CN109706146A

CN109706146A - Application of the finger-print of tiny RNA composition in the Cancerous Pleural Effusion diagnosing and treating of people

Info

Publication number: CN109706146A
Application number: CN201711014853.0A
Authority: CN
Inventors: 朱苗骏; 谭若颖; 徐静; 张新
Original assignee: Shanghai Xiang Qiong Biological Technology Co Ltd
Current assignee: Shanghai Xiang Qiong Biological Technology Co Ltd
Priority date: 2017-10-25
Filing date: 2017-10-25
Publication date: 2019-05-03
Anticipated expiration: 2037-10-25
Also published as: CN109706146B

Abstract

The present invention relates to application of the finger-print of tiny RNA composition in the Cancerous Pleural Effusion diagnosing and treating of people.By being screened to nearly 2,000 miRNA, finds the miRNA combination of a series of specificity, can effectively distinguish Cancerous Pleural Effusion and Benign pleural effusions.

Description

The finger-print of tiny RNA composition is in the Cancerous Pleural Effusion diagnosing and treating of people Using

Technical field

The present invention relates to biomedical, bioengineering and detection technique fields, and in particular, to the fingerprint of tiny RNA composition Application of the map in the Cancerous Pleural Effusion diagnosing and treating of people.

Background technique

Pleural effusion also known as be hydrothorax, is a kind of clinical common Diagnosis of Chest Damage in Patients, a variety of diseases such as tuberculous pleurisy, the heart It declines, Hypoproteinemia and tumour etc. can lead to generation (the Light RW. of hydrothorax；2002).Although clinical common, the cause of disease Complicated and changeable, and etiological diagnosis determines treatment method.And show have in adult pleural effusion according to related data 38%-52% or so is malignant pleural effusion (Konstantin A.Dimitriadis；2001).Malignant pleural effusion (Malignant pleural effusion, MPE) refers to the malignant tumour of the malignant tumour or other positions that are primary in pleura It is transferred to pleural effusion caused by pleura.According to statistics, until 2014, the number of the infected of the annual MPE in the U.S. is more than 150,000 people.Almost All malignant tumours may occur in which MPE.Wherein lung cancer is the most common cause of disease, accounts for about the 1/3 of MPE, and breast cancer is taken second place, lymph Tumor is also the major reason for causing MPE occur, in recent years, oophoroma and gastrointestinal cancer occur MPE patient it is quite a few see, there are about The MPE patient of 5%-10% can not find primary tumo(u)r lesion (Xu Yajun et al.；2011).Occur MPE show tumour diffusion or To advanced stage, patient's life expectancy will significantly shorten.Since MPE calculate making a definite diagnosis, and median survival interval is 3-12 months, this with Primary tumor types and by stages related.Evidence show, MPE patient survival caused by lung cancer is most short, ovarian cancer patients longest, Can not find the MPE patient survival of primary tumor between it is above-mentioned between the two.

The treatment and prognosis of Benign pleural effusions and both malignant pleural effusions are completely different, thus the good pernicious mirror of hydrothorax It does not diagnose particularly significant.Such as associative BCI-algebra and Malignant Pleural can behave as courage and uprightness, be difficult area with appearance and routine inspection Point, but the treatment and prognosis of the two are completely different.The antidiastole mode of pleural effusion has very much, such as by clinical condition Shape, the routine inspection of pleural effusion and biochemical analysis to distinguish diffusate and omission timber, and combine imaging diagnosis and clinical doctor Raw judgement etc. determines the property of hydrothorax.However, these method specificity are relatively low；And Imaging Method is to pleural effusion The etiologic diagnosis of matter still has deficiency, is only capable of judging the property of pleural effusion indirectly according to lung, pleura, mediastinum and heart disease Matter.Diagnosis for MPE nowadays relies on thoracentesis and obtains pleural effusion progress cytological analysis.However, pleural effusion Cytological analysis only has 50-70% (Ong K C et al for the sensitivity for diagnosing MPE；2000).In addition, not having in hydrothorax It is found the case of cancer cell, thoracoscope biopsy of pleura can also be carried out, specify pathology.But this method is invasive surgical, and There are infection, the risk of the complication such as bleeding and chronic ache^[7].For patients in poor condition of health and with other complication, It is difficult to carry out.

From this, the malignant and benign lesion of pleural effusion is still a great problem clinically faced.Particularly with those Tumour merges heart failure, and tumour secondary obstructive pneumonia or atelectasis, tumor invading lymphatic vessel or mediastinal lymph nodes cause lymph The hydrothorax of the generations such as reflux obstacle is more difficult to differentiate between good pernicious.In addition, the cell component of pleural effusion is relatively complicated, In be contaminated with mesothelial cell, megacaryocyte, lymphocyte, neutrophil leucocyte or malignant cell etc..Wound, inflammation and tumour Etc. the hyperplasia for leading to mesothelial cell, and sometimes the degree of hyperproliferative cell between it is good it is pernicious between, thus it is thin Born of the same parents are difficult to distinguish benign disease, celiothelioma and adenocarcinoma metastatic on learning.

Therefore, there is an urgent need in the art to explore other more sensitive diagnostic methods (as deeply excavated Cancerous Pleural Effusion Fall ill relevant miRNAs) antidiastole pleural effusion is assisted, this is pre- for the diagnosis and treatment and assessment of instructing Cancerous Pleural Effusion There is important directive significance afterwards.

Summary of the invention

The purpose of the present invention is to provide effective tiny RNA finger-print, for carcinous malignant pleural effusion (preferably, Lung cancer type malignant pleural effusion) diagnosis, tumor grade and prognostic evaluation；And/or distinguish Cancerous Pleural Effusion and benign pleural Hydrops (preferably, distinguishing lung cancer type malignant pleural effusion and Benign pleural effusions).

In the present invention, first aspect provides a kind of isolated miRNA, the miRNA are as follows:

(I) sequence miRNA as shown in SEQ ID NO:n, wherein n is the positive integer selected from 1-32；

(II) miRNA complementary with sequence shown in SEQ ID NO:n；

Two or more combination of (III) sequence in the miRNA as shown in SEQ ID NO:1-32；Or (IV) with Two or more combination in the miRNA of sequence complementation shown in SEQ ID NO:1-32.

Second aspect of the present invention provides a kind of miRNA collection or combination, the miRNA collection or combination are as follows:

(a) two or more the combination in sequence miRNA as shown in SEQ ID NO:1-32；

(b) two or more the combination in the miRNA complementary with sequence shown in SEQ ID NO:1-32；Or

(c) at least one is from sequence miRNA as shown in SEQ ID NO:1-32 and at least one comes from and SEQ ID The combination that the miRNA of the complementation of sequence shown in NO:1-32 is constituted, wherein coming from sequence miRNA as shown in SEQ ID NO:1-32 Sequence and the sequence from the miRNA complementary with sequence shown in SEQ ID NO:1-32 be not mutually complementation.

In another preferred example, the miRNA collection or combination includes sequence sequence as shown in SEQ ID NO:1-32 In 4 kinds of miRNA.

In another preferred example, the miRNA collection or combination includes sequence 10 as shown in SEQ ID NO:1 and 5-13 Kind miRNA.

In another preferred example, the miRNA collection or combination includes sequence such as SEQ ID NO:1,14-15 and 18-21 Shown in 7 kinds of miRNA.

In another preferred example, the miRNA collection or combination include sequence such as SEQ ID NO:1,4,9,13,16-17, 12 kinds of miRNA shown in 22-23 and 29-32.

In another preferred example, the miRNA is isolated from people.

In another preferred example, the sequence can be expressed by chemical synthesis or building eukaryotic expression vector It obtains.

In another preferred example, the miRNA collection or combination is the tiny RNA finger-print being made of 4 miRNAs, is used In the diagnosis, tumor grade and prognostic evaluation of carcinous malignant pleural effusion (preferably, lung cancer type malignant pleural effusion)；And/or Distinguish Cancerous Pleural Effusion and Benign pleural effusions (preferably, distinguishing lung cancer type malignant pleural effusion and Benign pleural effusions)；

4 miRNAs are HSA-MIR-141, HSA-MIR-429, HSA-MIR-200a and HSA-MIR-96.

In another preferred example, the miRNA collection or combination is the tiny RNA finger-print being made of 10 miRNAs, For the diagnosis of carcinous malignant pleural effusion, tumor grade and prognostic evaluation；And/or distinguish Cancerous Pleural Effusion and benign pleural Hydrops；

10 miRNAs be HSA-MIR-141, HSA-MIR-140-5P, HSA-MIR-29c#, HSA-MIR-708, HSA-MIR-98, HSA-MIR-196b, HSA-MIR-106b, HSA-MIR-361-3P, HSA-MIR-412 and HSA-MIR- 642a。

In another preferred example, the miRNA collection or combination is the tiny RNA finger-print being made of 7 miRNAs, is used In the diagnosis of lung cancer type malignant pleural effusion, tumor grade and prognostic evaluation；And/or distinguish lung cancer type malignant pleural effusion and good Property pleural effusion；

7 miRNAs are HSA-MIR-141, HSA-MIR-145, HSA-MIR-29c, HSA-MIR-133a, HSA- MIR-708, HSA-MIR-149 and HSA-MIR-199b-5p.

In another preferred example, the miRNA collection or combination is the tiny RNA finger-print being made of 12 miRNAs, For the diagnosis of adenocarcinoma of lung type malignant pleural effusion, tumor grade and prognostic evaluation；And/or distinguish adenocarcinoma of lung type malignant pleural product Liquid and Benign pleural effusions；

12 miRNAs are HSA-MIR-141, HSA-MIR-429, HSA-MIR-98, HSA-MIR-106b, HSA- MIR-29b, HSA-MIR-328, HSA-MIR-20a, HSA-MIR-17, HSA-MIR-140-3p, HSA-MIR-139-3p, HSA- MIR-125b-2# and HSA-MIR-106a.

A kind of third aspect present invention provides separation or artificial constructed precursor miRNA, the precursor miRNA energy It is sheared in people's cell and is expressed as miRNA described in first aspect present invention.

The present invention also provides a kind of separation or artificial constructed precursor miRNA collection or combination, the precursor miRNAs Precursor miRNA in collection or combination can be sheared in people's cell and be expressed as the set of miRNA described in second aspect of the present invention Or the miRNA in combination.

Fourth aspect present invention provides a kind of isolated polynucleotides, and the polynucleotides can be transcribed by people's cell Precursor miRNA, the precursor miRNA can be sheared in people's cell and be expressed as miRNA described in first aspect present invention；

Preferably, the polynucleotides have structure shown in Formulas I:

Seq forward direction-X-Seq inverse type I,

In Formulas I,

Seq forward direction is the nucleotide sequence that the miRNA can be expressed as in people's cell,

Seq is reversed to be substantially complementary or the nucleotide sequence of complete complementary with Seq forward direction；

X be positioned at Seq is positive and Seq it is reversed between intervening sequence, and the intervening sequence and Seq is positive and Seq It is reversed complementary,

And structure shown in Formulas I forms secondary structure shown in Formula II after being transferred to people's cell:

Formula II,

In Formula II, Seq is positive, Seq is reversed and X is as defined above and states,

| | indicate the base pair complementarity relationship formed between Seq is positive and Seq is reversed.

The present invention also provides a kind of isolated polynucleotides collection or combination, the polynucleotides collection or combination includes The polynucleotides of precursor miRNA can be transcribed by people's cell, the precursor miRNA can be sheared and be expressed as in people's cell MiRNA collection or combination described in second aspect of the present invention.

In another preferred example, one of described polynucleotides collection or combination or a variety of polynucleotides have Formulas I Shown in structure:

Seq forward direction-X-Seq inverse type I,

In Formulas I,

Formula II,

Fifth aspect present invention provides a kind of carrier, it contain miRNA isolated described in first aspect present invention or Polynucleotides described in miRNA collection or combination or fourth aspect present invention described in second aspect of the present invention.

Sixth aspect present invention provides miRNA or second aspect of the present invention isolated described in first aspect present invention The purposes of the miRNA collection or combination, is used to prepare chip or kit；The chip or kit are used for:

(1) diagnosis, tumor grade and the prognosis of carcinous malignant pleural effusion (preferably, lung cancer type malignant pleural effusion) are commented Valence；

(2) Cancerous Pleural Effusion and Benign pleural effusions are distinguished；

(3) lung cancer type malignant pleural effusion and Benign pleural effusions are distinguished；

(4) adenocarcinoma of lung type malignant pleural effusion and Benign pleural effusions are distinguished.

Seventh aspect present invention provides a kind of miRNA chip, and the miRNA chip includes:

Solid phase carrier；And

The oligonucleotide probe being orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to The sequence some or all of shown in the SEQ ID NO:1-32.

In another preferred example, the oligonucleotide probe contains:

Complementary combined area；And/or

The bonding pad being connected with solid phase carrier.

In another preferred example, the oligonucleotide probe specifically corresponds to complete shown in SEQ ID NO:1-4 Portion's sequence.

In another preferred example, the oligonucleotide probe specifically corresponds to shown in SEQ ID NO:1 and 5-13 Full sequence.

In another preferred example, the oligonucleotide probe specifically corresponds to SEQ ID NO:1,14-15 and 18- Full sequence shown in 21.

In another preferred example, the oligonucleotide probe specifically corresponds to

SEQ ID NO:1,4,9,13, full sequence shown in 16-17,22-23 and 29-32.

Eighth aspect present invention provides the purposes of miRNA chip described in seventh aspect present invention, is used to prepare differentiation Cancerous Pleural Effusion and Benign pleural effusions are (preferably, distinguish lung cancer type malignant pleural effusion and Benign pleural effusions；More preferably Adenocarcinoma of lung type malignant pleural effusion and Benign pleural effusions are distinguished in ground) kit.

Ninth aspect present invention provides a kind of kit, containing described in seventh aspect present invention in the kit MiRNA chip and/or detection reagent for miRNA collection or combination described in second aspect of the present invention.

In another preferred example, the kit is also containing miRNA collection or combination described in second aspect of the present invention For positive control.

It in another preferred example, further include specification in the kit, the specification, which describes, utilizes this hair The method that bright miRNA chip tests sequence shown in SEQ ID NO.:1-32.

Tenth aspect present invention provides a kind of screening anti-lung cancer type or adenocarcinoma of lung type malignant pleural effusion drug candidate Method the described method comprises the following steps:

(a) in experimental group, the lung cancer of lung cancer type or adenocarcinoma of lung type malignant pleural effusion is cultivated in the presence of test substance Cell (or lung adenocarcinoma cell)；And in control group, it is identical as the experimental group condition but be not present the test substance In the case where cultivate the lung carcinoma cell (or lung adenocarcinoma cell) of identical lung cancer type or adenocarcinoma of lung type malignant pleural effusion；

(b) measuring the lung carcinoma cell of lung cancer type in the experimental group or adenocarcinoma of lung type malignant pleural effusion, (or adenocarcinoma of lung is thin Born of the same parents) one or more miRNA expression, and the lung cancer with lung cancer type in control group or adenocarcinoma of lung type malignant pleural effusion The expression of the miRNA of cell (or lung adenocarcinoma cell) is compared；

Wherein, if compared with the control group, the expression of the miRNA in the experimental group is tended to In the variation of the expression of benign pleural ponding cell, then show that the test substance is that anti-lung cancer type or adenocarcinoma of lung type are pernicious The drug candidate of pleural effusion.

In another preferred example, the method also includes steps (c): the drug candidate being further processed and suffers from lung cancer type Or the non-human mammal of adenocarcinoma of lung type malignant pleural effusion, to measure the drug candidate to the non-human mammal The influence of lung cancer type or adenocarcinoma of lung type malignant pleural effusion.

In another preferred example, the miRNA is isolated miRNA described in claim 1.

In another preferred example, the miRNA is miRNA collection or combination as claimed in claim 2.

In another preferred example, " variation for being intended to the expression of benign pleural ponding cell has occurred " refers to For a certain miRNA, meet following formula:

Q≤0.6

Wherein, Q=abs (A1-A0)/abs (A2-A0)

In formula, A0 is miRNA expression described in benign pleural ponding cell；A1 is the miRNA table of experimental group Up to level；A2 is the miRNA expression of control group；Abs indicates absolute value.

In another preferred example, when the miRNA be lung cancer type (or adenocarcinoma of lung type) malignant pleural effusion up-regulation type (i.e. A2-A0 > 0) when, then A1-A0≤0 or Q≤0.6 (preferably≤0.5).

In another preferred example, when the miRNA be lung cancer type (or adenocarcinoma of lung type) malignant pleural effusion downward type (i.e. A2-A0 < 0) when, then A1-A0 >=0 or Q≤0.6 (preferably≤0.5).

In another preferred example, in the method, further include positive controls in step (a), i.e., with the reality Test that group condition is identical and there is no the test substances but there are known treatment lung cancer type or adenocarcinoma of lung type malignant pleural effusion medicines In the case where object, the lung carcinoma cell of identical lung cancer type or adenocarcinoma of lung type malignant pleural effusion is cultivated；

It also, further include by the one or more of lung carcinoma cell in the experimental group or lung adenocarcinoma cell in step (b) The expression of miRNA, and carried out with the expression of the miRNA of lung carcinoma cell or lung adenocarcinoma cell in the positive controls Compare.

In another preferred example, the miR is selected from: HSA-MIR-141, HSA-MIR-429, HSA-MIR-200a and HSA- MIR-96。

In another preferred example, the miR is selected from: HSA-MIR-141, HSA-MIR-140-5P, HSA-MIR-29c#, HSA-MIR-708, HSA-MIR-98, HSA-MIR-196b, HSA-MIR-106b, HSA-MIR-361-3P, HSA-MIR-412 and HSA-MIR-642a。

In another preferred example, the miR is selected from: HSA-MIR-141, HSA-MIR-145, HSA-MIR-29c, HSA- MIR-133a, HSA-MIR-708, HSA-MIR-149 and HSA-MIR-199b-5p.

In another preferred example, the miR is selected from: HSA-MIR-141, HSA-MIR-429, HSA-MIR-98, HSA- MIR-106b、HSA-MIR-29b、HSA-MIR-328、HSA-MIR-20a、HSA-MIR-17、HSA-MIR-140-3p、HSA- MIR-139-3p, HSA-MIR-125b-2# and HSA-MIR-106a.

Tenth one side of the invention provide it is a kind of it is external it is nondiagnostic judge cell or tissue whether be lung cancer type or Method of adenocarcinoma of lung type malignant pleural effusion or its tissue, comprising steps of the present invention the in the measurement cell or its tissue The expression of miRNA in miRNA collection or combination described in isolated miRNA or second aspect of the present invention described in one side, When the miRNA expression is compared with normal tissue, there is significant difference, then illustrate that the cell or tissue is lung cancer type Adenocarcinoma of lung type malignant pleural effusion or its tissue.

The twelfth aspect of the present invention provides a kind of method of diagnosing type or adenocarcinoma of lung type malignant pleural effusion sample, Comprising steps of miRNA collection described in miRNA or second aspect of the present invention isolated described in first aspect present invention in measurement sample Close or combination in the expression of miRNA there is significant difference when the miRNA expression is compared with normal sample, Then illustrate that the sample is lung cancer type or adenocarcinoma of lung type malignant pleural effusion sample.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is shown through SVM model and leave one cross validation method to 10 selected miRNA (table 2) to training set In Cancerous Pleural Effusion distinguish: A is that leave one cross validation result is based on SVM illustraton of model, and abscissa indicates each sample, institute There is sample (N=77) to be divided into two parts (cancer specimen 53 and benign sample 24), ordinate indicates each sample predictions For the probability value (>=0.5 be cancer,<0.5 is benign) of cancer, black color dots indicate that judicious sample, red point indicate to sentence The sample of dislocation accidentally；B is to obtain to cross-check the ROC curve figure obtained a result based on leaving-one method by the method for SVM model.

Fig. 2 is shown through SVM model and leave one cross validation method to 7 selected miRNA (table 2) in training set Lung cancer type malignant pleural effusion distinguish: A is that leave one cross validation result is based on SVM illustraton of model, and abscissa indicates each sample This, all samples (N=60) are divided into two parts (lung cancer sample 36 and benign sample 24), and ordinate indicates each sample It is predicted as the probability value (>=0.5 be cancer,<0.5 is benign) of cancer, black color dots indicate judicious sample, red point table Show the sample of misjudgment；B is to obtain to cross-check the ROC curve obtained a result based on leaving-one method by the method for SVM model Figure.

Fig. 3 is shown through SVM model and leave one cross validation method to 12 selected miRNA (table 2) to training set In gland cancer type malignant pleural effusion distinguish: A is that leave one cross validation result is based on SVM illustraton of model, and abscissa indicates each Sample, all samples (N=51) are divided into two parts (adenocarcinoma samples 27 and benign sample 24), and ordinate indicates each sample Originally it is predicted as the probability value (>=0.5 be cancer,<0.5 is benign) of cancer, black color dots indicate judicious sample, red point Indicate the sample of misjudgment；B is to show that the ROC to obtain a result based on leaving-one method crosscheck is bent by the method for SVM model Line chart.

Fig. 4 is shown through SVM model and leave one cross validation method to 10 selected miRNA (table 2) to test set In Cancerous Pleural Effusion judging result: be that leave one cross validation result is based on SVM illustraton of model, abscissa indicates each sample This, all samples (N=159) are divided into two parts (cancer specimen 94 and benign sample 65), and ordinate indicates each sample Originally it is predicted as the probability value (>=0.5 be cancer,<0.5 is benign) of cancer, black color dots indicate judicious sample, red point Indicate the sample of misjudgment.

Fig. 5 is shown through SVM model and leave one cross validation method to 7 selected miRNA (table 2) in test set Lung cancer type malignant pleural effusion judging result: be that leave one cross validation result is based on SVM illustraton of model, abscissa indicates each Sample, all samples (N=140) are divided into two parts (lung cancer sample 75 and benign sample 65), and ordinate indicates each Sample predictions are the probability value (>=0.5 be cancer,<0.5 is benign) of cancer, and black color dots indicate judicious sample, red Point indicates the sample of misjudgment.

Fig. 6 is shown through SVM model and leave one cross validation method to 12 selected miRNA (table 2) to test set In adenocarcinoma of lung type malignant pleural effusion judging result: be leave one cross validation result be based on SVM illustraton of model, abscissa indicate Each sample, all samples (N=122) are divided into two parts (57, adenocarcinoma of lung sample and benign sample 65), ordinate table Show probability value that each sample predictions are cancer (>=0.5 be cancer,<0.5 is benign), black color dots indicate judicious sample Product, red point indicate the sample of misjudgment.

Specific embodiment

The present inventor is sifted out several by extensive and in-depth research by screening to nearly 2,000 miRNA for the first time The miRNA of specificity, inspection proves that, the miRNA marker of these specificity is carried out certain combination can be effectively Distinguish Cancerous Pleural Effusion and Benign pleural effusions.The miRNA that the present inventor also filters out several specificity for the first time can be very It is effective to distinguish lung cancer type malignant pleural effusion and adenocarcinoma of lung type malignant pleural effusion.The invention also provides miRNA compositions Application of the finger-print in Cancerous Pleural Effusion diagnosis.On this basis, the present invention is completed.

MiRNA and its precursor

The present invention provides a new class of miRNA found from people.As used herein, " miRNA " refers to one Kind RNA molecule is processed from the transcript that can form miRNA precursor.Mature miRNA usually has 18-26 nucleotide (nt) (more particularly about 19-22nt) is also not excluded for the miRNA molecule with other number nucleotide.MiRNA usually can quilt Northern trace detects.

The miRNA in people source can be separated from people's cell.As used herein, " separation " refers to substance from its original ring (if it is crude, primal environment is natural surroundings) is separated in border.Under the native state in active somatic cell Polynucleotide and polypeptide do not isolate and purify, but same polynucleotide or polypeptide are deposited together such as from native state Other substances in separate, then isolate and purify.

MiRNA can be processed from precursor miRNA (Precursor miRNA, Pre-miRNA), the precursor miRNA It is can be folded into a kind of stable stem ring (hair clip) structure, the loop-stem structure length is generally between 50-100bp.Described Precursor miRNA can be folded into stable loop-stem structure, and the stem two sides of loop-stem structure include the two sequences being substantially complementary.Institute The precursor miRNA stated can be natural or artificial synthesized.

Precursor miRNA, which can be sheared, generates miRNA, and the miRNA can be at least part of the mRNA of encoding gene Sequence is substantially complementary.As used herein, it " is substantially complementary " and refers to that the sequence of nucleotide is enough complementations, it can be with one kind Foreseeable mode interacts, and such as forms secondary structure (such as loop-stem structure).In general, the core of two " being substantially complementary " Nucleotide sequence from each other at least 70% nucleotide be complementary；Preferably, at least 80% nucleotide is complementary； It is furthermore preferred that at least 90% nucleotide is complementary；It is further preferred that at least 95% nucleotide is complementary； Such as 98%, 99% or 100%.Generally, most 40 unmatched nucleosides be can have between two molecules complementary enough Acid；Preferably, there are most 30 unmatched nucleotide；It is furthermore preferred that having most 20 unmatched nucleotide；Into one Step is preferred, has most 10 unmatched nucleotide, such as has 1,2,3,4,5,8,11 unmatched nucleotide.

As used herein, " stem ring " structure is also referred to as " hair clip " structure, refers to a kind of nucleic acid molecule, can form one Kind includes the secondary structure of double-stranded region (stem), and the double-stranded region (is located at same by two regions of the nucleic acid molecule On one molecule) it is formed, the two sides of column double stranded section are divided in two regions；It further includes at least one " ring " structure, including incomplementarity Nucleic acid molecule, i.e. single-stranded regions.Even if two regions of the nucleic acid molecule are not complete complementary, the double-strand of nucleotide Part can also keep double-stranded state.For example, insertion, missing, substitution etc. can lead to not complementary or zonule of a zonule Itself forms the secondary structure of loop-stem structure or other forms, however, two regions can be still substantially complementary, and is being contemplated that Mode in interact, form the double-stranded region of loop-stem structure.Loop-stem structure be it is well-known to those skilled in the art, Usually after the nucleic acid for obtaining a nucleotide sequence with primary structure, those skilled in the art can determine the nucleic acid Whether loop-stem structure can be formed.

MiRNA of the present invention has the sequence as shown in SEQ ID NO:n, and wherein n is the positive integer selected from 1-32.

In order to improve the stability or other properties of miRNA, also at least one can be added at least one end of the miRNA A protectiveness base, such as " TT ".

Herein, miRNA, miRN, tiny RNA, microRNA, miR have the same meaning.

For the miRNA of disclosed Cancerous Pleural Effusion specificity, conventional miRNA chip skill can be passed through Art is verified, and for example including miRNA is extracted with conventional method or conventional kit, is then detected.Representative reagent Box includes (but being not limited to): the miRNAs extraction agent box extracting of Qiagen or Ambion company.

In addition, can also pass through specific amplification and detect amplified production detectable signals such as (or corresponding) fluorescence signals come Detect or verify the miRNA of Cancerous Pleural Effusion specificity of the invention.Preferred highly sensitive and high specific technology Including (but being not limited to): the technology disclosed in CN10267663A.In general, the specific binding region of the primer can basis The sequence of the known miRNA of required detection is designed, preferably expand when the primer specific binding region be usually with The complementary series of miRNA complete complementary.

Antisense oligonucleotides

Provided miRNA sequence according to the present invention can be designed that their antisense oligonucleotides, the antisense Oligonucleotides can lower the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotides (antisense-ol Igonucleotides, AS-Ons or ASO) " be also known as " GEM 132 ", refer to length be about 18-26nt (more particularly about DNA molecular or RNA molecule or its analog 19-22nt).

In the present invention, " antisense oligonucleotides " further includes using as based on nucleic acid lock or nucleic acid chains backbone modification The modified GEM 132 that the means such as technology obtain, the modification do not change the activity of antisense oligonucleotides substantially, more Goodly, described to modify the stability, activity or therapeutic effect that antisense oligonucleotides can be improved.Nucleic acid locks (locked nucleic Acid, LNA) typically refer to the modification skill for the 2' oxygen atom of ribose and 4' carbon atom being connected by a methylene bridge Art.LNA can extend the serum half-life of miRNA, improve to target compatibility, reduce the range and degree of effect of missing the target.It is based on The antisense drug that the modification technique of nucleic acid chain backbone develops has greatly improvement in solubility, nuclease-resistant degradation etc., and easily In a large amount of synthesis.There are many backbone modification methods of oligonucleotides, including thio method, such as by deoxynucleotide chain thio-modification For thio deoxynucleotide chain.This method is to substitute the oxygen atom of the phosphate bond on DNA skeleton with sulphur atom, can resist nucleic acid Enzyme degradation.It should be understood that any largely or entirely active modification for being able to maintain the antisense oligonucleotides is included in this In invention.

As preferred embodiment of the invention, nucleic acid lock modification is carried out to antisense oligonucleotides；More preferably also carry out thio repair Decorations.

After antisense oligonucleotides of the present invention transfer into the human body, they can obviously lower related miRNA's Expression.

Polynucleotides construction

Provided people's miRNA sequence according to the present invention, can be designed can be processed to influence after being imported into accordingly MRNA expression miRNA polynucleotides construction namely the polynucleotides construction can raise in vivo accordingly The amount of miRNA.Therefore, the present invention provides a kind of isolated polynucleotides (construction), the polynucleotides (construction) Precursor miRNA can be transcribed by people's cell, the precursor miRNA can be sheared by people's cell and be expressed as the miRNA.

As a kind of preferred embodiment of the invention, the polynucleotides construction contains structure shown in Formulas I:

Seq_{It is positive}-X-Seq_ReverselyFormulas I,

In Formulas I,

Seq_{It is positive}For the nucleotide sequence that can be expressed as the miRNA in cell, Seq_ReverselyFor with Seq_{It is positive}Substantially mutually The nucleotide sequence of benefit；Alternatively, Seq_ReverselyFor the nucleotide sequence that can be expressed as the miRNA in cell, Seq_{It is positive}For with Seq_{It is positive}The nucleotide sequence being substantially complementary；

X is positioned at Seq_{It is positive}And Seq_ReverselyBetween intervening sequence, and the intervening sequence and Seq_{It is positive}And Seq_ReverselyNot mutually It mends；

Structure shown in Formulas I forms secondary structure shown in Formula II after being transferred to cell:

Formula II,

In Formula II, Seq_{It is positive}、Seq_ReverselyIt is as defined above and states with X；

| | it indicates in Seq_{It is positive}And Seq_ReverselyBetween the base pair complementarity relationship that is formed.

In general, the polynucleotides construction is located on expression vector.Therefore, the invention also includes a kind of carrier, it Contain the miRNA or the polynucleotides construction.The expression vector usually also contains promoter, replicates Point and/or marker gene etc..Method well-known to those having ordinary skill in the art can be used to construct the required expression vector of the present invention.This A little methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The expression vector preferably includes One or more selected markers, to provide the phenotypic character for selecting the host cell of conversion, such as kalamycin, celebrating Big mycin, hygromycin, amicillin resistance.

Chip

MiRNA chip of expression spectrum usually contains up to several hundred a probes, covers a variety of miRNA, homologous mutually using DNA double chain The principle of benefit detects the content of contained various miRNA in sample in full-length genome level.It therefore, can be in the same time to be measured The transcriptional level of miRNA in sample within the scope of full-length genome is detected.

Using miRNA sequence of the present invention, corresponding miRNA chip can also be prepared, and then studies its express spectra And the regulative mode of miRNAs.

On the other hand, the present invention also provides a kind of for analyzing the chip of miRNA express spectra, and the chip can be used for Distinguish Cancerous Pleural Effusion and Benign pleural effusions.

The miRNA chip of the invention includes:

Solid phase carrier；And

The oligonucleotide probe being orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to In the sequence shown in SEQ ID NO:1-32 it is at least one kind of (such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32 kind).

Specifically, can miRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, formed " oligonucleotide arrays "." oligonucleotide arrays " refer to addressable point (i.e. with distinctive, addressablely The position that location is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to need It wants, oligonucleotide arrays can be divided into multiple sub- battle arrays.

The various common used materials in genetic chip field, such as, but not limited to nylon membrane can be used in the solid phase carrier, through work Property group (such as aldehyde radical, amino) slide or silicon wafer, unmodified slide, plastic sheet etc. for modifying.

The conventional manufacturing method of biochip known in the art can be used in the preparation of the miRNA chip.For example, such as For fruit solid phase carrier using modification slide or silicon wafer, amido modified poly- dT string is contained at the end 5' of probe, can be by oligonucleotides Probe is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence or array, Then it is fixed by standing overnight, so that it may obtain miRNA chip of the invention.If nucleic acid is without amido modified, system Preparation Method can also refer to: " the gene diagnosis technology-on-radiation operation manual " of Wang Shenwu chief editor；J.L.erisi, V.R.Iyer,P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.Science,1997；278:680 and Ma Li people, Jiang Zhonghua edit biology core The Beijing piece: Chemical Industry Press, 2000,1-130.

On the other hand, the present invention also provides a kind of sides that miRNA express spectra in people's tissue is detected by miRNA chip Method, comprising steps of

(1) RNA sample for being isolated from people's tissue, the setting flag object on the RNA are provided；

(2) RNA that step (1) obtains is contacted with the miRNA chip, is made on the RNA and solid phase carrier Hybridization reaction occurs for oligonucleotide probe, to form " oligonucleotide probe-RNA " binary complex on solid phase carrier；

(3) marker for the binary complex that detecting step (2) is formed, so that it is determined that corresponding miRNA in people's tissue Express spectra.

Extracting the method for RNA from people's tissue is method well known to those skilled in the art, including Trizol method.

It is furthermore preferred that after isolating RNA sample in people's tissue tissue, being fitted to RNA sample in step (1) Work as processing, to be enriched with the RNA with certain length, the length (small fragment RNA) generally between 10-100.By above-mentioned After processing, subsequent hybridization is carried out using these small fragment RNAs, the accuracy of chip capture miRNA can be improved in this way.This field Personnel are convenient to isolate the RNA with certain fragment length, for example gel electrophoresis can be used to separate.

It is also method well known to those skilled in the art that RNA, which is marked, can be special with RNA by being added in hybridization The method for the marker that the opposite sex combines realizes that the marker is such as labelling groups.The labelling groups include but unlimited In: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and its derivative biomolecule (FITC etc.), other fluorescence point Sub (such as Cy3, Cy5), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc..These labels and its labeling method are all It is routine techniques well-known in the art.

When above-mentioned RNA is hybridized with miRNA chip, first miRNA chip and pre-hybridization buffer can be carried out Prehybridization.

Solid-phase hybridization between RNA and miRNA chip of the present invention is carried out according to the classical way of this field, ability Domain general staff is empirically easy to determine related buffer, probe and concentration of specimens, prehybridization temperature, hybridization temperature with timely Between equal optimum condition.Or it is also referred to described in " Molecular Cloning:A Laboratory guide ".

Then the acquisition of information such as position, intensity according to marking signal on miRNA chip wait for measurement information.If amplified production It is marked with fluorophor, fluorescence detection device (such as laser confocal scanner Scanarray 3000) can also directly be used to obtain To measurement information.

Detection kit

The present invention also provides a kind of kit, chip of the invention is contained in the kit.The kit It can be used for detecting the express spectra of miRNA；Or for distinguishing Cancerous Pleural Effusion and Benign pleural effusions (preferably, lung cancer type is disliked Property pleural effusion and Benign pleural effusions, more preferably, adenocarcinoma of lung malignant pleural effusion and Benign pleural effusions).

It is furthermore preferred that in the kit also containing for labeled RNA sample marker, and with the marker Corresponding substrate.

In addition, may also include in the kit for various reagents needed for extracting RNA, PCR, hybridization, colour developing etc., Including but not limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion, antibody etc..Can also it contain in amplification liquid There are fluorescent dye, such as EvaGreen, SYBRGreen.It may also include primer in the kit.

In addition, may also include operation instructions and/or chip image analysis software in the kit.

The feature that the features described above or embodiment that the present invention mentions are mentioned can be in any combination.Disclosed in this case specification All features can be used in combination with any composition form, each feature disclosed in specification, can by it is any provide it is identical, The alternative characteristics of impartial or similar purpose replace.Therefore except there is special instruction, revealed feature is only impartial or similar spy The general example of sign.

Main advantages of the present invention include:

(1) the present invention provides the miRNA finger-prints that one kind can be used for preferably distinguishing Cancerous Pleural Effusion；

(2) the present invention provides the miRNA finger-prints that one kind can be used for distinguishing Cancerous Pleural Effusion well；

(3) miRNA finger-print of the invention can effectively distinguish Cancerous Pleural Effusion (such as lung cancer thoracic cavity product Liquid) and normal pleural effusion, high sensitivity, high specificity；

(4) miRNA finger-print of the invention can effectively distinguish Cancerous Pleural Effusion (such as adenocarcinoma of lung type thoracic cavity Hydrops) and normal pleural effusion, high sensitivity, high specificity；

(5) miRNA finger-print of the invention can be effectively used for diagnosis, parting and the prognostic evaluation of Cancerous Pleural Effusion.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and Number is calculated by weight.

Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art Justice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen Zhong The preferred implement methods and materials are for illustrative purposes only.

Unless stated otherwise, otherwise material used in description of the invention and reagent are commercial product.

Embodiment 1

The collection and information analysis of sample

Sample follows marine mountain Hospital Ethical Committee approval, and in the signature of Informed choice letter of consent, according to The method that upper marine mountain hospital pathology department collects pleural effusion obtains.236 pleural effusion samples are obtained from patient thoracic cavity in total It obtains and is stored in -80 DEG C.Sample is divided into two parts, and a part of sample is as training set, for establishing analysis model (77), Another part sample is used for double-blind comparative study (159), see Table 1 for details for sample information as double-blind comparative study collection.Male to female ratio in sample About 1.5:1, sample mean age are about 61.6 years old.

1 sample information of table

Embodiment 2

Total serum IgE extracting

Above-mentioned fresh pleural effusion is collected by centrifugation pretreatment, sample pellet and supernatant are respectively put into -80 degree refrigerators It freezes.

Hydrothorax sample is referring to miRNeasy MiniKit (Qiagen, 217004) specification, extracted total RNA.Concrete operations Step is carried out according to total serum IgE method in Xiang Qiong company extracting hydrothorax.

Electrophoresis detection quality calculates RNA concentration with determined by ultraviolet spectrophotometry OD260nm and OD280nm.- 80 DEG C of guarantors It deposits.

Embodiment 3

Add PolyA tail and reverse transcription

Above-described embodiment 2 is extracted to obtained total serum IgE uses the 0.1x RNA containing 0.1%Tween-20 (Sigma) to store Buffer (Ambion, USA) is diluted to 125ng/ul.

It is produced with Xiang Qiong companyMiRNA cDNA synthesizes box (Xiang fine jade Products number: 9000004) The tail of PolyA is added for miRNA, and reverse transcription is cDNA.Specific steps are as follows:

1.5ml centrifuge tube is placed in the total serum IgE for being added that 66 microlitres of concentration are 125ng/ul on ice, 33 microlitres of Xiang Qiong companies ProductionMiRNA cDNA synthesis reaction solution I (Xiang fine jade Products number: 9000005) 11 microlitres of Xiang Qiong companies ProductionMiRNA cDNA synthesis reaction solution II (Xiang fine jade Products number: 9000006)), and removes nuclease Ultrapure water to 165 microlitres.The final concentration of 50ng/ul of total serum IgE amount is distributed into the PCR pipe of 3 0.2ml, often after soft mixing Pipe 50ul.It is put into ABI9700PCR instrument after 1000rpm centrifugation 10s to be reacted, response procedures: 37 DEG C of 15min, 25 DEG C of 25min, 37 DEG C of 30min, 85 DEG C of 5min, 4 DEG C of holdings.

PCR pipe is taken out, 3 pipe RT products are merged, saves or be directly used in qPCR reaction for -20 DEG C after concussion centrifugation.

Embodiment 4

Fluorescence quantitative PCR detection

The method with reference to disclosed in CN10267663A detects tiny RNA.

The reverse transcription product that 24 microlitres of embodiments 3 obtain, 600 microlitres of fluorescent quantitation are added in 15 milliliters of centrifuge tube PCR enzyme reaction solution (Xiang fine jade Products number: 9000008,2x Universal qPCR Master Mix High Rox), 216 microlitres are gone nuclease ultrapure water (Xiang fine jade Products number: 9000015), soft to mix.

The diagnosis of pleural effusion that Xiang Qiong company is produced tiny RNA reaction template, SharpvueTMHuman miRNA (Xiang fine jade Products number: 1100001) taking out from -20 DEG C of refrigerators Array- hydrothorax, and packaging is opened after being returned to room temperature Bag, is placed on centrifuge, and 2000g is centrifuged 5min (Thermo, ST16R, rotary head model: M-20).Totally 93 tiny RNA reaction solutions, There are 2 positive controls and 1 blank control in reaction template.Carefully unlock sealer.

The mixed liquor that abovementioned steps are obtained pours into loading slot, is distinguished mixed liquor line by line using 12 continuous liquid-moving machines It is added in above-mentioned tiny RNA reaction template, every hole 7ul.Check whether each boreliquid amount is uniform after sample-adding.

Using being mixed by inversion after quantitative sealing plate film (ABI, 4711971) sealing plate, room temperature 1000g is centrifuged 5min.

It is put into quantitative PCR apparatus (ABI, 7900Ht Fast) and does quantitative PCR.Program are as follows: 95 DEG C of 10min, rear 95 DEG C of operation 5s, 58 DEG C of 1min, 3 circulations；95 DEG C of 5s later, 60 DEG C of 5s, 37 circulations, solubility curve.Reporter fluorescence is set as SYBR, reference Fluorescence is set as Rox.

Data are collected, bioinformatic analysis is carried out.

Embodiment 5

The calculating of miRNA biometric data is analyzed

We predict that patient suffers from the general of cancer by support vector machines (support vector machine, abbreviation SVM) Rate, support vector machines are a kind of sorting algorithms, and learning machine generalization ability is improved by seeking structuring least risk, realize warp The minimum of risk and fiducial range is tested, to reach in the case where statistical sample amount is less, can also obtain good statistics rule The purpose of rule, it is a kind of two classification model.

First we detection 1888 miRNA and two internal reference positive controls (HSA-RNU6B and HSA-RNU48) with One negative control (water), is further analyzed 1888 kinds of miRNA.The miRNA of detection pleural effusion has been determined Panel (1 piece of 96 orifice plate includes 93 miRNA and 3 internal references).The ct value and average value of each miRNA is by subtractive background, normalizing Change, uniform data ct value is less than or equal to 32.

We filter out the miRNA that can preferably distinguish cancer patient and non-cancer patient from 96 miRNA later Marker.It is contemplated that the miRNA additional amount to each sample may be different, need first to measure the miRNA of each sample Value is standardized, we use the difference of each sample miRNA detected value two-by-two as new variable, in this way when we are from 96 After selecting a subset in miRNA, the standardization of this sub- intensive data will be only related to the variable in this subset.Standardization We share C (96,2)+96=4560 new variables afterwards.We are with these variables of T checking computation in cancer patient's sample and non- Then conspicuousness between cancer patient's sample chooses most significant 20 new variables of difference.We finally wish to find quantity Variable of the miRNA as model within 12, so being selected within 12 from the miRNA set that this 20 new variables include All combinations of miRNA calculate the accuracy of every kind of combined prediction training set with SVM model in these combinations.Finally select The highest miRNA combination of accuracy is as final variables collection M.

E1071 software package of our the SVM model from R, 53 cancer patients that training data confirms from doctor With 24 non-cancer patients.Because control group and experimental group sample size differ greatly during model training, class.weights 53/24 is set by the weight of non-cancer clinical samples in variable, kernel function selects Radial basis kernel function (kernel=" Radial ") because wanting prediction probability, probability=TRUE is set.It is cancer and non-cancer to sample with predict function The probability of disease predicted, the calculating of accuracy is with staying a cross-validation method (LLO-CV).

The name of miRNA is the miRNA database according to miRBase Version 20, in contradictory situation, it then follows MiRNA database.

32 miRNA are chosen as the biomarker of diagnosis Cancerous Pleural Effusion of specificity, and the results are shown in Table 2.

Table 2

Embodiment 6

4 key miRNA are selected from miRNA variable, are preferably diagnosed to be Cancerous Pleural Effusion and lung cancer type malignant pleural Hydrops

When choosing sequence shown in SEQ ID NO.:1-4,4 miRNA are with the statistical method of SVM to training set The AUC that Cancerous Pleural Effusion obtains in (77 samples) is 0.897, accuracy 83.1%；Sensitivity is 84.9%, and specificity is 79.2%；Test set (only testing 61 samples) medium sensitivity is 63.1%, and specificity is 95.7%.4 miRNA are to instruction Practicing the AUC that lung cancer malignant pleural effusion obtains in collection (77 samples) is 0.898, accuracy 85%, sensitivity 77.8%, Specificity is 95.8%；It is sensitive in test set (61 samples only being tested, including 31 lung cancer type malignant pleural effusions) Degree is 64.5%, and specificity is 95.7%.

Embodiment 7

Crucial miRNA is further selected from miRNA variable, is diagnosed to be Cancerous Pleural Effusion

Permutation and combination is carried out to miRNA selected in embodiment 5, screens and test out specificity, sensitivity and accurate Degree can reach the horizontal best miRNA combination of clinical detection.By data statistic analysis, 5 groups of best miRNA combinations are found out, As a result, it has been found that can effectively be diagnosed to be Cancerous Pleural Effusion after part specificity miRNA is combined, medium sensitivity with Specificity is more excellent.Its five groups of miRNA combinations are as shown in table 3.

Table 3

Preferred result is as follows: when choose SEQ ID NO.:1 and 5-13 shown in sequence when, be labeled as cancer group, this 10 The ROC curve that miRNA obtains training set with the statistical method of SVM, as shown in Figure 1, AUC is 0.972, accuracy 93.5%, the sensitivity of selected label analyte detection Cancerous Pleural Effusion is 94.3%, and specificity is 91.7%；This 10 MiRNA carries out double-blind comparative study to test set, as shown in figure 4, obtained sensitivity is 71.3%, specificity is 87.7%；Test Collecting double blind accuracy is 78.0%.

Embodiment 8

Crucial miRNA is further selected from miRNA variable, is diagnosed to be lung cancer type malignant pleural effusion

Permutation and combination is carried out to miRNA selected in embodiment 5, screens and test out specificity, sensitivity and accurate Degree can reach the miRNA combination of clinical detection level.By data statistic analysis, 5 groups of best miRNA combinations are found out, are tied After part specificity miRNA is combined by fruit discovery, it can be effectively diagnosed to be lung cancer type malignant pleural effusion, wherein sensitive Degree and specificity are more excellent.Its five groups of miRNA combinations are as shown in table 4.

Table 4

Preferred result is as follows: when choosing SEQ ID NO.:1,8, sequence shown in 14-15 and 19-21, being labeled as lung cancer Group, the ROC curve which obtains training set with the statistical method of SVM, as shown in Fig. 2, AUC is 0.964, it is selected The sensitivity for the label analyte detection lung cancer type malignant pleural effusion selected is 91.7%, and specificity is 100%；7 miRNA are to survey Examination collection carries out double-blind comparative study, as shown in figure 5, obtained sensitivity is 84%, specificity is 84.6%；Test set accuracy is 84.3%.

Embodiment 9

Crucial miRNA is further selected from miRNA variable, is diagnosed to be adenocarcinoma of lung type malignant pleural effusion

To miRNA further progress permutation and combination selected in embodiment 5, specificity, sensitivity are screened and tested out The miRNA combination of clinical detection level can be reached.By data statistic analysis, 5 groups of best miRNA combinations are found out, as a result It was found that the combination of 10 miRNA composition can more effectively be diagnosed to be adenocarcinoma of lung type malignant pleural effusion.Its five groups of miRNA combinations are such as Shown in table 5:

Table 5:

Preferred result is as follows:

When choosing SEQ ID NO.:1,4,9,13, sequence shown in 16-17,22-23 and 29-32, it is labeled as adenocarcinoma of lung Group, the ROC curve which obtains training set with the statistical method of SVM, as shown in figure 3, AUC is 0.961, it is quasi- The sensitivity of exactness 92.2%, selected label analyte detection Cancerous Pleural Effusion is 85.2%, and specificity is 100%；This 12 A miRNA carries out double-blind comparative study to test set, as shown in fig. 6, obtained sensitivity is 91%, specificity is 89.2%；Test Collecting accuracy is 89.9%.

Conclusion

When selecting combination of the SEQ ID NO.:1-4 as miRNA finger-print, carcinous thoracic cavity can be preferably diagnosed Hydrops；And after optimizing the combination of miRNA in finger-print, when selecting SEQ ID NO.:1 and 5-13 to refer to as miRNA When the combination of line map, it can achieve and meet clinical requirement diagnosis Cancerous Pleural Effusion；And when selection SEQ ID NO.:1,8, When combination of the 14-15 and 19-21 as miRNA finger-print, it can more effectively be diagnosed to be lung cancer type malignant pleural effusion, and spirit Sensitivity and specificity are more excellent；When select SEQ ID NO.:1,4,9,13,16-17,22-23 and 29-32 as miRNA fingerprint image When the combination of spectrum, it can effectively be diagnosed to be adenocarcinoma of lung type malignant pleural effusion.On this basis, it is seen that miRNA fingerprint of the present invention Map can extremely efficiently distinguish Cancerous Pleural Effusion and Benign pleural effusions.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Bibliography

1. Xu second place, Che Feng, Cai Xiaodong etc., pleural effusion tumor marker detect in good malignant pleural effusion antidiastole It is worth the Hainan [J] medicine, 2011,22 (4): 51-52.

2.Konstantin A.DIMITRIADIS:Malignant pleural effusions.Archive of Oncology 2001；9(1):2.

Light RW.Clinical practice.Pleural effusion[J].N Engl J Med,2002,346: 1971-1977.

3.Bartel DP(2004)MicroRNAs:genomics,biogenesis,mechanism,and function.Cell 116:281-297.

4.Huang Y,Shen XJ,Zou Q,Zhao QL(2010)Biological functions of microRNAs.Bioorg Khim 36:747-752.

5.Mezzanzanica D,Bagnoli M,De Cecco L,Valeri B,Canevari S(2010)Role of microRNAs in ovarian cancer pathogenesis and potential clinical implications.Int J Biochem Cell Biol 42:1262-1272.

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<120>application of the finger-print of tiny RNA composition in the Cancerous Pleural Effusion diagnosing and treating of people

<130> P2017-0381

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Claims

1. a kind of isolated miRNA, which is characterized in that the miRNA are as follows:

(II) miRNA complementary with sequence shown in SEQ ID NO:n；

Two or more combination of (III) sequence in the miRNA as shown in SEQ ID NO:1-32；Or

Two or more combination in (IV) miRNA complementary with sequence shown in SEQ ID NO:1-32.

2. a kind of miRNA collection or combination, which is characterized in that the miRNA collection or combination are as follows:

(a) two or more the combination in sequence miRNA as shown in SEQ ID NO:1-32；

(c) at least one is from sequence miRNA as shown in SEQ ID NO:1-32 and at least one comes from and SEQ ID NO: The combination that the miRNA of the complementation of sequence shown in 1-32 is constituted, wherein the sequence from sequence miRNA as shown in SEQ ID NO:1-32 Column are not mutually complementation with the sequence from the miRNA complementary with sequence shown in SEQ ID NO:1-32.

3. a kind of separation or artificial constructed precursor miRNA, which is characterized in that the precursor miRNA can be in people's cell It shears and is expressed as miRNA described in claim 1.

4. a kind of isolated polynucleotides, which is characterized in that the polynucleotides can be transcribed into precursor miRNA by people's cell, The precursor miRNA can be sheared in people's cell and be expressed as miRNA described in claim 1.

5. a kind of carrier, which is characterized in that it contains isolated miRNA or as claimed in claim 2 described in claim 1 MiRNA collection or combination or polynucleotides as claimed in claim 4.

6. the purposes of isolated miRNA described in claim 1 or miRNA collection or combination as claimed in claim 2, special Sign is, is used to prepare chip or kit；The chip or kit are used for:

(1) diagnosis, tumor grade and the prognostic evaluation of carcinous malignant pleural effusion (preferably, lung cancer type malignant pleural effusion)；

7. a kind of miRNA chip, which is characterized in that the miRNA chip includes:

Solid phase carrier；And

The oligonucleotide probe being orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to Sequence some or all of shown in SEQ ID NO:1-32.

8. the purposes of miRNA chip as claimed in claim 7, which is characterized in that be used to prepare distinguish Cancerous Pleural Effusion and Benign pleural effusions are (preferably, distinguish lung cancer type malignant pleural effusion and Benign pleural effusions；More preferably, adenocarcinoma of lung type is distinguished Malignant pleural effusion and Benign pleural effusions) kit.

9. a kind of kit, which is characterized in that contain miRNA chip as claimed in claim 7 and/or use in the kit In the detection reagent of miRNA collection or combination as claimed in claim 2.

10. a kind of method of screening anti-lung cancer type or adenocarcinoma of lung type malignant pleural effusion drug candidate, which is characterized in that the side Method the following steps are included:

(a) in experimental group, the lung carcinoma cell of lung cancer type or adenocarcinoma of lung type malignant pleural effusion is cultivated in the presence of test substance (or lung adenocarcinoma cell)；And in control group, in feelings identical as the experimental group condition but there is no the test substance The lung carcinoma cell (or lung adenocarcinoma cell) of identical lung cancer type or adenocarcinoma of lung type malignant pleural effusion is cultivated under condition；

(b) lung carcinoma cell (or lung adenocarcinoma cell) of lung cancer type or adenocarcinoma of lung type malignant pleural effusion in the experimental group is measured The expression of one or more miRNA, and the lung carcinoma cell with lung cancer type in control group or adenocarcinoma of lung type malignant pleural effusion The expression of the miRNA of (or lung adenocarcinoma cell) is compared；

Wherein, if compared with the control group, the expression of the miRNA in the experimental group have occurred be intended to it is good The variation of the expression of property hydrothorax cell, then show that the test substance is anti-lung cancer type or adenocarcinoma of lung type malignant pleural The drug candidate of hydrops.

11. it is a kind of it is external it is nondiagnostic judge cell or tissue whether be lung cancer type or adenocarcinoma of lung type malignant pleural effusion or its The method of tissue, which is characterized in that comprising steps of separation described in claim 1 in the measurement cell or its tissue The expression of miRNA in miRNA collection or combination described in miRNA or claim 2, when the miRNA expression with Normal tissue is compared, and is had significant difference, is then illustrated that the cell or tissue is lung cancer type or adenocarcinoma of lung type malignant pleural effusion Or its tissue.