CN108753962A - Purposes of the hsa-miR-130a in non-small cell lung cancer prognosis - Google Patents
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Abstract
The present invention relates to clinical medicine, and in particular to purposes of the hsa-miR-130a in non-small cell lung cancer prognosis.The purposes of the primer of hsa-miR-130a expressions and/or probe in preparing the reagent for non-small cell lung cancer prognosis in subject's sample is specifically detected by one or more.What is filtered out can be used as the noninvasive marker of non-small cell lung cancer prognosis with the relevant miRNA of non-small cell lung cancer prognosis, and important theoretical foundation is provided for its clinical application.What is filtered out can be used as the noninvasive marker of non-small cell lung cancer prognosis with the relevant miRNA of non-small cell lung cancer prognosis, and important theoretical foundation is provided for its clinical application.Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will be also understood by the person skilled in the art by research and practice to the present invention.
Description
Technical field
The present invention relates to clinical medicine, and in particular to purposes of the hsa-miR-130a in non-small cell lung cancer prognosis.
Background technology
Lung cancer is in occupation of the tumor invasion first in world wide including China.Lung cancer is divided into cellule by pathological classification
Lung cancer and non-small cell lung cancer, wherein non-small cell lung cancer（NSCLC 85% or so of whole lung cancer) is accounted for.Nearly ten years, lung cancer
Diagnosis and treatment have epoch-making development, in addition to classical three big treatments such as operation, radiotherapy, chemotherapy, from EGFR, ALK base
Because the treatment of rearrangement etc. etc. lung cancer enters the new targeting epoch, Gefitinib, Tarceva, Conmana, a gram azoles replace
Buddhist nun, step it is auspicious replace Buddhist nun（AZD9291）Etc. drug development so that the survival rate of lung cancer has been greatly improved, however for
For medium and advanced lung cancer, 5 years survival rates still allow of no optimist.Current diagnosis is the patients with lung cancer of III b or IV phases, most important to control
Treatment means are still chemicotherapy, and certainly new molecular targeted therapy also comes into first-line treatment.However the effect of chemotherapy is still
It is barely satisfactory, advanced lung cancer patient's Progression free survival time（PFS) it is only 3-5 months, OS is about at 8-10 months.At present
The medical treatment of tumour has been enter into the epoch precisely treated, and has and clearly drives genetic test, and receives corresponding molecular targeted therapy and make
The remission rate for obtaining advanced lung cancer patient is up to 72% or so, and 5 years survival rates of patients with lung cancer patient wish to increase substantially, still
Also there is research to confirm at present, drug resistance often occurs when treating or so November in the treatment of lung cancer EGFR-TKI, therefore to improve
The life span and quality of life of patients with lung cancer, gene protein group, epigenetics carry out it is like a raging fire when, from
Gene angle looks for the diagnosis and treatment of potential lung cancer morbidity mechanism and suitable therapy target for lung cancer with vital
Meaning.
There is the largely research about miRNAs at present, it is that a kind of novel tumor marker is possible to prompt miRNAs.miRNAs
Also there are oncogene and tumor suppressor gene function, it is now recognized that the occurrence and development of miRNAs and tumour have close relationship, therefore
Further investigate the relationship of miRNAs and lung cancer morbidity mechanism, it is possible to provide novel targets for the diagnosis and treatment of lung cancer.MiR-130a is close
The miRNA molecule found over year inhibits forefront the study found that miR-130a can influence MAPK, AR signal path activity
The proliferation of gland cancer, invasion transfer, play the function of tumor suppressor gene.More studies have shown that miR-130a can also inhibit breast
Gland cancer, the proliferation of liver cancer cells, invasion transfer.
MiRNA- 130a are located at chromosome 11q12, close with the relevant regions 11q13 [ 6 ] of cancer, Wang XC
Etc. taking the lead in having detected 200 Patients with Non-small-cell Lung with quantitative reverse transcription polymerase chain reaction,PCR (qRT-PCR) technology
MiR -130a expressions in cancerous tissue and cancer beside organism, as a result, it has been found that cancerous tissue miR -130a expressions are compared with cancer
Side tissue significantly increases.
The present invention filters out the miRNA molecules of differential expression on lung carcinoma cell and control group by genetic chip, selects
Its expression in human lung cancer tissue is detected after miR-130a and analyzes its relationship between clinicopathologic characteristics of lung cancer；And it grinds
Study carefully biological functions and pathogenesis of the miR-130a in lung cancer.
Traditional screening methods, such as airway wall, fine needle aspirate cytology and Imaging Method such as enhance computerized tomography and sweep
Retouch (CT), magnetic resonance imaging (MRI) is that invasive, complication is high or typical indication is insufficient, therefore, it is difficult to become non-small cell
The effective ways of patients with lung cancer early screening.Therefore, accurate there is an urgent need to find the Noninvasive of early diagnosis and prognosis evaluation
Biomarker, be used for Patients with Non-small-cell Lung diagnosing and treating.
Invention content
It is an object of the invention to provide one kind to be to screen non-small cell lung cancer prognosis molecule marker, instructs clinical individual
Change treatment, is purposes of the hsa-miR-130a in non-small cell lung cancer prognosis.
The purpose of the present invention is be accomplished by the following way：
Existed by the primer and/or probe of hsa-miR-130a expressions in one or more specifically detection subject's samples
Prepare the purposes in the reagent for non-small cell lung cancer prognosis.
Preferably, the wherein nucleotide sequence 5 '-of the mature rna of hsa-miR-130a
CAGUGGAAUGUUAUAUGAGCAU-3’。
Preferably, wherein the non-small cell lung cancer is primary lung cancer.
Preferably, wherein subject's sample is cancerous tissue sample, preferred Non-Small Cell Lung Carcinoma sample
This.
Preferably, wherein the Non-Small Cell Lung Carcinoma sample is obtained by way of biopsy, or
It is obtained by way of surgical excision.
Preferably, wherein the subject is mammal.
Preferably, wherein the mammal is people.
Preferably, wherein hsa-miR-130a expressions improve and indicate that the expression of squamous carcinoma tissue is less than gland cancer,
Prompt has good effect in terms of non-small cell lung cancer prognosis guidance.
Preferably, wherein the prognosis refers to the survival region of subject's untreated.
Preferably, it includes for specifically detecting drawing for hsa-miR-130a expressions in subject's sample
Object and/or probe.
The present invention includes at least following advantageous effect：What is filtered out can with the relevant miRNA of non-small cell lung cancer prognosis
As the noninvasive marker of non-small cell lung cancer prognosis, important theoretical foundation is provided for its clinical application.It is filtered out
It can be used as the noninvasive marker of non-small cell lung cancer prognosis with the relevant miRNA of non-small cell lung cancer prognosis, be its clinical application
Provide important theoretical foundation.Part is illustrated to embody by further advantage, target and the feature of the present invention by following, part
It will be also understood by the person skilled in the art by research and practice to the present invention.
Description of the drawings
Fig. 1 is miRNA chip scanning schematic diagrames；
Fig. 2 is that miRNA chips detect matrix diagram；
Fig. 3 is that miRNA chips are shown in lung cancer cell line and normal bronchial epithelial cell line comparison diagram；
Fig. 4 is that RT-PCR detects expression figures of the miR-130a in normal bronchial epithelial cell line/lung cancer cell line；
Fig. 5 is that miR-130a expresses figure compared with control group in cancerous lung tissue；
Fig. 6 is miRNA-130a amplification curves；
Fig. 7 is miRNA-130a solubility curves；
Fig. 8 is expressions and clinical schematic diagram of the miR-130a in cancerous lung tissue；
Fig. 9 is to transfect A549 and Calu-3 cell strains and the miR-130a expression figures of NC groups；
Figure 10 is the influence diagram that CCK-8 detects miR-130a to lung cancer cell line proliferative capacity；
Figure 11 obviously inhibits A549 proliferation of lung cancer cells that can try hard to after being transfection miR-130a；
Figure 12 significantly inhibits Calu-3 proliferation of lung cancer cells after being transfection miR-130a to try hard to；
Figure 13 is the invasion transfer ability figure that Transwell detects lung carcinoma cell.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
Especially by using expression of the Real_time quantitative detection miR-130a in human lung cancer tissue, hsa-miR-130a at
The nucleotide sequence 5 '-CAGUGGAAUGUUAUAUGAGCAU-3 ' of ripe RNA is turned miR-130a mimic using liposome method
Lung cancer cell types and Calu-3 are contaminated, CCK-8 experiments, flow cytometry, the detection of EdU cell Proliferations, Transwell are passed through
The methods of experiment observation miR-130a studies itself and non-small cell lung cancer to influences such as lung cancer cell growth, proliferation, invasion
Correlation.
It should be noted that experimental method described in following embodiments is unless otherwise specified conventional method, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.First part：Non-small cell lung cancer correlation miRNA's
Bioinformatic analysis and screening.
Materials and methods
The acquisition of miRNA and gene expression data
The row radical-ability or palliative resction that selection Lishui People's Hospital's thoracic surgery is accepted for medical treatment in December, 2012 in January, 2014
Through 2 Pathologis according to U.S.'s NCCN standards after the fresh lung Non-Small Cell Lung Carcinoma 60 of art, slice and HE dyeing
Pulmonary cancer diagnosis and pathological grading are carried out, clinical information is as shown in table 1-1.Corresponding adjacent tissues materials apart from 5 cm of cancerous tissue with
On, patient makes a definite diagnosis by pathology, and agrees to through patient or its family members, and research approach obtains Lishui People's Hospital's ethics
The approval of the committee.It is place immediately into liquid nitrogen after the cancerous lung tissue of operation excision and corresponding normal cancer beside organism, after being used for
Continuous experiment.
Experimental method
The total serum IgE of these tissue blocks is extracted using Trizol, and further total serum IgE was carried out using biotin labeling reagent box
Column purification.Then use the methods of spectrophotometer or Qubit fluorescent quantitations quantitative, with agarose gel electrophoresis or
The methods of Agilent2100 detects its integrality.MiRNA array experiment data are extracted and analysis, chip pass through Agilent cores
Piece scanner is scanned, and obtains hybridization picture.Hybridization picture is analyzed using Agilent Feature Extraction softwares
And data are obtained, the statistical procedures of other data use 19.0 softwares of SPSS.Real-PCR confirms real after reaction
The amplification curve and melt curve analysis of time PCR.The Ct values of sample take the average value repeated three times.With Δ Δ Ct methods to purpose base
Because carrying out relative quantification.The relative expression quantity of target gene=2- Δ Δ Ct, Δ Δ Ct=experimental group（Target gene Ct- internal reference bases
Because of Ct）Control group（Target gene Ct- reference genes Ct）.Experimental data is indicated with i ± s, using t check analyses, p<0.05
It is significant.
MiRNA chip results
Through scanning visible sample RNA punctate fluorescence signals after microarray hybridization, illustrate that chip results are miRNA chips detection matrixes
Figure（See Fig. 1）.
It is very bright that experimental result shows that miRNAs express spectras exist between lung cancer cell line and normal lung epithelial cell line
Aobvious difference（See Fig. 2, Fig. 3）.
X axis in Fig. 2 represents control group data or experimental group data with Y axis.Point except 45 ° of lines, represents this spy
Pin mark in two chips signal value difference Fold Change values in 2 times or more.
Red in Fig. 3 to indicate up-regulation, green indicates to lower, compared with normal lung epithelial system, hsa- in lung cancer cell line
MiR-25, hsa-miR-222, hsa-miR-30e, hsa-miR-320e, hsa-miR-22, hsa-miR-16 etc. 24
The expression of miRNA significantly raises（Fold change values are more than 2 times）, and hsa-miR-9, hsa-miR-130a, hsa-
19 miRNA such as miR-21, hsa-miR-205 are significantly lowered（Fold change <0.5）.
Real-time PCR detection cancerous lung tissue miRNA-130a expression due between miRNA family members crossing instances compared with
More, to ensure the correctness of experimental result, we test the miRNA of differential expression in chip results using the method for PCR
Card, PCR methods measure the expression of the miR-130a that selects in 11 pairs of cancerous lung tissue/Carcinoma side normal tissues, while miR-130a
Expression on lung cancer cell line and normal bronchial in skin system cell is analysed and compared（It tests internal reference and uses people U6
RNA）.As a result as follows：
Compared with normal lung epithelial system, hsa-miR-25, hsa-miR-222, hsa-miR-30e, hsa- in lung cancer cell line
The expression of 24 miRNA such as miR-320e, hsa-miR-22, hsa-miR-16 significantly raises（Fold change values
More than 2 times）, and under 19 miRNA such as hsa-miR-9, hsa-miR-130a, hsa-miR-21, hsa-miR-205 are notable
It adjusts（Fold change <0.5）.
MiR-130a in lung cancer cell line/normal bronchial epithelial cell line is expressed as Fig. 4.RT-PCR experiment knots
Fruit prompts：Expression of the miR-130a in lung cancer cell line (A549, Calu-3) is in significantly downward（P<0.05）.With chip knot
Fruit is almost the same（See Fig. 4）.
The expression of miRNA-130a in cancerous lung tissue and normal lung tissue
The prompt of RT-PCR experimental results compares normal lung tissue, and miRNA-130a expresses apparent lower in cancerous lung tissue（Fig. 5）.
Its amplification curve and volume curve are clear, and flexion is clearly demarcated, illustrate PCR amplification credible result, high specificity（See Fig. 6, Fig. 7）.
Expression of the miR-130a in the cancerous lung tissue of histological types
For the expression of the miR-130a in more different types of cancerous lung tissue, we take out the tracheae of 25 Small Cell Lung Cancer again
Mirror sample does quantitative fluorescent PCR inspection, the results show that with normal structure ratio, in cancerous lung tissue the expression of miR-130a obviously drop
It is low（0.24±0.10）, difference is statistically significant；Histological types relatively in, Small Cell Lung Cancer and non-small cell lung cancer
Between miR-130a expressions without apparent gap（data not shown）（P > 0.05）；Non-small cell lung squamous cancer tissue miR-
130a is expressed（0.68±0.13）Less than adenocarcinoma of lung（P < 0.05）；In addition, lymphatic metastasis group（N1）The miR-130a tables of patient
It reaches（0.41±0.06）Significantly lower than the non-transfer group of lymph node (N0)（P < 0.05）, and with the raising of clinical staging of lung cancer,
MiR-130a is expressed in III/IV phase patient tissue（0.52±0.14）Occur being significantly lower than I/II（P < 0.05）（See Fig. 8）.
It prompts the expression in lung squamous cancer low compared with gland cancer, indicates that prognosis squamous carcinoma is preferable compared to gland cancer.
Second part：Pass through the technical research such as immunoblotting, quantitative PCR, Transwell and luciferase reporting experiment
Effects of the miR-130a in lung cancer occurrence and development
Case selection and sample are collected
This research passes through the approval of Lishui People's Hospital's Medical Ethics Committee, the equal signed informed consent of every subject
Book is agreed to use sample and clinical data.Sample is same as above.
The culture and passage and transfection of the conventional cell strain of progress, specification is transfected by Lipofectamine 3000
Carry out miR-130a mimic groups（A549 and Calu-3 groups of cells）With normal pulmonary epithelial cells control group (normal
Control groups) transfection, Trizol methods extraction transfection miR-130a mimic each group cells total serum IgE, reverse transcription
CDNA, fluorescence quantitative RT-RCR detect the expression of miR-130a in each group cell.RT-PCR methods detect each group cell
The cell strain of miR-130a expression real-time quantitative RT-PCR method detection transfection miR-130a in strain（A549 and Calu-3）With
The expression quantity of NC groups.From shown in Fig. 2-1 as it can be seen that the expression quantity of miR-130a is relatively low in NC group cell strains, and two kinds transfected
MiR-130a expression quantity is higher in lung cancer cell line, has statistical significance（P < 0.05）, prompt to be overexpressed miR-
The lung carcinoma cell of 130a can raise miR-130a expression（See Fig. 9）.
Influences of the miR-130a to proliferation of lung cancer cells ability
CCK-8 experimental results show that after being overexpressed miR-130a, the proliferative capacity of lung cell A549, Calu-3 receives
Inhibit, shows that miR-130a can inhibit the proliferation of lung carcinoma cell, the low expression in cancerous lung tissue may be promotion lung cancer
One of the reason of occurrence and development（See Figure 10）.
EdU experimental results are shown：
The ability of cell proliferation for being overexpressed 2 groups of lung carcinoma cells of miR-130a is remarkably decreased compared with cellular control unit.There are statistics
Learn difference（* P < 0.01）（See Figure 11,12）.
Transwell detects lung carcinoma cell invasion transfer ability
The results show that after miR-130a up-regulated expressions, A549 cell migration abilities are decreased obviously Transwell, pass through cell film
Cell number be less than NC groups (46.5 ± 5.30 vs 74.6 ± 4.37), Calu-3 cell migration abilities are significantly reduced, lower room
Face cell number is less than NC groups (34.0 ± 6.3vs 67.5 ± 3.10), and difference is statistically significant（P < 0.05)；Matrigel
The results show that across the A549 cell numbers of the cells Transwell film（15.8±2.1vs 33.5±1.50）It is thin with Calu-3
Born of the same parents' number（8.00±0.62 vs 22.8±2.47）It is decreased obviously, this is the result shows that miR-130a can inhibit lung cancer
The invasion of cell are shifted（See Figure 13）.
MiR-130a induces Increase Apoptosis of Lung Cancer Cells
Apoptosis detection finds that the Increase Apoptosis of Lung Cancer Cells quantity for being overexpressed miR-130a is apparent compared with cellular control unit and increases
Add.MiR-130a is prompted to can induce Increase Apoptosis of Lung Cancer Cells.
Soft agar plate clone, which forms experiment, confirms that miR-130a inhibits the growing multiplication soft agar tablet gram of lung carcinoma cell
It is grand formed as a result, it has been found that, transfected miR-130a lung carcinoma cell cloning efficiency be far below cellular control unit（The equal < of P
0.05）.The result shows the overexpressions of miR-130a can significantly inhibit the growing multiplication of cell.
Influences of the miR-130a to the lung carcinoma cell period
Flow Cytometry detects influences of the miR-130a to the lung carcinoma cell period, the results show that compared with NC groups, miR-130a
There are the G1/S phases and blocks in the lung carcinoma cell of mimics groups, show after miR-130a expression increases by regulate and control the G1/S phases convert come
Inhibit the growth of lung carcinoma cell.
Claims (10)
1. the primer of hsa-miR-130a expressions and/or probe are being made in one or more specifically detection subject's samples
The purposes being ready for use in the reagent of non-small cell lung cancer prognosis.
2. purposes according to claim 1, the wherein nucleotide sequence 5 '-of the mature rna of hsa-miR-130a
CAGUGGAAUGUUAUAUGAGCAU-3’。
3. purposes according to claim 1, wherein the non-small cell lung cancer is primary lung cancer.
4. purposes according to claim 1, wherein subject's sample is cancerous tissue sample, preferably non-small cell lung
Cancerous tissue sample.
5. purposes according to claim 4, wherein the Non-Small Cell Lung Carcinoma sample is obtained by way of biopsy
, or obtained by way of surgical excision.
6. purposes according to claim 1, wherein the subject is mammal.
7. purposes according to claim 6, wherein the mammal is people.
8. according to claim 1-7 any one of them purposes, wherein hsa-miR-130a expressions improve instruction squamous carcinoma group
The expression knitted is less than gland cancer, prompts have good effect in terms of non-small cell lung cancer prognosis guidance.
9. according to claim 1-7 any one of them purposes, wherein the prognosis refer to subject's untreated existence it is pre-
Afterwards.
10. one kind being used for non-small cell lung cancer prognosis kit, it includes for specifically detecting hsa- in subject's sample
The primer and/or probe of miR-130a expressions.
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