CN104726454B - Effects and its application of the hsa miR 1280 in non-small cell lung cancer - Google Patents

Abstract

The invention discloses effects and its application of a kind of hsa miR 1280 in Diagnosis of Non-Small Cell Lung.It is contemplated that the diagnosis of early stage is carried out to non-small cell lung cancer using conspicuousness differential expressions of the hsa miR 1280 in cancerous lung tissue and distal end normal structure.MiRNA1280 is compared in terms of cancerous tissue and distal end normal structure Sensitivity and Specificity, AUC=0.772, susceptibility during best cut point is 46.4%, specificity is 95.7%, therefore with very strong feasibility.

Description

Effects and its application of the hsa-miR-1280 in non-small cell lung cancer

Technical field

The present invention relates to applications of the miRNA in cancer diagnosis, and it is used as the diagnostic uses of non-small cell lung cancer.

Background technology

Lung cancer is one of most common malignant tumour of China, and its morbidity and mortality is respectively positioned on first of malignant tumour.It is non- ED-SCLC (Non-Small Cell Lung Cancer, NSCLC) account for its 80%, though curative effect was improved in recent years, Long-term survival rate is still more relatively low, and most patients have been cancer of late stage when making a definite diagnosis, and can only take palliative therapy, prognosis compared with Difference.

Invasion and attack and transfer are the processes that cancer cell is transferred to distal site formation tumour from primary site, and it is more than one Step, the complex biological process of multifactor participation, are to cause NSCLC to recur and influence the key factor of prognosis, are also to cause The main cause of death, the invasion and attack and metastasis to NSCLC are unclear at present.Think always tumour be by What a series of oncogene and Tumor Suppressor Gene Mutations were gradually formed, but with the discovery of non-coding RNA, this traditional concept is changed Become.It has recently been demonstrated that during member's microRNA molecule wide participation in non-coding RNA invasion and metastasis of tumor Gene expression regulation, this be found to be metastases diagnosis and treatment provide new thinking.

MicroRNA is the non-coding tiny RNA that 19-24nt is about by endogenous gene, can be heavy by forming RNA inductions Silent complex (RNA induced silencing complex, RISC) and the end of target gene 3 ' non-translational region (untranslated Region, UTR) complementary combination, induce the regulation mechanism suppression target gene of mRNA cutting degraded, Translational repression or other forms Expression.The mankind microRNA having now been found that is up to kind more than 1000, and the microRNA assignments of genes gene mapping more than 50% are in tumour Related chromosomal loci or its susceptibility loci, and confirm that part microRNA unconventionality expressions and specific tumour have close Relation, and take part in multiple processes of malignant progression, including cell differentiation, propagation, invasion and attack and transfer.

MicroRNA serves vital in NSCLC invasion and attack and transfer.Grawford etc. turns miRNA-126 Contaminate into Lung Squamous Carcinoma Cells system, with blank control group and compared with being transfected into unordered pre-microRNA cell, the former cell is invaded Attack and decline with transfer ability.Yu etc. is by high risk (death risk ratio>1) microRNA precursors and protectiveness (death risk Than<1) microRNA precursors are each separately transfected into the lung cancer cell line of low invasion and high invasion, and discovery is transferred to high-risk Property microRNA lung cancer cell line penetrate into the invasion enhancing of matrix glued membrane, and transfect protectiveness microRNA lung carcinoma cell System's invasion reduction, shows that these microRNA are related to the invasion and attack transfer of lung cancer.Zhang etc. is real-time using stem ring shape reverse transcription PCR method (stem-loop qRT-PCR) is quantified to 20 couples of NSCLC and cancer beside organism miR-21, and is utilized Western blot technology for detection miR-21 target gene PTEN expression, it is found that miR-21 has higher in NSCLC Expression, and the protein expression situation of Anti-oncogene PTEN then on the contrary, then will be loaded with miR-21 Transcription inhibitions plasmid turn NSCLC cells are contaminated, the growth and invasion and attack effect of cell is as a result significantly reduced, shows that miR-21 expression reduces tumor suppressor gene PTEN expression, promotes the growth and invasion and attack of NSCLC cells.Zhu etc. research is, it was also found that miR-21 is multiple swollen by targetting Tumor metastasis suppressor, regulation and control breast cancer, the invasion and attack and transfer of lung cancer.Wang etc. has been filtered out with microRNA chip technologies There is the miR-183 of notable differential expression in two kinds of cells by lung cancer and cancer, and find the metastatic potential of miR-183 and lung carcinoma cell It is negatively correlated, thus it is speculated that it is potential tumor metastasis suppressor gene, further investigation revealed that, miR-183 overexpression inhibits lung cancer The transfer and invasion and attack of cell.

MicroRNA research is not only that tumor development mechanism is verified there is provided new idea and method, Er Qie Very high application value is shown in terms of clinic diagnosis and prognosis evaluation.MicroRNA take part in each rank of tumor development Section, each stage has corresponding microRNA to change.Moreover, microRNA has good tissue specificity, warp Examination detects that 48 microRNA determine the tissue-derived rate of accuracy reached 90% of tumour.Meanwhile, microRNA again there is tumour to send out Raw phase specificity, occurring development difference, the stage has different microRNA express spectras to same tumour by stages.Therefore, MicroRNA can be used for as a kind of effective tumor markers the early diagnosing of cancer, pernicious classification, personalized diagnosis and treatment and Index for diagnosis etc., it might even be possible to as the target spot of oncotherapy, instruct clinical application.

Although tumor tissues microRNA express spectras are related to tumor invasion and prognosis, detection technique is complicated, wound Greatly, it is difficult to which really for clinical diagnosis, and peripheral blood serum is easier to obtain and detected, clinical practice is convenient, beneficial to popularization, and MicroRNA can exist steadily in the long term in serum, be difficult to be degraded by RNase, boil, multigelation, acid or alkali environment, long-term The extreme conditions such as preservation do not result in serum microRNA loss.Importantly, microRNA express spectras in serum Change and the tissue of tumour are closely related, can point out the state of tumour.Chen etc. studies have shown that is in non-small cell lung cancer blood In clear, miR-25 and the notable up-regulation of miR-223 expression.Ng etc. can diagnose I~IV using serum miR-92 as molecular marker The colorectal cancer of phase, sensitivity is up to 89%, and specificity reaches 70%.This has fully demonstrated serum microRNA in tumour diagnosis and treatment In application value.

It is contemplated that using hsa-miR-1280 in terms of cancerous tissue and distal end normal structure Sensitivity and Specificity Compare, assess it as the feasibility of the early diagnosis of clinically non-small cell lung cancer.

The content of the invention

The present invention builds up tissue samples storehouse and serum sample by setting up NSCLC patient's pathology and postoperative follow-up data storehouse The example of storehouse more than 200.Wherein there are 30 to have been achieved with serum after preoperative, postoperative and recurrence, every three months is paid a return visit, and record corresponding Follow-up Data.Complete by 10 pairs of NSCLC cancers and cancer on the basis of the microRNA chip analysis of sample, this research will be screened MicroRNA with differential expression, and its target gene is determined based on bioinformatic analysis and RIP experimental techniques, verify Regulatory mechanisms of the microRNA in NSCLC is attacked and is shifted, meanwhile, during the different pathologic stage of examination NSCLC patient, serum Middle NSCLC shifts special microRNA marks, provides early stage effective monitoring means for NSCLC transfers.

It is an object of the invention to provide a kind of miRNA, i.e. hsa-miR-1280, it can be used in non-small cell lung cancer Detection.

Technical scheme is as follows：

(1) screening NSCLC organizes the microRNA with corresponding cancer beside organism's differential expression；

(2) Bioinformatics Prediction and microRNA target gene is identified；

(3) influence of difference microRNA cell growths is investigated；

(4) effect of research difference microRNA and its correspondence target gene in NSCLC invasion and metastasis of tumor.

(5) microRNA of checking NSCLC serum and non-cancer control serum differential expression, assesses it as serum N SCLC Candidate markers, the possibility for NSCLC early stage transfers diagnosis, curative effect monitoring.

Further, technical scheme is as follows：

(1) consolidate and improve NSCLC databases

A sets up and arranged NSCLC patient's pathological data and postoperative follow-up data database, collects and arrange the pathology of patient The data such as archives, aftertreatment scheme and effect, post-operative survival rates situation；

B sets up and arranges NSCLC tissue samples storehouse (having completed 200 NSCLC tissue specimen collections)；

C sets up and arranges NSCLC patient and compare the blood sample sample storehouse of crowd.

(2) NSCLC and cancer beside organism difference microRNA screening

A samples are collected：The NSCLC patient 10 of clinical definite is chosen, cancer and corresponding cancer beside organism is taken, totally 20 marks This；

B sample Total RNAs extractions：The total serum IgE of 20 samples is extracted using method for extracting total RNA；

C utilizes the microRNA in microRNA extracts kit separation and Extraction total serum IgEs；

D utilizes mammal microRNA chips (Affymetrix), to the microRNA samples for 20 samples being collected into Product carry out screening experiment, and 20 chips are done altogether；

The microRNA of E application chip statistical analysis softwares, analysis cancerous tissue and cancer beside organism's difference.

(3) enlarged sample experimental verification and microRNA further screening

A utilizes cancer and the microRNA of cancer beside organism's significant difference in Real-timePCR technical identification array experiments to tie Really, bioinformatic analysis is carried out to these microRNA, chooses the 8-10 microRNAs related to invasion and metastasis of tumor, Expand cancerous lung tissue sample to verify to 100 Duis；

B is analyzed 8-10 microRNA of selection according to the pathologic data of above-mentioned NSCLC cases, is investigated Expression quantity distribution situation in the NSCLC patient of different pathological stagings.

(4) RIP technologies determine difference microRNA target gene, bioinformatics inquiry and reference literature report, prediction MicroRNA function, chooses 1-2 and the microRNA progress follow-up study related with transfer is attacked to NSCLC.

(5) microRNA lentivirus over-express vectors system/ASON suppresses micorRNA expression in fact Test and NSCLC test cell lines

A builds microRNA Lentivirus over-express vectors, is designed with reporter gene.

B is using surely turn technology is transfected into NSCLC cells by microRNA over-express vectors.The monitoring of transwell technologies is each The character mutation of cell line；Real-timePCR technical Analysis microRNA and correspondence target gene expression quantity situation；Western The protein expression change of target gene is investigated in experiment；

ASON after C modifications transfects NSCLC cells, the monitoring of transwell technologies using Lipofectamine The character mutation of each cell line；Real-timePCR technical Analysis microRNA and correspondence target gene expression quantity situation； The protein expression change of target gene is investigated in Western experiments.

(6) animal model experiment

NSCLC cell subcutaneous vaccination nude mouses after the microRNA carriers transfection that A Lentivirus are overexpressed；

B observes the Mice Body inner cell after inoculating cell into knurl situation and metastases situation.

(7) microRNA serum test, assesses difference microRNA as the possibility of NSCLC early stage blood serum marks

A collect that patient is corresponding preoperative, postoperative and recurrence after peripheral blood sample；

B isolates serum in mono- hour；

Free microRNA expression quantity in C Real-timePCR technology for detection serum；

D is associated analysis to serum microRNA situations of change and corresponding pathology Follow-up Data, investigates patient in difference Pathologic stage, corresponding microRNA expression quantity situation of change in serum.Confirm candidate microRNA as NSCLC early stages The possibility of blood serum designated object, and the practicality monitored applied to clinical NSCLC.

The present invention is advantageous in that：

1. couple hsa-miR-1280 is compared in terms of cancerous tissue and distal end normal structure Sensitivity and Specificity, AUC =0.772, susceptibility during best cut point is 46.4%, and specificity is 95.7%, therefore has very strong feasibility, preferably Application prospect and promotional value.

2.hsa-miR-1280 the early diagnosis of cancer can be used for as a kind of effective tumor markers, it might even be possible to As the target spot of oncotherapy, clinical application is instructed.

3. prediction hsa-miR-1280 can be used for Virus monitory, although tumor tissues microRNA express spectras are sent out with tumour Disease and prognosis are related, but detection technique is complicated, wound is big, it is difficult to which really for clinical diagnosis, and peripheral blood serum is easier to obtain And detect, clinical practice is convenient, beneficial to popularization, and microRNA can exist steadily in the long term in serum, be difficult by RNA Enzyme is degraded, and is boiled, multigelation, acid or alkali environment, long-term the loss that extreme condition does not result in serum microRNA such as is preserved.

Brief description of the drawings

Fig. 1 is NSCLC patient's RNA quality testing electrophoretograms.

Fig. 2 is microRNA chip scanning figures.

Fig. 3 is the differential expression microRNA between different pathological by stages distal end normal structure.

A：Ia phases and the comparison of IIa phases；B：IIa phases and the comparison of IIIa phases；C：Ia phases and the comparison of IIIa phases.

Fig. 4 is the differential expression microRNA between different pathological by stages cancer beside organism.

A：Ia phases and the comparison of IIa phases；B：IIa phases and the comparison of IIIa phases；C：Ia phases and the comparison of IIIa phases

Fig. 5 is the differential expression microRNA between different pathological by stages cancerous tissue.

A：Ia phases and the comparison of IIa phases；B：IIa phases and the comparison of IIIa phases；C：Ia phases and the comparison of IIIa phases.

Fig. 6 is distal end normal structure, the differential expression microRNA between cancer beside organism and cancerous tissue.

A：Distal end normal structure and the comparison of cancer beside organism；B：Cancer beside organism and the comparison of cancerous tissue；C：Distal end normal group Knit the comparison with cancerous tissue.

Fig. 7 is distal end normal structure, the clustering of the differential expression microRNA between cancer beside organism and cancerous tissue.

Fig. 8 is the clustering figure of the differential expression microRNA between lymphatic metastasis group and non-lymphatic metastasis group.

Fig. 9 is differential expressions of the hsa-miR-1280 in cancerous lung tissue and distal end normal structure.

Figure 10 is that hsa-miR-1280 is compared in terms of cancerous tissue and distal end normal structure Sensitivity and Specificity.

Embodiment

Embodiment 1：

NSCLC tissue samples and plasma specimen storehouse are improved, patient's pathological data and postoperative follow-up data is collected and arrange；

(1) clinical and pathological data of 120 NSCLC patients and the postoperative follow-up data database of some patientss are established；

(2) the tissue samples storehouse of 120 NSCLC patients is established；

(3) the periphery blood plasma Sample Storehouse 30 of NSCLC patient and part patients with recurrent is established.

Embodiment 2：

The microRNA array experiments of NSCLC tissue samples

(1) screening of microRNA array experiments sample

For ensure cDNA microarray sample histopathology data homogeneity, from the NSCLC tissue samples having built up Selection meets the case 9 of following inclusion criteria in storehouse：Male；- 70 years old 50 years old；Gland cancer；It is normal that every case includes distal end Tissue, cancer beside organism and cancerous tissue.Wherein clinical stages be respectively the Ia phases 3 (sample number is 511587,541056 respectively, 492827), IIa phases 3 (sample number is 435942,575901,473871 respectively) and (the sample number difference of IIIa phases 3 It is 399409,399514,509185).Every case takes its distal end normal structure respectively, and cancer beside organism and cancerous tissue are carried out MicroRNA chips are detected, are respectively labeled as N, P, C.Meanwhile, 9 cases include 4 non-lymphatic metastasis samples, and (sample is compiled Numbers 435942,492827,511587,541056) and 5 lymphatic metastasis samples (sample number 399409,473871, 509185,399514,575901).

(2) sample total RNA are extracted

Using mirVana^TMMiRNA Isolation Kit (Cat#AM1560, Ambion, Austin, TX, US), according to The Standard Operating Procedure that production firm provides carries out the total RNA extractings of sample, and extracting gained total RNA are through Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US) electrophoresis quality inspection, wherein A260/ A280>1.8, RIN values>5.0,28S/18S>=0.7 RNA samples be allowed for access next round experiment.After testing, needed for this research 27 RNA samples be satisfied by subsequent experimental requirement.

As a result as shown in Figure 1.

Embodiment 3：

Array experiment

Chip used project is Agilent human miRNA (8*60K) V16.0 chips (design ID：31181), altogether There are 27 samples, it is necessary to complete 27 said chips, commission biochip Shanghai National Engineering Research Centre is completed.Specific steps Including：

1) sample RNA mark

Laboratory sample RNA is using the supporting kit of Agilent miRNA chips, miRNA Complete Labeling And Hyb Kit (Cat#5190-0456, Agilent technologies, Santa Clara, CA, US), are grasped according to standard The mark part for making flow carries out fluorescence labeling to the miRNA molecule in sample.

2) chip hybridization

According to the Standard Operating Procedure and matched reagent box of the supporting offer of Agilent miRNA chips, miRNA Complete Labeling and Hyb Kit(Cat#5190-0456,Agilent technologies,Santa Clara, CA, US) hybridization portion, carry out sample hybrid experiment.In hybrid heater is rolled, Hybridization Oven (Cat# G2545A, Agilent technologies, Santa Clara, CA, US), 55 DEG C, 20rpm rolls hybridization 20 hours.Hybridization After the completion of developed a film in cylinder staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) are washed, develop a film Reagent used is Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent technologies,Santa Clara,CA,US)。

3) result is scanned

Chip results use Agilent Microarray Scanner (Cat#G2565BA, Agilent Technologies, Santa Clara, CA, US) it is scanned, with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) reads data, software design patterns Scan resolution=5 μ M, PMT 100%, 5% is last using Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US) it is normalized, algorithm used is Quantile.

As a result as shown in Figure 2.

Embodiment 4：

Array experiment Quality Control situation

The coefficient of variation (CV values) that the technology of all 27 chips is repeated is<10, illustrate that the chip detection architecture is stable；Respectively Chip recall rate scope is 20%-40%, meets normal recall rate requirement；The box traction substation analytic explanation point of each chip hybridization data Cloth and degree of scatter are preferable.Each quality control standard illustrates that chip results stability is good above, and sensitivity is high, reliable results.

Embodiment 5：

1) the differential expression microRNA of different pathological by stages between the normal structure of distal end

Compare the differential expression microRNA between IaN, IIaN, 3 groups of IIIaN, find not find between IaN and IIaN Differential expression microRNA；There are the microRNA of 11 differential expressions, including hsa-miR- between IaN+IIaN groups and IIIaN groups 205, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p, hsa-miR-376a, hsa-miR-429, hsa-miR- 449a, hsa-miR-449b, hsa-miR-495, hsa-miR-500a, hsa-miR-650.Primary Study illustrates Ia the and IIa phases The distal end normal structure of patient does not occur significant microRNA changes, but the distal end normal structure of IIIa phase patients has been opened There is the change of microRNA expression quantity in beginning.

As a result as shown in Figure 3.

2) the differential expression microRNA of different pathological by stages between cancer beside organism

Compare the differential expression microRNA between IaP, IIaP, 3 groups of IIIaP, find there are 3 between IIaP and IIIaP MicroRNA differential expressions；But have 9 microRNA differential expressions between IaP and IIaP, there are 23 between IIIaP MicroRNA differential expressions；Primary Study illustrates that compared with Ia phase patients the cancer beside organism of IIa phases and patient IIIa have begun to There is the change of microRNA expression quantity.

As a result as shown in Figure 4.

3) the differential expression microRNA of different pathological by stages between cancerous tissue

Differential expression microRNA between 3 groups of IaC, IIaC, IIIaC is unlike distal end normal structure and cancer beside organism The microRNA of group difference expression is more, and only hsv2-miR-H7-3p expression is variant between wherein IIaC and IIIaC；hsa- Expression quantity of the miR-23a* in IIaC and IIIaC is far above IaC.

As a result as shown in Figure 5.

4) distal end normal structure, the differential expression microRNA between cancer beside organism and cancerous tissue

Almost do not occur the change of microRNA expression quantity between distal end normal structure and cancer beside organism, only find hsa- MiR-338-5p is substantially less than distal end normal structure in the expression quantity of cancer beside organism；But have 36 between cancer beside organism and cancerous tissue Individual microRNA expression quantity there occurs significant changes；The microRNA of differential expression between distal end normal structure and cancerous tissue There are 55, illustrate that cancerous tissue microRNA expression quantity compared with the normal structure of distal end there occurs extensive change.With above-mentioned sieve 55 microRNA selected are object, carry out clustering to 27 samples, as a result show according to the differential expression 27 samples can be divided into cancerous tissue and the major class of non-cancer tissue two well by microRNA express spectras.

As a result such as accompanying drawing 6, shown in accompanying drawing 7.

Especially, a kind of hsa-miR-1280 genes very related to non-small cell lung cancer are screened.

The sequence of the hsa-miR-1280 genes is：5 '-UCCCACCGCUGCCACCC-3 ', it is in cancerous tissue and distal end Normal group is woven with extremely significant expression quantity difference.

5) the differential expression microRNA between lymphatic metastasis group and non-lymphatic metastasis group

It was found that microRNA3 of significant changes, including hsa-miR- occur for expression quantity in lymphatic metastasis group 1260b, hsa-miR-423-3p, hsa-miR23a-5p, as shown in Figure 8.

Using above-mentioned 3 microRNA filtered out as object, clustering is carried out to 9 cancerous tissue samples, as a result shown 9 samples can be divided into well by lymphatic metastasis group according to the microRNA express spectras of the differential expression and non-lymph is carried down The major class of shifting group two.

Embodiment 6：

Expand tumor sample checking microRNA array experiment results

(1) RNA extractings and quality control

78 NSCLC patient tissues samples are selected to carry out hsa-miR-145, hsa-miR-182 and hsa-miR-213 The checking work of microRNA chips, RNA extractings and method of quality control are ibid.

(2) reverse transcription and Real-PCR

Reverse transcription is carried out using ReverAid First cDNA strand kit (Fermentas).Take 1ug total RNA, adds 1ul random hexamer primer, and moisturizing to 12ul after 65 DEG C of water-bath 5min, is immediately placed on ice.Plus Enter 4ul 5*reaction buffer, RiboLock Rnase inhibitor 1ul, 10mM dNTP Mix 2ul, ReverAid M-MuLV Reverse Transcriptase 1ul.After 25 DEG C of 5min, 42 DEG C of 60min terminate after 70 DEG C of 5min Reaction, reverse transcription reaction is carried out in the PCR instruments of ABI 9700.Product is put in -20 DEG C of short-term preservations or -70 DEG C long-term preservation.Make Used time, by 20 times of cDNA product dilutions.MicroRNA primers are synthesized by ABI companies.System response system：2ul cDNA, 10ulPremix Ex TaqTM II (2 ×), 0.4ul 10uM upstream and downstream primers, 0.4ulROX II are added water to 20ul.95 DEG C of 5s of 40 circulations after amplified reaction, 95 DEG C of 10sec, 60 DEG C are carried out on the quantitative real time PCR Instruments of ABI 7900 34sec。

(3) experimental result

Distal end normal structure, cancer beside organism and cancerous tissues of difference 3 microRNA of quantitative analysis in 78 NSCLC patients In expression quantity；Research finds that expression quantity of the hsa-miR-21 in 66 distal end normal structures and cancer beside organism is substantially less than cancer Expression quantity in tissue, expression quantity of the hsa-miR-145 in 75 distal end normal structures and cancer beside organism is significantly higher than cancer group Expression quantity in knitting, the result of study is consistent substantially with chip results, therefore we plan to select after this 2 microRNA progress Continuous research.

Embodiment 7：

Differential expressions of the hsa-miR-1280 in cancerous lung tissue and distal end normal structure

Q-PCR checkings are carried out in 72 pairs of non-small cell cancerous tissues and its distal end normal structure, miR-1280 is as a result shown Expression quantity in Non-Small Cell Lung Carcinoma is significantly higher than distal end normal structure.This proof can be by detecting hsa- in tissue MiR-1280 expression quantity detects non-small cell lung cancer.

As a result it is as shown in table 1.

Table 1

Differential expressions of the hsa-miR-1280 in cancerous lung tissue and distal end normal structure is as shown in accompanying drawing 9.To hsa- MiR-1280 is compared in terms of cancerous tissue and distal end normal structure Sensitivity and Specificity, AUC=0.772, optimal critical Susceptibility during point is 46.4%, and specificity is 95.7%, as a result as shown in Figure 10.

Embodiment 8：

Analysis is associated to expression quantity situations of change of the serum miR-1280 in serum and corresponding pathology Follow-up Data, The blood serum designated object that prediction miR-1280 can be diagnosed as NSCLC.

The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.

Claims (1)

1. a kind of hsa-miR-1280 gene related to non-small cell lung cancer is in Diagnosis of Non-Small Cell Lung kit is prepared Application, the nucleotides sequence of the hsa-miR-1280 is classified as：5’-UCCCACCGCUGCCACCC-3’.