CN109825585A - A kind of biomarker of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation - Google Patents
A kind of biomarker of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation Download PDFInfo
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- CN109825585A CN109825585A CN201910164346.8A CN201910164346A CN109825585A CN 109825585 A CN109825585 A CN 109825585A CN 201910164346 A CN201910164346 A CN 201910164346A CN 109825585 A CN109825585 A CN 109825585A
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Abstract
The present invention provides RNA the and/or APLN albumen of APLN as biomarker is preparing the application in nasopharyngeal carcinoma diagnosis and/or prognosis evaluation reagent, kit or detection device.The research of the invention finds that there are differential expressions in Nasopharyngeal Carcinoma Patients and Healthy People for RNA the and APLN albumen of APLN, the reference frame of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation can be used as, there is great importance to survival is improved.The application and a kind of nasopharyngeal carcinoma detection kit that the present invention also provides the products of the RNA and/or APLN protein expression of detection APLN in nasopharyngeal carcinoma diagnosis and/or prognosis evaluation.
Description
Technical field
The present invention relates to field of biotechnology, mark more particularly to the biology of a kind of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation
Remember object.
Background technique
Nasopharyngeal carcinoma refers to the malignant tumour betided at the top of nasopharyngeal cavity with side wall, is one of high-incidence malignant tumour in China, hair
Sick rate is first of ear,nose & throat malignant tumour.The site of pathological change of nasopharyngeal carcinoma is hidden, and early symptom is unobvious, it is difficult to early detection,
Sing misdiagnosis and mistreatment rate is high.Therefore, screening and diagnosis are carried out to nasopharyngeal carcinoma, especially traumatic low, the convenient body fluid inspection of materials is right
Early treatment is carried out, survival is improved and is of great significance.
Summary of the invention
To solve the above problems, the present invention provides a kind of nasopharyngeal carcinoma diagnosis and/or the biomarkers of prognosis evaluation.
In a first aspect, the present invention provides RNA the and/or APLN albumen of APLN as biomarker in preparation nasopharyngeal carcinoma
Application in diagnosis and/or prognosis evaluation reagent, kit or detection device.
Optionally, the amino acid sequence of the APLN albumen is as shown in SEQ NO:1.Further, the RNA of the APLN
Sequence is as shown in SEQ NO:2.
Optionally, the RNA of the APLN includes the cfRNA of APLN in body fluid.Further, the body fluid include blood,
Saliva, sweat or urine.Specifically, can be, but not limited to be easy to acquire and detect for peripheral blood.
In the present invention, inventor has found and is good in the research of the biomarker of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation
Health people compares, RNA the and APLN protein expression up-regulation of APLN, the expression quantity of the RNA and APLN albumen of APLN in Nasopharyngeal Carcinoma Patients
There is close relevance with nasopharyngeal carcinoma.Therefore, RNA the and/or APLN albumen of APLN is preparing nasopharynx as biomarker
Application in cancer diagnosis and/or prognosis evaluation reagent, kit or detection device is of great significance.
In the present invention, the RNA of APLN includes the cfRNA of APLN in body fluid (cell free RNA, dissociate RNA).
CfRNA is generated by the approach such as cell active secretion, apoptosis and death, and wherein the cfRNA of cell active secretion is less, apoptosis and
Death is one of the approach of cell release cfRNA.Inventor studies in the biomarker of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation
Middle discovery, compared with Healthy People, the cfRNA expression quantity up-regulation of APLN in Nasopharyngeal Carcinoma Patients, therefore, the inspection to the cfRNA of APLN
The states such as the apoptosis that can be very good reflection cell and death are surveyed, as a kind of novel effective nasopharyngeal carcinoma diagnosis and/or prognosis
The biomarker of assessment, the present invention are equivalent to the new direction provided about nasopharyngeal carcinoma detection.At the same time, the hair of nasopharyngeal carcinoma
Sick position is hidden, its unpredictable site of pathological change, needless to say is analysed whether by detecting diseased region with nasopharyngeal carcinoma, and sends out
There are significant differences in Nasopharyngeal Carcinoma Patients and healthy population for the cfRNA expression quantity of APLN in bright people's research discovery body fluid, and
Body fluid extracts simple and convenient；Therefore, by extracting body fluid, extraction cfRNA analyzes the expression quantity of APLN, can be to nasopharyngeal carcinoma
Diagnosis and/or prognosis evaluation provide important reference frame.In actual clinical diagnosis, and can be in conjunction with other existing inspections
Survey means improve the accuracy rate of nasopharyngeal carcinoma diagnosis, further increase the survival rate of patient.
To sum up, RNA the and/or APLN albumen of APLN is preparing nasopharyngeal carcinoma diagnosis and/or prognosis is commented as biomarker
The application estimated in reagent, kit or detection device is of great significance.
Second aspect, the present invention provides the products of the RNA and/or APLN protein expression of detection APLN in nasopharyngeal carcinoma diagnosis
And/or the application in prognosis evaluation.
Optionally, the RNA of the APLN includes the cfRNA of APLN in body fluid.Further, the body fluid includes for blood
Liquid, saliva, sweat or urine.Specifically, can be, but not limited to be easy to acquire and detect for peripheral blood.
Optionally, the product of the RNA and/or APLN protein expression of the detection APLN can be, but not limited to as PCR reagent
Box, chip, immunoconjugates kit.
Optionally, the product of the RNA and/or APLN protein expression of the detection APLN passes through PCR, immune detection, original position
The expression of the RNA of hybridization or chip detection APLN.
Further, it is described by PCR detect APLN RNA expression be used for nasopharyngeal carcinoma diagnosis, treatment and in advance
The product assessed afterwards includes the primer pair of at least a pair of RNA for specific amplification APLN.
Further, the expression by immune detection APLN albumen is to be used for nasopharyngeal carcinoma diagnosis, treatment and pre-
The product assessed afterwards includes the antibody in conjunction with APLN protein-specific.
Further, the expression of the RNA that APLN is detected by situ hybridization is to be used for nasopharyngeal carcinoma diagnosis, treatment
Product with prognosis evaluation includes the probe hybridized with the RNA of APLN.
Further, the expression of the RNA and/or APLN albumen that APLN is detected by chip is to be used for nasopharyngeal carcinoma
The product of diagnosis, treatment and prognosis evaluation includes genetic chip and/or protein chip.Further, the genetic chip packet
The probe hybridized with the RNA of APLN is included, the protein chip includes the antibody in conjunction with APLN protein-specific.
Optionally, the amino acid sequence of the APLN albumen is as shown in SEQ NO:1.Further, the RNA of the APLN
Sequence is as shown in SEQ NO:2.
Specifically, the primer pair can be, but not limited to as F1:5 '-GTCTCCTCCATAGATTGGTCTGC-3 ', R1:
5 '-GGAATCATCCAAACTACAGCCAG-3 ' and F2:5 '-AATCCAGCCCACTTACAGGTTTTC-3 ', R2:5 '-
At least one set in TAGCAGAAGACACCCACCAAGG-3 '.
Specifically, the sequence of the probe can be, but not limited to as the sequence as shown in SEQ NO:3.Specifically, using such as
Probe sequence shown in SEQ NO:3 carries out the expression of the RNA of in situ hybridization detection APLN.
In the present invention, the primer pair and the length of the probe determine that it cannot detect the miRNA in cfRNA,
But for detecting the mRNA in cfRNA.In general, it is that mRNA is come by a chain of DNA as template transcription, carry it is hereditary
A kind of singlestranded RNA that protein can be instructed to synthesize of information, most mRNA are located at into the cell, and only a few is because thin
The apoptosis of born of the same parents and death dissociate to extracellular, and therefore, the mRNA in cfRNA carries hereditary information, but be free in it is extracellular,
Wherein the expression of the cfRNA of APLN can more reflect nasopharyngeal carcinoma situation.
The third aspect, the present invention provides a kind of nasopharyngeal carcinoma detection kits, including RNA the and/or APLN egg with APLN
The detectable substance of white specific binding.
Optionally, the RNA of the APLN includes the cfRNA of APLN in body fluid.Further, the body fluid include blood,
Saliva, sweat or urine.Specifically, can be, but not limited to be easy to acquire and detect for peripheral blood.
Optionally, the detectable substance includes primer pair in conjunction with RNA the and/or APLN protein-specific of APLN, probe
At least one of with antibody.
Further, the primer pair is F1:5 '-GTCTCCTCCATAGATTGGTCTGC-3 ', R1:5 '-
GGAATCATCCAAACTACAGCCAG-3 ' and F2:5 '-AATCCAGCCCACTTACAGGTTTTC-3 ', R2:5 '-
At least one set in TAGCAGAAGACACCCACCAAGG-3 '.
Further, the sequence of the probe includes the sequence as shown in SEQ NO:3.
Optionally, the amino acid sequence of the APLN albumen is as shown in SEQ NO:1.Further, the RNA of the APLN
Sequence is as shown in SEQ NO:2.
Optionally, the nasopharyngeal carcinoma detection kit further includes that cfRNA extracts reagent.Further, the cfRNA is extracted
Reagent includes cleaning solution, digestive juice, elution column, absorption tube.
Optionally, RNA the and/or APLN albumen of the APLN expresses up-regulation in tissues of nasopharyngeal carcinoma.
RNA the and/or APLN albumen relative expression quantity of APLN is detected using nasopharyngeal carcinoma detection kit provided by the invention,
It, then can auxiliary diagnosis when 3 times or more that the RNA and/or APLN albumen relative expression quantity of APLN is healthy population average level
Nasopharyngeal carcinoma.
Beneficial effects of the present invention:
The research of the invention finds that RNA the and APLN albumen of APLN in Nasopharyngeal Carcinoma Patients and Healthy People there are differential expression,
Using RNA the and/or APLN albumen of APLN as biomarker be applied to prepare nasopharyngeal carcinoma diagnosis and/or prognosis evaluation reagent,
In kit or detection device, the products application of the RNA and/or APLN protein expression of detection APLN in nasopharyngeal carcinoma diagnosis and/or
Nasopharyngeal carcinoma detection reagent is made in detectable substance in prognosis evaluation, and in conjunction with RNA the and/or APLN protein-specific of APLN
Box can provide important reference frame to nasopharyngeal carcinoma screening and diagnosis, improve survival；Meanwhile this is easy to operate, spirit
Quick property is good, and specificity is high.
Detailed description of the invention
Fig. 1 is the relative expression quantity result figure of the cfRNA for the APLN that the embodiment of the present invention 1 provides in blood；
Fig. 2 is the relative expression quantity result figure of the cfRNA for the APLN that the embodiment of the present invention 2 provides in blood；
Fig. 3 is the relative expression quantity result figure of the cfRNA for the APLN that the embodiment of the present invention 3 provides in blood；
Fig. 4 is the relative expression quantity result figure of the cfRNA for the APLN that the embodiment of the present invention 4 provides in blood.
Specific embodiment
As described below is the preferred embodiment of the embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, without departing from the principles of the embodiments of the present invention, several improvements and modifications can also be made, these improvement
Also it is considered as the protection scope of the embodiment of the present invention with retouching.
Embodiment 1
Experimental group: its peripheral blood blood plasma 10mL is acquired to Nasopharyngeal Carcinoma Patients (9 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Control group: its peripheral blood blood plasma 10mL is acquired to health volunteer (10 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Experimental group and control group are subjected to following experiment:
(1) cfRNA extraction is carried out using Quick-cfRNA Serum & Plasma kit.Specific steps are as follows: whole blood
It is centrifuged 15min under the conditions of 12000g, removes cell；Deng than be added the digestive juice containing Proteinase K, stand 1h-2h remove removing protein；
The binding soln containing ethyl alcohol is added, is sufficiently mixed；Air driven pump is connected using the suction filtering tube that kit provides, is filtered at 80KPa；
Step elution is provided by kit, obtains cfRNA.
(2) qPCR array analysis is carried out after the cfRNA extracted freezes.Use following formula: △ Ct=CtTarget gene-
CtHouse-keeping gene；△ △ Ct=△ CtExperimental group-△CtControl group；Relative expression quantity=2-△△Ct, calculate gene expression.Through screening 99 genes
CfRNA after obtain multiple alternate labels objects, by analysis as shown in Figure 1, the cfRNA expression quantity of APLN in Nasopharyngeal Carcinoma Patients and
Significant difference in health volunteer.
Embodiment 2
Experimental group: its peripheral blood blood plasma 10mL is acquired to Nasopharyngeal Carcinoma Patients (6 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Control group: its peripheral blood blood plasma 10mL is acquired to health volunteer (6 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Above-mentioned two groups of cfRNA is extracted using Quick-cfRNA Serum & Plasma kit, then carries out qPCR, is adopted
With primer pair F1:5 '-GTCTCCTCCATAGATTGGTCTGC-3 ', R1:5 '-GGAATCATCCAAACTACAGCCAG-3 ',
The reaction condition of middle qPCR is 95 DEG C of 10mi n, 40 circulation (95 DEG C of 15s；60℃20s；72℃45s).Through detection and analysis,
As a result as shown in Figure 2, it can be seen that the cfRNA of APLN is expressed there are significant difference in Nasopharyngeal Carcinoma Patients and health volunteer,
APLN expression quantity in Nasopharyngeal Carcinoma Patients is high.
Embodiment 3
Experimental group: its peripheral blood blood plasma 10mL is acquired to Nasopharyngeal Carcinoma Patients (13 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Control group: its peripheral blood blood plasma 10mL is acquired to health volunteer (7 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Above-mentioned two groups of cfRNA is extracted using Quick-cfRNA Serum&Plasma kit, then carries out qPCR, is used
Primer pair F2:5 '-AATCCAGCCCACTTACAGGTTTTC-3 ', R2:5 '-TAGCAGAAGACACCCACCAAGG-3 ', wherein
The reaction condition of qPCR is 95 DEG C of 10min, 60 circulation (95 DEG C of 15s；60℃20s；72℃45s).Through detection and analysis, as a result
As shown in Figure 3, it can be seen that there are significant difference, APLN for the cfRNA expression of APLN in Nasopharyngeal Carcinoma Patients and health volunteer
Expression quantity is high in Nasopharyngeal Carcinoma Patients.
Embodiment 4
Experimental group: its peripheral blood blood plasma 10mL is acquired to Nasopharyngeal Carcinoma Patients (29 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Control group: its peripheral blood blood plasma 10mL is acquired to health volunteer (7 people), is placed in collection tube.Uniformly shake with
Be sufficiently mixed anti-coagulants and RNase inhibitor, blood sampling be placed on 4 DEG C it is spare.
Above-mentioned two groups of cfRNA is extracted using Quick-cfRNA Serum&Plasma kit, then carries out qPCR, is used
Primer pair F2:5 '-AATCCAGCCCACTTACAGGTTTTC-3 ', R2:5 '-TAGCAGAAGACACCCACCAAGG-3 ', wherein
Reaction condition 95 DEG C of 10min, 60 circulation (95 DEG C of 15s of qPCR；60℃20s；72℃45s).Through detection and analysis, as a result such as
Shown in Fig. 4, it can be seen that the cfRNA of APLN is expressed there are significant difference in Nasopharyngeal Carcinoma Patients and health volunteer, and APLN exists
Relative expression quantity is 3 times or more of control group average level in Nasopharyngeal Carcinoma Patients, can auxiliary diagnosis nasopharyngeal carcinoma, operation letter
Single, sensitivity is good, and specificity is high.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Jiangsu Prov. Tumour Hospital
<120>biomarker of a kind of nasopharyngeal carcinoma diagnosis and/or prognosis evaluation
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Met Asn Leu Arg Leu Cys Val Gln Ala Leu Leu Leu Leu Trp Leu Ser
1 5 10 15
Leu Thr Ala Val Cys Gly Gly Ser Leu Met Pro Leu Pro Asp Gly Asn
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Gly Leu Glu Asp Gly Asn Val Arg His Leu Val Gln Pro Arg Gly Ser
35 40 45
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caccuggugc agcccagagg gucaaggaau gggccagggc ccuggcaggg aggucggagg 180
aaauuccgcc gccagcggcc ccgccucucc cauaagggac ccaugccuuu cuga 234
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<213> Artificial Sequence
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gcaaaagggg agaaagcccc ctccaaggaa tggctggtga tggcccctca cggaagctag 60
ggcctcccgg ggagagggtg ctattcctgc tgcacttcct cccatctttc tttccttcct 120
tctgttcctt tgctttcttt tcttccctcc ttccttctgc ccttcccttc cttcttctcc 180
cctctccgcc tcccccaaag gaaaagccct ggaaggaagg tccggtcaac acgaagggaa 240
ggccatggag tccagtgatt gaaggctacc tcggactcct gaaaaccacc ctggggttga 300
gcggtagtct cagtgcctga gccgccccta ttagagtacc ctgggtctgg gaatgctgcc 360
agttatgggg gcagctggcc agttatggaa ccttccagcc cagctggggg aatggtgcag 420
caggggtagg tcagggaggt gggagcagct 450
Claims (10)
- RNA the and/or APLN albumen of 1.APLN as biomarker prepare nasopharyngeal carcinoma diagnosis and/or prognosis evaluation reagent, Application in kit or detection device.
- 2. application as described in claim 1, which is characterized in that the RNA of the APLN includes the cfRNA of APLN in body fluid.
- 3. detecting application of the product of the RNA and/or APLN protein expression of APLN in nasopharyngeal carcinoma diagnosis and/or prognosis evaluation.
- 4. a kind of nasopharyngeal carcinoma detection kit, which is characterized in that including in conjunction with RNA the and/or APLN protein-specific of APLN Detectable substance.
- 5. nasopharyngeal carcinoma detection kit as claimed in claim 4, which is characterized in that the detectable substance includes the RNA with APLN And/or at least one of primer pair, probe and antibody that APLN protein-specific combines.
- 6. nasopharyngeal carcinoma detection kit as claimed in claim 5, which is characterized in that the primer pair is F1:5 '- GTCTCCTCCATAGATTGGTCTGC-3 ', R1:5 '-GGAATCATCCAAACTACAGCCAG-3 ' and F2:5 '- At least one set in AATCCAGCCCACTTACAGGTTTTC-3 ', R2:5 '-TAGCAGAAGACACCCACCAAGG-3 '.
- 7. nasopharyngeal carcinoma detection kit as claimed in claim 4, which is characterized in that the amino acid sequence of the APLN albumen is such as Shown in SEQ NO:1.
- 8. nasopharyngeal carcinoma detection kit as claimed in claim 7, which is characterized in that the RNA sequence of the APLN such as SEQ NO: Shown in 2.
- 9. nasopharyngeal carcinoma detection kit as claimed in claim 4, which is characterized in that the RNA of the APLN includes in body fluid The cfRNA of APLN.
- 10. nasopharyngeal carcinoma detection kit as claimed in claim 9, which is characterized in that the nasopharyngeal carcinoma detection kit is also wrapped It includes cfRNA and extracts reagent.
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