CN109694866A - Long-chain non-coding RNA ST8SIA6-AS1 and its application - Google Patents
Long-chain non-coding RNA ST8SIA6-AS1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of long-chain non-coding RNA that can be used as tumor markers and therapy target, the base sequence of the corresponding cDNA of the long-chain non-coding RNA is as shown in SEQ ID NO:1；And base sequence antisense lock nucleic acid as shown in SEQ ID NO:2 or SEQ ID NO:3；And a kind of purposes that can strike the substance for dropping the expression of above-mentioned long-chain non-coding RNA in the drug of preparation treatment tumour；And a kind of drug for treating tumour, the substance that can strike the expression for dropping above-mentioned long-chain non-coding RNA is contained in drug.For ST8SIA6-AS1 in the present invention as new tumor cells marker, height expression can indicate the poor prognosis of tumour；As cancer target, two antisense lock nucleic acids can specifically strike the expression of drop ST8SIA6-AS1, efficiently, specifically cause tumour cell that apoptosis occurs, and inhibit tumour growth.
Description
Technical field
The present invention relates to genetic engineering and oncology, especially a kind of long-chain non-coding RNA ST8SIA6-AS1；
The invention further relates to purposes of the above-mentioned RNA in tumor cells diagnosing and treating.
Background technique
Rejuvenation and the high trend of morbidity and mortality is presented in malignant tumour in the past 20 years in China.2015 annual China
Malignant tumour new cases 429.2 ten thousand, dead 281.4 ten thousand, seriously affect people's health.Wherein China's breast cancer incidence
Growth rate is the whole world more than twice, has become the first place of China's tumour registration area female malignant.On the other hand, lung
The China Ai Ju Death Cause for Malignant Tumors is the first, and morbidity and mortality persistently increase.
The method of clinical treatment tumour mainly operation excision, radiation and chemotherapy at present, but Radiotherapy chemotherapy is swollen in kill
While oncocyte, serious damage is also brought to human normal cell.Therefore researcher is tried hard in unremitting exploration
By targeted therapy efficiently, specifically kill tumour cell.Clinically has the targeted drug for HER2 molecule at present
Herceptin is used for the treatment of tumour, but tumour a small amount of on the whole for targeted drug Avastin of VEGF molecule etc.
Targeted drug is far from satisfying the demand of clinical treatment.Meanwhile there are certain blindness for oncotherapy clinical at present, only
Stage and step can be carried out to tumour or simply be classified using limited molecular marker to tumour, can not effectively screened pre-
Good and poor prognosis tumour afterwards, the preferable tumour of part prognosis may be by over-treatment, therefore urgently develops for judging tumour
The molecular marker of prognosis.
In recent years using non-coding RNA be the epigenetic regulation mechanism of core in vital movement effect increasingly by
Pay attention to.Long-chain non-coding RNA is non-coding RNA molecule of a kind of transcript length in 200nt or more, is typically considered to not have
Encode protein function.It is more and more researches show that the expression of long-chain non-coding RNA different tissues have high degree of specificity, no
Only the different phase of its expression and tumor development, different tissues type are closely related, can be used as the spy of lesion detection
Different marker, and important regulating and controlling effect of the long-chain non-coding RNA in tumor development also shows it and controls as tumour
A possibility that treating target spot.
Summary of the invention
In view of the deficiencies of the prior art, present inventor it has been investigated that, long-chain non-coding RNA ST8SIA6-AS1
Expression is apparently higher than related normal tissue in kinds of tumors tissue, the ST8SIA6- in the tumours such as breast cancer and lung cancer
AS1 high expression is closely related with patient's poor prognosis, can be used as the marker of tumor prognosis judgement.Data disclosed in the prior art
In there is no report of the ST8SIA6-AS1 as disease marker, also have no the report of correlative study at present to its function.
As the first aspect of the invention, the present invention provides a kind of long-chain that can be used as tumor markers and therapy target
Non-coding RNA (i.e. ST8SIA6-AS1), the base sequence such as SEQ ID NO:1 institute of the corresponding cDNA of the long-chain non-coding RNA
Show.Present inventor's studies have shown that in breast cancer and lung cancer the high expression of ST8SIA6-AS1 it is bright with patient's poor prognosis
Aobvious correlation, can be used as the marker of tumor patient Index for diagnosis.Present inventor's studies have shown that in breast cancer and lung cancer
In strike drop ST8SIA6-AS1, in vitro and in vivo experiment display can significantly induce tumour cell occur apoptosis；In view of
High expression ST8SIA6-AS1 special in tumor tissues, its expression of targeted inhibition can produce apparent antitumous effect；In view of just
ST8SIA6-AS1 expression quantity is lower in normal histocyte, inhibits its expression to have no obvious apoptosis and growth inhibition effect, we recognize
It is an efficient, special oncotherapy target for ST8SIA6-AS1.
Preferably, the marker is the corresponding mRNA of base sequence shown in SEQ ID NO:1.
Preferably, the tumour is breast cancer or lung cancer.Wherein, tumour can also be liver cancer, cancer of pancreas, prostate cancer,
Bladder cancer, kidney etc..
As the second aspect of the invention, the present invention provides a kind of antisense lock nucleic acid (locked nucleic
Acid, LNA), the base sequence of the antisense lock nucleic acid is as shown in SEQ ID NO:2 or SEQ ID NO:3.
Present inventor carries out test of many times discovery using the lock nucleic acid of a variety of base sequences, only by such as SEQ
Antisense lock nucleic acid shown in ID NO:2 or SEQ ID NO:3 in tumor cell line and transplanted tumor in nude mice to ST8SIA6-AS1 into
Row strikes drop, can obviously lower the expression of ST8SIA6-AS1, and cause the apoptosis of tumour cell, so that it is swollen to reach inhibition
The effect of tumor growth.It should be noted that antisense lock nucleic acid includes but is not limited to as shown in SEQ ID NO:2 or SEQ ID NO:3
Base sequence, as long as the base sequence of antisense lock nucleic acid can strike drop ST8SIA6-AS1 expression.
As the third aspect of the invention, the present invention provides a kind of can strike to drop the expression of above-mentioned long-chain non-coding RNA
Purposes of the horizontal substance in the drug of preparation treatment tumour.
Preferably, the substance is using base sequence shown in SEQ ID NO:4 or SEQ ID NO:5 as the anti-of target sequence
Adopted lock nucleic acid.Preferably, the base sequence of the antisense lock nucleic acid is as shown in SEQ ID NO:2 or SEQ ID NO:3.
Preferably, the tumour is breast cancer or lung cancer.Wherein tumour can also be liver cancer, cancer of pancreas, prostate cancer, wing
Guang cancer, kidney etc..
As the fourth aspect of the invention, the present invention provides a kind of drug for treating tumour, contain in the drug
The substance for dropping the expression of above-mentioned long-chain non-coding RNA can be struck.
Preferably, antisense lock nucleic acid, the base sequence of the antisense lock nucleic acid such as SEQ ID NO:2 are contained in the drug
Or shown in SEQ ID NO:3.Wherein, in base sequence shown in SEQ ID NO:2 and SEQ ID NO:1 such as SEQ ID NO:
Base sequence shown in 4 (referring to the sequence in SEQ ID NO:1 with underscore) complementary pairing；Shown in SEQ ID NO:3
The base sequence as shown in SEQ ID NO:5 in base sequence and SEQ ID NO:1 in SEQ ID NO:1 (under having
The sequence of scribing line) complementary pairing.
Preferably, the tumour is breast cancer or lung cancer.Wherein tumour can also be liver cancer, cancer of pancreas, prostate cancer, wing
Guang cancer, kidney etc..
As the fifth aspect of the invention, the present invention provides above-mentioned long-chain non-coding RNAs tumor patient prognosis
The purposes of the marker of judgement, the tumour are breast cancer or lung cancer.
In conclusion the invention has the benefit that
For ST8SIA6-AS1 in the present invention as new tumor cells marker, height expression can indicate the bad of tumour
Prognosis；As cancer target, two antisense lock nucleic acids can specifically strike the expression of drop ST8SIA6-AS1, efficient, special
Strange land causes tumour cell that apoptosis occurs, and inhibits tumour growth.
Detailed description of the invention
Fig. 1 is long-chain non-coding RNA ST8SIA6-AS1 highly expressed result figure in breast cancer cell line；Wherein, left
Side MCF-10A, 76N, HMLE are normal or immortalize galactophore epithelial cell system, remaining right side is breast cancer cell line；
Fig. 2 is that in situ hybridization detects expression result chart of the ST8SIA6-AS1 by the cancer in normal breast epithelial；Wherein right side
Figure is the enlarged drawing of rectangle in left hand view；
Fig. 3 be in situ hybridization detect ST8SIA6-AS1 with the expression of results in the breast cancer tissue of the same case of Fig. 2
Figure；Wherein, dark colour is the colour developing of ST8SIA6-AS1 hybridization signal；Right part of flg is the enlarged drawing of rectangle in left hand view；
Fig. 4 is that long-chain non-coding RNA ST8SIA6-AS1 expresses the result figure increased in kinds of tumors；Wherein, it swells for every kind
The upper surface of tumor is related normal tissue, is below tumor tissues, and horizontal axis is the relative expression quantity of ST8SIA6-AS1；
Fig. 5 is long-chain non-coding RNA ST8SIA6-AS1 (probe: 242350_at) and tumor prognosis in breast cancer
Relation schematic diagram；
Fig. 6 is the pass of long-chain non-coding RNA ST8SIA6-AS1 (probe: 242350_at) and tumor prognosis in lung cancer
It is schematic diagram；
Fig. 7 is that antisense lock nucleic acid strikes drop effect picture to long-chain non-coding RNA ST8SIA6-AS1；
Fig. 8 is that flow cytometer detection strikes after drop long-chain non-coding RNA ST8SIA6-AS1 to the shadow of breast cancer MCF7 Apoptosis
Ring result figure；Wherein, upper quadrant is non-viable apoptotic cell on the right side of streaming figure, and lower quadrant is viable apoptotic cell；
Fig. 9 is that flow cytometer detection strikes influence to lung cancer A549 cell apoptosis after drop long-chain non-coding RNA ST8SIA6-AS1
Result figure；Wherein, upper quadrant is non-viable apoptotic cell on the right side of streaming figure, and lower quadrant is viable apoptotic cell；
Figure 10 shows the ratio of breast cancer MCF7 apoptotic cell in Fig. 8, mean ± SD；
Figure 11 shows the ratio of lung cancer A549 apoptotic cell in Fig. 9, mean ± SD；
Figure 12 is the life for inhibiting human breast cancer in nude mice MCF7 cell transplantation tumor after striking drop long-chain non-coding RNA ST8SIA6-AS1
Long result (gross tumor volume) figure；
Figure 13 is the life for inhibiting human breast cancer in nude mice MCF7 cell transplantation tumor after striking drop long-chain non-coding RNA ST8SIA6-AS1
Long result (tumor weight) figure；
Figure 14 is the growth for inhibiting lung cancer in nude mice A549 cell transplantation tumor after striking drop long-chain non-coding RNA ST8SIA6-AS1
As a result (gross tumor volume) figure；
Figure 15 is the growth for inhibiting lung cancer in nude mice A549 cell transplantation tumor after striking drop long-chain non-coding RNA ST8SIA6-AS1
As a result (tumor weight) figure.
Specific embodiment
It is intended to illustrate the present invention embodiment (rather than limiting the invention) referring now to following and describes the present invention.Unless
It specializes, experimental methods of molecular biology and immunodetection used in the present invention, substantially with reference to J.Sambrook
Et al., molecular cloning: laboratory manual, second edition, CSH Press, 1989 and F.M.Ausubel et al., essence
Partial numerator biological experiment guide, the 3rd edition, method described in John Wiley&Sons, Inc., 1995 carries out；Apoptosis detection
The condition that the use of kit is recommended according to goods producer.As known to those skilled in the art, embodiment describes by way of example
The present invention, and it is not intended to limit scope of the present invention.
The present invention is directed to find long-chain non-coding RNA ST8SIA6-AS1 as new tumor markers and therapy target,
It has specificity and high efficiency as the target spot in diagnosing tumor and/or treatment, will judge and treat for tumor prognosis to dislike
Property tumour provides strong means.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
The expression of embodiment 1qPCR detection long-chain non-coding RNA ST8SIA6-AS1
1.1 design of primers: finding the sequence of long-chain non-coding RNA ST8SIA6-AS1 from gene library NCBI, uses
Primer software Design primers are as follows:
Forward:
5’-ttcagtggcatggttcagtc-3’(SEQ ID NO:6)
Reverse:
5’-ctcagaagcgacagggtttc-3’(SEQ ID NO:7)
1.2qPCR detection:
As shown in Figure 1, long-chain non-coding RNA ST8SIA6-AS1 is expressed in breast cancer cell line and is obviously increased.
2 in situ hybridization of embodiment detects long-chain non-coding RNA ST8SIA6-AS1 in normal breast and breast cancer tissue
Expression
Detection method: the breast tissue of paraffin embedding prepares organization chip, carries out routine paraffin wax slice, de- through dimethylbenzene
Wax, graded ethanol aquation.0.05% trypsase room temperature 10min enzymolysis protein, 4% paraformaldehyde 20min are fixed, PBS
It rinses.Then it is sliced and is handled 2 hours in prehybridization solution, abandon prehybridization solution, with 52 DEG C of the probe overnight incubations of 20nM.Second day
It is washed 2 times with 2 × SSC, every to wash 3 times all over 5min, 2 × SSC containing 50% formamide, every all over 25min, then PBST is washed 5 times, every time
5min.It is closed 1 hour with 10% Normal Goat Serum room temperature, then biotin labeling anti digoxin antibody (1:1000) 4 is spent
Night.Third day PBST is washed 5 times, and SA-AP (1:500) buffer is added and is incubated at room temperature 30min, then is washed 3 times with PBST, then uses AP
Buffer is washed 3 times, every all over 5min.NBT/BCIP develops the color 2 hours, then is washed 3 times with PBST, and core is red fastly to redye 10min, and mounting is shown
Micro- microscopic observation result.
Testing result: as shown in Figures 2 and 3, coloration result is shown in breast cancer tissue microarray, long non-coding RNA
The expression of ST8SIA6-AS1 is apparently higher than corresponding normal tissue.
Embodiment 3
The case where inquiring the expression quantity of long non-coding RNA ST8SIA6-AS1 in various tumours on the website LncRNAtor
(data source is in TCGA).
Increase as Fig. 4 shows that long-chain non-coding RNA ST8SIA6-AS1 is expressed in kinds of tumors.
Embodiment 4
Long-chain non-coding RNA ST8SIA6-AS1 (probe: 242350_at) is inquired on the website KM-plotter in mammary gland
In cancer and lung cancer with the relationship of tumor prognosis.
If Figures 5 and 6 are shown, in breast cancer and lung cancer compared with long-chain non-coding RNA ST8SIA6-AS1 low expression group,
The prognosis of high expression group is obviously poor, and P value has statistical significance；Show that long-chain non-coding RNA ST8SIA6-AS1 can be used as cream
The Index for diagnosis marker of gland cancer and non-small cell lung cancer.
Embodiment 5
With antisense lock nucleic acid transfection tumor cell, qPCR detects antisense lock nucleic acid to long non-coding RNA after 24 hours
ST8SIA6-AS1's strikes drop effect.
If Fig. 7 is shown, two antisense lock nucleic acids (as shown in SEQ ID NO:2, SEQ ID NO:3) can be significantly reduced
The expression quantity of long-chain non-coding RNA ST8SIA6-AS1.
6 flow cytometer detection of embodiment strikes influence to apoptosis of tumor cells after drop long-chain non-coding RNA ST8SIA6-AS1
Detection method: using targeting long-chain non-coding RNA ST8SIA6-AS1 antisense lock nucleic acid (LNA-1:
GACATGACAATGGTA, LNA-2:TTTGGCCATGATGGAT) transiently transfect tumour cell MCF7 and A549 can dramatically increase
The apoptosis of tumour cell.Using LNA-1 and LNA-2 transfection tumor cell for 24 hours after, carry out AnnexinV/PI dyeing, be incubated on ice
30min is detected with flow cytometer, counts the apoptosis rate of the AnnexinV/PI positive.
Testing result: as shown in Fig. 8~11, human breast cancer cell line MCF7 and Lines are shown in
Drop long-chain non-coding RNA ST8SIA6-AS11 is struck in A549 causes tumour cell that apoptosis largely occurs；It is dropped by antisense lock nucleic acid
The expression quantity of low long-chain non-coding RNA ST8SIA6-AS1 can obviously increase the apoptosis about 25~40% of tumour cell.
Influence to growth of transplanted human after drop long-chain non-coding RNA ST8SIA6-AS1 is struck in 7 zoopery of embodiment detection
Detection method: it when evaluating influence of the long-chain non-coding RNA ST8SIA6-AS1 for tumour cell tumor growth, swells
Oncocyte is with 2 × 106It is subcutaneous that/0.1ml PBS is inoculated in every nude mice back part, and every group 9.The 7th day after tumor cell inoculation,
When accessible tumour, antisense lock nucleic acid is injected intraperitoneally with the dosage of 10mg/kg to tumor-bearing mice, and injection in every 3 days thereafter is primary,
Continuous treatment three weeks.
Testing result: it as shown in Figure 12~15, is moved after striking drop long-chain non-coding RNA ST8SIA6-AS1 in MCF7 and A549
It plants in tumor and significantly inhibits tumour growth；Statistical chart shows mean+SD (mean plus-minus standard deviation), shows to move A549, MCF7
The tumor bearing nude mice intraperitoneal injection for planting tumor can obviously inhibit swollen for the antisense lock nucleic acid of long-chain non-coding RNA ST8SIA6-AS1
The growth of tumor.
To sum up, we combine non-coding RNA database data, are tested and are found with analyses such as flow cytometer detection, zooperies, long
Chain non-coding RNA ST8SIA6-AS1 is expressed in kinds of tumors to be increased, and can be used as the independent prognosis mark of tumor development
Will object is reduced the expression quantity of long non-coding RNA ST8SIA6-AS1 by antisense lock nucleic acid, can obviously increase tumour cell
Apoptosis significantly inhibits tumour growth in MCF7 and A549 transplantable tumor.Therefore, long-chain non-coding RNA ST8SIA6-AS1 makees
For new tumor markers and therapy target, new method can be provided for the diagnosis or treatment of tumour.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Sun Yat-sen Memorial Hospital
<120>long-chain non-coding RNA ST8SIA6-AS1 and its application
<130> 2017
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<170> PatentIn version 3.5
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ttcagtggca tggttcagtc 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<400> 7
ctcagaagcg acagggtttc 20
Claims (10)
1. a kind of long-chain non-coding RNA that can be used as tumor markers and therapy target, which is characterized in that the long-chain non-coding
The base sequence of the corresponding cDNA of RNA is as shown in SEQ ID NO:1.
2. a kind of antisense lock nucleic acid, which is characterized in that the base sequence of the antisense lock nucleic acid such as SEQ ID NO:2 or SEQ ID
Shown in NO:3.
3. a kind of substance for dropping the expression of long-chain non-coding RNA as described in claim 1 that can strike is in preparation treatment tumour
Drug in purposes.
4. purposes according to claim 3, which is characterized in that the substance is with SEQ ID NO:4 or SEQ ID NO:5
Shown in base sequence be target sequence antisense lock nucleic acid.
5. purposes according to claim 4, which is characterized in that the base sequence of the antisense lock nucleic acid such as SEQ ID NO:
Shown in 2 or SEQ ID NO:3.
6. purposes according to claim 3, which is characterized in that the tumour is breast cancer or lung cancer.
7. a kind of drug for treating tumour, which is characterized in that drop long-chain as described in claim 1 can be struck by containing in the drug
The substance of the expression of non-coding RNA.
8. drug according to claim 7, which is characterized in that contain antisense lock nucleic acid, the antisense lock in the drug
The base sequence of nucleic acid is as shown in SEQ ID NO:2 or SEQ ID NO:3.
9. drug according to claim 7, which is characterized in that the tumour is breast cancer or lung cancer.
10. a kind of purposes of marker of long-chain non-coding RNA as described in claim 1 as tumor patient Index for diagnosis,
It is characterized in that, the tumour is breast cancer or lung cancer.
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| CN110123829A (en) * | 2019-05-26 | 2019-08-16 | 苏州大学 | Long-chain non-coding RNA is preparing the application in Tumor angiogenesis inhibitor |
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| CN113355411A (en) * | 2020-03-02 | 2021-09-07 | 中山大学孙逸仙纪念医院 | Tumor immunotyping method based on lncRNA marker |
| CN113549690A (en) * | 2020-12-02 | 2021-10-26 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as molecular marker of multiple myeloma cell |
| CN114934052A (en) * | 2022-06-14 | 2022-08-23 | 贵州医科大学附属医院 | Application of long-chain non-coding RNA AABR07017227 |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110123829A (en) * | 2019-05-26 | 2019-08-16 | 苏州大学 | Long-chain non-coding RNA is preparing the application in Tumor angiogenesis inhibitor |
| CN113355411A (en) * | 2020-03-02 | 2021-09-07 | 中山大学孙逸仙纪念医院 | Tumor immunotyping method based on lncRNA marker |
| CN113355411B (en) * | 2020-03-02 | 2022-05-10 | 中山大学孙逸仙纪念医院 | Tumor immunotyping method based on lncRNA marker |
| CN111793642A (en) * | 2020-07-15 | 2020-10-20 | 中山大学孙逸仙纪念医院 | Cloning vector for efficiently and stably overexpressing long-chain non-coding RNA and application thereof |
| CN111793642B (en) * | 2020-07-15 | 2022-08-05 | 中山大学孙逸仙纪念医院 | Cloning vector for efficiently and stably overexpressing long-chain non-coding RNA and application thereof |
| CN113549690A (en) * | 2020-12-02 | 2021-10-26 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as molecular marker of multiple myeloma cell |
| CN113549690B (en) * | 2020-12-02 | 2022-07-01 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as molecular marker of multiple myeloma cell |
| CN114934052A (en) * | 2022-06-14 | 2022-08-23 | 贵州医科大学附属医院 | Application of long-chain non-coding RNA AABR07017227 |
| CN114934052B (en) * | 2022-06-14 | 2023-05-02 | 贵州医科大学附属医院 | Application of long-chain non-coding RNA AABR07017227 |
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