CN111100863B - Application of fingerprint spectrum composed of small RNA in diagnosis and treatment of lung cancer - Google Patents

Abstract

The invention relates to application of a fingerprint spectrum composed of small RNA in diagnosis and treatment of lung cancer in alveolar lavage fluid of a human. By screening nearly two thousand mirnas, a series of specific miRNA combinations was found that can very effectively differentiate lung cancer from benign from human alveolar lavage fluid.

Description

Application of fingerprint spectrum composed of small RNA in diagnosis and treatment of lung cancer

Technical Field

The invention relates to the technical fields of biomedicine, bioengineering and detection, in particular to application of a fingerprint spectrum composed of small RNAs in diagnosis and treatment of lung cancer by human bronchoalveolar lavage fluid.

Background

Since it is a very difficult task to accurately diagnose lung cancer from other diseases of the lung, lung cancer is usually found when advanced symptoms occur, and therefore about 75% of lung cancer patients are diagnosed already in stage IIIB or stage IV. This is also a major cause of low five year survival (15%) in lung cancer patients. And early diagnosis of lung cancer can improve the five-year survival rate of patients to 85 percent.

CT is a major means of primary screening to distinguish between benign and malignant pulmonary nodules, but its significantly different false positive and false negative results (44% false positive and 17% false negative) have significant limitations as a diagnostic means.

Tumor, interstitial lung disease, granulomatous disease, and certain infectious diseases all require definitive diagnosis by bronchoscopy, which is one of the most common examination items. The bronchoscope combines bronchobiopsy, brushing and flushing liquid, and the detection sensitivity can reach 75% from 25%. However, due to the limitations of the apparatus, the bronchoscopic needle cannot be extended to detect surrounding pulmonary nodules but has low sensitivity (55% of brushing sensitivity, 46% of fiberbronchoscopic biopsy sensitivity, 43% of flushing or bronchoalveolar lavage fluid detection sensitivity) and can be used for detecting airway epithelial cells by directly or indirectly flushing the tumor with bronchoalveolar lavage fluid, and immune cells and cell secretions can be collected for detection. However, markers for lung cancer for diagnosis or prognosis have been rarely reported. In addition, cytological examination of bronchoalveolar lavage samples is also less sensitive to lung cancer diagnosis. The expression of these extracellular matrices, including miRNAs, has been reported in the literature to be potentially useful as biomarkers for lung cancer.

Accordingly, there is a great need in the art to provide an agent and method for diagnosing and treating lung cancer using bronchoalveolar lavage fluid.

Disclosure of Invention

The invention aims to provide an effective small RNA fingerprint spectrum for helping diagnosis, tumor grading and prognosis evaluation of lung cancer in bronchoalveolar lavage fluid. ;

In particular, the invention aims to find out the miRNAs fingerprint spectrum which can help the bronchoscope to diagnose lung cancer by analyzing the miRNAs expression spectrum of bronchoalveolar lavage fluid. The invention utilizes miRNA real-time quantitative PCR detection platform of Shanghai Xiangqiong biotechnology limited company to detect and verify the expression condition of lung cancer patients with large sample amount bronchoalveolar lavage fluid and genome miRNAs of non-cancer control, and discovers that a fingerprint spectrum composed of 8 miRNAs can well distinguish lung cancer samples from the non-cancer control. As a bronchoscope auxiliary means, the fingerprint spectrum composed of 8 miRNAs has high detection sensitivity to surrounding lung cancer patients. Especially for some surrounding lung cancer patients, the bronchoscope can not carry out biopsy or operation biopsy, and the bronchoalveolar lavage liquid can be used for guiding the diagnosis and treatment of lung cancer. The method has important guiding significance for diagnosis and treatment and evaluation prognosis of lung cancer by using bronchoalveolar lavage fluid.

In a first aspect of the invention, there is provided an isolated miRNA:

A miRNA with a sequence shown as SEQ ID NO: n, wherein n is a positive integer selected from 1-17;

(ii) a miRNA complementary to the sequence shown in SEQ ID NO: n;

(iii) a combination of two or more miRNAs having the sequence shown in SEQ ID NOS.1-17; or (b)

(Iv) combinations of two or more of miRNAs complementary to the sequences shown in SEQ ID NOS.1-17.

In a second aspect of the invention, there is provided a set or combination of mirnas that are:

(a) Two or more miRNAs with sequences shown as SEQ ID NO. 1-17;

(b) A combination of two or more miRNAs complementary to the sequences shown in SEQ ID NOs 1 to 17; or (b)

(C) At least one miRNA from the sequences shown in SEQ ID NOS.1-17 and at least one miRNA from the sequences complementary to the sequences shown in SEQ ID NOS.1-17, wherein the sequences from the miRNAs of the sequences shown in SEQ ID NOS.1-17 and the sequences from the miRNAs complementary to the sequences shown in SEQ ID NOS.1-17 are not complementary to each other.

In another preferred embodiment, the collection or combination of miRNAs comprises 8 miRNAs in the sequence as shown in SEQ ID NOs 1-8.

In another preferred embodiment, the miRNA is isolated from a human.

In another preferred embodiment, the sequence may be obtained by expression by chemical synthesis or construction of eukaryotic expression vectors.

In another preferred embodiment, the miRNA pool or combination is a small RNA fingerprint consisting of 8 miRNAs for diagnosis, tumor fractionation and prognosis evaluation of lung cancer; and/or distinguishing lung cancer bronchoalveolar lavage from benign bronchoalveolar lavage; ;

And the 8 miRNAs are HSA-MIR-137-3p, HSA-MIR-182, HSA-MIR-210, HSA-MIR-181a, HSA-MIR-125b-2#, HSA-MIR-146b-5p, HSA-MIR-224 and HSA-MIR-146a.

In another preferred embodiment, the diagnosis, tumor grading and prognosis of lung cancer is performed using bronchoalveolar lavage fluid.

In another preferred embodiment, the lung cancer includes central lung cancer and peripheral lung cancer.

In a third aspect of the invention, there is provided an isolated or artificially constructed precursor miRNA that is capable of cleaving and expressing in a human cell a miRNA according to the first aspect of the invention.

The invention also provides an isolated or artificially constructed precursor miRNA set or combination, wherein the precursor miRNA in the precursor miRNA set or combination can be sheared and expressed in human cells as miRNA in the miRNA set or combination according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided an isolated polynucleotide which is transcribed by a human cell into a precursor miRNA which is sheared within the human cell and expressed as a miRNA according to the first aspect of the invention;

preferably, the polynucleotide has a structure represented by formula I:

Seq Forward-X-Seq reverse formula I,

In the formula I, the compound (I),

Seq forward is the nucleotide sequence capable of expressing said miRNA in human cells,

The reverse of a Seq is a nucleotide sequence that is substantially complementary or fully complementary to the forward of the Seq;

x is a spacer sequence located between the forward direction of the Seq and the reverse direction of the Seq, and said spacer sequence is non-complementary to the forward direction of the Seq and the reverse direction of the Seq,

And the structure shown in formula I forms a secondary structure shown in formula II after being transferred into human cells:

In formula II, the definition of the forward direction of the Seq, the reverse direction of the Seq and X are as above,

The term "complementary base pairing" refers to the base pairing between the forward and reverse directions of Seq.

The invention also provides an isolated set or combination of polynucleotides comprising polynucleotides capable of being transcribed by a human cell into a precursor miRNA, said precursor miRNA being capable of being sheared and expressed in a human cell as set or combination of mirnas according to the second aspect of the invention.

In another preferred embodiment, one or more polynucleotides of the set or combination of polynucleotides has a structure represented by formula I:

Seq Forward-X-Seq reverse formula I,

In the formula I, the compound (I),

Seq forward is the nucleotide sequence capable of expressing said miRNA in human cells,

The reverse of a Seq is a nucleotide sequence that is substantially complementary or fully complementary to the forward of the Seq;

x is a spacer sequence located between the forward direction of the Seq and the reverse direction of the Seq, and said spacer sequence is non-complementary to the forward direction of the Seq and the reverse direction of the Seq,

And the structure shown in formula I forms a secondary structure shown in formula II after being transferred into human cells:

In formula II, the definition of the forward direction of the Seq, the reverse direction of the Seq and X are as above,

The term "complementary base pairing" refers to the base pairing between the forward and reverse directions of Seq.

In a fifth aspect of the invention there is provided a vector comprising an isolated miRNA according to the first aspect of the invention, or a collection or combination of mirnas according to the second aspect of the invention, or a polynucleotide according to the fourth aspect of the invention.

In a sixth aspect of the invention there is provided the use of an isolated miRNA of the first aspect of the invention, or a miRNA collection or combination of the second aspect of the invention, for the preparation of a chip or kit; the chip or kit is used for:

(1) Diagnosis, tumor grading and prognosis of lung cancer, preferably lung cancer using bronchoalveolar lavage fluid;

(2) Lung cancer bronchoalveolar lavage fluid and benign bronchoalveolar lavage fluid are distinguished.

In a seventh aspect of the present invention, there is provided a miRNA chip comprising:

a solid phase carrier; and

An oligonucleotide probe which is orderly immobilized on the solid phase carrier, wherein the oligonucleotide probe specifically corresponds to part or all of the sequences shown in SEQ ID NOs 1-17.

In another preferred embodiment, the oligonucleotide probe comprises:

A complementary binding region; and/or

A junction region attached to the solid support.

In another preferred embodiment, the oligonucleotide probe specifically corresponds to the entire sequence shown in SEQ ID NOS.1-8.

In an eighth aspect of the invention, there is provided the use of a miRNA chip according to the seventh aspect of the invention for preparing a kit for:

(1) Diagnosis, tumor grading and prognosis of lung cancer, preferably lung cancer using bronchoalveolar lavage fluid;

(2) Lung cancer bronchoalveolar lavage fluid and benign bronchoalveolar lavage fluid are distinguished.

In a ninth aspect of the present invention, there is provided a kit comprising a miRNA chip according to the seventh aspect of the present invention and/or a detection reagent for a miRNA pool or combination according to the second aspect of the present invention.

In another preferred embodiment, the kit further comprises a collection or combination of mirnas according to the second aspect of the invention for use in a positive control.

In another preferred embodiment, the kit further comprises instructions describing a method for testing the sequence shown in SEQ ID No. 1-17 using the miRNA chip.

In a tenth aspect of the present invention, there is provided a method of screening for a candidate drug for treating lung cancer, the method comprising the steps of:

(a) In the experimental group, culturing lung cancer cells in the presence of a substance to be tested; culturing the same lung cancer cells in a control group under the same conditions as the experimental group but in the absence of the test substance; and culturing benign lung cells in a placebo group under the same conditions as the experimental group;

(b) Determining the expression level of the isolated miRNA of the first aspect of the invention or the miRNA of the collection or combination of mirnas of the second aspect of the invention in the experimental group of lung cancer cells and comparing the expression level of the miRNA with the expression level of the miRNA of the lung cancer cells in the control group;

Wherein if the expression level of the miRNA in the experimental group is changed toward the expression level of benign cells of the lung in the blank group compared with the control group, the substance to be tested is indicated to be a candidate drug for treating lung cancer.

In another preferred embodiment, the lung cancer cells are lung cancer cells in bronchoalveolar lavage fluid.

In another preferred embodiment, the method further comprises step (c): further administering the candidate drug to a non-human mammal having lung cancer, thereby determining the effect of the candidate drug on lung cancer in the non-human mammal.

In another preferred embodiment, the miRNA is an isolated miRNA according to the first aspect of the present invention.

In another preferred embodiment, the miRNA is a miRNA collection or combination according to the second aspect of the present invention.

In another preferred embodiment, the "change in expression level of benign lung cells in a blank group" means that for a certain miRNA, the following formula is satisfied:

Q≤0.5

Wherein q=abs (A1-A0)/abs (A2-A0)

Wherein, A0 is the expression level of miRNA in benign cells of lung in a blank group; a1 is the miRNA expression level of the experimental group; a2 is the miRNA expression level of the control group; abs represents the absolute value.

In another preferred embodiment, when the miRNA lung cancer bronchoalveolar lavage fluid is up-regulated (i.e., A2-A0 > 0), then A1-A0.ltoreq.0 or Q.ltoreq.0.5.

In another preferred embodiment, when the miRNA is lung cancer bronchoalveolar lavage fluid down-regulated (i.e., A2-A0 < 0), then A1-A0 is greater than or equal to 0 or Q is greater than or equal to 0.5.

In another preferred embodiment, in the method, in step (a), a positive control group is further included, i.e., the same lung cancer cells are cultured under the same conditions as the experimental group and in the absence of the test substance but in the presence of a known drug for treating lung cancer;

and, in step (b), comparing the expression level of one or more mirnas of the lung cancer cells in the experimental group with the expression level of the mirnas of the lung cancer cells in the positive control group.

In another preferred embodiment, the miR is selected from: HSA-MIR-137-3p, HSA-MIR-182, HSA-MIR-210, HSA-MIR-181a, HSA-MIR-125b-2#, HSA-MIR-146b-5p, HSA-MIR-224 and HSA-MIR-146a.

In an eleventh aspect of the invention, there is provided a method of non-diagnostic in vitro determination of whether a cell or tissue is a lung cancer cell or tissue, comprising the steps of: determining the expression level of an isolated miRNA of the first aspect of the invention or an miRNA of the collection or combination of mirnas of the second aspect of the invention in said cell or tissue, when said miRNA expression level differs significantly from that of normal tissue, indicates that the cell or tissue is a lung cancer cell or tissue.

In a twelfth aspect of the invention, there is provided a method of diagnosing a lung cancer bronchoalveolar lavage fluid sample comprising the steps of: determining the expression level of the isolated miRNA of the first aspect of the present invention or the miRNA of the collection or combination of mirnas of the second aspect of the present invention in a sample, wherein a significant difference in the expression level of the miRNA compared to a normal sample indicates that the sample is a lung cancer bronchoalveolar lavage sample.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows lung cancer bronchoalveolar lavage fluid in the training set differentiated from 12 selected mirnas (table 2) by SVM model and leave-one-out cross-validation.

Fig. 1A is a graph of a leave-one-out cross-validation result based on an SVM model, the abscissa represents each sample, all samples (n=106) are divided into two parts (53 cases of lung cancer samples and 53 cases of benign samples), the ordinate represents probability values of each sample predicted as cancer (> =0.5 for cancer, <0.5 for benign), the black dot represents samples judged correctly, and the red dot represents samples judged incorrectly;

FIG. 1B is a graph of ROC derived by a method of SVM model based on leave-one-out cross-checking.

Figure 2 shows lung cancer bronchoalveolar lavage fluid determination in test sets for 8 selected mirnas (table 3) by SVM model and leave-one-out cross-validation. Specifically, the leave-one-out cross-validation result is based on an SVM model diagram, the abscissa represents each sample, all samples (n=133) are divided into two parts (lung cancer samples 104 and benign samples 29), the ordinate represents a probability value of predicting cancer for each sample (> =0.5 is cancer, <0.5 is benign), the black dot represents a sample judged to be correct, and the red dot represents a sample judged to be incorrect.

Detailed Description

Through extensive and intensive studies, the inventor screens nearly two thousands of miRNAs, screens out a plurality of specific miRNAs for the first time, and tests prove that lung cancer and benign can be very effectively distinguished by combining specific miRNA markers to a certain extent. The inventor also screens out a plurality of specific miRNAs for the first time, so that lung cancer can be diagnosed very effectively in bronchoalveolar lavage fluid. The invention provides application of a fingerprint spectrum composed of miRNA in diagnosis of bronchoalveolar lavage fluid, and the fingerprint spectrum can be used as an auxiliary diagnosis means of bronchoscope to help diagnose lung cancer. On this basis, the present invention has been completed.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

MiRNA and precursor thereof

The present invention provides a novel class of mirnas found in humans. As used herein, the term "miRNA" refers to an RNA molecule that is processed from transcripts that can form a precursor of the miRNA. Mature mirnas typically have 18-26 nucleotides (nt) (more particularly about 19-22 nt), nor are miRNA molecules with other numbers of nucleotides excluded. mirnas are generally detectable by Northern blotting.

Mirnas of human origin can be isolated from human cells. As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and polypeptide are not isolated or purified in vivo, the same polynucleotide or polypeptide is isolated or purified from other naturally occurring substances.

Mirnas can be processed from precursor mirnas (Precursor miRNA, pre-mirnas) that can be folded into a stable stem-loop (hairpin) structure, typically between 50-100bp in length. The precursor miRNA can be folded into a stable stem-loop structure, and the two sides of the stem-loop structure comprise two sequences which are basically complementary. The precursor miRNA can be natural or synthetic.

The precursor miRNA may be sheared to generate a miRNA that may be substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure). Typically, two "substantially complementary" nucleotide sequences are at least 70% complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. Typically, there may be up to 40 mismatched nucleotides between two sufficiently complementary molecules; preferably, there are up to 30 mismatched nucleotides; more preferably, there are up to 20 mismatched nucleotides; it is further preferred to have at most 10 mismatched nucleotides, such as 1,2,3, 4, 5, 6, 7, 8 mismatched nucleotides.

As used herein, a "stem-loop" structure, also referred to as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure that includes a double-stranded region (stem) formed by two regions of the nucleotide molecule (on the same molecule) that are flanked by double-stranded portions; it also includes at least one "loop" structure comprising a non-complementary nucleotide molecule, i.e., a single-stranded region. The double-stranded portion of the nucleotide can remain double-stranded even if the two regions of the nucleotide molecule are not fully complementary. For example, insertions, deletions, substitutions, etc. may result in the non-complementation of a small region or the small region itself forming a stem-loop structure or other form of secondary structure, however, the two regions may still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. The stem-loop structure is well known to those skilled in the art, and usually after obtaining a nucleic acid having a nucleotide sequence of primary structure, the skilled person is able to determine whether the nucleic acid is capable of forming a stem-loop structure.

The miRNA has a sequence shown as SEQ ID NO: n, wherein n is a positive integer selected from 1-17.

To improve the stability or other properties of the miRNA, at least one protective base, such as "TT" or the like, may also be added to at least one end of the miRNA.

In this context, miRNA, miRN, small RNA, microRNA, miR have the same meaning.

For the lung cancer bronchoalveolar lavage fluid specific miRNA disclosed by the invention, verification can be performed by a conventional miRNA chip technology, for example, the miRNA is extracted by a conventional method or a conventional kit, and then detection is performed. Representative kits include (but are not limited to): the Qiagen or Ambion company's miRNAs extraction kit.

In addition, the lung cancer bronchoalveolar lavage fluid specific miRNA of the present invention can also be detected or verified by specifically amplifying and detecting the amplified product (or a corresponding detectable signal such as a fluorescent signal). Preferred high sensitivity and high specificity techniques include (but are not limited to): the technology disclosed in CN10267663 a. Typically, the specific binding region of the primer used may be designed according to the sequence of the known miRNA to be detected, preferably the specific binding region of the primer used in amplification is typically a complementary sequence that is fully complementary to the miRNA.

Antisense oligonucleotides

According to the miRNA sequences provided by the invention, antisense oligonucleotides of the miRNA sequences can be designed, and the antisense oligonucleotides can down-regulate the expression of corresponding miRNAs in vivo. As used herein, an "antisense oligonucleotide" (AS-oligonucleotides, AS-Ons or ASO) "is also referred to AS an" antisense nucleotide "and refers to a DNA molecule or RNA molecule or analog thereof that is about 18-26nt in length (more particularly about 19-22 nt).

In the present invention, the "antisense oligonucleotide" also includes modified antisense nucleotides obtained by means such as nucleic acid lock-based or nucleic acid strand backbone modification techniques, wherein the modification does not substantially alter the activity of the antisense oligonucleotide, and more preferably, the modification increases the stability, activity or therapeutic effect of the antisense oligonucleotide. Nucleic acid locks (locked nucleic acid, LNA) generally refer to modification techniques in which the 2 'oxygen atom and the 4' carbon atom of ribose are linked by a methylene bridge. LNA can prolong serum half-life of miRNA, improve affinity to target, and reduce scope and degree of off-target effect. The antisense medicine developed based on the modification technology of nucleic acid chain skeleton has greatly improved solubility, nuclease degradation resistance and other aspects, and is easy to synthesize in great amount. There are various methods for backbone modification of oligonucleotides, including thio methods, such as thio modification of a deoxynucleotide chain to a thio deoxynucleotide chain. The method is to replace oxygen atoms of phosphate bonds on the DNA skeleton with sulfur atoms, and can resist nuclease degradation. It is to be understood that any modification capable of retaining most or all of the activity of the antisense oligonucleotide is encompassed by the present invention.

As a preferred mode of the invention, the antisense oligonucleotide is subjected to nucleic acid lock modification; more preferably also thio-modifications.

After the antisense oligonucleotides are transferred into human body, they can obviously reduce the expression of miRNA.

Polynucleotide constructs

According to the human miRNA sequences provided herein, polynucleotide constructs can be designed that, after being introduced, can be processed into mirnas that can affect the expression of the corresponding mRNA, i.e., the amount of the corresponding miRNA that the polynucleotide construct is capable of up-regulating in vivo. Thus, the present invention provides an isolated polynucleotide (construct) that can be transcribed by a human cell into a precursor miRNA that can be sheared by the human cell and expressed as the miRNA.

As a preferred embodiment of the present invention, the polynucleotide construct comprises a structure represented by formula I:

Seq _{Forward direction} -X-Seq _{Reverse direction} A is a compound of formula I,

In the formula I, the compound (I),

Seq _{Forward direction} is the nucleotide sequence of a miRNA that can be expressed in a cell and Seq _{Reverse direction} is the nucleotide sequence substantially complementary to Seq _{Forward direction} ; or Seq _{Reverse direction} is a nucleotide sequence which can be expressed in a cell as said miRNA, and Seq _{Forward direction} is a nucleotide sequence which is substantially complementary to Seq _{Reverse direction} ;

x is a spacer sequence located between Seq _{Forward direction} and Seq _{Reverse direction} , and the spacer sequence is not complementary to Seq _{Forward direction} and Seq _{Reverse direction} ;

after the structure shown in the formula I is transferred into cells, a secondary structure shown in the formula II is formed:

In formula II, seq _{Forward direction} 、Seq _{Reverse direction} and X are as defined above;

The term "complementary base pairing" refers to the base pairing between Seq _{Forward direction} and Seq _{Reverse direction} .

Typically, the polynucleotide construct is located on an expression vector. Thus, the invention also includes a vector comprising said miRNA, or said polynucleotide construct. The expression vector typically also contains a promoter, origin of replication, and/or marker gene, etc. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as calicheamicin, gentamicin, hygromycin, ampicillin resistance.

Chip

MiRNA expression profiling chips typically contain up to several hundred probes covering a variety of mirnas, and detect the content of each miRNA contained in a sample at the whole genome level using the principle of DNA double strand homologous complementation. Thus, the transcript level of the miRNA in the whole genome range in the sample to be tested can be detected at the same time.

The miRNA sequence can be used for preparing corresponding miRNA chips, so that the expression profile and the regulation mode of miRNAs can be studied.

In another aspect, the invention also provides a chip for analyzing miRNA expression profiles, which can be used to distinguish lung cancer bronchoalveolar lavage from benign.

The miRNA chip of the invention comprises:

a solid phase carrier; and

Oligonucleotide probes that are sequentially immobilized on the solid support, the oligonucleotide probes specifically corresponding to at least 1 (e.g., 1,2,3, 4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17) of the sequences shown in SEQ ID NOs 1-17.

Specifically, suitable probes can be designed according to the miRNAs of the present invention and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays, as desired.

The solid phase carrier can be made of various common materials in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon wafers modified by active groups (such as aldehyde groups, amino groups and the like), unmodified glass slides, plastic sheets and the like.

The preparation of the miRNA chip can be carried out by adopting a conventional manufacturing method of a biochip known in the art. For example, if a modified slide or a silicon wafer is used as the solid phase carrier, and the 5' -end of the probe contains an amino-modified poly dT string, the oligonucleotide probe can be prepared into a solution, then spotted on the modified slide or the silicon wafer by a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, so that the miRNA chip of the invention can be obtained. If the nucleic acid does not contain amino modifications, the preparation method can also be referred to as: wang Shenwu "Gene diagnosis technology-nonradioactive handbooks ";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 Liren, incorporated by reference" biological chip ", beijing: chemical industry Press 2000,1-130.

In another aspect, the present invention also provides a method for detecting a miRNA expression profile in human tissue by a miRNA chip, comprising the steps of:

(1) Providing an RNA sample isolated from human tissue, and providing a marker on said RNA;

(2) Contacting the RNA obtained in the step (1) with the miRNA chip to enable the RNA to carry out hybridization reaction with the oligonucleotide probe on the solid phase carrier, thereby forming an oligonucleotide probe-RNA binary complex on the solid phase carrier;

(3) Detecting the markers of the binary complex formed in step (2), thereby determining the expression profile of the corresponding miRNA in human tissue.

Methods for extracting RNA from human tissue are well known to those skilled in the art and include the Trizol method.

More preferably, in step (1), after isolation of the RNA sample from human tissue, the RNA sample is suitably treated to enrich for RNA having a length, typically between 10 and 100 (small fragment RNA). After the treatment, the small fragment RNAs are used for subsequent hybridization, so that the accuracy of capturing miRNA by the chip can be improved. The person skilled in the art can conveniently isolate RNA having a certain fragment length, for example by gel electrophoresis.

Labelling of RNA is also a well known method to the person skilled in the art and can be achieved by adding a label, such as a labelling group, which specifically binds to RNA during hybridization. Such labeling groups include, but are not limited to: digoxin molecules (DIG), biotin molecules (Bio), fluorescein and its derivative biomolecules (FITC, etc.), other fluorescent molecules (e.g., cy3, cy5, etc.), alkaline Phosphatase (AP), horseradish peroxidase (HRP), etc. These labels and their labeling methods are all well known in the art.

When hybridizing the RNA with the miRNA chip, the miRNA chip and a prehybridization buffer solution can be prehybridized.

The solid phase hybridization between RNA and miRNA chips according to the present invention is performed according to classical methods in the art, and the person skilled in the art can easily determine the optimum conditions for buffer, probe and sample concentrations, prehybridization temperature, hybridization temperature, time, etc., according to experience. Or may be as described in the guidelines for molecular cloning experiments.

And then obtaining information to be detected according to the position, the intensity and other information of the marking signal on the miRNA chip. If the amplified product is marked by a fluorescent group, a fluorescence detection device (such as a laser confocal scanner Scanarray 3000) can be directly used for obtaining the information to be detected.

Detection kit

The invention also provides a kit, which contains the chip of the invention. The kit can be used for detecting the expression profile of miRNA; or to differentiate lung cancer from benign bronchoalveolar lavage (preferably, to differentiate between peripheral lung cancer, more preferably, to differentiate between central lung cancer).

More preferably, the kit further comprises a marker for marking the RNA sample and a substrate corresponding to the marker.

In addition, various reagents required for extracting RNA, PCR, hybridization, color development, etc. can be included in the kit, including but not limited to: extract, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, antibody, etc. Fluorescent dyes such as EvaGreen, SYBRGreen and the like can also be contained in the amplification solution. The kit can also comprise a primer. Other detection means include biochips or may be determined by the probe method (Taqman) or the like.

In addition, the kit can also comprise instructions for use and/or chip image analysis software.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

The main advantages of the invention include:

(1) The invention provides a miRNA fingerprint spectrum which can be used for well detecting lung cancer in bronchoalveolar lavage fluid;

(2) The invention provides a miRNA fingerprint spectrum which can be used for better assisting bronchoscopy in bronchoalveolar lavage fluid to detect surrounding lung cancer;

(3) The miRNA fingerprint can effectively distinguish lung cancer from benign bronchoalveolar lavage fluid, and has high sensitivity and strong specificity;

(4) The miRNA fingerprint can effectively assist a bronchoscope to detect surrounding lung cancer and benign bronchoalveolar lavage fluid, and has high sensitivity and strong specificity;

(5) The miRNA fingerprint can be effectively used for diagnosing, typing and prognosis evaluation of bronchoalveolar lavage fluid on lung cancer.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Unless otherwise indicated, materials and reagents used in the description of the present invention are commercially available products.

Example 1

Sample collection and information analysis

The collection of the bronchoalveolar lavage fluid of the non-cancer control group and the lung cancer patients was completed by the double-denier affiliated Zhongshan hospital, and the training set contained 106 samples in total, which contained bronchoalveolar lavage fluid of 53 inflammatory patients as benign controls, bronchoalveolar lavage fluid of 53 lung cancer patients, 27 lung adenocarcinoma patients, 17 lung squamous carcinoma patients, 3 lung small cell lung cancer patients, 1 complex neuroendocrine cancer patient (lung small cell lung cancer and large cell cancer mixed), and 4 lung cancer (subtype unknown) patients. A total of 133 bronchoalveolar lavage samples from the test set were also collected. If the training set samples are divided according to the cancer occurrence parts, the 106 training set samples comprise 68 central cases (30 cases of lung cancer and 38 cases of benign control), 38 peripheral cases (23 cases of lung cancer and 15 cases of benign control); the 133 double blind validation samples included 84 central (64 lung cancers and 20 benign controls), 49 peripheral (40 lung cancers and 9 benign controls). Samples of 239 bronchoalveolar lavage fluids were collected prior to treatment and stored at-80 degrees within 1 hour. All samples obtained by the invention were approved by the ethical committee of the secondary university affiliated Zhongshan hospital. All samples were classified according to WHO criteria for clinical characterization and pathology, as detailed in table 1 below.

Table 1: sample information and classification

Example 2

Bronchoalveolar lavage fluid sample preparation:

Bronchoalveolar lavage samples were collected at room temperature in sterile 15ml BD tubes and pre-treated within 1 hour.

Bronchoalveolar lavage fluid was centrifuged at 2000g at 20℃for 15min and the resulting supernatant was stored at-80℃or tested.

Example 3

Total RNA extraction of bronchoalveolar lavage sample supernatant:

1000ul QIAzol Lysis Reagent reagents (QIAGEN MIRNEASY MINI KIT; qiagen, 217004) were added to a 200ul bronchoalveolar lavage supernatant sample and total RNA extraction was performed following the relevant procedure.

5 Nanomolar exogenous cel-miR-39 is added into the total RNA extraction process of the supernatant of bronchoalveolar lavage fluid, and serves as an endogenous control to control the extraction of a quality control sample.

Example 4

Isolation of miRNA and cDNA Synthesis:

the total RNA obtained by extraction can be detected by agarose gel electrophoresis, and the total RNA quality obtained by separation can be analyzed by ultraviolet spectrophotometry Biomate (Thermo Scientific).

The preparation method comprises the steps of adding a Poly A tail at the 3' end of miRNA by adopting Sharpvue ^TM miRNA reverse transcription kit Sharpvue ^TM MIRNA FIRST STRAND KIT (Biovue, 9000004), then converting into cDNA by a reverse transcription primer, and operating according to the kit instruction. The RT reaction is completed in GENE AMP PCR, 9700 and Thermocycler (Applied Biosystems) under the reaction condition of 37 ℃ for 60min; inactivating at 95deg.C for 10 min.

Example 5

Selection of array of miRNAs in training set:

Through preliminary experiments, 1920 miRNAs of a cancer sample are subjected to primary screening, and through real-time quantitative PCR reaction, unsupervised clustering and bioinformatic analysis and comparison of literature reports, a miRNAs array suitable for liquid biopsy is screened. The array of miRNAs screened was then tested by 25 bronchoalveolar lavage samples (11 lung cancer patients and 14 negative controls), all by real-time quantitative PCR, quantile-Mean method was used for bioinformatic statistical analysis. Only those miRNAs with expression changes of > 2 and p <0.05 were selected. And comparing the miRNAs with the miRNAs reported in the literature, and finally screening out a miRNAs array relevant to diagnosis of bronchoalveolar lavage lung cancer. So far, this is also the first such large-scale detection of miRNAs in bronchoalveolar lavage using real-time fluorescent quantitative PCR, and the first study on such large-scale detection of 1920 miRNAs in connection with lung cancer diagnosis. The array of 96 miRNAs bronchoalveolar lavage fluid candidate markers was scaled down. And (3) performing real-time fluorescent quantitative PCR detection on 53 lung cancer patients and 53 negative control samples by using the screened 96 miRNAs bronchoalveolar lavage fluid candidate marker arrays, and establishing a bronchoalveolar lavage fluid lung cancer diagnosis model.

Example 6

Double-blind validation set biomarker test:

To verify the circulating miRNAs of these bronchoalveolar lavages found in the study as fingerprints for lung cancer diagnosis, a double blind verification test was subsequently performed in 133 samples. These bronchoalveolar lavage samples included samples from 48 lung adenocarcinoma patients, 32 lung squamous carcinoma patients, 18 lung small cell lung carcinoma patients, 1 lung adenosquamous carcinoma patient, 2 large cell neuroendocrine carcinoma patients, 3 lung carcinoma (unknown subtype) patients, and 29 negative controls.

Example 7

Fluorescent quantitative PCR detection

The method disclosed in reference to CN10267663a detects small RNAs.

Into a 15 ml centrifuge tube, 24. Mu.l of the reverse transcription product obtained in example 3 and 600. Mu.l of a fluorescent quantitative PCR enzyme reaction solution (product number: 9000008 of Xiangqiong Co.),2XUniversal qPCR Master Mix High Rox), 216 μl of nuclease ultrapure water (product number of Xiangqiong Co., ltd.): 9000015 Gently mixing.

The small RNA reaction template for diagnosis of bronchoalveolar lavage fluid produced by Xiangqiong corporation, sharpvue ^TM Human MIRNA ARRAY-chest water (product number: 1100001 of Xiangqiong corporation) is taken out from a refrigerator at-20 ℃, the packaging bag is opened after the temperature returns to room temperature, and the mixture is placed on a centrifuge, and 2000g is centrifuged for 5min (Thermo, ST16R, model of turning head: M-20). A total of 93 small RNA reactions were performed with 2 positive controls and 1 blank control on the reaction template. Carefully unpack the film.

Pouring the mixed solution obtained in the previous step into a sample adding groove, and adding the mixed solution into the small RNA reaction template line by using a 12-channel continuous pipettor, wherein each hole is 7ul. After the sample addition, the liquid amount of each hole is checked to be uniform.

The mixture was then sealed with a quantitative seal plate membrane (ABI, 4711971) and mixed upside down and centrifuged at 1000g for 5min.

The sample was put into a quantitative PCR apparatus (ABI, 7900Ht Fast) to perform quantitative PCR. The procedure is: after 10min at 95℃for 5s at 95℃and 1min at 58℃for 3 cycles; after 95℃for 5s,60℃for 5s,37 cycles, dissolution profile. The reporter fluorescence was SYBR and the reference fluorescence was Rox.

Data were collected for bioinformatics analysis.

Example 8

Computational analysis of miRNA biometric data

The probability of cancer of a patient is predicted by a support vector machine (support vector machine, SVM for short), which is a classification algorithm, the generalization capability of a learning machine is improved by seeking the minimum structural risk, and the minimization of experience risk and confidence range is realized, so that the aim of obtaining good statistical rules under the condition of less statistical sample size is fulfilled, and the model is a class II classification model.

First, 1888 mirnas and two internal reference positive controls (HSA-RNU 6B and HSA-RNU 48), and one negative control (water) were detected in cancer and normal samples using real-time fluorescent quantitative PCR for further analysis and screening. MIRNA PANEL (1 96-well plate containing 93 mirnas and 3 internal controls) were determined for detection of bronchoalveolar lavage. The ct value and average value of each miRNA are subtracted from background and normalized, and the unified data ct value is less than or equal to 32.

The 96 miRNAs were then screened for miRNA markers that better distinguish between cancer and non-cancer patients. Considering that the amount of miRNA added may be different for each sample, the measured miRNA value for each sample needs to be normalized first, and the difference between the detected miRNA values for each sample is used as a new variable, so that when a subset is selected from 96 mirnas, the normalization of the data in the subset will be related to only the variables in the subset. There were a total of C (96,2) +96=4560 new variables after normalization. The significance of these variables between cancer and non-cancer patient samples was calculated using T-test, and then the 20 new variables with the most significant differences were selected. The inventors finally hoped to find a number of mirnas within 12 as variables of the model, so all combinations of mirnas within 12 were selected from the set of mirnas contained in these 20 new variables, and these combinations were used to calculate the accuracy of each combination prediction training set with the SVM model. Finally, the miRNA combination with highest accuracy is selected as a final variable set M.

The SVM model of the present invention is derived from the e1071 software package of R, and the training data is derived from 53 cancer patients and 53 cancer patients confirmed by doctors. In the model training process, the sample number difference of the control group is consistent with that of the experimental group, and a radial basis function (kernel= "radial") is selected as a kernel function, and probability is set to be predicted, wherein probability = TRUE. The predict function was used to predict the probability that the sample was cancerous and non-cancerous, and the accuracy was calculated using leave-one-out cross-validation (LLO-CV).

Since the problem of normalization of liquid biopsy sample miRNA data has been controversial, a two-to-two method was performed for analysis of stably expressed miRNAs in all bronchoalveolar lavages. The value of each miRNA was compared to the other 95 miRNAs, and 4560 ratios were used for subsequent bioinformatic analysis and comparative analysis with the biological significance of the clinical samples. The results also show that the use of the miRNAs ratio is an easy-to-apply method, has potential for general clinical application, and avoids the need for large-scale, high-throughput screening, so that the method can also be used for developing fingerprint patterns as biomarkers for clinical liquid biopsies.

The naming of mirnas is according to the mirnas database of miRBase Version 20, followed in contradictory cases.

The results of 17 mirnas selected as specific biomarkers for diagnosing lung cancer in bronchoalveolar lavage fluid are shown in table 2.

TABLE 2

Example 9

The lung cancer bronchoalveolar lavage fluid and negative control were validated using risk assessment:

The miRNAs selected in example 8 are arranged and combined, and the best miRNA combination with the specificity, sensitivity and accuracy reaching the clinical detection level can be screened and tested. According to data statistical analysis, the best 10 groups of miRNA combinations are found, and as a result, after partial specific miRNAs are combined, lung cancer bronchoalveolar lavage fluid can be effectively diagnosed, wherein the sensitivity and the specificity are better. The 10 groups of miRNA combinations are shown in Table 3.

TABLE 3 Table 3

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When the fingerprint spectrum composed of the sequences shown in SEQ ID No. 1-8 is selected, lung cancer patients in experimental samples can be well distinguished. The most obvious miRNAs for these changes are detailed in table 2, area under roc curve (AUC) of 0.919, with 86.8% accuracy and 88.7% specificity. In addition, the accuracy of predictive analysis of bronchoalveolar lavage fluid samples (including 9 lung adenocarcinomas, 14 lung squamous carcinomas, 3 lung small cell carcinomas, 1 composite neuroendocrine carcinoma, 3 non-typed lung carcinomas and 38 negative controls) of 68 patients with central type can reach 85.3%, and the specificity is 86.7%; the accuracy of predictive analysis of 38 peripheral bronchoalveolar lavage fluid samples (18 lung adenocarcinomas, 3 lung squamous carcinomas, 1 non-small cell lung cancer, 1 non-typed lung cancer and 15 negative controls) was 89.5% with a specificity of 82.6% (FIG. 1).

Example 10

Double-blind verification of selected miRNA variables:

When the sequences shown in SEQ ID No. 1-8 were selected, double-blind verification was performed on 133 bronchoalveolar lavage fluid samples (48 lung adenocarcinomas, 32 non-squamous carcinomas, 18 lung small cell carcinomas, 1 lung adenosquamous carcinoma, 2 large cell neuroendocrine carcinomas, 3 non-parting lung carcinomas and 29 negative controls), thereby judging the correctness of the obtained miRNAs fingerprint for lung cancer bronchoalveolar lavage fluid diagnosis.

To verify the accuracy and specificity of the fingerprint composed of these 8 miRNAs for lung cancer determination, double-blind verification was performed in 133 samples. The test result shows that the accuracy can reach 85 percent and the specificity is 83.7 percent. 84 central bronchoalveolar lavage fluid samples (including 17 lung adenocarcinomas, 28 lung squamous carcinomas, 10 lung small cell carcinomas, 1 neuroendocrine carcinoma, 2 non-typed lung carcinomas and 20 negative controls) are predicted with an accuracy of 89.3% and a specificity of 85.9%; whereas 49 surrounding bronchoalveolar lavage fluid samples (31 lung adenocarcinomas, 4 lung squamous carcinomas, 2 lung small cell carcinomas, 1 lung adenosquamous carcinoma, 1 large cell neuroendocrine carcinoma, 1 non-typed lung carcinoma and 9 negative controls) were predicted to be 77.6% accurate with 80% specificity (fig. 2).

Conclusion(s)

When SEQ ID NO. 1-8 is selected as the combination of miRNA fingerprint patterns, lung cancer bronchoalveolar lavage fluid can be well diagnosed; the combination of the miRNA fingerprint patterns can well reach clinical requirements for diagnosing lung cancer; can also effectively diagnose surrounding lung cancer, and has better sensitivity and specificity. Based on the above, the miRNA fingerprint can be used for distinguishing lung cancer from benign from bronchoalveolar lavage fluid very efficiently.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai Xiangqiong biotechnology Co., ltd

Application of fingerprint spectrum composed of <120> small RNA in diagnosis and treatment of lung cancer

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<170> PatentIn version 3.5

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Claims (8)

1. And the miRNA collection or combination consists of 8 miRNAs in a sequence shown as SEQ ID NO. 1-8.

2. A vector comprising the collection or combination of mirnas of claim 1.

3. Use of the collection or combination of mirnas of claim 1 for the preparation of a chip or kit; the chip or kit is used for:

(1) Diagnosing lung cancer; or (b)

(2) Lung cancer bronchoalveolar lavage fluid and benign bronchoalveolar lavage fluid are distinguished.

4. The use of claim 3, wherein the chip or kit is for diagnosis of lung cancer using bronchoalveolar lavage.

5. A miRNA chip, characterized in that the miRNA chip comprises:

a solid phase carrier; and

An oligonucleotide probe orderly immobilized on the solid support, the oligonucleotide probe specifically corresponding to the miRNA collection or combination of claim 1.

6. The use of the miRNA chip of claim 5 for preparing a kit for:

(1) Diagnosing lung cancer; or (b)

(2) Lung cancer bronchoalveolar lavage fluid and benign bronchoalveolar lavage fluid are distinguished.

7. The use according to claim 6, wherein the kit is for diagnosis of lung cancer using bronchoalveolar lavage.

8. A kit comprising the miRNA chip of claim 5 and/or a reagent for detecting the miRNA assembly or combination of claim 1.