CN107475379A - A kind of molecular marker and its detection primer and kit detected for diagnosing tumor with prognosis - Google Patents
A kind of molecular marker and its detection primer and kit detected for diagnosing tumor with prognosis Download PDFInfo
- Publication number
- CN107475379A CN107475379A CN201710679293.4A CN201710679293A CN107475379A CN 107475379 A CN107475379 A CN 107475379A CN 201710679293 A CN201710679293 A CN 201710679293A CN 107475379 A CN107475379 A CN 107475379A
- Authority
- CN
- China
- Prior art keywords
- prss3
- primer
- seq
- nucleotide sequence
- splicing variants
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of molecular marker detected for diagnosing tumor with prognosis, and the molecular marker is PRSS3 splicing variants；Wherein, the PRSS3 splicing variants are PRSS3 V1, PRSS3 V2, PRSS3 V3 and/or PRSS3 V4, and its nucleotide sequence is respectively as shown in SEQ ID No.1 4.Invention further provides the RT qPCR primers and kit for detecting above-mentioned molecular marked compound.The present invention, which is used for the primer for detecting PRSS3 splicing variants or kit, can specifically detect PRSS3 splicing variants transcriptional levels in cell, tissue, body fluid and juice equal samples, and the testing result obtained by the present invention can provide evaluation method for the Index for diagnosis of patient tumors Individual Diagnosis and drug therapy.In addition, detection method is easy to operate, accurate stabilization, there is clinical application significance.
Description
Technical field
The present invention relates to biological technical field.More particularly, to a kind of for diagnosing tumor and the molecule of prognosis detection
Mark and its detection primer and kit.
Background technology
Cancer is one of the main reason for global disease is lethal.Because the relatively special mark of shortage, or sensitiveness are low
Tissue specificity is poor, a late period, and due to the limitation of monitoring treatment intervention means, cause death rate pole when most of patients is medical
It is high.Although increasing evidence shows that many genes have differential expression in tumour, do not find reliable biological marker yet
Thing carrys out the Precise Diagnosis and monitoring for tumour.
Therefore, the high molecular marker of the strong specificity of sensitiveness is screened, and establishes effective monitoring means and establishes accurate
Detection method, to diagnosing tumor and prognosis detection there is important clinical value.
The content of the invention
First purpose of the present invention is to provide a kind of molecular marker detected with prognosis for diagnosing tumor.
Second object of the present invention be to provide above-mentioned molecular marked compound prepare the medical diagnosis on disease such as tumour or infection with
Application in the reagent of prognosis detection.
Third object of the present invention is to provide a kind of examination for the medicals diagnosis on disease such as tumour or infection and prognosis detection
Agent box.
To reach above-mentioned purpose, the present invention uses following technical proposals：
The present invention research find PRSS3 genes mRNA transcription after, translation before through produced by different splice modes
Different splicing variants, the expression of these splicing variants have a tissue specificity, and under environmental stimulus, medicine effect or
During tumor development, it may occur that transcriptional level changes, and is presented so as to produce differential expression splicing variants different type
Histocyte is specific expressed.The specific expressed change of splicing variants and tumour or infectious diseases are in close relations.Cause
This, the molecular marked compound that these can be detected as tumour or inflammation infection diagnosis with prognosis.
Found based on more than, be used for diagnosing tumor and the molecular marker of prognosis detection present invention firstly provides a kind of,
The molecular marker is PRSS3 splicing variants；Wherein described PRSS3 splicing variants be PRSS3-V1, PRSS3-V2,
PRSS3-V3 and/or PRSS3-V4, its nucleotide sequence is respectively as shown in SEQ ID No.1-4.
The invention provides above-mentioned molecular marked compound to prepare the application of diagnosing tumor and the preparation of prognosis detection.
Present invention also offers the material for detecting above-mentioned molecular marked compound to prepare diagnosing tumor and the preparation of prognosis detection
In application.
Preferably, the material is detects the primer of above-mentioned molecular marked compound, and the reagent comprising above-mentioned primer or examination
Agent box.
Invention further provides a kind of RT-qPCR primers for being used to detect above-mentioned molecular marked compound, including for examining
Survey PRSS3-V1, PRSS3-V2, PRSS3-V3 and PRSS3-V4 universal primer Vc1 or Vc2；
Wherein, the nucleotide sequence of the sense primer of the primer Vc1 is as shown in SEQ ID No.6, the core of anti-sense primer
Nucleotide sequence is as shown in SEQ ID No.7；The nucleotide sequence of the sense primer of the primer Vc2 as shown in SEQ ID No.8,
The nucleotide sequence of anti-sense primer is as shown in SEQ ID No.9；Preferably, the universal primer is primer Vc1.
The present invention by above-mentioned universal primer Vc1 or Vc2 be for PRSS3 splicing variants PRSS3-V1, PRSS3-V2,
PRSS3-V3 and PRSS3-V4 consensus sequences (as shown in SEQ ID No.5) design, detectable or screening sample is expanded by PCR
It whether there is PRSS3 splicing variants in this, in favor of further determining that the type of PRSS3 splicing variants.Therefore can enter
It whether there is the examination of PRSS3 splicing variants in row cell, tissue, body fluid and juice equal samples.
Further, present invention also offers a kind of RT-qPCR primers for being used to detect above-mentioned molecular marked compound, including：
PRSS3-V1 specific primer V1, the nucleotide sequence of its sense primer is as shown in SEQ ID No.10, downstream
The nucleotide sequence of primer is as shown in SEQ ID No.14；
PRSS3-V2 specific primer V2, the nucleotide sequence of its sense primer is as shown in SEQ ID No.11, downstream
The nucleotide sequence of primer is as shown in SEQ ID No.14；
PRSS3-V3 specific primer V3, the nucleotide sequence of its sense primer is as shown in SEQ ID No.12, downstream
The nucleotide sequence of primer is as shown in SEQ ID No.14；
PRSS3-V4 specific primer V4, the nucleotide sequence of its sense primer is as shown in SEQ ID No.13, downstream
The nucleotide sequence of primer is as shown in SEQ ID No.14.
Due to PRSS3 splicing variants V1-V4 primer be respectively for PRSS3 splicing variants PRSS3-V1,
PRSS3-V2, PRSS3-V3 and PRSS3-V4 distinguished sequence sequence (as shown in SEQ ID No.1-4) design, PCR can be passed through
Amplification can determine that the particular type that the expression of PRSS3 splicing variants in sample be present.Present invention discover that PRSS3 splicing variants are not
Same type presentation histocyte is specific expressed, and this specific expressed change and relation between tumor are close.Therefore the present invention
Cell, tissue, body fluid and juice equal samples further can detect by the specific primer of above-mentioned each PRSS3 splicing variants
The middle type that PRSS3 splicing variants be present, contributes to diagnosing tumor and Index for diagnosis.
It should be noted that the above-mentioned universal primer of the present invention and specific primer can be used alone, also may be used in combination.
Present invention also offers a kind of RT-qPCR kits for being used to detect above-mentioned molecular marked compound, including above-mentioned RT-
QPCR primers.
Further, the RT-qPCR kits also include the internal control primer, positive control template, cDNA for uniforming
Synthetic agent and PCR reaction reagents.
Wherein, the internal control primer for uniforming is the primer using GAPDH as internal reference, its nucleotide sequence such as SEQ
Shown in ID No.15 and SEQ ID No.16；
The positive control template is the PRSS3 splicing variants pcr amplification products crossed through sequence verification, or sequence verification
The PRSS3 splicing variants cloned plasmids crossed；Preferably, PRSS3 splicing variants cloned plasmids include PRSS3 splicing variants
Consensus sequence, the consensus sequence is as shown in SEQ ID No.5；
The negative control template is deionized water.
The present invention is used for the method for detecting above-mentioned molecular marked compound, comprises the following steps：
1) above-mentioned RT-qPCR primers or RT-qPCR kits are utilized, while by the use of GAPDH as homogenization with reference to primer pair
Reverse transcription synthesizes sample cDNA to be detected and carries out qPCR amplifications, obtains amplified production；
2) result interpretation：
When being detected using above-mentioned universal primer, gel electrophoresis visible PCR amplificates, or the dissolving for passing through qPCR
The specificity of curve observation pcr amplification product, Ct values are obtained by amplification curve<Or=33, then illustrate at least exist in sample
A kind of expression of PRSS3 splicing variants；
Without pcr amplification product, the visible PCR of a certain specific primer (such as V1, V2, V3 or V4) gel electrophoresis expands for gel electrophoresis
Increase production thing, or solubility curve is not observed by qPCR or non-specific solubility curve occurs, is without amplification curve or bent by expanding
Line obtains Ct values ＞ 33, then illustrates that PRSS3 splicing variants are not expressed in the sample.
When being detected using above-mentioned specific primer, a certain specific primer (such as V1, V2, V3 or V4) gel electrophoresis
Visible PCR amplificates, or a certain specific primer (such as V1, V2, the V3 or V4) PCR for the solubility curve observation for passing through qPCR expand
Increase production the specificity of thing, Ct values are obtained by amplification curve<Or=33, then illustrate corresponding in the presence of PRSS3 montages change in sample
The expression (PRSS3-V1, PRSS3-V2, PRSS3-V3 or PRSS3-V4) of allosome.
A certain specific primer (such as V1, V2, V3 or V4) gel electrophoresis is without pcr amplification product, or the dissolving for passing through qPCR
Curve does not observe a certain specific primer (such as V1, V2, V3 or V4) pcr amplification product or non-specific solubility curve, nothing occurs
Amplification curve obtains Ct values ＞ 33 by amplification curve, then illustrate in sample the PRSS3 splicing variants (PRSS3-V1,
PRSS3-V2, PRSS3-V3 or PRSS3-V4) do not express.
The present invention is established based on RT-qPCR method to detect different type PRSS3 montages in kinds of tumors histocyte
The differential expression of variant.The specific amplified of PRSS3 splicing variants is analyzed by solubility curve, sequencing further demonstrate
The specificity of PCR primer.By being expanded by the PCR that template is carried out of the PRSS3-V1 plasmids of gradient dilution, CT values are obtained with copying
Linear relationship between shellfish number, it is determined that the specificity and sensitiveness of this method.
In addition, the present invention can effectively identify the types of PRSS3 splicing variants, the effect for being PRSS3 in tumour provides
Molecular basis, contribute to Transformation Application of the PRSS3 splicing variants in the molecule Clinics and Practices of tumour.These researchs are found
The occurrence and development for not being only tumour provide new mechanism, also should in the clinic that tumour Individual Diagnosis is treated for splicing variants
With providing new clue, thus, there is important scientific value and clinical meaning.
Beneficial effects of the present invention are as follows：
The difference of PRSS3 splicing variants expression of the present invention can be effectively used for diagnosing tumor and prognosis and medication curative effect
Detection.The present invention, which is used for the primer for detecting PRSS3 splicing variants or kit, can specifically detect cell, tissue, body fluid
And PRSS3 splicing variants transcriptional level in juice equal samples, the testing result obtained by the present invention can swell for patient
Knurl Individual Diagnosis and the Index for diagnosis of drug therapy provide evaluation method.In addition, detection method is easy to operate, accurate
It is stable, there is clinical application significance.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 shows the structural representation of PRSS3 genetic transcription variants, and RT-qPCR primer locations such as arrow indicates：Vc1
And Vc2：PRSS3 universal primers；V1-V4 represents PRSS3 splicing variants 1-4 RT-qPCR specific primers respectively.
Fig. 2 shows RT-qPCR analysis universal primer Vc1 and Vc2 amplification human pancreatic cancer cells SW1990, lung cancer cell line
A549 and the PRSS3 in glioma cell line U87 mRNA are expressed and electrophoretic analysis.A.RT-qPCR results, RT-qPCR internal references
For GAPDH；B. agarose gel electrophoresis analyzes the amplified production of Vc1 and Vc2 in human pancreatic cancer cell SW1990.
Fig. 3 shows expression of the RT-qPCR methods detection PRSS3 in HCC cell lines and the normal liver cell system immortalized.
Fig. 4 shows the RT-qPCR analyses of PRSS3 splicing variants in pancreatic carcinoma SW1990.A.PRSS3 montages become
Heterologous express is analyzed；B.RT-qPCR methods detect PRSS3 splicing variants in human lung cancer cell line A549 and brain glioblastoma cell
It is the expression in U87；Vc1:PRSS3 universal primers；V1-V4:PRSS3 splicing variants 1-4；RT-qPCR internal references are GAPDH.
C. gel electrophoresis analysis RT-qPCR amplified productions；Vc1 and Vc2：PRSS3 universal primers；V1-V4 represents PRSS3 montages change respectively
Allosome 1-4 RT-qPCR specific primers；RT-qPCR internal references are GAPDH.
Fig. 5 A show 10 times of gradient dilutions of gel electrophoresis analysis PRSS3 plasmids；PRSS3 splicing variants are shown respectively in B and C
The solubility curve and amplification curve of qPCR amplifications；D. gel electrophoresis analysis qPCR amplified productions.
Fig. 6 shows the corresponding relation of PRSS3 splicing variants V1 plasmid copy numbers and Ct values.
Fig. 7 shows PRSS3 splicing variants expressions in RT-qPCR detection people's GC cell lines；V1-V4:PRSS3 montages
Variant 1-4；RT-qPCR internal references are GAPDH.
Fig. 8 shows the type of RT-qPCR detection people GC cell line expression PRSS3 splicing variants；V1-V4:PRSS3 montages
Variant 1-4；RT-qPCR internal references are GAPDH.
Fig. 9 shows the expression of PRSS3 splicing variants V1 in being organized by RT-qPCR detection human gastric cancers；2N,5N,7N,
8N,9N：Tissue sample is numbered；GAPDH:RT-qPCR internal references.
Figure 10 shows methylation status of PTEN promoter (MSP) detection people GC cell lines and immortalized in normal gastric mucosa cell line
PRSS3 genosome methylation state of DNA；IVD：The DNA of methylating in vitro, as the positive control to methylate；NL：Normal circumference
Lymphocyte DNA, as the non-negative control to methylate；U：It is non-to methylate；M：Methylate.
Figure 11 A show to vulcanize PRSS3 genosome methylation states in sequence verification people's GC cell lines；B. methylation-specific
PCR and vulcanization sequencing primer position.Filled circles represent the CpG islands site to methylate；Open circles represent the non-CpG islands position to methylate
Point；TSS：Transcription initiation site；Vulcanization sequencing region is that+308bp arrives+549bp, covers MSP amplification regions.
Figure 12 shows that Semiquatitative RT-PCR assay (A) and RT-qPCR (B) analysis GC cell lines and the normal gastric mucosa immortalized are thin
Born of the same parents system through 5-Aza/TSA before and after the processing PRSS3 expression change；5-Aza：5-aza-2’-deoxycytidine；TSA：
TrichostatinA；GAPDH：RT-PCR and RT-qPCR internal references；The apparent medicine untreated fish group cell of "-"；The apparent medicine processing of "+"
Group cell.
Figure 13 shows the PRSS3 expressions in RT-qPCR detection transfections PRSS 3BGC823 and SGC7901 cells；
GAPDH:RT-qPCR internal references.
Figure 14 shows the unloaded growth ability with being overexpressed PRSS3 GC cell lines of MTT detections；A.BGC823 cell lines；
B.SGC7901 cell lines；Summarize experimental data three times to present with mean+SD, * * P<0.01.
Figure 15 is shownDetect every square millimeter of (mm2) the unloaded cell with being overexpressed PRSS3 GC cell lines
Number；A.BGC823 cell line unit area inner cell numbers change over time situation；B.SGC7901 cell line unit area inner cells
Number changes over time situation；C.BGC823 representative schematic diagrams；D.SGC7901 representative schematic diagrams；Summarize experimental data three times
Presented with mean+SD, * * P<0.01.
Figure 16 shows that the detection of Clone formation method is unloaded and is overexpressed the clonality of PRSS3 GC cell lines；A.
Representative Clone formation figure；B. the quantization figure of experimental result three times is summarized；Presented with mean+SD, * * P<0.01.
Figure 17 shows that the detection of cut Healing Experiments is unloaded and is overexpressed the transfer ability of PRSS3 GC cell lines；Left side：
BGC823 cell lines；Right side：SGC7901 cell lines.
Figure 18 shows that Transwell migration experiment detections are unloaded and are overexpressed the transfer ability of PRSS3 GC cell lines；A.
Representative migration results；B. quantify figure, summarize experimental data three times and presented with mean+SD, * * P<0.01.
Embodiment
In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings
It is bright.Similar part is indicated with identical reference in accompanying drawing.It will be appreciated by those skilled in the art that institute is specific below
The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
The foundation of the PRSS3 gene splicing body RT-qPCR detection methods of embodiment 1
1st, the design of primer
Arranged in the PRSS3 gene orders presented from NCBI PRSS3 splicing variants PRSS3-V1, PRSS3-V2,
PRSS3-V3 and PRSS3-V4 mRNA sequence (its nucleotide sequence is as shown in SEQ ID No.1-4), according to four kinds of spliceosomes
The different sequence in 5' ends (its nucleotide sequence is as shown in SEQ ID No.1-4) designs PRSS3-V1, PRSS3-V2, PRSS3-V3
With PRSS3-V4 special primer V1, V2, V3 and V4 (SEQ ID No.6-10)；It is identical according to four kinds of spliceosome 3' ends
4 exon sequences (its nucleotide sequence is as shown in SEQ ID No.5) design PRSS3 splicing variants universal primer Vc1 and
Vc2 (its nucleotide sequence is as shown in SEQ ID No.11-14), as shown in figure 1, the primer of the PRSS3 splicing variants is：
PRSS3 splicing variants universal primers Vc1 (pcr amplified fragment size is 122bp):
- the CTTCCTTGAGGGAGGCAAGG-3 ' of sense primer 5 ' (SEQ ID No.6)
- the CTCCAGGCCTGTTCTTCCAG-3 ' of anti-sense primer 5 ' (SEQ ID No.7)
PRSS3 splicing variants universal primers Vc2 (pcr amplified fragment size is 101bp):
- ATT CTG GCT CCC ACT TCT the GC-3 ' (SEQ ID No.8) of sense primer 5 '
- CTC TCC CAG TCT CAC CTG the GA-3 ' (SEQ ID No.9) of anti-sense primer 5 '
PRSS3 splicing variants 1 (trypsinogen transcript variant 1, PRSS3-V1) primer V1 (PCR
Amplified fragments size is 124/2521bp):
- the CTGCGAGGCGCTGGG-3 ' of sense primer 5 ' (SEQ ID No.10)
PRSS3 splicing variants 2 (trypsinogen transcript variant 2, PRSS3-V2) primer V2 (PCR
Amplified fragments size is 129bp):
- the ATCCTTGCCTTTGTGGGAGC-3 ' of sense primer 5 ' (SEQ ID No.11)
PRSS3 splicing variants 3 (trypsinogen transcript variant 3, PRSS3-V3) primer V3 (PCR
Amplified fragments size is 140bp):
- the GTGCGCCATTGGTTTTCCAT-3 ' of sense primer 5 ' (SEQ ID No.12)
PRSS3 splicing variants 4 (trypsinogen transcript variant 4, PRSS3-V4) primer V4 (PCR
Amplified fragments size is 131bp):
- the CGACTCGCATGGGACCTG-3 ' of sense primer 5 ' (SEQ ID No.13)
PRSS3 splicing variants 1-4 general reverse primers:
- the GCAGAAGTGGGAGCCAGAAT-3 ' of anti-sense primer 5 ' (SEQ ID No.14)
2nd, the screening and checking of universal primer
Cellar culture pancreatic carcinoma SW 1990 in the culture mediums of RPMI 1640, lung cancer cell line A549 and human liver cancer are thin
Born of the same parents system and human glioma cells U87.Human pancreas cancer SW1990, lung cancer A549 and glioma U87 cell mRNAs are extracted, is passed through
RT-qPCR methods compare primer Vc1 and Vc2 amplification efficiency.As a result as shown in Figure 2 A, PRSS3 is presented in three plants of cells
Expression status, and PRSS3 universal primers Vc1 and the similar amplification efficiency of Vc2 presentations；Through further electrophoretic analysis (Fig. 2 B), choosing
The universal primer that Vc1 is selected as amplification PRSS3 controls primer as the internal reference of detection PRSS3 expression.
Expression water of the PRSS3 in the normal liver cell system of more plants of HCC cell lines and immortalization is compared by RT-qPCR
It is flat, checking PRSS3 universal primers Vc1 reliability.As a result show, with LO2Cell is compared, and PRSS3 is in HCC cell lines BEL-
7402nd, BEL-7404, BEL-7405, HepG2 and PLC/PRF/5 present silence, SNU-449, SK-Hep-1, SNU-182,
Expression in SMMC-7721, SNU-387, QGY-7701, HuH1, HuH7, LM3 gradually increases, with HuH1, HuH7 and
The expression of LM3 cells significantly (Fig. 3), shows internal reference controls of the PRSS3 universal primers Vc1 as detection PRSS3 expression
Reliability.
3rd, the specificity of special primer and amplified production fail-safe analysis
Using RT-qPCR primer V1, V2, V3 and V4 for tetra- kinds of splicing variants of PRSS3-V1, V2, V3 and V4, divide
Its expression in human pancreas cancer SW1990, lung cancer A549 and glioma U87 cells is not detected.As a result show, in SW1990
In, tetra- kinds of splicing variants of PRSS3-V1, V2, V3 and V4 have expression (Fig. 4 A)；In cell line A549 and U87, only primer
Obvious amplification is presented in Vc1 and V1, and the splicing variants for showing mainly to express in cell line A549 and U87 are V1 (Fig. 4 B).
As a result differential expression of the PRSS3 different splicing variants in tumour cell is shown.
The reliability of specificity and amplified production for checking primer, enters to RT-qPCR amplified productions in SW1990 first
Row electroresis appraisal, Fig. 4 C electrophoresis results show that RT-qPCR amplified productions are single band；By the way that recovery band is further entered
Row cloning and sequencing, NCBI blast analyze its homology and shown, what PRSS3-V1, V2, V3 and V4 RT-qPCR primers were expanded
Product sequencing sequence is respectively 99% (Vc1 with the splicing variants sequence homology degree matched in NCBI data bank：
BC06949.1), 100% (V1:NM_07343.3), 95% (V2:NM_002771.3), 99% (V3:NM_001197097.2)
And 97% (V4：NM_001197098.1) (table 1), show to expand the special of PRSS3-V1, V2, V3 and V4 RT-qPCR primers
Property and RT-qPCR amplification precision.Result above also furthermore present the RT- to PRSS3-V1, V2, V3 and V4 expression
QPCR detection methods.
Table 1.PRSS3 splicing variants draw the homologous degree analysis of pcr amplification product sequencing result
4th, PRSS3 splicing variants RT-qPCR detection methods sensitiveness and stability analysis
PRSS3 plasmids pLenti6-PRSS3 is built as standard sample template, standard sample template concentrations is established, that is, leads to
10 times of μ g plasmids of gradient dilution 1 are crossed to 10-11(numbering is respectively 1~12) (table 2).Fig. 5 A show that molecular weight is 11000bp's
PRSS3 plasmids visible obvious band in 1 μ g (numbering 1) original samples loading capacity, it is 10 in V1 plasmids extension rate-1
When (i.e. plasmid quality is 100ng, numbering 2), it is seen that faint band, be consistent with estimating.
Vc1 primers are selected, and using extension rate as 10-2~10-11The PRSS3 plasmids of (numbering is 3~12) are template, are seen
The scope of RT-qPCR amplifications is surveyed, determines the sensitiveness and stability of this method.The amplification curve that Fig. 5 B are shown shows the steady of amplification
Qualitative and masterplate multiple proportion；Fig. 5 C solubility curves then show the specificity and its purity of PCR primer in each sample；Fig. 5 C's
Agarose gel electrophoresis analysis further shows that V1 plasmids minimum template quality is 1 × 10 in RT-qPCR results-4Pg (numberings
For 12) when it is still visible with it is expected that consistent single amplified band, with reference to its amplification curve and melting curve, shows that qPCR is expanded
The susceptibility of V1 plasmids can as little as 1 × 10-4pg.These results illustrate feasibility of the V1 plasmid templates as positive control, and can
Stability, specificity and sensitiveness as the detection Vc1 primers RT-qPCR amplifications of standard items gradient.
According to the calculation formula (being presented in materials and methods) between plasmid quality and copy number, PRSS3 plasmids are analyzed
Relation (table 2) in amplification between copy number and Ct values, the effective amplification scope for showing RT-qPCR methods is 1 × 10-3pg–1
×104Pg (black matrix font in table 2), and make corresponding linear relationship chart, wherein R2=0.99387, slope M are -3.343, are expanded
Increasing Efficiency E=1.99 (1≤E≤2).The plyability (Fig. 6) of reproducible results three times, show RT-qPCR methods to detecting PRSS3 tables
The accuracy reached and repeatability.Determine therefrom that the stability, specificity and sensitivity of RT-qPCR detection PRSS3 splicing variants
Property.
Table 2 analyzes relation of the PRSS3 splicing variants V1 plasmids in amplification between copy number and Ct values
5th, result interpretation
RT-qPCR carries out data analysis using calibration curve method, and the special simple spike of solubility curve illustrates primer specific
Property is good, so as to ensure that the specificity of product.
When being detected using above-mentioned universal primer, gel electrophoresis visible PCR amplificates (Fig. 4 C), or pass through qPCR
Solubility curve observation pcr amplification product specificity (Fig. 5 B), Ct values are obtained by amplification curve<Or=33 (Fig. 5 C), then
Illustrate a kind of expression of PRSS3 splicing variants in sample at least be present；
Gel electrophoresis without pcr amplification product, solubility curve do not observed by qPCR or occur non-specific solubility curve,
Ct values ＞ 33 is obtained without amplification curve or by amplification curve, then illustrates that PRSS3 splicing variants are without expression in sample.
When being detected using above-mentioned specific primer, a certain specific primer (such as V1, V2, V3 or V4) gel electrophoresis
Visible PCR amplificates (Fig. 4 C), or observed by qPCR solubility curve a certain specific primer (such as V1, V2, V3 or
V4) specificity of pcr amplification product, Ct values are obtained by amplification curve<Or=33, then illustrate to correspond to exist in sample to be somebody's turn to do
The expression of PRSS3 splicing variants (PRSS3-V1, PRSS3-V2, PRSS3-V3 or PRSS3-V4).
A certain specific primer (such as V1, V2, V3 or V4) gel electrophoresis is without pcr amplification product, or the dissolving for passing through qPCR
Curve does not observe a certain specific primer (such as V1, V2, V3 or V4) pcr amplification product or non-specific solubility curve, nothing occurs
Amplification curve obtains Ct values ＞ 33 by amplification curve, then illustrate in sample the PRSS3 splicing variants (PRSS3-V1,
PRSS3-V2, PRSS3-V3 or PRSS3-V4) do not express.
Embodiment 2 detects the composition of the RT-qPCR kits of the molecular marker for diagnosing tumor and prognosis detection
A kind of kit for detecting the molecular marker for diagnosing tumor and prognosis detection, i.e., for cell, tissue, body
The detection of PRSS3 splicing variants transcriptional levels in liquid and juice equal samples, it is medical diagnosis on disease and the prognosis such as tumour or infection
And medication provides guidance.The kit includes PRSS3 gene splicing variant RT-qPCR primer (its nucleosides described in embodiment 1
Acid sequence is as shown in SEQ ID No.6 to 14), the internal control primer for homogenization, land contrast template, as negative control mould
Deionized water, cDNA synthetic agents and the PCR reaction reagents of plate；
Wherein, the internal control primer for being used to uniform is the primer using GAPDH as internal reference,
- CCA TGG AGA AGG CTG the GG-3 ' (SEQ ID No.15) of sense primer 5 '
- CGC CAC AGT TTC CCG the GA-3 ' (SEQ ID No.16) of anti-sense primer 5 '；
The positive control template is the PRSS3 splicing variants pcr amplification products crossed through sequence verification, or sequence verification
The PRSS3 splicing variants cloned plasmids crossed；Preferably, PRSS3 splicing variants cloned plasmids include PRSS3 splicing variants
Consensus sequence, the consensus sequence is as shown in SEQ ID No.5；
The negative control template is deionized water.
In the PRSS3 gene splicing body RT-qPCR detection methods detection stomach organization cell that the application of embodiment 3 is established
The expression of PRSS3 splicing variants and its commitment and biological function, the feasibility of verification method
1st, cell culture
(1) gastric carcinoma cell lines (BGC823, MGC803, SGC7901, NCI-N87, AGS), the normal gastric mucosa of immortalization are thin
Born of the same parents system GES-1,
2nd, RNA extractions, RT-qPCR
(1) extraction of intracellular total serum IgE：It is 90% to select degrees of fusion, and the good cell of growth conditions carries out carrying for total serum IgE
Take.
1) 1mL/10 is pressed6Individual cell adds Trizol reagents in Tissue Culture Dish, softly rocks uniformly, places on ice
10min.Liquid is then transferred to 1.5mL centrifuge tube.
2) chloroform of 1/5Trizol volumes is incorporated in above-mentioned 1.5mL centrifuge tube, turned upside down, fully mixed, ice
Upper standing 10min, 12000rpm, 4 DEG C of centrifugations 15min, supernatant liquid is transferred in new 1.5mL centrifuge tubes.
3) draw and mixed with the isometric isopropanol of chloroform in above-mentioned new 1.5mL centrifuge tubes and softly, placed on ice
12000rpm after 10min, 4 DEG C of centrifugation 15min, abandons supernatant.
4) 1mL 75% ethanol 7500rpm, 4 DEG C of centrifugation 10min, so as to be washed to obtained white precipitate, is abandoned
Supernatant.Repeat the step.
5) appropriate RNAse-free Water are added to dissolve RNA.
6) resulting RNA is carried out to measurement of concetration respectively and runs glue checking, according to RNA concentration, A260 and A280 ratio
It is worth and runs cementing fruit to judge RNA concentration and quality.
7) RNA is stored in -80 DEG C of refrigerators.
(2) RNA reverse transcriptions
The is carried out according to EasyScript First-Strand cDNA Synthesis SuperMix kit specifications
One chain cDNA synthesis：
1) following components is added in 0.2mL centrifuge tubes：
2) after flicking mixing, 65 DEG C of incubation 5min, then ice bath 2min.
3) other reactive components are added after brief centrifugation into 0.2mL centrifuge tubes：
4) after flicking mixing, 42 DEG C of metal baths, it is incubated 30min.
5) 85 DEG C of heating 1min inactivationsRT/RI。
(3)RT-qPCR
1) primer is for example foregoing.
2) reaction system for being used for RT-qPCR amplifications is as follows：
3) RT-qPCR reaction conditions are as follows：
95℃30s
95 DEG C/30s, 60 DEG C/30s 40cycles (collection fluorescence)
95 DEG C/15s, 60 DEG C/1min, 95 DEG C/15s, 60 DEG C/15s (Melt Curve Stage)
Synthesized using above-mentioned RT-qPCR primers, while by the use of GAPDH as homogenization with reference to primer with reverse transcription in sample
CDNA is that template carries out qPCR amplifications.
3rd, the result obtained according to the result interpretation of embodiment 1：
1) expression of the PRSS3 in people GC cell lines and the normal gastric mucosa cell line immortalized
First using PRSS3 universal primers Vc1, RT-qPCR detects PRSS3 in people GC cell lines and the normal gastric immortalized
Expression in mucosa cells system.As shown in fig. 7, detect PRSS3's in normal gastric mucosa cell line GES-1 is immortalized
Expression, by contrast, in detected GC cell lines, PRSS3 significantly reduces in BGC823, MGC803 and SGC7901 expression, and
The expression of higher level is then presented in NCI-N87 and AGS.
2) GC cell lines and the PRSS3 splicing variants types of stomach normal tissue expression are detected
According to the above results, further detect what is embodied in NCI-N87, AGS and the GES-1 cell of PRSS3 expression
Splicing variants type.As a result as shown in figure 8, being glued in GC the cell lines NCI-N87, AGS and immortalization normal gastric of PRSS3 expression
In theca cell system GES-1, significantly expression (P is presented in only splicing variants V1 (PRSS3-V1)<0.05).
Further detect the expression in PRSS3-V1 people's gastric tissue sample.RT-qPCR results are shown, normal in 5 people's stomaches
In tissue sample, PRSS3-V1 has the expression (Fig. 9) of higher level.
Embodiment 4 determines the differential expression of PRSS3 splicing variants as tumour by detecting PRSS3 apparent silence
The reliability of mark
1st, people GC cell lines and immortalize normal gastric mucosa cell line in PRSS3 genosome methylation state of DNA
For checking PRSS3-V1 express in GC reduction it is whether related to commitment, separately designed methylation-specific
PCR (Methylation-specific PCR, MSP) and sodium hydrogensulfite sequencing (Bisulfite sequencing) primer).
Methylation status of PTEN promoter (Methylation-specific PCR, MSP), detect the change that methylates of PRSS3-V1 in GC cells
Change.
1) design of primers：MSP primers are designed and synthesized according to PRSS3 gene body DNA sequence dnas, methylated so as to detect
Site (M) and non-methylation sites (U).Primer sequence is as follows：
1. MSP methylated primers sequences (primer size 152bp)：
- GGT ACG CGG ATA GGG AGG GGA TAT the C-3 ' (SEQ ID No.17) of sense primer 5 '
- TAA TAT ACG CAT CGA TAC CGC AAC the CCG-3 ' (SEQ ID No.18) of anti-sense primer 5 '
2. the non-methylated primers sequences (primer size 155bp) of MSP：
- GGG TAT GTG GAT AGG GAG GGG ATA the TT-3 ' (SEQ ID No.19) of sense primer 5 '
- AAT AAT ATA CAC ATC AAT ACC ACA ACC the CA-3 ' (SEQ ID No.20) of anti-sense primer 5 '
2) reaction system for being used for MS-PCR amplifications is as follows：
Each group of PCR reaction includes a positive control (Human Methylated DNA Standard) and a moon
Property control (Normal human peripheral's lymphocyte DNA).
3) MS-PCR reaction conditions are as follows：
95℃10min
95 DEG C/30s, 60 DEG C/30s, 72 DEG C/30s, 35cycles；
72℃7min
4℃
4) gel electrophoresis：MSP products are verified through 2% agarose gel electrophoresis.
2nd, sodium hydrogensulfite sequence verification MSP results
1) gastric carcinoma cell lines (BGC823 and SGC7901) and the normal gastric mucosa cell line GES-1 immortalized vulcanization
For example above-mentioned step of journey is consistent.
2) design of primers：The primer of sodium hydrogensulfite sequencing is located at the side for treating sequencing sequence, and covers MSP primers, PCR
The size of product is 241bp.Primer sequence is as follows：
- TTG TTTGT the TGGGATTTGTGG-3 ' of sense primer 5 ' (SEQ ID No.21)
- the ACCTTCCCCTCACCCTACAAC-3 ' of anti-sense primer 5 ' (SEQ ID No.22)
3) reaction system for being used for PCR amplifications is as follows：
4) PCR reaction conditions are as follows：
95℃10min
95 DEG C/30s, 60 DEG C/30s, 72 DEG C/30s, 35cycles；
72℃7min
4℃
5) PCR primer obtained by after 2% agarose gel electrophoresis confirms, in ultraviolet gel imager to it is single just
True band carries out glue reclaim.PCR primer after glue reclaim is stored in -20 DEG C.
6) connection and conversion of glue reclaim product
Glue reclaim product and pEASY-T1 cloning vectors are attached, according toCloning Kit explanations
Book carries out step operation：
The preparation of connection product, following components is added in 0.2mL centrifuge tubes, specific reaction system is such as
Under：
After mixing, 4 DEG C are placed in, being used for subsequent transformation after connection overnight tests.Obtained connection product is added on firm defrosting
50 μ L Trans1-T1 competent cells in, be placed in mixture of ice and water after flicking mixing and react 30min；Immediately by it
It is placed in heat shock 90s in 42 DEG C of water-baths, rapid ice bath 2min；The LB liquid training of 250 μ L non-resistants is added into competent cell
Support base, 200rpm, 37 DEG C of activation 1h；4 μ L, 1mol/L IPTG, 40 μ are added on to the LB solid mediums with ammonia benzyl resistance
L, 20mg/mL X-gal, and it is evenly distributed with spreading rod, it is inverted in 37 DEG C of constant incubators, 30min；By what is activated
Bacterium solution 4000rpm centrifuges 1min, retains 150 μ L liquid, culture plate is added to after slowly mixing, first 37 DEG C are just being placed in after being coated with uniformly
30min is cultivated in constant incubator, after be inverted again, continue cultivate 14h；With by autoclaved pipette tips picking blueness gram
Big, the white clone in grand side, is placed in the LB fluid nutrient mediums containing 0.1% ampicillin, 200rpm, 37 DEG C
Shake bacterium culture 14h.
7) plasmid extraction
Step operation is carried out according to Plasmid Mini Kit I specifications, concentration mensuration is carried out to obtained plasmid, and
It is stored in -20 DEG C.
8) recon identification and sequencing：
BamH I and Xoh I double digestion systems are added into 0.2mL centrifuge tubes, its is anti-to be identified to the plasmid of extraction
The system is answered to be：Its reaction condition is 30 DEG C, 30min；37 DEG C, 30min, as a result identified through 1% agarose gel electrophoresis, use M13R
Primer is sequenced, and analyzes sequencing result.
3rd, apparent medicine effect
When gastric carcinoma cell lines and the normal gastric mucosa cell line growth density immortalized reach 30%, added into culture dish
5-Aza simultaneously makes its concentration be 5 μM, and changes a subculture every 24h, while continuously adds 5-Aza, maintains its concentration to be
5 μM, 72h is acted on altogether.When cell density reaches 70%, TSA is added into culture medium, makes its final concentration of 1 μM, acts on 24h.
4th, the result obtained according to the result interpretation of embodiment 1：
GC cell lines (BGC823, MGC803, SGC7901, NCI-N87, AGS) and immortality are detected by MS-PCR method
PRSS3-V1 methylation states in the gastric mucosal cell system (GES-1) of change.As a result show, PRSS3-V1 is in BGC823, MGC803
Be in exhaustive methylation state in SGC7901；It is in non-methylation state in NCI-N87 and AGS；And it is in half first in GES-1
Base state (Figure 10).As a result the expression status with PRSS3 in GC cell lines and immortalization gastric mucosal cell system matches.
Further determine PRSS3-V1 in BGC823, SGC7901 and NCI-N87 using the method for vulcanization sequencing (BSSQ)
In methylation state, verify above-mentioned MSP results (Figure 11 A and B), it is in complete first in BGC823 and SGC7901 to show PRSS3
Base state, and be in non-methylation state in NCI-N87, it was demonstrated that MSP results.
Whether decline to observe expression of the PRSS3 in GC cell lines by apparent mechanism regulating, pass through demethyl chemical drug
Thing 5-Aza and/or acetylation of histone enzyme inhibitor TSA combination, examined using Semiquatitative RT-PCR assay and RT-qPCR method
Expression of the PRSS3 in GC cell lines and immortalization gastric mucosal cell system changes before and after surveying medication.Semiquatitative RT-PCR assay result shows
Show, BGC823, MGC803 and SGC7901 cell that PRSS3 expression reduces, apparent medicine 5-Aza and/or TSA can increase PRSS3's
Expression；And in expression PRSS3 NCI-N87, AGS and GES-1 cell, drug-treated has no significant change (Figure 12 A).RT-
QPCR further analyzes display, and apparent medicine can dramatically increase the expression (figure of PRSS3 in BGC823, MGC803 and SGC7901 cell
12B), show that the expression of the PRSS3 in GC cell lines declines by apparent mechanism regulating.In addition, qPCR is more simpler than regular-PCR
Just medication effect, is accurately assessed.
Biological function verifications of the PRSS3 of embodiment 5 as tumor markers
1st, cell biological function is tested
(1)MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)
Experiment
By in exponential phase, growing state it is good containing it is unloaded and be overexpressed PRSS3 BGC823 cell lines and
SGC7901 cell lines carry out digestion counting, adjust the concentration of cell suspension, and 100 μ L are added per hole in 96 porocyte culture plates,
BGC823 spreads 2000 cells per hole, and SGC7901 spreads 3000 cells per hole, sets 8 multiple holes, Continuous Observation 4 days；Often it is preordained
When 10 μ L MTT are added in every hole, make its final concentration of 5 μ g/mL, after co-culturing 4h, discard culture medium.After 4 days, add per hole
Enter 150 μ L dimethyl sulfoxide (DMSO)s (dimethyl sulfoxide, DMSO), after lucifuge jog 10min, in enzyme-linked immunosorbent assay instrument OD
Each hole light absorption value is detected at 490nm, the data obtained is analyzed.
(2)Long-time dynamic living cells imaging system
By in exponential phase, growing state it is good containing it is unloaded and be overexpressed PRSS3 BGC823 cell lines and
SGC7901 cell lines digest counting respectively, adjust the concentration of cell suspension, and 100 μ L are added per hole in 96 porocyte culture plates,
BGC823 spreads 1000 cells per hole, and SGC7901 spreads 500 cells per hole, sets 3 multiple holes, Continuous Observation 5 days；
IncuCyte ZOOM are shot once per 2h to 96 porocyte culture plates, observe ability of cell proliferation.
(3) colony formation
By in exponential phase, growing state it is good containing it is unloaded and be overexpressed PRSS3 BGC823 cell lines and
SGC7901 cell lines digest respectively counts and is inoculated in 60mm2In Tissue Culture Dish, each culture dish is thin containing 500 BGC823
Born of the same parents, 300 SGC7901 cells；A subculture is changed within every 3 days, continuous culture grows to macroscopic for two weeks to cell clone
After size, culture medium is discarded, 1 × PBS is for several times；30min is fixed with 4% paraformaldehyde, with 1 × PBS twice,
0.5% violet staining 30min, flushing are taken pictures after drying, statistics clone's number.
(4) migration experiment
By in exponential phase, growing state it is good containing it is unloaded and be overexpressed PRSS3 BGC823 cell lines and
SGC7901 cell lines digest counting respectively, are added separately to the upper chamber of the Transwell cells in 8.0 μm of apertures, and each upper chamber adds
Enter the cell suspension that 200 μ L are free of serum, 4 × 104 BGC823 cells, 2 × 104 SGC7901 cells are added per hole；Lower room
600 μ L complete mediums of middle addition；Cultivated in 37 DEG C of cell culture incubators, BGC823 cell culture 12h, SGC7901 cell culture
8h；Carefully cleaned for several times with 1 × PBS；4% paraformaldehyde fixes 30min, with 1 × PBS three times；0.5% violet staining
30min, it is colourless to rinse to rinsing liquid, and upper ventricular cell is carefully removed with swab stick, and it is thin to be placed in statistics migration under microscope (100 ×)
The number of born of the same parents.
(5) scratch experiment
By in exponential phase, growing state it is good containing it is unloaded and be overexpressed PRSS3 BGC823 cell lines and
SGC7901 cell lines digest counting respectively, add into 6 porocyte culture plates, and 6 × 10 are added per hole6Individual BGC823 cells, 5
×105Individual SGC7901 cells, every group sets 3 multiple holes；When cell fusion degree is up to 90%, using white pipette tips in 6 well culture plates
Bottom draws a straight line, and discards culture medium, with 1 × PBS twice；After after floating cells are cleaned, serum-free is added
DMEM, it is put into inspection box and continues to cultivate；6 porocyte culture plates are taken a picture per 12h, observe the speed of its cut healing.
(6) statistical analysis
Experiment repeats at least three times.Experimental result is examined using sided t, its result mean+SD table
Show, * p<0.05 representative has notable significant difference, * * p<0.01 representative has and extremely significantly has significant difference.
2nd, experimental result：
(1) exogenous high expression splicing variants V1 stably transfected cell line is established
PRSS3 splicing variants V1 plasmid construction overexpressing cell model is used in GC cell lines, with liposome bag
Dress system will be overexpressed PRSS3 splicing variants V1 and empty plasmid is transfected to BGC823 and SGC7901 cell lines, by two
After all blasticidin S screenings, exogenous high expression splicing variants V1 stably transfected cell line is established.Experimental result is as schemed
Shown in 13, PRSS3mRNA expression is had no in unloaded control cell；It can detect that in PRSS3 stably transfected cell lines higher
PRSS3mRNA is expressed.
(2) it is overexpressed influences of the PRSS3 to GC cell line growth abilities
Shadows of the exogenous expression PRSS3 to BGC823 and SGC7901 cell line growth abilities is detected using MTT experiment
Ring.As a result (Figure 14) is shown, compared with the unloaded BGC823 and SGC7901 cell lines of stable transfection, being overexpressed PRSS3 can be notable
Suppress the Cell growth ability (P of BGC823 and SGC7901 cell lines<0.01).Meanwhile further utilizeWhen long
Between dynamic living cells imaging system to containing unloaded and be overexpressed PRSS3 BGC823 cell lines and SGC7901 cell line unit planes
(every square millimeter/mm of product2) inner cell number of variations situation.Recorded once per 2h, Continuous Observation 5 days.As a result show, with zero load
Group is compared, and PRSS3 is stable, and the every square millimeter of cell number of BGC823 cell lines and SGC7901 cell lines being overexpressed substantially reduces
(Figure 15) (P<0.01) it is, consistent with MTT experiment result.
(3) it is overexpressed influences of the PRSS3 to GC cell line clone Forming abilities
Further analysis is overexpressed influences of the PRSS3 to BGC823 and SGC7901 clonalities.As a result show, with
Empty plasmid group is compared, and PRSS3 overexpressing cells system BGC823 and SGC7901 clonality significantly reduce (P<0.01)
(Figure 16), exogenous overexpression PRSS3 is prompted to suppress GC cell lines BGC823 and SGC7901 ability of cell proliferation.
(4) it is overexpressed influences of the PRSS3 to GC cell line transfer abilities
Using cut Healing Experiments shadows of the PRSS3 to BGC823 and SGC7901 cell line transfer abilities is overexpressed to detect
Ring, as a result such as Figure 17 is shown, compared with empty plasmid group, GC cell lines BGC823 and the SGC7901 migration that PRSS3 is overexpressed are fast
Rate is remarkably decreased.
The transfer ability of GC cell lines is further verified by transwell Cell migration assays, as a result shown
PRSS3 overexpression can significantly inhibit cell migration ability (Figure 18) (P of BGC823 and SGC7901 cell lines<0.01).
Above PRSS3 biological function verification experiment shows, PRSS3 is overexpressed in BGC823 and SGC7901 cells,
GC cell lines BGC823 and SGC7901 cytoactive, multiplication capacity and migration can be significantly inhibited, shows PRSS3 spliced variants
Body V1 plays the effect of tumor suppressor gene sample in gastric carcinoma cell lines.For PRSS3 splicing variants differential expression be applied to tumour or
Inflammation infection diagnoses has established theoretical foundation with the molecular marked compound of prognosis detection.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical scheme is extended out still in protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Jiaotong University
<120>A kind of molecular marker and its detection primer and kit detected for diagnosing tumor with prognosis
<130> JLC17I0394E
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 965
<212> DNA
<213>PRSS3-V1 sequences
<400> 1
ggagttcctg gggccagaga catctccaag ggaaggtcaa gggcctggag gatgtgcgga 60
cctgacgaca gatgccccgc acgctggccg ggaccgggaa gggcggtcaa gtgtggaaag 120
ggtctggcgg ctgccaggcc tggcagagtg gagcggggcg gggcgcagcg gggcggggcg 180
ggcctggagc tgcacccgct tctgggtgga cgcacttggc gagcggcgcg ggatgcagac 240
ggctgcgagg cgctgggcac agttgctgtc ccctttgacg atgatgacaa gattgttggg 300
ggctacacct gtgaggagaa ttctctcccc taccaggtgt ccctgaattc tggctcccac 360
ttctgcggtg gctccctcat cagcgaacag tgggtggtat cagcagctca ctgctacaag 420
acccgcatcc aggtgagact gggagagcac aacatcaaag tcctggaggg gaatgagcag 480
ttcatcaatg cggccaagat catccgccac cctaaataca acagggacac tctggacaat 540
gacatcatgc tgatcaaact ctcctcacct gccgtcatca atgcccgcgt gtccaccatc 600
tctctgccca ccacccctcc agctgctggc actgagtgcc tcatctccgg ctggggcaac 660
actctgagct ttggtgctga ctacccagac gagctgaagt gcctggatgc tccggtgctg 720
acccaggctg agtgtaaagc ctcctaccct ggaaagatta ccaacagcat gttctgtgtg 780
ggcttccttg agggaggcaa ggattcctgc cagcgtgact ctggtggccc tgtggtctgc 840
aacggacagc tccaaggagt tgtctcctgg ggccatggct gtgccggaag aacaggcctg 900
gagtctacac caaggtctac aactatgtgg actggattaa ggacaccatc gctgccaaca 960
gctaa 965
<210> 2
<211> 756
<212> DNA
<213>PRSS3-V2 sequences
<400> 2
acactctacc accatgaatc cattcctgat ccttgccttt gtgggagctg ctgttgctgt 60
cccctttgac gatgatgaca agattgttgg gggctacacc tgtgaggaga attctctccc 120
ctaccaggtg tccctgaatt ctggctccca cttctgcggt ggctccctca tcagcgaaca 180
gtgggtggta tcagcagctc actgctacaa gacccgcatc caggtgagac tgggagagca 240
caacatcaaa gtcctggagg ggaatgagca gttcatcaat gcggccaaga tcatccgcca 300
ccctaaatac aacagggaca ctctggacaa tgacatcatg ctgatcaaac tctcctcacc 360
tgccgtcatc aatgcccgcg tgtccaccat ctctctgccc accacccctc cagctgctgg 420
cactgagtgc ctcatctccg gctggggcaa cactctgagc tttggtgctg actacccaga 480
cgagctgaag tgcctggatg ctccggtgct gacccaggct gagtgtaaag cctcctaccc 540
tggaaagatt accaacagca tgttctgtgt gggcttcctt gagggaggca aggattcctg 600
ccagcgtgac tctggtggcc ctgtggtctg caacggacag ctccaaggag ttgtctcctg 660
gggccatggc tgtgccggaa gaacaggcct ggagtctaca ccaaggtcta caactatgtg 720
gactggatta aggacaccat cgctgccaac agctaa 756
<210> 3
<211> 1093
<212> DNA
<213>PRSS3-V3 sequences
<400> 3
ggagttcctg gggccagaga catctccaag ggaaggtcaa gggcctggag gatgtgcgga 60
cctgacgaca gatgccccgc acgctggccg ggaccgggaa gggcggtcaa gtgtggaaag 120
ggtctggcgg ctgccaggcc tggcagagtg gagcggggcg gggcgcagcg gggcggggcg 180
ggcctggagc tgcacccgct tctgggtgga cgcacttggc gagcggcgcg ggatgcagac 240
ggctgcgagg cgctgggcac aggttgccag gacaaccgtg aggctgcata aaaagaacct 300
atgacaggat gcacatgaga gagacaagtg gcttcacatt gaagaagggg aggagtgcgc 360
cattggtttt ccatcctcca gatgcactga ttgctgtccc ctttgacgat gatgacaaga 420
ttgttggggg ctacacctgt gaggagaatt ctctccccta ccaggtgtcc ctgaattctg 480
gctcccactt ctgcggtggc tccctcatca gcgaacagtg ggtggtatca gcagctcact 540
gctacaagac ccgcatccag gtgagactgg gagagcacaa catcaaagtc ctggagggga 600
atgagcagtt catcaatgcg gccaagatca tccgccaccc taaatacaac agggacactc 660
tggacaatga catcatgctg atcaaactct cctcacctgc cgtcatcaat gcccgcgtgt 720
ccaccatctc tctgcccacc acccctccag ctgctggcac tgagtgcctc atctccggct 780
ggggcaacac tctgagcttt ggtgctgact acccagacga gctgaagtgc ctggatgctc 840
cggtgctgac ccaggctgag tgtaaagcct cctaccctgg aaagattacc aacagcatgt 900
tctgtgtggg cttccttgag ggaggcaagg attcctgcca gcgtgactct ggtggccctg 960
tggtctgcaa cggacagctc caaggagttg tctcctgggg ccatggctgt gccggaagaa 1020
caggcctgga gtctacacca aggtctacaa ctatgtggac tggattaagg acaccatcgc 1080
tgccaacagc taa 1093
<210> 4
<211> 735
<212> DNA
<213>PRSS3-V4 sequences
<400> 4
ggttccgact cgcatgggac ctgcggggga ggttgctgtc ccctttgacg atgatgacaa 60
gattgttggg ggctacacct gtgaggagaa ttctctcccc taccaggtgt ccctgaattc 120
tggctcccac ttctgcggtg gctccctcat cagcgaacag tgggtggtat cagcagctca 180
ctgctacaag acccgcatcc aggtgagact gggagagcac aacatcaaag tcctggaggg 240
gaatgagcag ttcatcaatg cggccaagat catccgccac cctaaataca acagggacac 300
tctggacaat gacatcatgc tgatcaaact ctcctcacct gccgtcatca atgcccgcgt 360
gtccaccatc tctctgccca ccacccctcc agctgctggc actgagtgcc tcatctccgg 420
ctggggcaac actctgagct ttggtgctga ctacccagac gagctgaagt gcctggatgc 480
tccggtgctg acccaggctg agtgtaaagc ctcctaccct ggaaagatta ccaacagcat 540
gttctgtgtg ggcttccttg agggaggcaa ggattcctgc cagcgtgact ctggtggccc 600
tgtggtctgc aacggacagc tccaaggagt tgtctcctgg ggccatggct gtgccggaag 660
aacaggcctg gagtctacac caaggtctac aactatgtgg actggattaa ggacaccatc 720
gctgccaaca gctaa 735
<210> 5
<211> 703
<212> DNA
<213>PRSS3 splicing variants consensus
<400> 5
ttgctgtccc ctttgacgat gatgacaaga ttgttggggg ctacacctgt gaggagaatt 60
ctctccccta ccaggtgtcc ctgaattctg gctcccactt ctgcggtggc tccctcatca 120
gcgaacagtg ggtggtatca gcagctcact gctacaagac ccgcatccag gtgagactgg 180
gagagcacaa catcaaagtc ctggagggga atgagcagtt catcaatgcg gccaagatca 240
tccgccaccc taaatacaac agggacactc tggacaatga catcatgctg atcaaactct 300
cctcacctgc cgtcatcaat gcccgcgtgt ccaccatctc tctgcccacc acccctccag 360
ctgctggcac tgagtgcctc atctccggct ggggcaacac tctgagcttt ggtgctgact 420
acccagacga gctgaagtgc ctggatgctc cggtgctgac ccaggctgag tgtaaagcct 480
cctaccctgg aaagattacc aacagcatgt tctgtgtggg cttccttgag ggaggcaagg 540
attcctgcca gcgtgactct ggtggccctg tggtctgcaa cggacagctc caaggagttg 600
tctcctgggg ccatggctgt gccggaagaa caggcctgga gtctacacca aggtctacaa 660
ctatgtggac tggattaagg acaccatcgc tgccaacagc taa 703
<210> 6
<211> 20
<212> DNA
<213>Artificial synthesized Vc1 upstream primer sequences
<400> 6
cttccttgag ggaggcaagg 20
<210> 7
<211> 20
<212> DNA
<213>Artificial synthesized Vc1 downstream primer sequences
<400> 7
ctccaggcct gttcttccag 20
<210> 8
<211> 20
<212> DNA
<213>Artificial synthesized Vc2 upstream primer sequences
<400> 8
attctggctc ccacttctgc 20
<210> 9
<211> 20
<212> DNA
<213>Artificial synthesized Vc2 downstream primer sequences
<400> 9
ctctcccagt ctcacctgga 20
<210> 10
<211> 15
<212> DNA
<213>Artificial synthesized V1 upstream primer sequences
<400> 10
ctgcgaggcg ctggg 15
<210> 11
<211> 20
<212> DNA
<213>Artificial synthesized V2 upstream primer sequences
<400> 11
atccttgcct ttgtgggagc 20
<210> 12
<211> 20
<212> DNA
<213>Artificial synthesized V3 upstream primer sequences
<400> 12
gtgcgccatt ggttttccat 20
<210> 13
<211> 18
<212> DNA
<213>Artificial synthesized V4 upstream primer sequences
<400> 13
cgactcgcat gggacctg 18
<210> 14
<211> 20
<212> DNA
<213>PRSS3 V1-4 general reverse primers
<400> 14
gcagaagtgg gagccagaat 20
<210> 15
<211> 17
<212> DNA
<213>The upstream primer sequence of GAPDH internal references
<400> 15
ccatggagaa ggctggg 17
<210> 16
<211> 17
<212> DNA
<213>The downstream primer sequence of GAPDH internal references
<400> 16
cgccacagtt tcccgga 17
<210> 17
<211> 25
<212> DNA
<213>MSP methylates upstream primer sequence
<400> 17
ggtacgcgga tagggagggg atatc 25
<210> 18
<211> 27
<212> DNA
<213>MSP methylates downstream primer sequence
<400> 18
taatatacgc atcgataccg caacccg 27
<210> 19
<211> 26
<212> DNA
<213>The non-upstream primer sequences to methylate of MSP
<400> 19
gggtatgtgg atagggaggg gatatt 26
<210> 20
<211> 29
<212> DNA
<213>The non-downstream primer sequences to methylate of MSP
<400> 20
aataatatac acatcaatac cacaaccca 29
<210> 21
<211> 20
<212> DNA
<213>Sense primer is sequenced in sodium hydrogensulfite
<400> 21
ttgtttgttg ggatttgtgg 20
<210> 22
<211> 21
<212> DNA
<213>Anti-sense primer is sequenced in sodium hydrogensulfite
<400> 22
accttcccct caccctacaa c 21
Claims (10)
- It is 1. a kind of for diagnosing tumor and the molecular marker of prognosis detection, it is characterised in that the molecular marker is PRSS3 Splicing variants；Wherein, the PRSS3 splicing variants are PRSS3-V1, PRSS3-V2, PRSS3-V3 and/or PRSS3-V4, Its nucleotide sequence is respectively as shown in SEQ ID No.1-4.
- 2. the molecular marked compound described in claim 1 is preparing the application of diagnosing tumor and the preparation of prognosis detection.
- 3. the material of the molecular marked compound described in test right requirement 1 answering in preparation of the diagnosing tumor with prognosis detection is prepared With.
- 4. application according to claim 3, it is characterised in that the material is the molecule mark described in test right requirement 1 Remember the primer or reagent of thing.
- A kind of 5. RT-qPCR primers of molecular marked compound for described in test right requirement 1, it is characterised in that including：For PRSS3-V1, PRSS3-V2, PRSS3-V3 and PRSS3-V4 universal primer Vc1 or Vc2 are detected, wherein, the primer Vc1's The nucleotide sequence of sense primer is as shown in SEQ ID No.6, and the nucleotide sequence of anti-sense primer is as shown in SEQ ID No.7； The nucleotide sequence of the sense primer of the primer Vc2 is as shown in SEQ ID No.8, the nucleotide sequence such as SEQ of anti-sense primer Shown in ID No.9；Preferably, the universal primer is primer Vc1.
- A kind of 6. RT-qPCR primers of molecular marked compound for described in test right requirement 1, it is characterised in that including：PRSS3-V1 specific primer V1, the nucleotide sequence of its sense primer is as shown in SEQ ID No.10, anti-sense primer Nucleotide sequence as shown in SEQ ID No.14；PRSS3-V2 specific primer V2, the nucleotide sequence of its sense primer is as shown in SEQ ID No.11, anti-sense primer Nucleotide sequence as shown in SEQ ID No.14；PRSS3-V3 specific primer V3, the nucleotide sequence of its sense primer is as shown in SEQ ID No.12, anti-sense primer Nucleotide sequence as shown in SEQ ID No.14；PRSS3-V4 specific primer V4, the nucleotide sequence of its sense primer is as shown in SEQ ID No.13, anti-sense primer Nucleotide sequence as shown in SEQ ID No.14.
- 7. the RT-qPCR kits of a kind of molecular marked compound for described in test right requirement 1, it is characterised in that including power Profit require 5 and/or claim 6 described in RT-qPCR primers.
- 8. RT-qPCR kits according to claim 7, it is characterised in that also include for uniform internal control primer, Positive control template, negative control template, cDNA synthetic agents and PCR reaction reagents.
- 9. RT-qPCR kits according to claim 8, it is characterised in that it is described be used for uniform internal control primer be Primer using GAPDH as internal reference, its nucleotide sequence is as shown in SEQ ID No.15 and SEQ ID No.16；The positive control template is the PRSS3 splicing variants pcr amplification products crossed through sequence verification, or sequence verification is crossed PRSS3 splicing variants cloned plasmids；Preferably, PRSS3 splicing variants cloned plasmids include being total to for PRSS3 splicing variants There is sequence, the consensus sequence is as shown in SEQ ID No.5；The negative control template is deionized water.
- 10. the method for the molecular marked compound described in a kind of test right requirement 1, it is characterised in that comprise the following steps：1) using the RT-qPCR primers described in claim 5 and/or 6 or the RT-qPCR kits described in claim 7 to inverse Transcription synthesizes sample cDNA to be detected and carries out qPCR amplifications, obtains amplified production；2) result interpretation：Interpretation is carried out to amplified production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710679293.4A CN107475379B (en) | 2017-08-10 | 2017-08-10 | Molecular marker for tumor diagnosis and prognosis detection, detection primer and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710679293.4A CN107475379B (en) | 2017-08-10 | 2017-08-10 | Molecular marker for tumor diagnosis and prognosis detection, detection primer and kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107475379A true CN107475379A (en) | 2017-12-15 |
| CN107475379B CN107475379B (en) | 2020-06-02 |
Family
ID=60599152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710679293.4A Expired - Fee Related CN107475379B (en) | 2017-08-10 | 2017-08-10 | Molecular marker for tumor diagnosis and prognosis detection, detection primer and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475379B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265467A (en) * | 2021-06-24 | 2021-08-17 | 四川省医学科学院·四川省人民医院 | Multiple myeloma marker, primer, kit and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191320A (en) * | 2005-05-02 | 2011-09-21 | 东丽株式会社 | Composition and method for diagnosing esophageal cancer |
| CN106636366A (en) * | 2016-11-25 | 2017-05-10 | 苏州首度基因科技有限责任公司 | Gene detection kit for prognosing gastric cancer metastasis and use method of gene detection kit |
-
2017
- 2017-08-10 CN CN201710679293.4A patent/CN107475379B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191320A (en) * | 2005-05-02 | 2011-09-21 | 东丽株式会社 | Composition and method for diagnosing esophageal cancer |
| CN106636366A (en) * | 2016-11-25 | 2017-05-10 | 苏州首度基因科技有限责任公司 | Gene detection kit for prognosing gastric cancer metastasis and use method of gene detection kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265467A (en) * | 2021-06-24 | 2021-08-17 | 四川省医学科学院·四川省人民医院 | Multiple myeloma marker, primer, kit and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107475379B (en) | 2020-06-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103930563B (en) | For the method and apparatus predicting cancer return | |
| Ma et al. | Exosomal hsa-miR199a-3p promotes proliferation and migration in neuroblastoma | |
| CN104818334B (en) | Relevant Microrna is shifted with adenocarcinoma of lung | |
| CN110283908B (en) | Colorectal cancer auxiliary diagnosis SNP marker and application thereof | |
| CN109468382A (en) | Application of the lncRNA in adenocarcinoma of lung diagnosis and treatment | |
| CN109593848A (en) | A kind of tumour correlated series, long-chain non-coding RNA and its application | |
| Wang et al. | Molecular tracing of prostate cancer lethality | |
| Guo et al. | Selection of brain metastasis-initiating breast cancer cells determined by growth on hard agar | |
| Zhao et al. | Analysis of the expression profile of serum exosomal lncRNA in breast cancer patients | |
| CN106480033B (en) | One kind ring-type circRNA-005365 gene relevant to leukaemia and application thereof | |
| CN107586842A (en) | A kind of biomarker for clear cell carcinoma of kidney diagnosis and treatment | |
| CN105506156B (en) | Diagnose the molecular marker of osteosarcoma | |
| CN100371459C (en) | Kit with fluorescent quantitative RT-PCR detection technique used for cell keratin 19(Ck19)mRNA | |
| CN108034655A (en) | A kind of application of the long non-coding RNA and combinations thereof in diagnosis/treatment colorectal cancer | |
| CN107475379A (en) | A kind of molecular marker and its detection primer and kit detected for diagnosing tumor with prognosis | |
| CN106244688A (en) | A kind of mark assessing adenocarcinoma of colon risk | |
| CN105603117B (en) | MiR-3613 is used to distinguish lung squamous cancer transfer and non-diverting miRNA marker | |
| CN108998532A (en) | A kind of diagnosis and treatment marker of rectal adenocarcinoma | |
| CN108660211A (en) | A kind of and the relevant biomarker LINC01549 of hepatocellular carcinoma and its application | |
| CN105734155B (en) | Chondroblastic osteosarcoma Disease-causing gene and its application | |
| CN109456971A (en) | A kind of application of long-chain non-coding RNA in diagnoses and treatment cholangiocarcinoma | |
| CN109486817A (en) | A kind of application of the long-chain non-coding RNA and combinations thereof in diagnoses and treatment cholangiocarcinoma | |
| CN104774966A (en) | Lung adenocarcinoma miRNA marker | |
| CN106148337A (en) | Long non-coding RNA AY927503 and application thereof | |
| CN105505936A (en) | Metastasis of osteosarcoma resistant biological agent and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200602 Termination date: 20200810 |