CN107254521A - lnc‑PCDH9‑13:Application of 1 detection reagent in reagent/kit of diagnosing cancer of liver is prepared - Google Patents
lnc‑PCDH9‑13:Application of 1 detection reagent in reagent/kit of diagnosing cancer of liver is prepared Download PDFInfo
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Abstract
The invention belongs to biomedicine field, it is related to the reagent and kit of a kind of diagnosing liver cancer.Reagent/the kit is long-chain non-coding RNA (long non coding RNA, lncRNA) lnc PCDH9 13:1 detection reagent, the detection of the reagent is with saliva etc. for sample.The present invention searches out the sensitive special biomarker of diagnosing liver cancer in saliva first, and the early diagnosis to liver cancer is early controlled, and reduces the death rate, the medical burden for mitigating China is significant.
Description
Technical field
The invention belongs to biomedicine field, it is related to liver cancer marker lnc-PCDH9-13:1 detection reagent is preparing liver
Application in reagent/kit of cancer diagnosis, and with the reagent and kit of a kind of diagnosing liver cancer.
Background technology
China is liver cancer big country, and the morbidity and mortality of liver cancer all account for more than half global.The accurate of liver cancer is examined
It is disconnected to have important practical significance.
Now, the biomarker in the world commonly used to examination liver cancer is Serum AFP (alpha-fetoprotein).But its sensitiveness
Only 39% to 65%, specificity only 76% to 94%.Because it detects that the sensitiveness of liver cancer is not satisfactory with specificity, because
The treatment guidelines in this U.S. do not recommend Serum AFP to be used for the diagnosis of liver cancer.China all points out with the treatment guidelines in Europe, normal individual
The AFP levels of body should be less than 20ng/ml, and the diagnosis of liver cancer can be then pointed out more than 400ng/ml.And 20 to 400ng/ml then examine
Disconnected " gray zone ".Therefore it is easily caused Misdiagnosis using AFP examination liver cancer.
Non-coding RNA take part in the especially tumorigenic regulation process of a variety of diseases, be one of recent research focus.Such as
It is modern, it has been found that human body has more than 30,000 kinds of non-coding RNAs.Although non-coding RNA not encoding proteins, in life likewise of which
Very important effect.For example directly adjust the expression of other genes, the activation of such as gene, silence, trace.In transcriptional level
The expression of RNA or albumen, or structure etc. are adjusted with translation skill.Finally affect intracellular various activities.
Long-chain non-coding RNA refers to more than 200 nucleotides of length, the non-coding RNA expressed with controlling gene, more next
More lncRNA (Long non-conding, long non-coding RNA) molecules be found they not only to a variety of diseases have examine
Disconnected and prediction prognosis value, while structure and the expression of a variety of RNA of controllable and albumen, in the generation evolution of disease
Play an important role.Therefore lncRNA is expected to turn into the target spot of diagnosing tumor mark and oncotherapy thing.
Except blood sampling mode, another diagnostic mode is sample of tissue diagnosis.However, hepatic tissue sampling is broken to human body
Bad property is strong, therefore, need a kind of diagnostic method accurate without wound formula.
There are many capillary networks in human saliva gland, therefore, saliva is sanguimotor end productses.Deposited in blood
Molecular substance, such as DNA, RNA, albumen, medicine, virus, can be found in saliva.Thus saliva is considered as people
The mirror of body health status, can reflect the various interior ambient conditions of body, such as cancer, infection, systemic disease.
The food and FAD (FDA) in the U.S. had been approved by using saliva detection of drugs of abuse, HIV, hormonal readiness, in
The reagents such as poison are commercially applied, and have all been popularized in the whole America.In clinic, molecular marked compound is found in saliva
Optimal method because collect saliva exist it is simple, conveniently, it is noninvasive, economical, will not cause that patient is uneasy, operator is easy
The advantages of grasping.Research has confirmed that transcript, albumen, metabolin and other molecular substances in saliva can detect outlet
Other oral cavities such as chamber cancer, breast cancer, lung cancer, oophoroma, Sjogren syndrome or systemic disease.Internal long-chain non-coding
RNA (long non-coding RNA, lncRNA) and the kinds cancer including liver cancer morbidity are closely related with developing.
Research finds that a variety of lncRNA lack of proper care in Expression In Hepatocellular Carcinoma, and the lncRNA that imbalance is expressed in tissue can be as biomarker
There is preferably diagnostic value to liver cancer.Although existing large quantities of hepatocarcinoma mark thing is found at present, these labels are present in group
In tissue samples or blood sample, however, same label is sampled using saliva sample, it is often another result, it is impossible to use
Make diagnosing cancer of liver.The label that this is likely due to be secreted into saliva is inherently seldom, and exists in saliva big
The enzyme of amount, label is easily degraded.But whether saliva lncRNA can assist diagnosing liver cancer as biomarker, and report is there is no at present
Road.
The content of the invention
Present invention aims at provide lnc-PCDH9-13:1 detection reagent is in diagnosing cancer of liver reagent/kit is prepared
Application.
The present invention also aims to simultaneously there is provided a kind of reagent/kit of diagnosing cancer of liver.
The above-mentioned purpose of the present invention is realized by following technological means：
lnc-PCDH9-13:1 is newfound lncRNA in nearly 2 years, and total length is 972bp, by lnc-PCDH9-13 bases
Because coded, positioned at No. 13 chromosome h19 genome, sequence is from the 6454958th base to 64550555 bases.Without mutation
lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1.At present, the research report for not yet having correlation shows lnc-PCDH9-
13:1 is related to liver cancer.
On the one hand, the invention provides lnc-PCDH9-13:1 detection reagent is preparing diagnosing cancer of liver reagent/kit
In application；Preferably, described lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1.
On the one hand, the invention provides lnc-PCDH9-13:1 application in screening medicines resistant to liver cancer；
On the one hand, the invention provides one group of LncRNA primer, the LncRNA primers are lnc-PCDH9-13:1 it is anti-
Transcription primers, including SEQ ID NO：2 and SEQ ID NO：Primer pair shown in 3, or such as SEQ ID NO：4 and SEQ ID
NO：Primer pair shown in 5, or such as SEQ ID NO：6 and SEQ ID NO：Primer pair shown in 7.
On the one hand, the application the invention provides above-mentioned primer pair in diagnosing cancer of liver reagent/kit is prepared.
On the one hand, the present invention provides a kind of diagnosing cancer of liver reagent/kit, it is characterised in that described diagnostic reagent/examination
Agent box contains lnc-PCDH9-13 in detection sample:The reagent of 1 expression quantity.
In the present invention, the sample of described reagent/kit detection is tissue, blood, saliva；Preferably, it is saliva.Cause
For collect saliva exist it is simple, conveniently, it is noninvasive, economical, the advantages of patient is uneasy, operator is easy to grasp will not be caused.
Lnc-PCDH9-13 in described detection sample:1 expression quantity is to lnc-PCDH9- by qPCR, gene microarray
13:1 carries out quantitative analysis；As preferred embodiment, using qPCR to lnc-PCDH9-13:1 carries out quantitative analysis.
The testing result of described reagent/kit is：As the lnc-PCDH9-13 in sample:1 expression quantity is more than
When 1.85, liver cancer is positive, as the lnc-PCDH9-13 in sample:When 1 expression quantity is less than 1.85, liver cancer is negative；It is highly preferred that
As the lnc-PCDH9-13 in saliva:When 1 expression quantity is more than 1.85, liver cancer is positive；As the lnc-PCDH9-13 in saliva:1
Expression quantity when being less than 1.85, liver cancer is negative；Described lnc-PCDH9-13:1 expression quantity is by qPCR relative expression quantities
What method was determined, wherein internal reference is β-actin.
Described reagent/kit contains lnc-PCDH9-13:1 post transcription cloning primer, for detecting lnc-
PCDH9-13:1 expression quantity.It is used as a kind of embodiment, described amplimer such as SEQ ID NO：2 and SEQ ID NO：3
It is shown；As another embodiment, the amplimer such as SEQ ID NO：4 and SEQ ID NO：Shown in 5；As another
Embodiment, described amplimer such as SEQ ID NO：6 and SEQ ID NO：Shown in 7.The amplimer is examined by qPCR
Survey lnc-PCDH9-13:1 expression quantity.
Further, described reagent/kit also contains protease；Preferably, described protease is Proteinase K；
Further, described reagent/kit is also containing sour phenol chloroform mixture；Preferably, described sour phenol chloroform
Mixture is hydrochloric acid-phenol chloroform mixture；More preferably；The volume ratio of phenol and chloroform is 4.5-5.5:1；It is highly preferred that
Described sour phenol chloroform mixture pH value is 4.3-4.7；
Further, described reagent/kit also contains RNase inhibitor；Preferably, described RNase inhibitor
For pyrocarbonic acid diethyl ester (DEPC).
It is used as a kind of application method of reagent/kit：It includes extracting in saliva after total serum IgE, carries out lnc-PCDH9-
13:1 reverse transcription, then pass through qPCR reaction detections lnc-PCDH9-13:1 level.
Extract saliva lnc-PCDH9-13:1 method can use following steps：
S1. saliva sample is taken, protease is added, 60-65 DEG C is incubated to remove sialoprotein；
S2. add after sour phenol chloroform mixture, 10000-12000g is centrifuged 4-6 minutes, collect supernatant；
S3. the ethanol of 1.2-1.4 times of volume is added, then 20-60 seconds filter centrifugations are centrifuged through 5000-6000g, filtrate is abandoned；
S4. the RNA inhibitory enzyme water of preheating is added, 10000-12000g is centrifuged 20-60 seconds, collects RNA；
S5. to lnc-PCDH9-13:1 carries out reverse transcription, then passes through primer and qPCR reaction detections lnc-PCDH9-13:1
Level.
On the other hand, present invention also offers a kind of chip for detecting liver cancer, it is characterised by, described chip includes solid phase
Carrier and the biomarker lnc-PCDH9-13 being fixed on solid phase carrier:1 probe.
Described lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1.
On the other hand, present invention also offers a kind of diagnosing cancer of liver system：Characterized in that, described detecting system contains
Have：
A) detection means：Described detection means are to detect lnc-PCDH9-13 in sample to be tested:1 expression quantity；
B) result judges component：Described result judges that component is used for the lnc-PCDH9- detected according to detection means
13:1 expression quantity draws the risk height or risk value of liver cancer；
Described sample is tissue, blood, saliva；It is highly preferred that being saliva；
Described detection means are RTPCR components, qPCR components, gene microarray component, expressed sequence analysis
SAGE components, grand gene order-checking component；It is highly preferred that being qPCR components；
Described result judges component for software, and it contains input module, analysis module and output module；Input module is used
In input lnc-PCDH9-13:1 expression quantity；Analysis module is used for according to lnc-PCDH9-13:1 expression quantity, analyzes liver
The risk height or risk value of cancer；Output module is used for the analysis result for exporting analysis module；
In described diagnostic system, as the lnc-PCDH9-13 in sample:When 1 expression quantity is more than 1.85, diagnostic result
It is positive for liver cancer, as the lnc-PCDH9-13 in sample:When 1 expression quantity is less than 1.85, diagnostic result is that liver cancer is negative；It is more excellent
Selection of land, as the lnc-PCDH9-13 in saliva:When 1 expression quantity is more than 1.85, diagnostic result liver cancer is positive；When in saliva
lnc-PCDH9-13:When 1 expression quantity is less than 1.85, diagnostic result liver cancer is negative；Described lnc-PCDH9-13:1 expression
Amount is determined by qPCR relative expression quantities method, and wherein internal reference is β-actin.
The quantum jump of the present invention is, finds blood, the tissue samples in liver cancer patient, especially in saliva sample,
There is substantial amounts of lnc-PCDH9-13:1 (Fig. 4).lnc-PCDH9-13:1 it is stable and exist in large quantities, can be by high reappearance
Ground is detected.And in the sample of healthy population, the marker representation level is very low.This causes lnc-PCDH9-13:1 turns into one kind
Good sample especially saliva label, detection or diagnosis for liver cancer have breakthrough meaning.
The present invention carries out the detection of hepatocarcinoma mark thing using saliva as sample, and maximum difficult point is, it is necessary to search out
It can be secreted into saliva, and the label that can be stabilized in saliva.Although having large quantities of hepatocarcinoma mark thing quilts at present
It was found that, these labels are present in tissue sample or blood sample, however, same label is sampled using saliva sample,
Often another result, it is impossible to as diagnosing cancer of liver.This is likely due to the label that can be secreted into saliva in itself
It is just seldom, and there is substantial amounts of enzyme in saliva, label is easily degraded.And this label that the present invention is had found not only is deposited
It is in tissue, and is present in saliva and can stably be detected, this causes the present invention to have what is protruded very much to take
Sample advantage.
The present invention first, by U.S.'s Agilent genetic chips (Figure 1A) and qPCR accurate quantifications technology (Figure 1B), has found
lnc-PCDH9-13:1 significantly raises in Expression In Hepatocellular Carcinoma.Meanwhile, lnc-PCDH9-13 is had found by bioanalysis:1
The regulating and controlling effect (Fig. 2) of key is played in a variety of oncogenes such as liver cancer.Followed by quantitative polyase chain reaction technology
(qPCR) lnc-PCDH9-13 is more accurately detected:1, analyzed, found relative to normal healthy controls person, lnc- using biostatistics
PCDH9-13:The 1 notable expression rise in the blood plasma of liver cancer patient and saliva.Meanwhile, it is in tissue, blood plasma and saliva
Expression notable positive correlation (Fig. 3) each other, points out the lnc-PCDH9-13 in saliva:1 derives from the secretion of liver cancer tissue.Connect
Discovery relative with normal healthy controls person (50), HBVer (50), chronic active hepatitis B patient (50), hepatic sclerosis
Patient (50) and cancer patient's (removing liver cancer, men and women separately counts) in China and first 10 of Global mortality row, lnc-
PCDH9-13:1 in the saliva of liver cancer patient it is significantly high expression (Fig. 4, Fig. 5).Point out lnc-PCDH9-13:1 examines liver cancer
It is disconnected that there is good specificity.
ROC (Receiver Operating Characteristic) curve deduction of predictive diagnosis liver cancer is finally set up,
The value of healthy individuals should be less than 1.8530245.If by the saliva lnc-PCDH9-13 of inspection individual:1 expression is more than this value,
Then it is determined as that the sensitiveness of liver cancer is 85% by inspection individual, specificity is 98%.Saliva lnc-PCDH9-13:1 as assistance
There is good prospect in terms of the mark of examination HBV-associated hepatocellular carcinoma, the sampling mode for collecting saliva is simple, conveniently, nothing
Wound, it is inexpensive, will not cause that patient is uneasy, operator is easy to grasp, person under inspection is easy to receive, and accuracy rate is high, to the morning of liver cancer
Examine and early control, reduce the death rate, the medical burden for mitigating China is significant.
Brief description of the drawings
Fig. 1 is genetic chip (Figure 1A) and qPCR (Figure 1B) scientific discoveries lnc-PCDH9-13:1 is notable in liver cancer tissue
Height expression, wherein lnc-PCDH9-13 of the circle for normal structure:1 expression quantity, square frame is liver cancer tissue lnc-PCDH9-13:1 table
Up to amount.
Fig. 2 is that bioinformatics finds lnc-PCDH9-13:The oncogene of 1 pair of liver cancer plays important regulating and controlling effect.
Fig. 3 is tissue, blood plasma, saliva lnc-PCDH9-13:The correlation analysis of 1 expression quantity.
Fig. 4 is the lnc-PCDH9-13 in different crowd saliva sample:1 expression.
Fig. 5 is saliva lnc-PCDH9-13:1 arranges the different expression between preceding 10 cancer patients in the whole world in fatal rate.
Fig. 6 is saliva lnc-PCDH9-13:1 qPCR amplification curves (Fig. 6 A) and solubility curve (Fig. 6 B) and sequencing result
(Fig. 6 C).
Fig. 7 is saliva lnc-PCDH9-13:The ROC curve that 1 pair of diagnosing cancer of liver value is inferred.
Fig. 8 is ratio of the different Serum AFP groups in liver cancer patient.
Fig. 9 is the saliva lnc-PCDH9-13 of different AFP expressions liver cancer patients:1 detection level.
Embodiment
Technical scheme is further illustrated below by way of specific embodiment, specific embodiment is not represented to this hair
The limitation of bright protection domain.Some nonessential modifications and adjustment that other people are made according to theory of the present invention still fall within this hair
Bright protection domain.
In the present invention, " expression quantity " uses 2-ΔΔCtMethod is calculated and represented.Ct values are the number that qPCR is arrived to each Samples detection
Value.Δ Ct=lncRNA-β-the actin of each sample；The Δ Ct- healthy control group Δ Ct's of each samples of Δ Δ Ct=is averaged
Value.
The saliva of embodiment 1 gathers
Before saliva gathers, research individual needs fasting, prohibits and drink, ban on opium-smoking and the opium trade and forbid more than oral cleaning 2h.
For increase saliva collection amount, aseptic cotton carrier cotton head is moistened using 2% citric acid solution, then cotton head is put in
After the rear wall about 5s of the individual tongue side of research, discharge saliva, then cotton swab is put into the tongue rear wall of opposite side.It is so anti-
It is multiple to collect.Saliva collection amount need to reach more than 5ml, and collecting pipe is sterile without enzyme centrifuge tube using 50ml.4 DEG C, 3000g centrifugations 15min
Upper strata supernatant is taken to 1.5ml Eppendoff pipes, then supernatant is taken to abandon precipitation again after 4 DEG C, 12 000g centrifugations 10min.At sample
- 80 DEG C of preservations are put after reason.
The extraction of the saliva total serum IgE of embodiment 2
1. by 1ml salivas average mark mounted in two 1.5ml without on enzyme EP pipes, i.e., every EP pipe all fills 500 μ l saliva
Liquid, then adds Proteinase K (20mg/ml) 15 μ l to every EP pipe, mixes, and is put in 65 DEG C of water-bath overnight incubations.Because saliva
Containing albumen such as more digestive enzymeses in liquid, influence subsequent extracted saliva RNA effect, therefore this method can remove sialoprotein
Influence to experiment.
2. two EP pipes respectively add 0.5ml sour phenol chloroform mixture (main component is hydrochloric acid, phenol and chloroform) to arrive above-mentioned
In saliva mixed liquor.
The configuration of sour phenol chloroform
The volume ratio of 2.1 phenol and chloroform is 5:1
2.2 adjust pH value with concentrated hydrochloric acid, and pH value is scheduled on 4.5 or so, and this pH value is most useful for extraction RNA.Mix.
3. defensive position is added to shake even 30 to 60 seconds.
4. 10000g is centrifuged 5 minutes at room temperature.Middle level liquid need to compact after centrifugation, otherwise centrifuge again.
5. collecting supernatant, it is careful to need, it is impossible to stir lower floor's liquid phase.
6. 100% ethanol of 1.2-1.4 times of volume is added in supernatant.500 μ l supernatants are for example collected into, then add 625 μ
L absolute ethyl alcohols.
7. screen pipe is put into collecting pipe, said mixture is added in screen pipe (screen pipe and collecting pipe in the market
There is sale).
8. 6000g is centrifuged 30 seconds.
9. abandon filtered fluid.Repeat sky and throw away the heart 1 time.Retain collecting pipe.
10. 700 μ l 75% ethanol is added in screen pipe, centrifuge 15 seconds.Abandon filtered fluid.Screen pipe is reentered into
In collecting pipe originally.
11. repeating the 10th step, then it washed once.
12. abandoning filtered fluid, screen pipe is put into same collecting pipe.Sky throws away the heart 1 minute.
13. screen pipe is put into new collecting pipe.Add 95 DEG C of preheating DEPC water.Cover lid.Centrifugation 30 seconds.Collect
RNA。
14. collect eluent.Eluent is to be dissolved with the total serum IgE extracted from saliva.Total serum IgE after extraction is at -80 DEG C
Preserve below.
The saliva lnc-PCDH9-13 of embodiment 3:1 reverse transcription and expression quantity detection
Lnc-PCDH9-13 in saliva:1 expression quantity detects that relative quantification represents using qPCR instrument, wherein, internal reference be β-
actin。
1. saliva lnc-PCDH9-13:1 reverse transcription reaction
2 μ L RNA are taken to carry out reverse transcription.Ice bath 2min immediately after 65 DEG C of reaction 5min.
2 μ 5 × RT of l Buffer, 0.5 μ l Enzyme Mix, 0.5 μ l Primer are separately added into above-mentioned 2 μ L RNA
The nuclease-free water that Mix (being Toyobo Products, article No. FSK-100) and 5 μ l are handled through DEPC, is then proceeded by inverse
Transcription, reaction condition is 37 DEG C of 15min, 98 DEG C of 5min, 4 DEG C of ∞.
2. saliva lnc-PCDH9-13:1 expression quantity detection (qPCR reactions)
The μ L of reverse transcription product 3 are taken as template, with SYBR Premix Ex Taq II kit (TaKaRa companies, article No.
RB820A) carry out adding 9 μ L 5 × SYBR Premix Buffer in RT-qPCR detections, 20 μ L reaction systems, 0.8 μ L's is upper
Swim primer, 0.8 μ L anti-sense primer, and 6.4 μ L through DEPC handle without enzyme water.
Upstream primer sequence SEQ ID NO:2:
TCCAAGGGCAGCAAGTAGGAC
Downstream primer sequence SEQ ID NO:3
GCTGTGACTCGCTGGAGAATG；
All reactions use 3 multiple holes, and reaction condition is 95 DEG C of 2min, then 95 DEG C of 5s, 60 DEG C of 10s, 50 circulations.Dissolving
Curve is set：Melt curve:65.0to 95.0℃,increment 0.5℃for 0.05min+plate read.Using
Bio-Rad CFX real-time PCRs are detected.
The amplification curve of embodiment 4, solubility curve and sequencing analysis
Using saliva as experiment sample, after above-mentioned Total RNAs extraction, reverse transcription, qPCR reactions, expanded, dissolved
Curve and product sequencing analysis.
Draw lnc-PCDH9-13:1 qPCR amplification curves, solubility curve and sequencing result are as shown in Figure 6.QPCR is expanded
Curve, solubility curve and sequencing result such as Fig. 6 can be drawn：As a result show through above method detection saliva lnc-PCDH9-13:1,
It can succeed and specifically detect saliva lnc-PCDH9-13:1 expression.
Lnc-PCDH9-13 in the saliva sample of embodiment 5:1 expression is detected
Collection normal healthy controls individual (50), HBVer (50), chronic active hepatitis B patient (50), liver are hard
Changing patient (50), liver cancer patient (includes preoperative (50) and postoperative (50) and the advanced liver cancer (50 of early liver cancer patient
Example)) totally 350 saliva samples.M-F 1:1.
Detect saliva lnc-PCDH9-13:1 expression and otherness in each group.Saliva processing and detection method are such as
Shown in embodiment 3.
As a result it is as shown in Figure 4.Saliva lnc-PCDH9-13:1 expression in liver cancer patient is all significantly higher than other
Control group.
By statistical analysis, ROC curve is built, saliva lnc-PCDH9-13 can be drawn:The sensitiveness of 1 pair of diagnosing cancer of liver
85%, specificity is up to 98%.As a result it is as shown in Figure 7.
Test as a comparison, inventor also have detected the serum afp of collected liver cancer patient.Collected at us
In liver cancer patient, only 20% individual serum afp reaches the diagnostic level of liver cancer, and about 50% individual is in normally
Level.As a result it is as shown in Figure 8.
Lnc-PCDH9-13 in 10 cancer before the fatal rate ranking of embodiment 6:1 expression quantity detection
Ten cancer patient's (being specifically shown in Fig. 5) before collection China and Global mortality ranking, and healthy control group, men and women
Detection individual is separately counted.Liver cancer patient totally 100, each 50 of men and women.Normal healthy controls person totally 50, each 25 of men and women.It is other
Each 6 of cancer patient men and women.
Detect saliva lnc-PCDH9-13:1 expression and otherness in each group.Saliva processing and detection method are such as
Shown in embodiment 3.
Experimental result as shown in figure 5, arrange cancer patient's (men and women separately counts) of first 10 in China and Global mortality,
lnc-PCDH9-13:1 in the saliva of liver cancer patient it is significantly high expression prompting lnc-PCDH9-13:The diagnosis of 1 pair of liver cancer has
Good specificity.
The saliva sample testing result of other existing hepatocarcinoma mark things of comparative example
Serum AFP is the existing biomarker for assisting diagnosing cancer of liver in the world today.But its Sensitivity and Specificity
It is very low.Inventor further analyzes the saliva lnc-PCDH9-13 of different AFP expressions patients:1 detection level, as a result
As shown in Figure 9.AFP expressions regardless of liver cancer patient blood serum, saliva lnc-PCDH9-13:1 can detect liver cancer.
All there is conspicuousness height expression in the saliva that different AFP are grouped liver cancer patient in it.Saliva lnc-PCDH9-13:1 compares Serum AFP
Diagnostic value is had more to liver cancer.
SEQUENCE LISTING
<110>The Third Affiliated Hospital of Zhongshan University
<120> lnc-PCDH9-13:Application of 1 detection reagent in reagent/kit of diagnosing cancer of liver is prepared
<130> PT20170664
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 972
<212> DNA
<213> Homo sapiens
<400> 1
atggagcgac atgcagtaaa cagcaatacc tcaccacaga atactggttt agagagtgga 60
cagattctgc agcaggaaag gggttatgcc ttgatgtttc aagtcagtag aggcctgtta 120
aatcacacca aatgacaaca aatcattgtc caagctgtcc ctgagggaca aggctaaacc 180
atcctacaag tatctatgtt tgggacacag ccttgtagca gatatagttg gtcttgtgga 240
aatagatcga ggtctggggg aaaaaaagtg gaggatagag atacagaggg aaagacttta 300
caggttcagg gccaacaaga ccagactcag aagattgtca tccagcacgt gcagacagcc 360
ctcaccacca gccagacaca caaagaagca gattgctatc caagggcagc aagtaggaca 420
gatcatggtc taccagccag ttaatgcaga tggcaacatt ctccagcgag tcacagccac 480
catctcagca gccactttga cagtggcata gacttttcag acagcaagcc aataccaaca 540
caaccagcag tgggcaagga accatcactg taacactacc agtggcagat tatgtggtca 600
cttaaggaga gacagtcatg atggtgcctg ggctgtctct gtgactgcca cccaaagaat 660
ctctcacctg gaacagagat agaaagacag cctcccaatg tgaatgtgga taacaactag 720
tgacatcatc tttgttaaga ggatgtaagc ctgggctgaa ctagaggtac aaggaacatt 780
cctaagggaa aaaaaaaaaa aaatatatat atatatatat atatatatat atatatctgc 840
ataattcgtg gtatggcata aaagtgtggc agagttgaat gttttttttt cccaaaataa 900
aagaatagtc tccatatgca ggttgcagac caagtaaatg cagcagcaat ggcatgcaga 960
atttgagtgt tc 972
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
tccaagggca gcaagtagga c 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
gctgtgactc gctggagaat g 21
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<400> 4
gacaaggcta aaccatccta caag 24
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
ctttccctct gtatctctat cctcc 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
acagcaatac ctcaccacag aatac 25
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence
<400> 7
aggcctctac tgacttgaaa catc 24
Claims (11)
1.lnc-PCDH9-13:Application of 1 detection reagent in diagnosing cancer of liver reagent/kit is prepared.
2. application according to claim 1, it is characterised in that described lnc-PCDH9-13:1 sequence such as SEQ ID
NO：Shown in 1.
3.lnc-PCDH9-13:1 application in screening medicines resistant to liver cancer；
Preferably, described lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1.
4. one group of LncRNA primer, the LncRNA primers are lnc-PCDH9-13:1 reverse transcription primer, including SEQ ID
NO：2 and SEQ ID NO：Primer pair shown in 3, or SEQ ID NO：4 and SEQ ID NO：Primer pair shown in 5, or
SEQ ID NO：6 and SEQ ID NO：Primer pair shown in 7.
5. application of the primer in diagnosing cancer of liver reagent/kit is prepared described in claim 4.
6. a kind of diagnosing cancer of liver reagent/kit, it is characterised in that described diagnostic reagent/kit contains in detection sample
lnc-PCDH9-13:The reagent of 1 expression quantity；
Preferably, described lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1；
Preferably, described sample is tissue, blood, saliva；It is highly preferred that being saliva；
Preferably, lnc-PCDH9-13 in described detection sample:1 expression quantity is to lnc- by qPCR, gene microarray
PCDH9-13:1 carries out quantitative analysis；It is highly preferred that by qPCR to lncRNA lnc-PCDH9-13:1 carries out quantitative analysis；
Preferably, the testing result of described reagent/kit is：As the lncRNA lnc-PCDH9-13 in sample:1 table
When being more than 1.85 up to amount, liver cancer is positive, as the lnc-PCDH9-13 in sample:When 1 expression quantity is less than 1.85, liver cancer is negative；
It is highly preferred that as the lnc-PCDH9-13 in saliva:When 1 expression quantity is more than 1.85, liver cancer is positive；As the lnc- in saliva
PCDH9-13:When 1 expression quantity is less than 1.85, liver cancer is negative；
It is highly preferred that described lnc-PCDH9-13:1 expression quantity is determined by qPCR relative expression quantities method, wherein interior
Join as β-actin.
7. diagnosing cancer of liver reagent/kit according to claim 6, it is characterised in that contain lnc-PCDH9-13:1
Amplimer；
Preferably, described amplimer is for qPCR reactions；
Preferably, described amplimer such as SEQ ID NO：2 and SEQ ID NO：Shown in 3, or such as SEQ ID NO：4 Hes
SEQ ID NO：Shown in 5, or such as SEQ ID NO：6 and SEQ ID NO：Shown in 7.
8. diagnosing cancer of liver reagent/kit according to claim 7, it is characterised in that also contain protease；Preferably,
Described protease is Proteinase K；
Preferably, also containing sour phenol chloroform mixture；It is highly preferred that described sour phenol chloroform mixture is hydrochloric acid-phenol chloroform
Mixture；More preferably；The volume ratio of phenol and chloroform is 4.5-5.5:1；It is highly preferred that described sour phenol chloroform mixture pH
It is worth for 4.3-4.7；
Preferably, RNase inhibitor is also contained；It is highly preferred that described RNase inhibitor is pyrocarbonic acid diethyl ester DEPC.
9. diagnosing cancer of liver reagent/kit according to claim 8, the application method of described diagnostic kit is：
Extract in sample after total serum IgE, carry out lnc-PCDH9-13:1 reverse transcription, then pass through qPCR reaction detections lnc-PCDH9-
13:1 level；
Preferably, described sample is tissue, blood, saliva；It is highly preferred that being saliva；
Preferably, saliva sample lnc-PCDH9-13 is extracted:1 method can use following steps：
S1. saliva sample is taken, protease is added, 60-65 DEG C is incubated to remove removing protein；
S2. add after sour phenol chloroform mixture, 10000-12000g is centrifuged 4-6 minutes, collect supernatant；
S3. the ethanol of 1.2-1.4 times of volume is added, then 20-60 seconds filter centrifugations are centrifuged through 5000-6000g, filtrate is abandoned；
S4. the RNA inhibitory enzyme water of preheating is added, 10000-12000g is centrifuged 20-60 seconds, collects RNA.
10. a kind of chip for detecting liver cancer, is characterised by, described chip includes solid phase carrier and is fixed on solid phase carrier
Biomarker lnc-PCDH9-13:1 probe；
Described lnc-PCDH9-13:1 sequence such as SEQ ID NO：Shown in 1.
11. a kind of diagnosing cancer of liver system：Characterized in that, described detecting system contains：
A) detection means：Described detection means are to detect lnc-PCDH9-13 in sample to be tested:1 expression quantity；
B) result judges component：Described result judges that component is used for the lnc-PCDH9-13 detected according to detection means:1
Expression quantity draws the risk height or risk value of liver cancer；
Preferably, described sample is tissue, blood, saliva；It is highly preferred that being saliva；
Preferably, described detection means are qPCR components or gene microarray component；It is highly preferred that being qPCR components；
Preferably, described result judges component for software, and it contains input module, analysis module and output module；Input mould
Block is used to input lnc-PCDH9-13:1 expression quantity；Analysis module is used for according to lnc-PCDH9-13:1 expression quantity, analysis
Go out the risk height or risk value of liver cancer；Output module is used for the analysis result for exporting analysis module；
Preferably, in described diagnostic system, as the lnc-PCDH9-13 in sample:When 1 expression quantity is more than 1.85, diagnosis knot
Fruit is positive for liver cancer, as the lnc-PCDH9-13 in sample:When 1 expression quantity is less than 1.85, diagnostic result is that liver cancer is negative；More
Preferably, as the lnc-PCDH9-13 in saliva:When 1 expression quantity is more than 1.85, diagnostic result liver cancer is positive；When in saliva
lnc-PCDH9-13:When 1 expression quantity is less than 1.85, diagnostic result liver cancer is negative；
Preferably, described lnc-PCDH9-13:1 expression quantity is determined by qPCR relative expression quantities method, wherein internal reference
For β-actin.
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