CN108103202A - 7 kind serum excretion body miRNAs relevant with liver cancer diagnosis and treatment and its application - Google Patents
7 kind serum excretion body miRNAs relevant with liver cancer diagnosis and treatment and its application Download PDFInfo
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Abstract
The invention discloses 7 kind serum excretion body miRNAs relevant with liver cancer diagnosis and treatment and its applications.Present invention firstly discovers that hsa miR 122, hsa miR 125b, hsa miR 145, hsa miR 192, hsa miR 194, the hsa miR 29a and hsa miR 106a in serum excretion body can be as the biomarkers of liver cancer, with higher diagnostic value, fast and accurately diagnostic mode is provided for clinic, the diagnosis for making liver cancer is more convenient and easy.The present invention can inhibit hepatoma cell proliferation/growth it is further found that the special miRNA of inhibition, lay a good foundation for clinical treatment liver cancer.
Description
Technical field
The invention belongs to biotechnologys and diagnostic field, and in particular to 7 kinds of serum excretion bodies relevant with liver cancer diagnosis and treatment
MiRNAs and its application, 7 kinds of serum excretion body miRNAs be specially hsa-miR-122, hsa-miR-125b, hsa-miR-145,
Hsa-miR-192, hsa-miR-194, hsa-miR-29a and hsa-miR-106a.
Background technology
Liver cancer is that incidence occupies the whole world the 4th, the malignant tumour of the death rate second.Because diet, alcoholic liver disease are especially
The relation of hepatic sclerosis, hepatitis B, liver cancer have frequently-occurring and high risk sexual in China.Many liver cancer have been to face when being usually found
Late period is presented on bed so that 5 years survival rates of China's liver cancer are less than 10%.Therefore, the diagnosis of the hepatic sclerosis of early stage and liver cancer is extremely
It closes important.If currently used for the blood markers owner alpha-fetoprotein (AFP) of diagnosing cancer of liver, but its accuracy is not good enough, sensitive
Degree and specificity are insufficient for the early detection of liver cancer.
Excretion body refer to contain complicated RNA and protein, diameter 40-100nm plate-like vesica.Various kinds of cell exists
Excretion body can be secreted under normal and pathological state, intracellular lysosome particle is mainly derived from and invaginates the more vesicas to be formed
Body is discharged into after the external film of more vesicas and cell membrane fusion in extracellular matrix.The cell type of all cultures can be secreted outer
Body is secreted, and excretion body is naturally occurring in body fluid (including blood, saliva, urine, cerebrospinal fluid and milk).
With the development in liquid biopsy field, the biomarker in excretion body source is increasingly paid attention to by everybody.Excretion
Stability and convenience of the body in blood circulation are a kind of extraordinary circulating tumor markers, are noninvasive and effectively examine
Disconnected marker.MiRNA is a kind of non-coding RNA of endogenic, length in 22nt or so, it can be by combining target gene
3 ' UTR areas carry out expression and the function of controlling gene.Numerous studies show that miRNA can be as the diagnosis of tumour and other diseases
Marker.
The content of the invention
The object of the present invention is to provide 7 kind serum excretion body miRNAs relevant with liver cancer diagnosis and treatment and its applications.
Present invention protection inhibits application of the substance of special miRNA in product is prepared；
The function of the product is following (b1), (b2), (b3) or (b4)：
(b1) hepatoma cell proliferation is inhibited；
(b2) liver cancer cell growth is inhibited；
(b3) liver cancer cells invasion and attack are inhibited；
(b4) liver cancer is treated.
Inhibit the substance of hsa-miR-106a as the single stranded RNA shown in the sequence 31 of sequence table.
The present invention also protection is enclosed with application of the excretion body for the substance for inhibiting special miRNA in product is prepared；
The function of the product is following (b1), (b2), (b3) or (b4)：
(b1) hepatoma cell proliferation is inhibited；
(b2) liver cancer cell growth is inhibited；
(b3) liver cancer cells invasion and attack are inhibited；
(b4) liver cancer is treated.
Inhibit the substance of hsa-miR-106a as the single stranded RNA shown in the sequence 31 of sequence table.
Excretion body and the proportioning for the substance for inhibiting special miRNA can be：10 μ g excretions bodies (with albumen gauge)：20-
2000pmol inhibits the substance of special miRNA.Excretion body and the proportioning for the substance for inhibiting special miRNA can be：10 μ g excretion bodies
(with albumen gauge)：200pmol inhibits the substance of special miRNA.
The present invention also protects the single stranded RNA shown in the sequence 31 of sequence table.
The excretion body of single stranded RNA of the present invention also shown in the sequence 31 of protection package ordered list.
The present invention also protects applications of the special miRNA in product is prepared；
The function of the product is following (c1), (c2), (c3) or (c4)：
(c1) hepatoma cell proliferation is promoted；
(c2) liver cancer cell growth is promoted；
(c3) liver cancer cells invasion and attack are promoted；
(c4) liver cancer animal model is made.
The present invention also protects application of the analogies of special miRNA in product is prepared；
The function of the product is following (c1), (c2), (c3) or (c4)：
(c1) hepatoma cell proliferation is promoted；
(c2) liver cancer cell growth is promoted；
(c3) liver cancer cells invasion and attack are promoted；
(c4) liver cancer animal model is made.
The analogies of hsa-miR-106a is shown in the single stranded RNA as shown in the sequence 29 of sequence table and the sequences of sequence table 30
The double-stranded RNA that single stranded RNA is formed.
The present invention also protects to detect application of the substance of special miRNA in product is prepared；
The function of the product is following (d1) or (d2)：
(d1) identify or aid in identification liver cancer cells；
(d2) diagnosis or auxiliary diagnosis liver cancer.
It is described to be combined for detecting the substance of special miRNA for primer.
The primer is combined as primer sets A and/or primer sets B and/or primer sets C and/or primer sets D and/or primer sets E
And/or primer sets F and/or primer sets G；
Each primer sets are made of reverse transcriptase primer, sense primer and anti-sense primer；
In primer sets A, reverse transcriptase primer is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 26 of sequence table；
(e2) sequence 26 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 26
There is the DNA molecular of identical function；
In primer sets A, sense primer is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 27 of sequence table；
(e4) sequence 27 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 27
There is the DNA molecular of identical function；
In primer sets A, anti-sense primer is following (e5) or (e6)：
(e5) single strand dna shown in the sequence 28 of sequence table；
(e6) sequence 28 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 28
There is the DNA molecular of identical function；
In primer sets B, reverse transcriptase primer is following (f1) or (f2)：
(f1) single strand dna shown in the sequence 17 of sequence table；
(f2) sequence 17 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 17
There is the DNA molecular of identical function；
In primer sets B, sense primer is following (f3) or (f4)：
(f3) single strand dna shown in the sequence 18 of sequence table；
(f4) sequence 18 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 18
There is the DNA molecular of identical function；
In primer sets B, anti-sense primer is following (f5) or (f6)：
(f5) single strand dna shown in the sequence 19 of sequence table；
(f6) sequence 19 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 19
There is the DNA molecular of identical function；
In primer sets C, reverse transcriptase primer is following (g1) or (g2)：
(g1) single strand dna shown in the sequence 8 of sequence table；
(g2) sequence 8 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 8
The DNA molecular of identical function；
In primer sets C, sense primer is following (g3) or (g4)：
(g3) single strand dna shown in the sequence 9 of sequence table；
(g4) sequence 9 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 9
The DNA molecular of identical function；
In primer sets C, anti-sense primer is following (g5) or (g6)：
(g5) single strand dna shown in the sequence 10 of sequence table；
(g6) sequence 10 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 10
There is the DNA molecular of identical function；
In primer sets D, reverse transcriptase primer is following (h1) or (h2)：
(h1) single strand dna shown in the sequence 20 of sequence table；
(h2) sequence 20 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 20
There is the DNA molecular of identical function；
In primer sets D, sense primer is following (h3) or (h4)：
(h3) single strand dna shown in the sequence 21 of sequence table；
(h4) sequence 21 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 21
There is the DNA molecular of identical function；
In primer sets D, anti-sense primer is following (h5) or (h6)：
(h5) single strand dna shown in the sequence 22 of sequence table；
(h6) sequence 22 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 22
There is the DNA molecular of identical function；
In primer sets E, reverse transcriptase primer is following (i1) or (i2)：
(i1) single strand dna shown in the sequence 23 of sequence table；
(i2) sequence 23 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 23
There is the DNA molecular of identical function；
In primer sets E, sense primer is following (i3) or (i4)：
(i3) single strand dna shown in the sequence 24 of sequence table；
(i4) have by sequence 24 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 24
There is the DNA molecular of identical function；
In primer sets E, anti-sense primer is following (i5) or (i6)：
(i5) single strand dna shown in the sequence 25 of sequence table；
(i6) sequence 25 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 25
There is the DNA molecular of identical function；
In primer sets F, reverse transcriptase primer is following (j1) or (j2)：
(j1) single strand dna shown in the sequence 11 of sequence table；
(j2) sequence 11 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 11
There is the DNA molecular of identical function；
In primer sets F, sense primer is following (j3) or (j4)：
(j3) single strand dna shown in the sequence 12 of sequence table；
(j4) sequence 12 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 12
There is the DNA molecular of identical function；
In primer sets F, anti-sense primer is following (j5) or (j6)：
(j5) single strand dna shown in the sequence 13 of sequence table；
(j6) sequence 13 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 13
There is the DNA molecular of identical function；
In primer sets G, reverse transcriptase primer is following (k1) or (k2)：
(k1) single strand dna shown in the sequence 14 of sequence table；
(k2) sequence 14 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 14
There is the DNA molecular of identical function；
In primer sets G, sense primer is following (k3) or (k4)：
(k3) single strand dna shown in the sequence 15 of sequence table；
(k4) sequence 15 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 15
There is the DNA molecular of identical function；
In primer sets G, anti-sense primer is following (k5) or (k6)：
(k5) single strand dna shown in the sequence 16 of sequence table；
(k6) sequence 16 is had by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 16
There is the DNA molecular of identical function.
The primer combination falls within protection scope of the present invention.The function of primer combination be following (d1) or
(d2)：(d1) identify or aid in identification liver cancer cells；(d2) diagnosis or auxiliary diagnosis liver cancer.
Special miRNA described in any of the above is following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5)
And/or (a6) and/or (a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145。
The concretely kit of product described in any of the above.
The hsa-miR-106a is as shown in the sequence 7 of sequence table.
The hsa-miR-192 is as shown in the sequence 4 of sequence table.
The hsa-miR-122 is as shown in the sequence 1 of sequence table.
The hsa-miR-194 is as shown in the sequence 5 of sequence table.
The hsa-miR-29a is as shown in the sequence 6 of sequence table.
The hsa-miR-125b is as shown in the sequence 2 of sequence table.
The hsa-miR-145 is as shown in the sequence 3 of sequence table.
Excretion body described in any of the above is serum excretion body.
The substance of the special miRNA of detection described in any of the above is the substance of special miRNA in detection excretion body.
The substance of the special miRNA of detection described in any of the above is the substance of special miRNA in detection serum excretion body.
Present invention firstly discovers that hsa-miR-122, hsa-miR-125b, hsa-miR-145 in serum excretion body,
Hsa-miR-192, hsa-miR-194, hsa-miR-29a and hsa-miR-106a can have as the biomarker of liver cancer
Higher diagnostic value provides fast and accurately diagnostic mode for clinic, and the diagnosis for making liver cancer is more convenient and easy.The present invention also into
One step finds to inhibit special miRNA, can inhibit hepatoma cell proliferation/growth, lay a good foundation for clinical treatment liver cancer.
Description of the drawings
Fig. 1 is the exemplary photo that excretion body is observed using projection electron microscope.
Fig. 2 is the example results for the significant antigen that excretion body is detected using westernblot.
Fig. 3 is the result of HEPG2 Cells Cell Proliferations experiment.
Fig. 4 is the result of SMMC-7721 Cells Cell Proliferations experiment.
Fig. 5 is the result of cell invasion test.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Any skill for being familiar with this field
In the range of the careless mistake of the present invention, technique according to the invention scheme and design are replaced or change and belong to this hair art personnel
Bright protection category.Experimental method in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Test material is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantifying in following embodiment
Experiment, is respectively provided with three repeated experiments, results are averaged.Using T method of inspection statistical experiment the data obtaineds, P values are less than
0.05 and variation multiple be considered to have statistical significance more than 2 times.
The rabbit of anti-CD 63 is mostly anti-：Proteintech,25682-1-AP.
The rabbit of anti-CD9 is mostly anti-：Proteintech,20597-1-AP.
HEPG2 cells (Bel7402)：Chinese Academy of Sciences's cell bank (catalog number (Cat.No.)：TCHu 2).
SMMC-7721 cells (Bel7402)：Chinese Academy of Sciences's cell bank (catalog number (Cat.No.)：TCHu 52).
The discovery and verification of embodiment 1,7 kind of serum excretion body miRNAs liver cancer marker
Serum sample is 110 parts, wherein 80 parts are cured from December, 2015 in January, 2017 in University Of Suzhou attached first
(volunteer of informed consent is diagnosed as I-IV phase liver cancer patients through Histopathology, in being admitted to hospital for 80 liver cancer patients that institute makes a definite diagnosis
Take a blood sample afterwards before non-underwent operative and chemicotherapy), remaining 30 parts are carried out 30 Healthy People (informed consents that automatic self synchronizing carries out disorder in screening
Volunteer).The information of 80 patients and 30 Healthy Peoples is shown in Table 1.
Table 1
Each serum sample is detected respectively.
First, the extraction and identification of serum excretion body
1st, serum sample is taken, excretion body is extracted using the PEG-base precipitation method.
2nd, the excretion body that step 1 obtains is taken, is observed using projection electron microscope.Each serum sample obtains
Excretion body, exemplary photo are shown in Fig. 1.Excretion body is shown in the arrows in Fig. 1.
3rd, the excretion body that step 1 obtains is taken, total protein is extracted after being cracked with cell pyrolysis liquid.Total protein is taken, carries out SDS-
PAGE and westernblot.The primary antibody used resists for the rabbit of anti-CD 63 more or the rabbit of anti-CD9 is mostly anti-, and the secondary antibody used is horseradish
The goat antirabbit lgG of peroxidase labelling.Using serum sample as the control of excretion body.The excretion that each serum sample obtains
Body is CD63 and CD9 double positive, and example results are shown in Fig. 2.
2nd, the extracting of excretion body total serum IgE
The 1 of step 1 obtained excretion body is taken, extracts total serum IgE.
3rd, reverse transcription and qPCR detections
1st, the total serum IgE that step 2 is taken to obtain carries out reverse transcription using reverse transcriptase primer, obtains cDNA.
2nd, using the cDNA that step 1 obtains as template, quantitative fluorescent PCR is carried out, detects 7 kinds of miRNA respectively.
Reaction system (20 μ l)：By 10 μ l 2 × SYBR Green qPCR Mix, sense primer solution (20 μM), downstream
Primer solution (20 μM), template, rTaq archaeal dna polymerases solution (5U/ μ L) and dd H2O is formed.In reaction system, sense primer
Concentration with anti-sense primer is 0.10 μM, and the concentration of rTaq archaeal dna polymerases is 0.05U/ μ L, and template addition is 50ng.
Reaction condition：94℃3min；94 DEG C of 12s, 62 DEG C of 30s, 72 DEG C of 30s, 40 Xun Huans.
7 kinds of miRNA are as follows：
Hsa-miR-122 (sequence 1 of sequence table)：5’-uggagugugacaaugguguuug-3’；
Hsa-miR-125b (sequence 2 of sequence table)：5’-ucccugagacccuaacuuguga-3’；
Hsa-miR-145 (sequence 3 of sequence table)：5’-guccaguuuucccaggaaucccu-3’；
Hsa-miR-192 (sequence 4 of sequence table)：5’-cugaccuaugaauugacagcc-3’；
Hsa-miR-194 (sequence 5 of sequence table)：5’-uguaacagcaacuccaugugga-3’；
Hsa-miR-29a (sequence 6 of sequence table)：5’-acugauuucuuuugguguucag-3’；
Hsa-miR-106a (sequence 7 of sequence table)：5’-aaaagugcuuacagugcagguag-3’.
Using hsa-miR-16 as the internal reference of 7 kinds of miRNA.Hsa-miR-16 is the known stable content in serum excretion body
MiRNA.
hsa-miR-16：5’-uagcagcacguaaauauuggcg-3’.
Primer for detecting 7 kinds of miRNA and hsa-miR-16 is shown in Table 2.MiR122-RT (sequence 8 of sequence table),
Mir-122-FO (sequence 9 of sequence table) and miR-122-RE (sequence 10 of sequence table) is used to detect hsa-miR-122.
MiR125b-RT (sequence 11 of sequence table), miR-125b-FO (sequence 12 of sequence table) and miR-125b-RE be (sequence table
Sequence 13) it is used to detect hsa-miR-125b.MiR145-RT (sequence 14 of sequence table), the miR-145-FO (sequences of sequence table
15) and miR-145-RE (sequence 16 of sequence table) is used to detect hsa-miR-145.MiR192-RT (sequence 17 of sequence table),
MiR-192-FO (sequence 18 of sequence table) and miR-192-RE (sequence 19 of sequence table) is used to detect hsa-miR-192.
MiR194-RT (sequence 20 of sequence table), miR-194-FO (sequence 21 of sequence table) and the miR-194-RE (sequences of sequence table
22) it is used to detect hsa-miR-194.MiR29a-RT (sequence 23 of sequence table), miR-29a-FO (sequence 24 of sequence table) and
MiR-29a-RE (sequence 25 of sequence table) is used to detect hsa-miR-29a.MiR106a-RT (sequence 26 of sequence table), miR-
106a-FO (sequence 27 of sequence table) and miR-106a-RE (sequence 28 of sequence table) is used to detect hsa-miR-106a.miR-
16-RT, miR-16-FO and miR-16-RE are used to detect hsa-miR-16.FO represents sense primer, and RE represents anti-sense primer, RT
Represent reverse transcriptase primer.
Table 2
Using the content of hsa-miR-16 in serum excretion body as 1, the relative expression levels of 7 kinds of miRNA are calculated.
Compare the relative expression levels of 7 kinds of miRNA in liver cancer patient group and Healthy People group serum excretion body, the results are shown in Table 3.
In liver cancer patient group hsa-miR-122, hsa-miR-125b in serum excretion body, hsa-miR-145, hsa-miR-192,
Hsa-miR-194, hsa-miR-29a and hsa-miR-106a relative expression levels are significantly higher than Healthy People group (p<0.05).Into
Row ROC curve is analyzed, and the results are shown in Table 4.AUC areas are between 0.569-0.752, p ＜ 0.005.The result shows that in serum excretion body
Hsa-miR-122, hsa-miR-125b, hsa-miR-145, hsa-miR-192, hsa-miR-194, hsa-miR-29a and
The relative expression levels of hsa-miR-106a are related to liver cancer generation.
The relative expression levels of hsa-miR-122 are more than 0.0112 as the threshold value for being judged as liver cancer patient, accuracy rate
For 74.6%.The relative expression levels of hsa-miR-125b are more than 0.00196 as the threshold value for being judged as liver cancer patient, accurately
Rate is 65.0%.The relative expression levels of hsa-miR-145 are more than 0.001177 as the threshold value for being judged as liver cancer patient, it is accurate
True rate is 56.9%.The relative expression levels of hsa-miR-192 are more than 0.00128 as the threshold value for being judged as liver cancer patient,
Accuracy rate is 75.2%.The relative expression levels of hsa-miR-194 are more than 0.000376 as the threshold for being judged as liver cancer patient
Value, accuracy rate 73.8%.The relative expression levels of hsa-miR-29a are more than 0.0198 as the threshold for being judged as liver cancer patient
Value, accuracy rate 70.3%.The relative expression levels of hsa-miR-106a are more than 0.0156 as being judged as liver cancer patient
Threshold value, accuracy rate 70.4%.
Table 3
Table 4
| miRNA | AUC | 95% confidence interval |
| hsa-miR-122 | 0.746 | 0.650-0.842 |
| hsa-miR-125b | 0.650 | 0.526-0.744 |
| hsa-miR-145 | 0.569 | 0.422-0.649 |
| hsa-miR-192 | 0.752 | 0.658-0.846 |
| hsa-miR-194 | 0.738 | 0.638-0.838 |
| hsa-miR-29a | 0.703 | 0.597-0.809 |
| hsa-miR-106a | 0.704 | 0.534-0.873 |
Embodiment 2, cell proliferation test
Determinand is：Hsa-miR-106a analogies or hsa-miR-106a mortifiers.
Hsa-miR-106a analogies are single shown in the single stranded RNA as shown in the sequence 29 of sequence table and the sequence of sequence table 30
The double-stranded RNA that chain RNA is formed.
The sequence 29 of sequence table：5’-AAAAGUGCUUACAGUGCAGGUAG-3’；
The sequence 30 of sequence table：5’-ACCUGCACUGUAAGCACUUUUUU-3’.
Hsa-miR-106a mortifiers are the single stranded RNA shown in the sequence 31 of sequence table.
The sequence 31 of sequence table：5’-CUACCUGCACUGUAAGCACUUUU-3’.
Cell to be measured is：HEPG2 cells or SMMC-7721 cells.
1st, using DMEM culture solutions suspension cell to be measured, then by cell suspension inoculation into 96 orifice plates, per 100 μ l of hole
(contain 105A cell), when then 37 DEG C of quiescent cultures 24 are small.
2nd, the preparation method of every part of mixture：The excretion body of healthy person prepared by the 1 of the step of 20 μ l embodiment 1 one (contains
10 μ g excretion bodies, with albumen gauge) and the mixing of 200pmol determinands, obtain mixture.
3rd, after completing step 1,96 orifice plate is taken, supernatant is abandoned in suction, mixture prepared by 1 part of step 2 is added in per hole, then
37 DEG C of 0 moment of quiescent culture, 24 it is small when, 48 it is small when, 72 it is small when or 96 it is small when.It sets and only adds in excretion body, it is mixed to replace adding in
Close the control of object.
4th, after completing step 3, operated using CCK8 kits (Japan's east Renhua subject skill, CK04) and by specification, inspection
Survey cell Proliferation vigor.
Using the cell viability at 0 moment as 1, calculate 24 it is small when, 48 it is small when, 72 it is small when or 96 it is small when after cell it is relatively living
Power.
Three reprocessings are carried out, results are averaged.
HEPG2 cells the result is shown in Fig. 3.SMMC-7721 cells the result is shown in Fig. 4.The result shows that hsa-miR-106a moulds
Growth and the multiplication of liver cancer cells can be promoted by intending object, and hsa-miR-106a mortifiers then inhibit the growth and increasing of liver cancer cells
It grows.
Embodiment 3, cell invasion test
Determinand is the same as embodiment 2.
Cell to be measured is the same as embodiment 2.
1st, using DMEM culture solutions suspension cell to be measured, then by cell suspension inoculation into 96 orifice plates, per 100 μ l of hole
(contain 105A cell), when then 37 DEG C of quiescent cultures 24 are small.
2nd, the preparation method of every part of mixture：The excretion body of healthy person prepared by the 1 of the step of 20 μ l embodiment 1 one (contains
10 μ g excretion bodies, with albumen gauge) and the mixing of 200pmol determinands, obtain mixture.
3rd, after completing step 1,96 orifice plate is taken, supernatant is abandoned in suction, mixture prepared by 1 part of step 2 is added in per hole, then
When 37 DEG C of quiescent cultures 24 are small, cell is collected.It sets and only adds in excretion body, to replace adding in the control of mixture.
4th, matrigel is mixed in 4 DEG C of thawings, the matrigel that 100 μ l is taken to melt with 400 μ l serum-free DMEM culture solutions
It is even.
5th, take and wear film cell, the upper strata of each cell adds in the mixture that 500 μ l steps 4 obtain, and lower floor is added in containing 10%
The DMEM culture solutions of hyclone simultaneously make it touch the intermediate coat of cell.
6th, after completing step 5, take it is described wear film cell, the cell that step 3 is collected, 37 DEG C of quiescent cultures 24 are added on upper strata
Hour.
7th, after completing step 6, take it is described wear film cell, wipe intermediate coat towards the cell of the one side on upper strata, it is small by film is worn
Room, which is inverted, dries, and the one side of intermediate coat towards lower floor is carried out formaldehyde fixes, and then carries out violet staining.
Coloration result is shown in Fig. 5.The result shows that hsa-miR-106a analogies can promote the invasion and attack of liver cancer cells, and
Hsa-miR-106a mortifiers then inhibit the invasion and attack of liver cancer cells.
SEQUENCE LISTING
<110>Suzhou GenePharma Co., Ltd.
Shanghai JiMa pharmacy Technology Co., Ltd
<120>7 kind serum excretion body miRNAs relevant with liver cancer diagnosis and treatment and its application
<130> GNCYX180568
<160> 31
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> RNA
<213> Artificial sequence
<400> 1
uggaguguga caaugguguu ug 22
<210> 2
<211> 22
<212> RNA
<213> Artificial sequence
<400> 2
ucccugagac ccuaacuugu ga 22
<210> 3
<211> 23
<212> RNA
<213> Artificial sequence
<400> 3
guccaguuuu cccaggaauc ccu 23
<210> 4
<211> 21
<212> RNA
<213> Artificial sequence
<400> 4
cugaccuaug aauugacagc c 21
<210> 5
<211> 22
<212> RNA
<213> Artificial sequence
<400> 5
uguaacagca acuccaugug ga 22
<210> 6
<211> 22
<212> RNA
<213> Artificial sequence
<400> 6
acugauuucu uuugguguuc ag 22
<210> 7
<211> 23
<212> RNA
<213> Artificial sequence
<400> 7
aaaagugcuu acagugcagg uag 23
<210> 8
<211> 60
<212> DNA
<213> Artificial sequence
<400> 8
gtctgtatgg ttgttcacga ctccttcaca tccctatcca accatacaga ccaaacacca 60
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence
<400> 9
gatgctctgg agtgtgacaa tg 22
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence
<400> 10
tatggttgtt cacgactcct tcac 24
<210> 11
<211> 59
<212> DNA
<213> Artificial sequence
<400> 11
gtctgtatgg ttgttctgct ctctgtcaca tccctatcta caaccataca gactcacaa 59
<210> 12
<211> 25
<212> DNA
<213> Artificial sequence
<400> 12
actgataaat ccctgagacc ctaac 25
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<212> DNA
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<400> 13
tatggttgtt ctgctctctg tcac 24
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<211> 61
<212> DNA
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<400> 14
gtctgtatgg ttgttcacga gtccttcaca tccctatcca accatacaga cagggattcc 60
t 61
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence
<400> 15
tgccgagtcc agttttccc 19
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence
<400> 16
tatggttgtt cacgagtcct tcac 24
<210> 17
<211> 59
<212> DNA
<213> Artificial sequence
<400> 17
gtctgtatgg ttgttcacga ctccttcaca tccctatcca accatacaga cggctgtca 59
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<400> 18
agccgctgac ctatgaattg 20
<210> 19
<211> 24
<212> DNA
<213> Artificial sequence
<400> 19
tatggttgtt cacgactcct tcac 24
<210> 20
<211> 62
<212> DNA
<213> Artificial sequence
<400> 20
agtcgggtct cagagcaggg tccgaggtac acgttcgctc tgagacccga cttccacatg 60
ga 62
<210> 21
<211> 20
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<213> Artificial sequence
<400> 21
agcccgtgta acagcaactc 20
<210> 22
<211> 19
<212> DNA
<213> Artificial sequence
<400> 22
cagagcaggg tccgaggta 19
<210> 23
<211> 62
<212> DNA
<213> Artificial sequence
<400> 23
gtctgtatcc ttgttcacga ctccttcaca tccctatcca aggatacaga ctaaccgatt 60
tc 62
<210> 24
<211> 19
<212> DNA
<213> Artificial sequence
<400> 24
ctgccgtagc accatctga 19
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence
<400> 25
tatccttgtt cacgactcct tcac 24
<210> 26
<211> 61
<212> DNA
<213> Artificial sequence
<400> 26
gtctgtatgg ttcttgacga ctggttgaca tccctatcga accatacaga cctacctgca 60
c 61
<210> 27
<211> 21
<212> DNA
<213> Artificial sequence
<400> 27
ccgagcgaaa agtgcttaca g 21
<210> 28
<211> 24
<212> DNA
<213> Artificial sequence
<400> 28
tatggttctt gacgactggt tgac 24
<210> 29
<211> 23
<212> RNA
<213> Artificial sequence
<400> 29
aaaagugcuu acagugcagg uag 23
<210> 30
<211> 23
<212> RNA
<213> Artificial sequence
<400> 30
accugcacug uaagcacuuu uuu 23
<210> 31
<211> 23
<212> RNA
<213> Artificial sequence
<400> 31
cuaccugcac uguaagcacu uuu 23
Claims (10)
1. inhibit application of the substance of special miRNA in product is prepared；
The special miRNA be following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5) and/or (a6) and/or
(a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145；
The function of the product is following (b1), (b2), (b3) or (b4)：
(b1) hepatoma cell proliferation is inhibited；
(b2) liver cancer cell growth is inhibited；
(b3) liver cancer cells invasion and attack are inhibited；
(b4) liver cancer is treated.
2. application as described in claim 1, it is characterised in that：The substance for inhibiting hsa-miR-106a is the sequence 31 of sequence table
Shown single stranded RNA.
3. it is enclosed with application of the excretion body for the substance for inhibiting special miRNA in product is prepared；
The special miRNA be following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5) and/or (a6) and/or
(a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145；
The function of the product is following (b1), (b2), (b3) or (b4)：
(b1) hepatoma cell proliferation is inhibited；
(b2) liver cancer cell growth is inhibited；
(b3) liver cancer cells invasion and attack are inhibited；
(b4) liver cancer is treated.
4. application as claimed in claim 3, it is characterised in that：The substance for inhibiting hsa-miR-106a is the sequence 31 of sequence table
Shown single stranded RNA.
5. the single stranded RNA shown in the sequence 31 of sequence table.
6. wrap up the excretion body of the single stranded RNA shown in the sequence 31 of ordered list.
7. applications of the special miRNA in product is prepared；
The special miRNA be following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5) and/or (a6) and/or
(a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145；
The function of the product is following (c1), (c2), (c3) or (c4)：
(c1) hepatoma cell proliferation is promoted；
(c2) liver cancer cell growth is promoted；
(c3) liver cancer cells invasion and attack are promoted；
(c4) liver cancer animal model is made.
8. application of the analogies of special miRNA in product is prepared；
The special miRNA be following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5) and/or (a6) and/or
(a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145；
The function of the product is following (c1), (c2), (c3) or (c4)：
(c1) hepatoma cell proliferation is promoted；
(c2) liver cancer cell growth is promoted；
(c3) liver cancer cells invasion and attack are promoted；
(c4) liver cancer animal model is made.
9. for detecting application of the substance of special miRNA in product is prepared；
The special miRNA be following (a1) and/or (a2) and/or (a3) and/or (a4) and/or (a5) and/or (a6) and/or
(a7)：
(a1)hsa-miR-106a；
(a2)hsa-miR-192；
(a3)hsa-miR-122；
(a4)hsa-miR-194；
(a5)hsa-miR-29a；
(a6)hsa-miR-125b；
(a7)hsa-miR-145；
The function of the product is following (d1) or (d2)：
(d1) identify or aid in identification liver cancer cells；
(d2) diagnosis or auxiliary diagnosis liver cancer.
10. a kind of primer combination, is primer sets A and/or primer sets B and/or primer sets C and/or primer sets D and/or primer sets E
And/or primer sets F and/or primer sets G；
Each primer sets are made of reverse transcriptase primer, sense primer and anti-sense primer；
In primer sets A, reverse transcriptase primer is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 26 of sequence table；
(e2) there is phase by sequence 26 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 26
The DNA molecular of congenerous；
In primer sets A, sense primer is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 27 of sequence table；
(e4) there is phase by sequence 27 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 27
The DNA molecular of congenerous；
In primer sets A, anti-sense primer is following (e5) or (e6)：
(e5) single strand dna shown in the sequence 28 of sequence table；
(e6) there is phase by sequence 28 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 28
The DNA molecular of congenerous；
In primer sets B, reverse transcriptase primer is following (f1) or (f2)：
(f1) single strand dna shown in the sequence 17 of sequence table；
(f2) there is phase by sequence 17 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 17
The DNA molecular of congenerous；
In primer sets B, sense primer is following (f3) or (f4)：
(f3) single strand dna shown in the sequence 18 of sequence table；
(f4) there is phase by sequence 18 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 18
The DNA molecular of congenerous；
In primer sets B, anti-sense primer is following (f5) or (f6)：
(f5) single strand dna shown in the sequence 19 of sequence table；
(f6) there is phase by sequence 19 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 19
The DNA molecular of congenerous；
In primer sets C, reverse transcriptase primer is following (g1) or (g2)：
(g1) single strand dna shown in the sequence 8 of sequence table；
(g2) have by sequence 8 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 8 identical
The DNA molecular of function；
In primer sets C, sense primer is following (g3) or (g4)：
(g3) single strand dna shown in the sequence 9 of sequence table；
(g4) have by sequence 9 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 9 identical
The DNA molecular of function；
In primer sets C, anti-sense primer is following (g5) or (g6)：
(g5) single strand dna shown in the sequence 10 of sequence table；
(g6) there is phase by sequence 10 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 10
The DNA molecular of congenerous；
In primer sets D, reverse transcriptase primer is following (h1) or (h2)：
(h1) single strand dna shown in the sequence 20 of sequence table；
(h2) there is phase by sequence 20 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 20
The DNA molecular of congenerous；
In primer sets D, sense primer is following (h3) or (h4)：
(h3) single strand dna shown in the sequence 21 of sequence table；
(h4) there is phase by sequence 21 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 21
The DNA molecular of congenerous；
In primer sets D, anti-sense primer is following (h5) or (h6)：
(h5) single strand dna shown in the sequence 22 of sequence table；
(h6) there is phase by sequence 22 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 22
The DNA molecular of congenerous；
In primer sets E, reverse transcriptase primer is following (i1) or (i2)：
(i1) single strand dna shown in the sequence 23 of sequence table；
(i2) there is phase by sequence 23 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 23
The DNA molecular of congenerous；
In primer sets E, sense primer is following (i3) or (i4)：
(i3) single strand dna shown in the sequence 24 of sequence table；
(i4) there is phase by sequence 24 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 24
The DNA molecular of congenerous；
In primer sets E, anti-sense primer is following (i5) or (i6)：
(i5) single strand dna shown in the sequence 25 of sequence table；
(i6) there is phase by sequence 25 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 25
The DNA molecular of congenerous；
In primer sets F, reverse transcriptase primer is following (j1) or (j2)：
(j1) single strand dna shown in the sequence 11 of sequence table；
(j2) there is phase by sequence 11 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 11
The DNA molecular of congenerous；
In primer sets F, sense primer is following (j3) or (j4)：
(j3) single strand dna shown in the sequence 12 of sequence table；
(j4) there is phase by sequence 12 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 12
The DNA molecular of congenerous；
In primer sets F, anti-sense primer is following (j5) or (j6)：
(j5) single strand dna shown in the sequence 13 of sequence table；
(j6) there is phase by sequence 13 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 13
The DNA molecular of congenerous；
In primer sets G, reverse transcriptase primer is following (k1) or (k2)：
(k1) single strand dna shown in the sequence 14 of sequence table；
(k2) there is phase by sequence 14 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 14
The DNA molecular of congenerous；
In primer sets G, sense primer is following (k3) or (k4)：
(k3) single strand dna shown in the sequence 15 of sequence table；
(k4) there is phase by sequence 15 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 15
The DNA molecular of congenerous；
In primer sets G, anti-sense primer is following (k5) or (k6)：
(k5) single strand dna shown in the sequence 16 of sequence table；
(k6) there is phase by sequence 16 by the substitution of one or several nucleotide and/or missing and/or addition and with sequence 16
The DNA molecular of congenerous.
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Application publication date: 20180601 |
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