon makes patient to gefitinib.On the other hand, in treatment clinical course, patient produces resistance gradually.Further research display, the generation of this resistance is undergone mutation (T790M) due to EGFR the 20th exon and causes.Be directed to this, before patients with lung cancer carries out clinical treatment, conventional means that is clinical and personalized treatment become to its EGFR Mutation Screening, and has obtained good effect.As can be seen here, significant for Prevention and Curation cancer especially lung cancer for Related oncogene abrupt climatic change.But because the sudden change of other oncogenes such as EGFR only accounts for 30%-50% in lung cancer patient, also have the pathogeny of most patient and unclear, therefore, use of the new technology and find oncogene and detect its sudden change to seem extremely urgent.

Along with completing and the continuous innovation of sequencing technologies of mankind's genome sequencing work, genome sequencing has become the favourable weapon of Oncogenome research gradually.Ca ²⁺ion take part in much important physiology, biological process as second messenger requisite in cell.And calcium channel albumen plays vital effect in the process regulating calcium ion.Studied discovery in recent years, the generation of tumour, development and calcium associated protein have close contacting, and such as, gene C ACNA2D1 can maintain the tumor stem cell feature of liver cancer cell.

Gene C ACNA1E is the α 1E subunit of calcium channel albumen.It has 3 transcripts, and the albumen of these three transcript codings is made up of 2251,2270 and 2313 amino acid respectively.Have report, CACNA1E gene is high copy state in the nephroblastoma, its mRNA and the equal high expression level of albumen, and the recurrence positive correlation of this high expression level and the nephroblastoma before.But up to now, gene C ACNA1E expression unclear in other tumours, the relation of its sudden change in tumor sample and tumour is not also reported.Therefore, study the expression of CACNA1E in tumour and tumour relation and detect its suddenly change and suddenly change and the relation of tumour significant to finding new oncogene, Prevention and Curation tumour.

Summary of the invention

Contriver uses genome sequencing technology, degree of depth order-checking has been carried out to the cancer of 14 patients from the polycyclic aromatic hydrocarbons contaminated serious Lung Cancer With High Incidence area of China and cancer beside organism, data analysis finds, Calcium Signal path and calcium channel albumen there occurs mass mutation in these patient bodies.In genome sequencing result, calcium channel protein mutation not only obtains enrichment but also has very high mutation frequency, especially gene C ACNA1E.

Further, contriver is for each exon sequence feature of gene C ACNA1E, design the Auele Specific Primer of each exon, by polymerase chain reaction (PCR), in 164 routine patient specimens and 5 clones (L78 clone, 95D clone, Hela clone, NCI-H1975 clone and XLA-07 clone) by all exon amplifications of CACNA1E out, and by capillary electrophoresis check order and find out the sudden change causing CACNA1E amino acid change.Found that, in 164 routine patients, find that 16 routine patients undergo mutation altogether, wherein in the 79 routine patients in Xuanwei/natural resources area, have 15 examples to undergo mutation, mutation frequency reaches 19%; In the 85 routine patients in Guangzhou and non-Xuanwei/natural resources area, Yunnan, only have 1 routine patient to undergo mutation, mutation frequency is 1.2%.

The invention provides a kind of method detecting CACNA1E mutator gene, the method is full-length genome degree of depth sequencing or capillary electrophoresis sequencing.

Present invention also offers CACNA1E mutator gene.

Invention further provides a kind of detect CACNA1E mutator gene test kit and the application of mentioned reagent box in the prevention of lung cancer, detection and/or treatment.

Present invention also offers the application in the prevention of lung cancer, detection and/or treatment of said mutation gene and detection method.

According to an aspect of the present invention, the invention provides a kind of method detecting CACNA1E mutator gene, described method is full-length genome degree of depth sequencing or capillary electrophoresis sequencing.

Preferably, described method is capillary electrophoresis sequencing, said method comprising the steps of:

1) to each exon design Specific PCR primers of CACNA1E gene, performing PCR of going forward side by side increases;

Preferably, described PCR primer size controls between 400-1200bp;

Preferably, the sequence of described PCR primer is as shown in SEQ ID NO:1-SEQ ID NO:92;

2) order-checking of PCR primer purifying postcapillary electrophoresis detects, and the sequence obtained is compared with the gene order (GRCh37/hg19) of the CACNA1E in UCSC website, thus determines the mutational site of CACNA1E gene;

3) PCR primer obtained is translated according to normal reading frame, to determine the mutational site of CACNA1E.

Preferably, described nucleotide mutant site at least has following one or more: c.C356A, c.C507T, c.C535A, c.G746T, c.G823T, c.G1054T, c.G1173T, c.G1276T, c.G1637T, c.G2315T, c.G2385T, c.G2552T, c.C2773A, c.T3038A, c.G3981T, c.G4303A, c.G4994T, c.G5608T, c.C5825A, c.G6037T, c.C6871A, c.C6862A, c.G6865T.Further preferably, described mutational site is also positioned at c.A6515G, c.C253A.

Again on the one hand, the invention provides a kind of CACNA1E mutator gene, at least one mutational site of nucleotide sequence of wherein said CACNA1E mutator gene is positioned at c.C356A, c.C507T, c.C535A, c.G746T, c.G823T, c.G1054T, c.G1173T, c.G1276T, c.G1637T, c.G2315T, c.G2385T, c.G2552T, c.C2773A, c.T3038A, c.G3981T, c.G4303A, c.G4994T, c.G5608T, c.C5825A, c.G6037T, c.C6871A, c.C6862A, c.G6865T, c.A6515G, c.C253A.

Preferably, at least one amino acid mutation site of coded protein sequence of described CACNA1E mutator gene is positioned at p.P119H, p.L169F, p.H179N, p.R249L, p.A275S, p.G352W, p.E391D, p.D426Y, p.G546V, p.R772L, p.E795D, p.S851I, p.R925S, p.L1013Q, p.K1327N, p.E1435K, p.W1665L, p.A1870S, p.S1942Y, p.D2013Y, p.P2291T, p.R2288S, p.G2289W.Further preferably, described mutational site is also positioned at p.Y2172C, p.L85I.

Sudden change names all in the application is the nomenclature mo based on suddenling change in the world, contain three parts, amino acid after the amino acid position-sudden change of amino acid-sudden change before sudden change, as A275S, namely sudden change result in the 275th amino acid and becomes the Serine after sudden change (S) by the L-Ala (A) before suddenling change, and coding mutation in like manner.

Present invention also offers a kind of test kit detecting CACNA1E mutator gene, described test kit comprises the primer for CACNA1E mutational site of increasing, and preferably, described test kit comprises the primer shown in SEQ ID NO:1-SEQ ID NO:92.

Preferably, described test kit also comprises conventional PCR component, such as pcr amplification enzyme and corresponding damping fluid.

It should be noted that, the primer in test kit can design according to known aminoacid sequence, and this is ordinary skill in the art means.

The concrete using method of test kit provided by the invention is as follows:

1) design of each exon Specific PCR primers of CACNA1E:

Software Primer5 is used to design the Auele Specific Primer of CACNA1E 48 exons, totally 46 pairs of primers; During design primer, product size controlled between 400-1200bp, and be unicity by NCBI blast function blast result, in a preferred embodiment in accordance with this invention, described primer is as shown in table 1.

2) PCR primer amplification:

PCR reaction system is 20 μ l, and template DNA consumption is 10ng.

PCR program is 95 DEG C, 5 minutes, 95 DEG C, 30 seconds, 60 DEG C, 45 seconds, 72 DEG C, 1 minute, 35 circulations, and last 72 DEG C extend 10 minutes.

3) PCR primer carries out capillary electrophoresis order-checking, by the Gene sequence comparison of the sequence that obtains and CACNA1E, thus judges whether to there is mutational site.

In addition, also directly can design primer for certain or some mutational sites, such as primer can be one or more in the sequence shown in SEQ ID NO:1-SEQ ID NO:92.

Because mentioned reagent box can detect the catastrophe of CACNA1E gene, and CACNA1E gene is at lung cancer patient, especially have higher mutation rate in Chinese polycyclic aromatic hydrocarbons contaminated serious Lung Cancer With High Incidence area and clone, therefore this test kit can be applied in the prevention of lung cancer, detection and/or treatment.

Similarly, CACNA1E mutator gene provided by the invention and detection method also can be applied to the application in the prevention of lung cancer, detection and/or treatment.Such as, sudden change is there occurs owing to present invention finds gene C ACNA1E in the lung cancer in the polycyclic aromatic hydrocarbons contaminated serious area of China, and confirm that gene C ACNA1E occurs relevant to lung cancer by series of experiments, therefore gene C ACNA1E and mutational site thereof can as the therapy target of lung cancer.

Preferably, can pass through siRNA perturbation technique, CACNA1E is fallen in interference, thus reaches the object of one-tenth knurl ability or the Tumor suppression growth reducing tumour, is used for the treatment of and/or prevents lung cancer.

Therefore, present invention also offers the combination of a kind of siRNA, described siRNA is the specific siRNA for disturbing CACNA1E to express.Preferably, the sequence of described siRNA is

SEQ ID NO:93:5 '-CCUCCGGCUUCUAAGAAUA-3 '; (CACNA1E disturbed one)

SEQ?ID?NO:94：5’-CGCCAUUUGAGUACAUGAU-3’。(CACNA1E interference 2)

Present invention also offers a kind of test kit, described test kit comprises above-mentioned siRNA sequence.Pcr amplification enzyme and corresponding damping fluid is also comprised in described test kit.

The invention provides the using method additionally providing mentioned reagent box, preferably, described method is secondary interference method.

In a preferred embodiment, described secondary interference method is that method by comprising the following steps realizes:

A. the cell of experimenter is inoculated in 6 orifice plates, inoculum density is 1 × 10 ⁵individual cells/well, and use culture medium culturing;

Preferably, described substratum is the RPMI1640/DMEM substratum containing 10%FBS;

B. after one day, sop up substratum, add 800 μ l Opti-MEM substratum;

C. join in the pipe containing the RNase free of 200 μ l Opti-MEM by transfection reagent Hyperfect 2000 and 2.5 μ l siRNA (20 μMs), vortex oscillation mixes, the static 10-15 minute of room temperature;

D. mixed liquid is joined in 6 orifice plates, in cell culture incubator, cultivate 6 hours;

Hour e.6, after, transfection liquid is replaced with fresh culture;

F. the 3rd day repeating step a-e.

Because mentioned reagent box can disturb the expression of CACNA1E mutator gene, thus the propagation of T suppression cell, Clone formation, one-tenth knurl ability, and intracellular calcium ion concn and Intracellular phosphorylation EGFR, ERK, AKT are also declined thereupon, therefore this test kit can be applied in the prevention of lung cancer, detection and/or treatment.

The present invention provides theoretical basis for detecting oncogene CACNA1E sudden change, devises the Auele Specific Primer detecting sudden change.On this basis, the present invention discloses the catastrophe of gene C ACNA1E in the especially Chinese polycyclic aromatic hydrocarbons contaminated serious Lung Cancer With High Incidence area of lung cancer patient and clone by genome sequencing and the method such as PCR, capillary electrophoresis order-checking.In addition, the present invention tentatively discloses the effect of oncogene CACNA1E in lung cancer morbidity by the mode of interference.Contriver finds, disturb CACNA1E in the cell of band CACNA1E sudden change after, the propagation of cell, Clone formation, one-tenth knurl ability are all suppressed, and intracellular calcium ion concn and Intracellular phosphorylation EGFR, ERK, AKT also decline thereupon.

The present invention is applicable to the sudden change detecting oncogene CACNA1E, comprises tumor tissues and the clone in patient source.By detecting it, sudden change can be clinical treatment, medication provides molecule parting, therapy target scheduling theory basis, for Prevention and Curation tumour provides new thinking.

In lung cancer patient sample, gene C ACNA1E mutational site has been found, the effect of these mutational sites in lung cancer prevention, Treatment and diagnosis process by genome sequencing and based on specific PCR reaction.Design specificity small nucleic acids RNA interference sequence (siRNA) for CACNA1E, by interference mode CACNA1E sudden change and wild-type cell in by PROTEIN C ACNA1E interference fall, detect cell proliferation, the change of the change of clonality, Ca2+ oscillations and downstream signaling pathway.Result shows, in the cell of CACNA1E sudden change, can the obviously propagation of T suppression cell and clonality after interference CACNA1E, and intracellular calcium concentration can be caused to reduce.Meanwhile, detect its downstream signaling pathway and find, in the cell of band CACNA1E sudden change, interference CACNA1E, EGFR, ERK, AKT of phosphorylation lower.

Accompanying drawing explanation

Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:

Fig. 1 is the structural representation of CACNA1E protein mutation in lung cancer patient sample and clone; Wherein scheme the structural representation that A is CACNA1E protein mutation in lung cancer patient sample; Wherein scheme the structural representation that B is CACNA1E protein mutation in lung cancer cell line (XLA-07) and Hela cell;

In the Hela cell that Fig. 2 undergos mutation at gene C ACNA1E and lung carcinoma cell XLA-07, cell proliferation after interference CACNA1E, Clone formation and associated signal paths change; Wherein, A is that after Hela and XLA-07 cell interference CACNA1E, multiplication capacity is suppressed; B is that Hela cell interference CACNA1E rear clone Forming ability is suppressed; C is that after Hela and XLA-07 cell interference CACNA1E, p-EGFR, phosphorylation AKT and phosphorylated CREB are lowered;

Fig. 3 is that in the gene C ACNA1E Hela cell of undergoing mutation and lung carcinoma cell XLA-07, after interference CACNA1E, intracellular calcium concentration reduces; Wherein, X-coordinate represents fluorescence intensity, and intracellular calcium concentration is higher, and fluorescence is stronger; Ordinate zou is the cell proportion of emitting fluorescence; As shown in FIG., the cell fluorescence intensity in control group is higher than the cell after CACNA1E interference, and namely after CACNA1E interference, intracellular calcium concentration reduces;

Fig. 4 is in the gene C ACNA1E Hela cell of undergoing mutation, and after interference CACNA1E, cell becomes knurl ability to be suppressed.Wherein, figure A is the volume of the different time point measurement tumour in lotus knurl, and the tumor volume in control group is obviously greater than the tumor volume after CACNA1E interference; Figure B is the tumor photo after tumor being stripped down after putting to death mouse; Lotus knurl photo when figure C is mouse execution.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.

Unless specifically stated otherwise, the Hela clone adopted in following examples is purchased from consonance cell bank; XLA-07 clone is purchased from Kunming animal institute of the Chinese Academy of Sciences; L78 clone purchased from Chinese Academy of Sciences's Shanghai cell bank, 95D clone purchased from Chinese Academy of Sciences's Shanghai cell bank, NCI-H1975 clone purchased from ATCC;

Unless specifically stated otherwise, the nude mice adopted in following examples is Nude mouse, purchased from Department Of Medicine, Peking University.

Unless specifically stated otherwise, test method used in following examples is ordinary method used in this area.

Unless specifically stated otherwise, reagent used in following examples is analytical pure level reagent, and can be commercially available from regular channel.

embodiment 1CACNA1E design of primers

CACNA1E is R type voltage door dependency calcium channel protein alpha subunit 1E, belongs to surface of cell membrane albumen, enters in the cell of regulation and control calcium ion.Also take part in the cell physiological biochemical reaction of polycalcium ion dependence, as the transmission of Muscle contraction, neurotransmitter, gene expression regulation, cell movement, cell fission and necrocytosis etc. simultaneously.This gene finds that there is three transcripts at present, and its longest transcript is NM_001205293.1, comprises 48 exons, 2313 amino acid of encoding.Its protein structure contains three Ion_trans structural domains, a Ca_chan_IQ structural domain and a PKD_Channel structural domain.Log in the gene order that CACNA1E is downloaded in UCSC website, the Auele Specific Primer of each exon of CACNA1E is designed by software Primer 5, product size is controlled between 400-1200bp, and be unicity by NCBI blast function blast result, concrete primer sequence is as shown in table 1.

Table 1.CACNA1E exon specific primer sequence

the examination of embodiment 2CACNA1E mutator gene

The catastrophe (the results are shown in Figure 1) of gene C ACNA1E in the routine patient of examination 164 and 5 routine clones (L78 clone, 95D clone, Hela clone, NCI-H1975 clone and XLA-07 clone) is reacted by PCR.

2.1 examination object: in 164 routine patients with lung cancer, 79 examples are from the polycyclic aromatic hydrocarbons contaminated serious Lung Cancer With High Incidence area of China, i.e. Xuanwei/natural resources area, 85 examples are regional from contrast, i.e. non-Xuanwei, Guangdong/Yunnan, natural resources area.Wherein in order to 14 patients of genome sequencing from area, Xuanwei/natural resources, age range be 38-64 year.In 14 samples, according to the histological type of lung cancer, 11 is adenocarcinoma of lung, and 3 is lung squamous cancer; According to patient's whether smoking, 6 is smoking patients, and 8 is non-smoking patient.All 164 routine patients have cancerous tissue and corresponding cancer beside organism.

2.2DNA extract

The fragmentation of tissue:

1. get the cancerous lung tissue being less than 30mg, with historrhexis's instrument, it is broken;

2. add the lysate (RLT Buffer) of certain volume according to the weight of got tissue, lysis at room temperature 0.5-1 hour, centrifugal 3 minutes of maximum speed, siphons away supernatant carefully;

3. transfer in DNA pillar by lysate below, DNA post is placed in 2ml collection tube, lightly cover lid, centrifugal 30 seconds of 8000g;

The extraction of genomic dna:

1. the step 3 then in historrhexis, in DNA post, add 500 μ l AW1 damping fluids, DNA post is placed in new 2ml collection tube, lightly lid upper tube cap, and centrifugal 15 seconds of 8000g, abandons filtrate, and the object of this step is the filter membrane of rinsing DNA pillar;

2. in DNA post, add 500 μ l buffer A W2, lightly lid upper tube caps, high speed centrifugation 2 minutes, the filter membrane of rinsing DNA pillar;

3. DNA post is placed in new 1.5ml collection tube, to filter membrane, central authorities drip 100 μ l damping fluid EB, lid upper tube cap, and room temperature leaves standstill 1 minute, centrifugal 1 minute of 8000g, eluted dna.

2.3PCR amplification

PCR condition is 95 DEG C, 5 minutes, 95 DEG C, 30 seconds, 60 DEG C, 45 seconds, 72 DEG C, 1 minute, 35 circulations, and last 72 DEG C extend 10 minutes.

The reaction system of pcr amplification

2.4 interpretations of result:

PCR primer checks order through 3730-capillary electrophoresis;

Sequencing result Genious software comparison, finds out the base mutation inconsistent with reference sequences; Codon is connected according to the position of sudden change and three of contrast encoding histone, determine the corresponding amino acid number (the results are shown in Table 2) that suddenlys change, in 164 routine patients, find that 16 routine patients undergo mutation altogether, wherein, in the 79 routine patients in Xuanwei/natural resources area, have 15 examples to undergo mutation, mutation frequency reaches 19%; In the 85 routine patients in Guangzhou and non-Xuanwei/natural resources area, Yunnan, only have 1 routine patient to undergo mutation, mutation frequency is 1.2%.Particularly, be numbered 6 identical with the two routine patients position of undergoing mutation of 7, the guanine (G) being the 823rd sports thymus pyrimidine (T), and its amino acid mutation is the alanine mutation of 275 is Serine (referring to table 2); The patient being numbered 1 has two sites to undergo mutation, and the coding mutation of its correspondence and amino acid mutation are respectively c.C356A (p.P119H), c.C5825A (p.S1942Y); The patient being numbered 2 has four sites and undergos mutation, and the coding mutation of its correspondence and amino acid mutation are respectively c.C507T (p.L169F), c.C2773A (p.R925S), c.G4303A (p.E1435K), c.G6037T (p.D2013Y); The patient being numbered 4 has two sites to undergo mutation, and corresponding coding mutation and amino acid mutation are respectively c.G746T (p.R249L), c.G2315T (p.R772L); The patient being numbered 7 has four sites and undergos mutation, and the coding mutation of its correspondence and amino acid mutation are respectively c.G1054T (p.G352W), c.G1173T (p.E391D), c.G3981T (p.K1327N), c.C6862A (p.R2288S).

The distribution of table 2CACNA1E mutator gene

cell proliferation in embodiment 3CACNA1E mutator gene, clonality and coherent signal are logical road is studied

Disturbed by siRNA, in cell Hela and XLA-07 of band CACNA1E sudden change, detect cell proliferation, clonality and associated signal paths:

With CACNA1E minor interference sequence (SEQ ID NO:93 and SEQ ID NO:94 is used in combination with the ratio of the 1:1) (siCE of 50nM, Genepharma) and negative control sequence (siNC, Genepharma) process respectively band CACNA1E sudden change cell Hela (Y2172C) and XLA-07 (L85I).SiCE and siNC process cell again with 50nM after 24 hours.1 × 10 is got after 24 hours ⁵cell inoculates 6 orifice plates, collects cell respectively at the 2nd day, the 4th day, with after Trypan Blue to viable count, to find that in the cell of band CACNA1E sudden change cell proliferation after CACNA1E is fallen in interference and be suppressed.Meanwhile, get 1000 cells and be seeded to lower floor containing 0.6% agar, 10% foetal calf serum, in the 35mm culture dish of upper strata containing 0.3% agar, 10% foetal calf serum, at 37 DEG C, 5%CO ₂cell culture incubator in cultivate 15 days.Violet staining is used, statistics number of cell clones after 15 days.Result shows, and after in Hela (Y2172C) cell, CACNA1E is fallen in interference, its clonality is suppressed (Fig. 2 B).Western blotting detects downstream molecules change after CACNA1E interference in Hela (Y2172C), XLA-07 (L85I) cell.Result shows, and in the Hela (Y2172C) and XLA-07 (L85I) cell of band CACNA1E sudden change, after CACNA1E is fallen in interference, Intracellular phosphorylation EGFR, ERK, AKT level declines (Fig. 2 C).

calcium ion concn research in embodiment 4CACNA1E mutator gene

With cell Hela (Y2172C) and the XLA-07 (L85I) of siRNA SEQ ID NO:93 and 94 (SEQ ID NO:93 and SEQ ID NO:94 is used in combination with the ratio of 1:1) the process band CACNA1E sudden change of 50nM, random siRNA in contrast.The siRNA (CACNA1E) of 50nM and random these 2 cells of siRNA process are again used after 24 hours.Microscopic fluorescence is used to survey calcium systematic survey intracellular calcium concentration after 48 hours.Result shows, and in the Hela (Y2172C) and XLA-07 (L85I) cell of band CACNA1E sudden change, after CACNA1E is fallen in interference, intracellular calcium concentration declines (Fig. 3)

embodiment 5 nude mice becomes knurl capability study

With the cell Hela (Y2172C) of siRNA (SEQ ID NO:93 and SEQ ID NO:94 is used in combination with the ratio of 1:1) the process band CACNA1E sudden change of 50nM, random siRNA in contrast.The siRNA of 50nM (SEQ ID NO:93 and SEQ ID NO:94 is used in combination with the ratio of 1:1) and random siRNA process Hela (Y2172C) cell is again used after 24 hours.Get 1 × 10 respectively ⁶interference and compared with control cells are seeded to nude mice belly side, in latter 7th day of inoculation, the 10th day, within the 13rd day and the 16th day, measure gross tumor volume and mouse body weight.Result shows, and after in the cell Hela (Y2172C) of band CACNA1E sudden change, CACNA1E is fallen in interference, nude mice becomes knurl ability to be suppressed (Fig. 4).

Inject in nude mouse by hypodermic mode after Hela cell interference CACNA1E, different time point measurement gross tumor volume size.NC siRNA is as negative control.Result shows, and after interference CACNA1E, gross tumor volume obviously reduces.

Claims (9)

1. a CACNA1E mutator gene, at least one mutational site of wherein said CACNA1E mutator gene is positioned at c.C356A, c.C507T, c.C535A, c.G746T, c.G823T, c.G1054T, c.G1173T, c.G1276T, c.G1637T, c.G2315T, c.G2385T, c.G2552T, c.C2773A, c.T3038A, c.G3981T, c.G4303A, c.G4994T, c.G5608T, c.C5825A, c.G6037T, c.C6871A, c.C6862A, c.G6865T, c.A6515G, c.C253A;

Preferably, described mutational site is also positioned at c.A6515G, and wherein said CACNA1E mutator gene is the mutator gene in Hela clone;

Preferably, described mutational site is also positioned at c.C356A, and wherein said CACNA1E mutator gene is the mutator gene in LXA-07 clone.

2. detect a method for CACNA1E mutator gene as claimed in claim 1, described method is by full-length genome degree of depth sequencing or capillary electrophoresis sequencing determination mutator gene.

3. method according to claim 2, wherein, described mutational site is positioned Ion_trans structural domain, PKD_Channel structural domain, Ca_chan_IQ structural domain and/or other his nonfunctional area;

Preferably, described nucleotide mutant site at least has following one or more: c.C356A, c.C507T, c.C535A, c.G746T, c.G823T, c.G1054T, c.G1173T, c.G1276T, c.G1637T, c.G2315T, c.G2385T, c.G2552T, c.C2773A, c.T3038A, c.G3981T, c.G4303A, c.G4994T, c.G5608T, c.C5825A, c.G6037T, c.C6871A, c.C6862A, c.G6865T, c.A6515G, c.C253A;

Further preferably, described mutational site is also positioned at c.A6515G, c.C253A.

4. according to the method in claim 2 or 3, wherein, described method, by capillary electrophoresis sequencing determination mutator gene, comprises the following steps:

1) to each exon design Specific PCR primers of CACNA1E gene, performing PCR of going forward side by side increases;

Preferably, described PCR primer size controls between 400-1200bp;

Preferably, the sequence of described PCR primer is as shown in SEQ ID NO:1-SEQ ID NO:92;

2) order-checking of PCR primer purifying postcapillary electrophoresis detects, and the sequence obtained is compared with the gene order (GRCh37/hg19) of the CACNA1E in UCSC website, thus determines the mutational site of CACNA1E gene;

Preferably, described method also comprises to be translated the PCR primer obtained according to normal reading frame, to determine the mutational site of CACNA1E.

5., for detecting a test kit for CACNA1E mutator gene as claimed in claim 1, described test kit comprises the primer for CACNA1E mutational site of increasing;

Preferably, the corresponding restriction enzyme that described test kit also comprises pcr amplification enzyme, damping fluid, enzyme cut different mutational site;

Preferably, the sequence of described PCR primer is selected from one or more in the sequence shown in SEQ ID NO:1-SEQ ID NO:92.

6. the combination for the siRNA that disturbs CACNA1E mutator gene as claimed in claim 1 to express;

Preferably, the sequence of described siRNA is

SEQ ID NO:93:5 '-CCUCCGGCUUCUAAGAAUA-3 '; (CACNA1E disturbed one)

SEQ ID NO:94:5 '-CGCCAUUUGAGUACAUGAU-3 ' (CACNA1E interference 2).

7. the test kit for disturbing CACNA1E mutator gene as claimed in claim 1 to express, described test kit comprises siRNA sequence as claimed in claim 6;

Preferably, pcr amplification enzyme and corresponding damping fluid is also comprised in described test kit.

8. the using method of test kit as claimed in claim 7, described method is secondary interference method;

Preferably, described secondary interference method is that method by comprising the following steps realizes:

A. the cell of experimenter is inoculated in 6 orifice plates, inoculum density is 1 × 10 ⁵individual cells/well, and use culture medium culturing

Preferably, described substratum is the RPMI1640/DMEM substratum containing 10%FBS;

B. after one day, sop up substratum, add 800 μ l Opti-MEM substratum;

C. join in the pipe containing the RNase free of 200 μ l Opti-MEM by transfection reagent Hyperfect 2000 and 2.5 μ l siRNA (20 μMs), vortex oscillation mixes, the static 10-15 minute of room temperature;

D. mixed liquid is joined in 6 orifice plates, in cell culture incubator, cultivate 6 hours;

Hour e.6, after, transfection liquid is replaced with fresh culture;

F. the 3rd day repeating step a-e.

9. one group of primer for the CACNA1E mutator gene as claimed in claim 1 that increases and/or for disturb the SiRNA of CACNA1E mutator gene as claimed in claim 1 for the preparation of prevention, detect and/or purposes in the treatment medicine of lung cancer or test kit;

Preferably, the corresponding restriction enzyme that described test kit also comprises pcr amplification enzyme, damping fluid, enzyme cut different mutational site;

Preferably, described primer is selected from one or more in the sequence shown in SEQ ID NO:1-SEQ ID NO:92

Preferably, described siRNA is SEQ ID NO:93 and/or 94.