CN108192970A - A kind of diagnosing cancer of liver marker and its application - Google Patents

A kind of diagnosing cancer of liver marker and its application Download PDF

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CN108192970A
CN108192970A CN201810068446.6A CN201810068446A CN108192970A CN 108192970 A CN108192970 A CN 108192970A CN 201810068446 A CN201810068446 A CN 201810068446A CN 108192970 A CN108192970 A CN 108192970A
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mir
liver cancer
expression
liver
tool
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沈百荣
钱福良
谢宇锋
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Suzhou University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses a kind of diagnosing cancer of liver marker and its applications, belong to biomedicine field.The present invention detects and demonstrates respectively differential expressions of 4 microRNA in liver cancer cancerous tissue, liver cancer cells in liver cancer tissue sample, liver cancer cell lines by the method for microRNA tailing quantitative fluorescent PCRs, obtains the RNA molecule that can be used as liver cancer marker.

Description

A kind of diagnosing cancer of liver marker and its application
Technical field
The present invention relates to a kind of diagnosing cancer of liver marker and its applications, belong to biomedicine field.
Background technology
Liver cancer is one of higher malignant tumour of global incidence and the death rate, the annual whole world new hair of more than half China is happened at dead liver cancer patient.Wherein, most liver cancer belongs to hepatocellular carcinoma.Find suitable biomarker It is of great significance for the diagnose and treat of liver cancer.MicroRNA is the non-coding that a kind of length is about 22-24 nucleotide RNA, they can the level up regulation gene expression after transcription.Due to the particularity of function itself, microRNA has preferable Expression specificity can be stable in the presence of in tissue and blood, can be as the biological marker of medical diagnosis on disease, prognosis and treatment Object.
Invention content
Present invention firstly provides a kind of liver cancer marker, base sequence such as SEQ ID NO:1(miR-378a-3p)、SEQ ID NO:2(miR-490-3p)、SEQ ID NO:3 (miR-381-3p) or SEQ ID NO:Shown in 4 (miR-101-3p).
The liver cancer marker can be used for preparing the tool of auxiliary diagnosis liver cancer or be used to prepare auxiliary Index for diagnosis Tool is used to prepare the tool that the drug of liver cancer is treated in screening.
The present invention is by the method for microRNA tailing quantitative fluorescent PCRs respectively in liver cancer tissue sample (by 28 pairs of cancers, cancer Tissue), detect and demonstrate 4 microRNA (miR-101-3p, miR-378a-3p, miR- in liver cancer cell lines (8 kinds) 490-3p, miR-381-3p) differential expression in liver cancer cancerous tissue, liver cancer cells.In tissue specimen level, 25/28 cancer MiR-101-3p expression is organized to lower；23/25 cancerous tissue miR-378a-3p expression is lowered；26/28 cancerous tissue miR-490-3p tables Up to downward；18/28 cancerous tissue miR-381-3p expression is lowered.Under cell line level, 8/8 cancer cell miR-101-3p expression It adjusts；8/8 cancer cell miR-378a-3p expression is lowered；8/8 cancer cell miR-490-3p expression is lowered；5/8 cancer cell miR-381- 3p expression is lowered.By the method for in situ hybridization, the present invention analyzes miR-378a-3p in 90 paired liver cancer organization chips Expression and miR-378a-3p expressions and liver cancer patient clinical pathological factors and the correlation of prognosis, the results showed that, miR- Expression of the 378a-3p in liver cancer tissue substantially reduces, negatively correlated with tumor size, vessel invasion, TNM stage, and and liver Cancer patient's prognosis difference correlation.
Description of the drawings
Fig. 1 liver cancer tissue microRNAqPCR amplification curve representative diagrams.(a) 1# cases cancer beside organism microRNAqPCR Amplification curve；(b) 1# cases cancerous tissue microRNAqPCR amplification curves.
Fig. 2 liver cancer tissues microRNAqPCR dissolves peak representative diagram.(a) 1# cases cancer beside organism microRNAqPCR is molten Xie Feng；(b) 1# cases cancerous tissue microRNAqPCR dissolves peak.
Fig. 3 liver cancer tissue microRNAqPCR results (2-ΔΔTMethod).(a) respectively to match cancer group of the cancer beside organism as control Knit the relative expression of miR-378a-3p；(b) respectively to match opposite table of the cancer beside organism as the cancerous tissue miR-490-3p of control It reaches；(c) respectively to match relative expression of the cancer beside organism as the cancerous tissue miR-381-3p of control；(d) respectively to match by cancer It is organized as the relative expression of the cancerous tissue miR-101-3p of control；(e) respectively to match cancerous tissue of the cancer beside organism as control The relative expression of miR-221-3p；(f) respectively to match relative expression of the cancer beside organism as the cancerous tissue miR-25-3p of control.
Fig. 4 liver cancer tissue microRNA qPCR results (Δ T methods).(a) miR-378a-3p qPCR scatter plots；(b)miR- 490-3p qPCR scatter plots；(c) miR-381-3p qPCR scatter plots；(d) miR-101-3p qPCR scatter plots；(e)miR- 221-3p qPCR scatter plots；(f) miR-25-3p qPCR scatter plots miR-378a-3p：Compared with cancer beside organism, * * p<0.01； miR-490-3p：Compared with cancer beside organism, * p<0.05；miR-381-3p：Compared with cancer beside organism, * p<0.05；miR-101- 3p：Compared with cancer beside organism, * * p<0.01；miR-221-3p：Compared with cancer beside organism, * p<0.05.
Fig. 5 liver cancer cells microRNAqPCR amplification curve representative diagrams.(a) L-02 liver cells miRNAqPCR amplifications are bent Line；(b) SMMC-7721 liver cancer cells miRNAqPCR amplification curves.
Fig. 6 liver cancer cells microRNAqPCR dissolves peak representative diagram.(a) L-02 liver cells miRNAqPCR dissolves peak； (b) SMMC-7721 liver cancer cells miRNAqPCR dissolves peak.
Fig. 7 liver cancer cells microRNAqPCR results (2-ΔΔTMethod).(a) using normal liver cell L-02 as 8 kinds of livers of control The relative expression of cancer cell miR-378a-3p；(b) using normal liver cell L-02 as 8 kinds of liver cancer cells miR-490-3p of control Relative expression；(c) using normal liver cell L-02 as the relative expression of 8 kinds of liver cancer cells miR-381-3p of control；(d) with just Normal liver cell L-02 is the relative expression of 8 kinds of liver cancer cells miR-101-3p of control；(e) using normal liver cell L-02 as control 8 kinds of liver cancer cells miR-221-3p relative expression；(f) using normal liver cell L-02 as 8 kinds of liver cancer cells miR- of control The relative expression of 25-3p.miR-378a-3p：Compared with L-02, * p<0.05, * * p<0.01；miR-490-3p：With L-02 ratios Compared with * p<0.05, * * p<0.01；miR-381-3p：Compared with L-02, * p<0.05, * * p<0.01；miR-101-3p：With L-02 Compare, * p<0.05, * * p<0.01；miR-221-3p：Compared with L-02, * p<0.05, * * p<0.01；miR-25-3p：With L-02 Compare, * p<0.05, * * p<0.01.
The expression of miR-378a-3p in Fig. 8 microRNA in situ hybridizations detection liver cancer tissue.(a) liver cancer tissue chip (55# case liver cancer cancerous tissues miR-378a-3p is expressed as "-" to miR-378a-3p in situ hybridizations representative photo；7# case livers Cancer cancerous tissue miR-378a-3p is expressed as "+"；82# case liver cancer cancerous tissues miR-378a-3p is expressed as " ++ "；57# case livers Cancer cancerous tissue miR-378a-3p is expressed as " +++ ")；(b) liver cancer tissue miR-378a-3p in situ hybridizations signal strength scores； (c) liver cancer tissue miR-378a-3p high expression, low expression percentage column diagram are compared with normal cancer beside organism, * * p< 0.01。
Fig. 9 miR-378a-3p expressions and the correlation of liver cancer patient prognosis.With liver cancer cancerous tissue miR-378a-3p High expression group compares, * p<0.05.
Specific embodiment
The material information that 1 present invention of table uses
1st, the extraction of mRNA (microRNA)
The Total RNA of cell line and clinical tissue specimen samples extractions use the RNA extracts kits of QIAGEN companies, specifically Experimental procedure is as follows：
A. the lysate of 700 μ l is added in after attached cell digestion is collected by centrifugation, it is appropriate to shake；Group is weighed when extracting tissue It knits about 50mg and adds in the piping and druming of 1ml lysates, finally draw 700 μ l, stand homogenate 5 minutes at room temperature；
B. 15s is shaken after the chloroform of 140 μ l is often added in pipe, stands 4 DEG C of centrifugation 15min after 2-3min at room temperature (12000g)；
C. supernatant liquor is moved in new centrifuge tube, is mixed after adding in the absolute ethyl alcohol of 1.5 times of volumes (being typically 525 μ l) It is even；
D. the sample for drawing 700 μ l is transferred in RNeasy Mini columns, and be put into the collecting pipe of 2ml, room temperature centrifugation 8000g 15 seconds, discards the waste liquid in collecting pipe；
E. step D is repeated, collects remaining sample；
F. Buffer RWT, the 8000g centrifugations of 700 μ l are added in into centrifugal column, 15s abandons filtrate；
G. the Buffer RPE of 500 μ l are drawn in centrifugal column, 8000g centrifugations, 15s abandons filtrate；
H. the Buffer RPE of 500 μ l are drawn in centrifugal column, 8000g centrifugations, 2min abandons filtrate；
I. RNeasy Mini columns are transferred in the centrifuge tube of new 1.5ml, in the DEPC water that 30-50 μ l are added in column, 8000g centrifuges 1min, and sample total rna solution is collected into centrifuge tube；
J. each sample rna concentration is surveyed, -80 DEG C of refrigerators is put into and preserves for use.
2nd, tailing reverse transcription
RNA reverse transcriptions are tested into cDNA using the miScript II RT tailings Reverse Transcriptase kits of QIAGEN companies Step approximately as：
A. the RNA sample of said extracted is melted on ice, uses the miRNA reverse transcription reagent box of QIAGEN companies (miScript II RTKit) melts water, 10X miScript the Nucleics Mix and 5X of no RNase in room temperature MiScript HiSpec Buffer (15-25 DEG C), with hand tapping by solution mixing, of short duration centrifugation, which sinks solution to collect, arrives test tube Bottom is placed on for use on ice；
B. it is whole that reverse transcription reaction mixed solution is configured on ice；
Reaction system is as follows
5X miScript HiSpec Buffer 4μl
10X miScriptNucleics Mix 2μl
RNase-free water up to 20μl
miScript Reverse Transcriptase Mix 2μl
Template RNA 1μg
20 μ l of total volume
C. reaction condition：37 DEG C, 60min；95 DEG C, 5min；180 are added in the cDNA products of the of short duration backward every 20 μ l of centrifugation The pure water without RNase of μ l carries out 10 times of dilution, is placed in for use on ice.
3rd, real-time fluorescence quantitative PCR
Using the SYBR Green PCR quantification kits of QIAGEN companies by RNA reverse transcriptions into cDNA, experimental procedure is such as Under：
A. SYBR Green PCR quantification kits (the miScript SYBR Green PCR of QIAGEN companies are used Kit), 2X QuantiTect SYBR Green PCRMaster Mix, 10X miScript are melted in room temperature (15-25 DEG C) Universal Primer, microRNA specific primers (has-miR-25-3p：5’-cat tgc act tgt ctc ggt ctg a-3’；hsa-miR-101-3p：5’-tac agt act gtg ata act gaa-3’；hsa-miR-378a-3p：5’- act gga ctt gga gtc aga agg c-3’；hsa-miR-490-3p：5’-caa cct gga gga ctc cat gct g-3’；hsa-miR-381-3p：5’-tat aca agg gca agc tct ctg t-3’；hsa-miR-221-3p： 5’-agc tac att gtc tgc tgg gtt tc-3’；Internal reference U6：5’-gaa gga tga cac gca aat tcg- 3 '), cDNA and the pure water without RNase, it is for use on ice；
B. it is whole that PCR reaction mixtures are configured on ice；
Reaction system is as follows
10X miScript Universal Primer 1μl
1 μ l of 10uM miRNA (microRNA specific primers and internal control primer U6)
SYBR Green 5μl
Without 2 μ l of RNase pure water
1 μ l of cDNA templates (reverse transcription reaction product)
10 μ l of total volume
C. PCR reactions are carried out according to the following conditions；
PCR starting activation：95 DEG C of 15min,
3 steps recycle：94 DEG C, 15s；55 DEG C, 30s；70 DEG C, 30s；40 cycles of setting
D. relative quantification method analysis data, using people U6 as internal reference, pass through 2-ΔΔCTThe expression that algorithm calculates microRNA is poor It is different, the numerical value of clinical tissue Samples detection and with Δ CT=(CTU6-CTmicroRNA) method expression, each 4 multiple holes of experimental setup, In triplicate.
4th, microRNA in situ hybridizations
5 ', 3 ' the double target hsa-miR-378a-3p Probe In Situ Hybridizations (in of digoxin synthesized using Exiqon companies Situ hybridization, ISH) liver cancer tissue chip, experiment basic step approximately as：
A. routine paraffin wax slice dewaxing, is placed on deparaffnize in dimethylbenzene by slice, is vibrated with 50rpm, then use successively 100%, 96%, aquation 5min in 70% ethyl alcohol；
B. it is sliced and cleans 3 × 3min with PBS；
C. using 98 DEG C of effect 15min of citrate；
D. it is exposure mRNA nucleic acid fragments, makes an addition to slice to being completely covered using Proteinase K (10 μ g/ml) immediately, 37 DEG C effect 20min, then PBS rinse 3 × 10min；
E. 0.1mol/l triethanolamines containing 0.25% acetic anhydride is used to rinse 10min, then again with PBS rinsings 4 × 5min；
F. prehybridization：Slice is acted on into 60min for 37 DEG C as 0.2 × SSC+50% formamide prehybridizations liquid；
G. hybridize：Using the lock nucleic acid probe of the diluted digoxigenin labeled of Hybridization Dilution liquid to concentration 40nM, 30 μ l are added dropwise Hybridization solution is upper in slice and is covered with clean coverslip, 60 DEG C of hybridization 60min；
H. post-hybridization washing：Coverslip about 5min, 60 DEG C of 5 × SSC, which are washed away, with 5 × SSC of room temperature incubates 5min；
DEG C I.60 1 × SSC incubates 2 × 5min, and 60 DEG C of 0.2 × SSC incubates 2 × 5min, 0.2 × SSC drifts of room temperature Wash 5min；
J. confining liquid, 37 DEG C of effect 30min are added dropwise；
K. anti-Dig IgG primary antibodies (1 are added dropwise:200 dilutions), 37 DEG C of incubations 30min, PBS rinse 3 × 3min；
L. sheep anti mouse-Dig the secondary antibodies (1 of AP labels are added dropwise:5000 dilutions), 37 DEG C of incubation 30min, PBS rinsings 3 × 3min；
M. it is added drop-wise on slice using NBT/BCIP colour reagents, developing time is controlled under mirror about 10 minutes, PBS rinsings 3 ×3min；
N. hematoxylin slightly redyes 1-2min, and flowing water, which rinses, after developing the color fully terminates reaction；
O. 5min is dehydrated in 70%, 96%, 100% ethyl alcohol successively again；
P. after waiting for chip drying, with resinene mounting；
Q. judge that clear brown color is the positive according to staining power, no positive dyeing is negative "-"；According to stained positive Rate judgement "+" is positive cell number<25%th, " ++ " is positive cell number 26%-50%, " +++ " is positive cell number>50%.
5th, statistical analysis
Pearson ' s χ 2test, Student's t test, single factor test/dual factors are carried out using SPSS13.0 softwares ANOVA analytical statistics meanings, p<0.05 variant, the p that is considered as statistics<0.01 is considered as the significant difference of statistics.
The liver cancer clinical samples verification of 1 microRNA differential expressions of embodiment
SEQ ID NO:1(miR-378a-3p)：5’-ACUGGACUUGGAGUCAGAAGGC-3’
SEQ ID NO:2(miR-490-3p)：5’-CAACCUGGAGGACUCCAUGCUG-3’
SEQ ID NO:3(miR-381-3p)：5’-UAUACAAGGGCAAGCUCUCUGU-3’
SEQ ID NO:4(miR-101-3p)：5’-UACAGUACUGUGAUAACUGAA-3’
SEQ ID NO:5(miR-25-3p)：5’-CAUUGCACUUGUCUCGGUCUGA-3’
SEQ ID NO:6(miR-221-3p)：5’-AGCUACAUUGUCUGCUGGGUUUC-3’
In order to probe into miR-25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR-381-3p, miR- This differential expressions of 6 microRNA in human liver cancer of 221-3p, we utilize miR-25-3p, miR-101-3p, miR- 378a-3p, miR-490-3p, miR-381-3p, miR-221-3p specific forward primer, general reverse primer, pass through tailing Fluorescence quantitative PCR method, using U6 as internal reference, pairing cancerous tissue, the cancer side for analyzing 28 hepatocellular carcinoma (HCC) patient sources are normal The table of miR-25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR-381-3p, miR-221-3p in tissue It reaches.
By Fig. 1 amplification curves as it can be seen that miR-25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR- The amplification efficiency of 381-3p, miR-221-3p and internal reference U6 are basically identical.
By Fig. 2 solubility curves as it can be seen that miR-25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR- There is single dissolving peak in 381-3p, miR-221-3p and internal reference U6, prompt do not occur non-specificity in amplification procedure Amplification and primer dimer generate.
The results are shown in Figure 3 by microRNA qPCR：Compared with the cancer beside organism respectively matched, had in 23 liver cancer tissues MiR-378a-3p expression is lowered, and variation (Fig. 3 (a)) is raised or is not apparent from other 5 expression；Have in 26 liver cancer tissues MiR-490-3p expression is lowered, and variation (Fig. 3 (b)) is raised or is not apparent from other 2 expression；Have in 18 liver cancer tissues MiR-381-3p expression is lowered, and variation (Fig. 3 (c)) is raised or is not apparent from other 10 expression；Have in 25 liver cancer tissues MiR-101-3p expression is lowered, and other 3 expression up-regulations (Fig. 3 (d))；There is miR-221-3p in 22 liver cancer tissues to express Up-regulation, and variation (Fig. 3 (e)) is lowered or is not apparent from other 6 expression；There is miR-25-3p in 19 liver cancer tissues in 28 Up-regulated expression, and variation (Fig. 3 (f)) is lowered or is not apparent from other 9 expression.
By Fig. 4 furthermore, it can be seen that miR-378a-3p (Fig. 4 (a)), miR-101-3p (Fig. 4 (d)) are in HCC cancerous tissues Expression is substantially reduced (p<0.01), the expression of miR-490-3p (Fig. 4 (b)), miR-381-3p (Fig. 4 (c)) in HCC cancerous tissues Also (p is reduced in otherness<0.05)；And expression raising (ps of the miR-221-3p (Fig. 4 (e)) in HCC cancerous tissues<0.05).Though Smaller ascendant trend is still presented in right statistical test indifference, expression of the miR-25-3p in HCC cancerous tissues.
The miRNA expression verification result promptings of above-mentioned 28 pairs of HCC cancerous tissues that we select, cancer beside organism's clinical samples, MiR-378a-3p, miR-490-3p, miR-101-3p, miR-381-3p are reduced in Expression In Hepatocellular Carcinoma, and miR-221-3p Increase in Expression In Hepatocellular Carcinoma.
The liver cancer cell lines verification of 2 microRNA differential expressions of embodiment
HepG2, HuH7, SMMC-7721, Li-7, PLC/PRF5, SK-Hep- are analyzed using tailing fluorescence quantitative PCR method 1st, miR-25-3p, miR-101-3p, miR-378a-3p, miR-490- in 8 kinds of human hepatoma cell strains of MHCC97L, MHCC97H The expression of 3p, miR-381-3p, miR-221-3p (L-02 Human normal hepatocytes are as control).By Fig. 5, Fig. 6 as it can be seen that miR- The amplification effect of 25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR-381-3p, miR-221-3p and internal reference U6 Rate is also basically identical, and miR-25-3p, miR-101-3p, miR-378a-3p, miR-490-3p, miR-381-3p, miR- There is single dissolving peak in 221-3p and internal reference U6, does not occur non-specific amplification and primer dimer production in amplification procedure It is raw.MicroRNA tailings fluorescent quantitative PCR result (Fig. 7) shows that, using L-02 normal liver cells as control, miR-378a-3p exists Reduction (Fig. 7 (a)) (p is expressed in 8 kinds of liver cancer cells of all detections<0.01or p<0.05)；MiR-490-3p is in all inspections Being expressed in the 8 kinds of liver cancer cells surveyed also reduces (Fig. 7 (b)) (p<0.01or p<0.05)；MiR-381-3p is in HuH7, SMMC- 7721st, expression reduces (Fig. 7 (c)) (p in PLC/PRF5, MHCC97L, MHCC97H<0.01orp<0.05), and in HepG2, Li- 7th, but raising (Fig. 7 (c)) (p is expressed in SK-Hep-1<0.01)；MiR-101-3p tables in 8 kinds of liver cancer cells of all detections Up to reduction (Fig. 7 (d)) (p<0.01or p<0.05)；MiR-221-3p is expressed in 8 kinds of liver cancer cells of all detections and is risen Height (Fig. 7 (e)) (p<0.01or p<0.05)；MiR-25-3p HepG2, HuH7, Li-7, SK-Hep-1, MHCC97L, Expression raising (Fig. 7 (f)) (p in MHCC97H<0.01orp<0.05), (figure is reduced in SMMC-7721, PLC/PRF5 expression 7(f))(p<0.01or p<0.05)。
3 miR-378a-3p expressions of embodiment and liver cancer patient clinical pathological factors, the correlation analysis of prognosis
With reference to the preliminary knot of the tailing quantitative fluorescent PCR miR-378a-3p to 28 pairs of liver cancer tissues, 8 plants of liver cancer cells Fruit, and liver cancer tissue chip (90 cases, 90 pairs of liver cancer tissues, cancer beside organisms) is utilized, pass through the side of microRNA in situ hybridizations Method analyzes the expression (Fig. 8 (a)) of miR-378a-3p.In the cancer beside organism of 90 liver cancer pairings, miR-378a-3p high tables Up to having 54 (43 " ++ ", 11 " +++ ") for (++, +++), only 36 of low expression (- ,+) (10 "-", 26 “+”)；And in 90 liver cancer cancerous tissues, only 29 of miR-378a-3p high expression (++, +++) (20 " ++ ", 9 " ++ + "), low expression (- ,+) has 61 (15 "-", 46 "+") (Fig. 8 (b), 8 (c)) (p<0.01).In situ hybridization result It further confirms, expression of the miR-378a-3p in liver cancer is substantially reduced, be consistent with tailing fluorescence quantitative PCR detection result.
The expression of miR-378a-3p in 90 liver cancer cancerous tissues is divided into height by us according to the appraisal result of in situ hybridization Two groups of expression group (++, +++), low expression group (- ,+), analyze miR-378a-3p expression and liver cancer patient clinical pathological factors (gender, age, Hbs antigens, liver fibrosis, serum afp, tumor size, tumor number, vessel invasion, TNM stage) and The correlation of prognosis.As can be seen from Table 2, liver cancer tissue miR-378a-3p expressions and tumor size, vessel invasion, TNM stage Negatively correlated (p<0.05).As seen from Figure 9, miR-378a-3p expresses 5 years overall survivals of low liver cancer patient less than miR- 378a-3p high expresses patient, the poor (p of prognosis<0.05).It is prompted by table 2, Fig. 9 results, miR-378a-3p tables in liver cancer tissue The malignant progression of liver cancer may be importantly taken part in up to horizontal reduction.
2 miR-378a-3p expressions of table and the correlation of liver cancer patient clinical pathological factors
*p<0.05
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>University Of Suzhou
<120>A kind of diagnosing cancer of liver marker and its application
<160> 13
<170> PatentIn version 3.3
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<212> RNA
<213>People
<400> 5
cauugcacuu gucucggucu ga 22
<210> 6
<211> 23
<212> RNA
<213>People
<400> 6
agcuacauug ucugcugggu uuc 23
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
cattgcactt gtctcggtct ga 22
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
tacagtactg tgataactga a 21
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence
<400> 9
actggacttg gagtcagaag gc 22
<210> 10
<211> 22
<212> DNA
<213>Artificial sequence
<400> 10
caacctggag gactccatgc tg 22
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence
<400> 11
tatacaaggg caagctctct gt 22
<210> 12
<211> 23
<212> DNA
<213>Artificial sequence
<400> 12
agctacattg tctgctgggt ttc 23
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<400> 13
gaaggatgac acgcaaattc g 21

Claims (8)

  1. A kind of 1. liver cancer marker, which is characterized in that its base sequence such as SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:Shown in 4.
  2. 2. a kind of tool diagnosed or detection liver cancer is in progress, which is characterized in that including being used to detect and/or quantifying body fluid or organ The reagent of liver cancer marker shown in claim 1 in tissue.
  3. 3. a kind of tool diagnosed or detection liver cancer is in progress according to claim 2, which is characterized in that the body fluid includes Cell liquid, cerebrospinal fluid, sperm, urine, blood or saliva.
  4. 4. it is a kind of screen or exploitation have to liver cancer therapeutic efficiency drug tool, which is characterized in that including be used for detect and/or The reagent of liver cancer marker shown in claim 1 in quantitative body fluid or cell.
  5. 5. a kind of tool screened or develop the drug for having therapeutic efficiency to liver cancer according to claim 4, feature exist In the operation principle of the tool includes：Sandwich immunoassays, enzyme-linked immunosorbent assay, radiommunoassay Method, enzyme immunoassay, western blot, immune precipitation and the immunoassay based on particle.
  6. 6. a kind of tool screened or develop the drug for having therapeutic efficiency to liver cancer according to claim 5, feature exist In the immunoassay based on particle includes the use of gold, silver or latex particle, magnetic-particle.
  7. 7. liver cancer marker shown in claim 1 is screening or is developing the application in the drug for treating liver cancer.
  8. 8. liver cancer marker shown in claim 1 is screening or is developing to diagnose or detect the application in the reagent of liver cancer.
CN201810068446.6A 2018-01-24 2018-01-24 A kind of diagnosing cancer of liver marker and its application Pending CN108192970A (en)

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CN111518886A (en) * 2020-04-23 2020-08-11 浙江大学 MicroRNA related to sorafenib drug resistance of tumor cells and application thereof
CN114574489A (en) * 2022-03-03 2022-06-03 集美大学 MicroRNA regulated by docosahexaenoic acid and application thereof
CN114574489B (en) * 2022-03-03 2024-05-03 集美大学 MicroRNA regulated and controlled by docosahexaenoic acid and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111518886A (en) * 2020-04-23 2020-08-11 浙江大学 MicroRNA related to sorafenib drug resistance of tumor cells and application thereof
CN111518886B (en) * 2020-04-23 2021-08-17 浙江大学 MicroRNA related to sorafenib drug resistance of tumor cells and application thereof
CN114574489A (en) * 2022-03-03 2022-06-03 集美大学 MicroRNA regulated by docosahexaenoic acid and application thereof
CN114574489B (en) * 2022-03-03 2024-05-03 集美大学 MicroRNA regulated and controlled by docosahexaenoic acid and application thereof

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