CN109010831A - The application of LncRNA RET modulate tumor cellular radiosensitivity - Google Patents

Abstract

The invention discloses application of the LncRNA RET in modulate tumor cellular radiosensitivity.The present invention provides application of the RET relevant biological material in the product that preparation is used for modulate tumor cellular radiosensitivity；The RET relevant biological material is RET or can promote/inhibit the substance of RET expression.Growth of tumour cell under radiation pressure can be inhibited, increase radiosensitivity for tumor cell by lowering RET expression, and mechanism is that tumour cell Epithelial and stromal caused by promoting the radiation-induced apoptosis of tumour cell, the radiation-induced DNA damage reparation of reduction, reverse radiation converts.The radiosensitivity that RET passes through adjusting PTEN modulate tumor cell.Under radiation pressure, PTEN can reverse growth of tumour cell caused by RET silencing, apoptosis increase, DNA damage reparation and the generation of EMT, to adjust radiosensitivity for tumor cell caused by RET.

Description

The application of LncRNA RET modulate tumor cellular radiosensitivity

Technical field

The present invention relates to field of biotechnology, and in particular to LncRNA RET modulate tumor cellular radiosensitivity application.

Background technique

Full-length genome transcript profile is studies have shown that there are a large amount of non-coding RNA (ncRNA), non-coding RNA (non- Coding RNA, ncRNA) refer to the RNA molecule for not translating into protein in transcript profile, including microRNA (microRNA, MiRNA), endonuclear tiny RNA (small nuclear RNA, snRNA), kernel microRNA (small nucleolar RNAs, snoRNA), interference tiny RNA (small interfering RNA, siRNA), with the RNA of piwi protein-interacting (piwi-interactingRNA, piRNA) etc., long non-coding RNA (long non-codingRNA, LncRNA) etc..Nearest Research estimation is about 15000 in the quantity of the intracorporal lncRNA of people, and it was found that the expression pattern of most of lncRNA has Tissue specificity.According to the position of LncRNA and feature, 5 seed types can be classified as: just LncRNA (sense LncRNA), between antisense LncRNA (antisense LncRNA), introne LncRNA (intronic LncRNA), gene LncRNA (intergenic LncRNA), two-way LncRNA (bidirectional LncRNA).More and more research tables Bright, LncRNA plays a significant role in extensive bioprocess, including stress reaction, development, embryonic stem cell versatility, fixed Position, alternative splicing, chromatin remodeling and mRNA decay.In addition, lncRNA can influence many cell processes, as the cell cycle, Cell survival, cell migration and cell metabolism etc..

More and more evidences show that many lncRNA are similar with miRNA, and hair is expressed in the various types of cancers of the mankind Change is given birth to, and the change of lncRNA expression can be used as tumor suppressor gene or oncogene functions.Although most of The detailed mechanism of lncRNAs is still indefinite, but lncRNA has become the new participant of the cancer after miRNAs.

Nearest researches show that LncRNAs, as miRNAs, play important regulation person's in gene regulatory network Effect, and play a significant role in cancer progression.LncRNAs has become the participant of new tumour, and examines in clinic Potential effect is shown in disconnected and treatment.Although having studied the biological function for reporting some LncRNA, have Many effects and functional mechanism are still not clear.In future, the base motifs for further studying LncRNA and second level or three-level are also needed Structure fully states the various regulatory mechanisms of LncRNAs in terms of structure and function, and utilizes the means such as bioinformatic analysis New, effective method is found for predicting the target gene of LncRNAs.It is mentioned for the LncRNAs complicated gene regulatory network being related to New strategy is provided for new opinion, and finally for cancer diagnosis and treatment.

Summary of the invention

The object of the present invention is to provide LncRNA RET and its new applications of relevant biological material.LncRNA RET is invention People's early stage induces highly expressed new LncRNA, LOC90024 with the radiation that genetic chip screens, and is named as LncRNA RET(LncRNA radiation-elevated transcript)。

In a first aspect, claimed LncRNA RET relevant biological material answering in following (A1) or (A2) With:

(A1) preparation is used for the product of modulate tumor cellular radiosensitivity；

(A2) modulate tumor cellular radiosensitivity；

The LncRNA RET relevant biological material is the LncRNA RET, or can promote the LncRNA RET The substance of expression, or it is able to suppress the substance of the LncRNA RET expression.

Wherein, the modulate tumor cellular radiosensitivity is the radiation-sensitive by target protein PTEN modulate tumor cell Property.

Second aspect, the claimed substance for being able to suppress LncRNA RET expression following (a)-(e) at least A kind of application in:

(a) product for inhibiting the growth of tumour cell under radiation pressure is prepared, or inhibits tumour under radiation pressure thin The growth of born of the same parents；

In a specific embodiment of the invention, described radiate is specially⁶⁰The irradiation of Co gamma-rays, dosage 4Gy.

(b) product for increasing radiosensitivity for tumor cell is prepared, or increases radiosensitivity for tumor cell；

In a specific embodiment of the invention, described radiate is specially⁶⁰The irradiation of Co gamma-rays, dosage 2-8Gy.

(c) product for increasing radiation-induced apoptosis of tumor cells is prepared, or increases radiation-induced tumour cell Apoptosis；

In a specific embodiment of the invention, described radiate is specially⁶⁰The irradiation of Co gamma-rays, dosage 4Gy.

(d) preparation is for inhibiting radiation that the product of DNA of tumor cell injury repair, or inhibition radiation is caused to cause tumour cell DNA damage reparation；

In a specific embodiment of the invention, described radiate is specially⁶⁰The irradiation of Co gamma-rays, dosage 4Gy.

In a specific embodiment of the invention, the inhibition radiation causes DNA of tumor cell injury repair to be embodied as Promote the expression of γ-H2AX in tumour cell under radiation condition.

(e) product or reverse radiation of the preparation for the generation of the tumour cell Epithelial and stromal conversion of reverse radiation induction The generation of the tumour cell Epithelial and stromal conversion of induction.

In a specific embodiment of the invention, described radiate is specially⁶⁰The irradiation of Co gamma-rays, dosage 6Gy.

Wherein, the substance for being able to suppress LncRNA RET expression can be able to suppress what LncRNA RET was expressed to be any Substance is such as able to suppress siRNA, shRNA or miRNA or the LncRNA RET expression inhibiting agent of LncRNA RET expression Deng.

In a specific embodiment of the invention, it is described be able to suppress LncRNA RET expression substance be siRNA or shRNA；The sequence of the siRNA is as shown in SEQ ID No.2；The sequence of the shRNA is to replace the T in SEQ ID No.3 It is changed to resulting sequence after U.

Further, the product can be drug or health care product etc..

The third aspect, claimed LncRNA RET or the substance that LncRNA RET can be promoted to express are such as Under (a ')-(e ') it is at least one in application:

(a ') prepares the product for promoting the growth of tumour cell under radiation pressure, or promotes tumour under radiation pressure thin The growth of born of the same parents；

(b ') prepares the product for mitigating radiosensitivity for tumor cell, or mitigates radiosensitivity for tumor cell；

(c ') prepares the product for reducing radiation-induced apoptosis of tumor cells, or reduces radiation-induced tumour cell Apoptosis；

(d ') preparation is for promoting radiation that the product of DNA of tumor cell injury repair, or promotion radiation is caused to cause tumour cell DNA damage reparation；

The product of generation of (the e ') preparation for promoting radiation-induced tumour cell Epithelial and stromal to convert, or promote radiation The generation of the tumour cell Epithelial and stromal conversion of induction.

Wherein, the substance that LncRNA RET can be promoted to express can be following any: can be transcribed into described The DNA of LncRNA RET, expression cassette, recombinant vector or recombinant cell containing the DNA.

Further, the product for promoting tumour cell under radiation pressure can increase for growth ability under radiation pressure Strong tumor models, or it is used to prepare the substance for the tumor models that growth ability enhances under radiation pressure.Institute The tumor models that the product for mitigating radiosensitivity for tumor cell can weaken for radiosensitivity are stated, or are used for Prepare the substance for the tumor models that radiosensitivity weakens.The production for being used to reduce radiation-induced apoptosis of tumor cells The tumor models that product can weaken for radiation-induced lower apoptosis capacity, or be used to prepare radiation-induced lower apoptosis capacity and subtract The substance of weak tumor models.The product for promoting radiation to cause DNA of tumor cell injury repair can lure for radiation Under the conditions of leading DNA damage repair ability enhance tumor models, or be used to prepare it is radiation-induced under the conditions of DNA damage The substance of the tumor models of repair ability enhancing.It is described to be used to that radiation-induced tumour cell Epithelial and stromal to be promoted to convert The product of generation can be the tumor models of the radiation-induced lower ability enhancing that Epithelial and stromal conversion occurs, or for making The substance of the tumor models of the standby radiation-induced lower ability enhancing that Epithelial and stromal conversion occurs.

Fourth aspect, the claimed substance for being able to suppress pten protein expression have in following function in preparation Application at least one product:

(A) growth of tumour cell is suppressed under radiation pressure caused by reverse is expressed due to inhibition LncRNA RET Situation；

(B) radiosensitivity for tumor cell caused by due to inhibiting the phenomenon that LncRNA RET expression is reversed to enhance；

(C) the radiation-induced increased feelings of resulted tumour Apoptosis caused by due to inhibiting LncRNA RET expression are reversed Condition；

(D) radiation-induced resulted tumour cell epithelia mesenchymal transformation caused by due to inhibiting LncRNA RET expression is reversed The case where being suppressed.

Wherein, the amino acid sequence of the pten protein is as shown in SEQ ID No.4.It is described to be able to suppress pten protein table The substance reached is siRNA shown in SEQ ID No.5.

In above-mentioned first to fourth aspect, the LncRNA RET is concretely following any:

(a1) RNA shown in SEQ ID No.1；

(a2) by nucleotide sequence shown in SEQ ID No.1 by one or several nucleotide residues substitution and/or Deletion and/or addition and RNA with the same function；

(a3) there is 99% or more, 95% or more, 90% or more, 85% with nucleotide sequence defined by (a1) or (a2) Above or 80% or more homology and RNA with the same function.

In above-mentioned first to fourth aspect, the tumour cell can be cervical cancer cell or lung adenocarcinoma cell etc..

In a specific embodiment of the invention, the tumour cell is specially cervical cancer cell HeLa or lung adenocarcinoma cell A549。

It is demonstrated experimentally that growth of tumour cell under radiation pressure can be inhibited, increase tumour cell by lowering RET (RET) expression Radiosensitivity, mechanism be promote the radiation-induced apoptosis of tumour cell, reduce radiation-induced DNA damage reparation, The conversion of tumour cell Epithelial and stromal caused by reverse radiation.The radiosensitivity that RET passes through adjusting PTEN modulate tumor cell.Spoke Under injection pressure, PTEN can reverse growth of tumour cell caused by RET silencing, apoptosis increase, DNA DSB reparation and EMT Occur, to adjust radiosensitivity for tumor cell caused by RET.

Detailed description of the invention

Fig. 1 is the influence that LncRNA RET is expressed in ionising radiation.A is the expression of HeLa cell LncRNA RET；B For the expression of A549 cell LncRNA RET.

Fig. 2 is the variation of LncRNA RET after irradiation.A is the variation of HeLa cell LncRNA RET；B is A549 cell The variation of LncRNA RET.

Fig. 3 is that RT-qPCR detects LncRNA RET knockout effect (P < 0.01 * *).A is that HeLa cell LncRNA RET strikes Except effect；B is that A549 cell LncRNA RET knocks out effect.

Fig. 4 is influence of the LncRNA RET knockout to growth of tumour cell under radiation pressure.A is that HeLa cell grows feelings Condition；B is A549 cell growth status.

Fig. 5 is influence of the LncRNA RET knockout to radiosensitivity for tumor cell.A is to knock out LncRNA RET to HeLa The influence of cellular radiosensitivity；B is the influence for knocking out LncRNA RET to A549 cellular radiosensitivity.

Fig. 6 is that formula cell art detects apoptosis and apoptosis rate statistical result.A is Apoptosis by Flow Cytometry；B is Apoptosis rate statistical chart.

Fig. 7 is the effect of slow virus infected cell.A is luciferase expression situation；B is to knock out compliance test result.

Fig. 8 is the variation of the content of γ-H2AX.A and C is respectively the variation of γ-H2AX in HeLa and A549 cell；B and D The quantization that γ-H2AX changes respectively in HeLa and A549 cell.

Fig. 9 is the variation of EMT under different condition.A is Western Blot testing result；B is Immunofluorescence test result.

Figure 10 is the variation of PTEN after irradiation.A is the variation of HeLa cell PTEN；B is the variation of A549 cell PTEN.

Figure 11 is the influence for knocking out LncRNA RET to PTEN.A is that mRNA level in-site detects LncRNA RET to the shadow of PTEN It rings；B is that protein level detects influence of the LncRNA RET to PTEN.

Figure 12 is the knockout effect of PTEN in tri- groups of cells of siNC, siRET and siRET+siPTEN.A is HeLa cell PTEN content detection；B is A549 cell PTEN content detection.

Figure 13 is the influence for knocking out PTEN to growing under radiation pressure caused by LncRNA RET silencing.A is HeLa cell Growth curve；B is A549 cell growth curve.

Figure 14 is the influence for knocking out PTEN to radiosensitivity caused by LncRNA RET silencing.A is HeLa radiotherapy Sensibility；B is A549 cellular radiosensitivity.

Figure 15 is the influence for knocking out PTEN to Apoptosis radiation-induced caused by LncRNA RET silencing.A is HeLa Apoptosis situation；B is A549 Apoptosis situation.

Figure 16 is the influence for knocking out PTEN to radiation-induced EMT caused by LncRNA RET silencing.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The Effect study of embodiment 1, LncRNA RET in radiosensitivity for tumor cell

LncRNA RET is that screened with genetic chip early stage one of inventor is radiation-induced highly expressed new It is named as LncRNA RET (LncRNA radiation-elevated transcript) by LncRNA.LncRNA RET The full length sequence of (http://www.ncbi.nlm.nih.gov/nuccore/NR_033874.1) is as shown in SEQ ID No.1.

One, the influence that LncRNA RET is expressed in ionising radiation

1, the dose-effect relationship expressed after LncRNA RET raying in tumour cell

The present invention has detected the dose-effect relationship expressed after LncRNA RET raying first, and HeLa cell and A549 are thin Born of the same parents use⁶⁰The irradiation of Co gamma-rays, the dosage of setting are 0Gy, 2Gy, 4Gy, 6Gy, 8Gy.48h after irradiation extracts RNA, uses RT-qPCR Detect the expression of LncRNA RET.It is specific as follows:

(1) it is ready to test required cell, is inoculated with, is inoculated in the culture dish of 6cm after cell count, every ware 8 ×10⁵A cell.

(2) preferable to second day cell state, carry out Co⁶⁰Gamma-rays treatment with irradiation, divide 0Gy (not irradiating), 2Gy, 4Gy, 6Gy, 8Gy are irradiated, and continue to cultivate 48h after irradiation.

(3) after 48h, cell is collected, extracts total serum IgE, carries out RT-qPCR.

Primer sequence for detecting β-actin (internal reference) is as follows:

F:5 '-GAATCAATGCAAGTTCGGTTCC-3 '；

R:5 '-TCATCTCCGCTATTAGCTCCG-3 '.

Primer sequence for detecting the coding DNA of LncRNA RET is as follows:

F:5 '-GGGACAGTTGACACCCATCT-3 '；

R:5 '-AACATTTGGGCCACTATTGC-3 '.

Experimental result as shown in Figure 1, in two kinds of cells gradually increasing with dosage, the content of LncRNA RET also by Edge up height, it was demonstrated that LncRNA RET to irradiation have dose dependent, difference have statistical significance (* P < 0.05, * * P < 0.01)。

2, the Time-effect relationship expressed after LncRNA RET raying in tumour cell

Then the Time-effect relationship expressed after LncRNA RET irradiation after irradiating, HeLa and A549 cell are had detected⁶⁰Co Gamma-rays 6Gy irradiation, LncRNA RET changes of contents when RT-qPCR detects 0,0.5,1,3,6,12,24,48h.

Primer sequence for detecting β-actin (internal reference) is as follows:

F:5 '-GAATCAATGCAAGTTCGGTTCC-3 '；

R:5 '-TCATCTCCGCTATTAGCTCCG-3 '.

Primer sequence for detecting the coding DNA of LncRNA RET is as follows:

F:5 '-GGGACAGTTGACACCCATCT-3 '；

R:5 '-AACATTTGGGCCACTATTGC-3 '.

As shown in Fig. 2, ascendant trend, after irradiation 6h is totally presented in the content of the irradiation intracellular LncRNA RET of latter two When reach highest, difference has statistical significance (P < 0.001 * P < 0.05, * *).

Two, it establishes LncRNA RET and strikes low cell line

SiRET- and siNC is transferred to HeLa and A549 cell with lip2000, after culture 48 hours, to LncRNARET's Effect is knocked out to be detected.

SiRET-1:5 '-GAGGAGACGGGAGAAACAUTT-3 ' (SEQ ID No.2)；

SiNC:5 '-UUCUCCGAACGUGUCACGUTT-3 '.

The expression of LncRNA RET in two kinds of cells is detected with the method for RT-qPCR, concrete operations are as follows: to cell It is cleaned cell 3 times behind confluent cultures ware bottom with PBS, adds 200 μ l chloroforms after adding the abundant lytic cell of 1ml Trizol, acutely shake After swinging, 4 DEG C of 12000rpm are centrifuged 15min, suct a layer transparent aqueous phase, add 500 μ l of isopropanol, be mixed by inversion, be stored at room temperature 10min, 4 DEG C of 12000rpm are centrifuged 10min, it is seen that and tube bottom white RNA precipitate abandons supernatant, adds 75% ethyl alcohol 1ml, and 4 DEG C 8000rpm is centrifuged 5min, blots net ethyl alcohol, obtains the total serum IgE of each cell, utilize PrimeScript^TMRT Master Mix examination Agent box reverses RNA for cDNA, using cDNA as template SYBR Premix Ex Taq^TMII kit carries out RT-PCR reaction.

Primer sequence for detecting β-actin (internal reference) is as follows:

F:5 '-GAATCAATGCAAGTTCGGTTCC-3 '；

R:5 '-TCATCTCCGCTATTAGCTCCG-3 '.

Primer sequence for detecting the coding DNA of LncRNA RET is as follows:

F:5 '-GGGACAGTTGACACCCATCT-3 '；

R:5 '-AACATTTGGGCCACTATTGC-3 '.

As shown in figure 3, in HeLa and A549 cell, the expression of siRET-1 and siRET-2 group LncRNA RET with SiNC is compared and is remarkably decreased (P < 0.01).It should be the result shows that LncRNA RET knockout effect be obvious.

Three, LncRNA RET knocks out the growth for inhibiting tumour cell under radiation pressure

SiRET-1 and siNC (after rapid two) of sequence synchronization transfect HeLa and A549 cell respectively, are carried out⁶⁰Co gamma-rays shines It penetrates (dosage is set as 4Gy), 1,2,3,4 day detection light absorption value after irradiation is distinguished, during which with mtt assay respectively to HeLa and A549 The growing state of the shNC and shRET group of cell is detected.

As a result as shown in figure 4, after recombinant slow virus infection cell, cell is carried out⁶⁰The irradiation of Co gamma-rays, respectively after irradiation 1,2,3,4 days detection light absorption values, compared with the control, the 2nd, 3,4 day light absorption value substantially reduces shRET group after irradiation, and with The increase of number of days, gap be more and more obvious, difference have statistical significance (P < 0.05 *).As a result prompt: LncRNA RET strikes Except the growth for inhibiting tumour cell under radiation pressure.

Four, LncRNA RET knocks out the radiosensitivity for increasing tumour cell

SiRET-1 and siNC (after rapid two) of sequence synchronization transfect HeLa and A549 cell respectively, are carried out⁶⁰Co gamma-rays shines It penetrates, the influence for knocking out LncRNA RET to radiosensitivity for tumor cell is detected with colony formation.Concrete operations are as follows:

With the radiosensitivity of colony formation detection cell by predetermined quantity cell inoculation in 60mm Tissue Culture Dish In, every group is all provided with 0,2,4,6 and 8Gy, 5 dosage groups, 3 repetitions of each dosage group.

(1) cell after irradiating is placed in culture in cell incubator.

(2) culture is terminated after cell culture 2 weeks, is removed culture solution, is cleaned twice with PBS, then fixed with 75% ethyl alcohol 30~60min.

(3) Giemsa dye liquor dyes 1h or so after natural drying, and clear water is cleaned, and counts containing clones more than 50 cells Number counts cloning efficiency (planting efficiency, PE) and survival rate (survival ratio):

Cloning efficiency=number of cell clones/inoculating cell number × 100%

Cell survival rate=exposure cell cloning efficiency/control cell cloning efficiency × 100%

Colony formation the results are shown in Table 1 and table 2, draws curve, sees Fig. 5.ShNC and shRET group cell is carried out⁶⁰Co Cell is inoculated with by gamma-rays irradiation, exposure dose 0Gy, 2Gy, 4Gy, 6Gy, 8Gy after irradiation according to certain cell number.14 After it, number of cell clones is counted, cell survival rate is calculated according to number of cell clones.Learnt through statistical analysis: in HeLa and In A549 cell, shRET group compared with shNC group 4,6, the cell survival rate of 8Gy be decreased obviously, it is poor and with the increase of metering Different increasing, difference has statistical significance (P < 0.05).As a result it prompts: after knocking out LncRNA RET, cell survival rate drop Low, cellular radiosensitivity increases.

1 HeLa cell various dose of table irradiation after survival rate (%,)

cell	0Gy	2Gy	4Gy	6Gy	8Gy
						shNC	100	78.96±6.25	57.67±0.10	26.99±1.19	14.79±0.44
shRET	100	68.03±5.57	28.56±2.40	11.63±1.33	5.83±0.07

2 A549 cell various dose of table irradiation after survival rate (%,)

cell	0Gy	2Gy	4Gy	6Gy	8Gy
						shNC	100	76.75±10.17	60.22±2.56	30.63±4.28	16.16±0.97
shRET	100	59.37±7.64	36.50±4.65	13.98±1.47	6.94±0.07

Five, LncRNA RET, which is knocked out, increases radiation-induced apoptosis of tumor cells

SiRET-1 and siNC (after rapid two) of sequence synchronization transfect HeLa and A549 cell respectively, are subjected to Co after 48h⁶⁰γ Radiation exposure processing, exposure dose 4Gy.Continue to cultivate after irradiation, receive cell afterwards for 24 hours, with Annexin V, FITC kit After handling cell, the Apoptosis situation of the shNC group of two kinds of cells and shRET group is analyzed respectively with flow cytometry. It is specific as follows:

1. Apoptosis by Flow Cytometry

(1) cell culture fluid is collected in the Ep pipe of 10mL, is washed cell 2 times with PBS, is collected cleaning solution.

(2) it with the trypsin digestion and cell of no EDTA, and is transferred in the 10mL Ep pipe of previous step.1000rpm centrifugation 3min abandons supernatant.

(3) 1mL PBS is added, 1000rpm is centrifuged 3min, abandons supernatant.It repeats this operation one time.

(4) 500 1 × Annexin of μ l, V Binding Solution is added, is made final concentration of 1 × 10⁶cells/mL Cell suspension.

(5) cell suspension in 100 μ l steps 5 is taken, is added in new streaming pipe.

(6) by specification is successively added 5 μ l Annexin V in cell suspension, FITC conjugate, 5 μ l PISolution。

(7) it is protected from light at room temperature and is incubated for 15min.

(8) 400 1 × Annexin of μ l, V Binding Solution is added in last every pipe, the upper machine testing in 1h.

Flow cytometry apoptosis result is as shown in Figure 6.It is learnt through statistical analysis: in HeLa and A549 cell, Compared with shNC group, shRET group apoptosis rate is significantly raised, and difference has statistical significance (P < 0.05 *).Prompt: it knocks out LncRNA RET increases radiation-induced apoptosis of tumor cells.

Six, LncRNA RET stablizes the building of interference cell line

ShRET:5 '-GAGGAGACGGGAGAAACATTTCAAGAGAATGTTTCTCCCGTCTCCTCTTTTTTG-3 ' (SEQ ID No.3)；

ShNC:5 '-TTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACTTCGGAGAA TTTTTTG-3 '.

LncRNA RET knocks out target sequence (shRET) and control sequence (shNC) by the limited public affairs of Suzhou Ji Ma gene share Take charge of be responsible for design, and by the said firm be packaged into recombinant slow virus shNC and shRNA (specific packing method step be this field Routine operation), titre is 1 × 10⁹TU/mL (band GFP label).Virus liquid is dispensed with the amount of every 20 μ l of pipe, is then frozen It is stored in -80 DEG C of refrigerators (to avoid multigelation).Use slow virus liquid infected cell, the specific steps are as follows:

1, target cell infection preliminary experiment

(1) experiment the previous day is inoculated with every hole 1 × 10⁴For a target cell in 24 well culture plates, added culture medium is 500 μ l. When carrying out virus infection, the density of target cell is 40-60% or so, and cell state is preferable, and inoculation is unsuitable overstocked, can not agglomerate.

(2) virus liquid and Polybrene are taken out, is melted on ice.

(3) prepare virus infection liquid: 1) preparing 4 EP pipe, be separately added into 500 μ l complete culture solutions (according to cell state, It can be not added dual anti-)；Wherein one is not added virus liquid, as infected group, for compareing.2) in other three according to 1:1000's Volume ratio is separately added into 0.5 μ l of Polybrene；3) 1:10, the volume ratio of 1:100,1:500, virus liquid needed for calculating are distinguished again (50 μ l, 5 μ l, 1 μ l), is added in above-mentioned EP pipe；4) it mixes gently, avoids bubble.

(4) cell is taken out, old culture solution is removed, PBS is cleaned 3 times, exhausts supernatant.The virus liquid point that previous step is prepared It is not added drop-wise in cell gently, marks (time, target cell type, viral liquid proportional).Cell is put back in incubator.

(5) observe cell state after 12h, if cell state be infected group no significant difference, show slow virus to thin Born of the same parents do not have overt toxicity effect.Continue to cultivate, is changed to new complete medium afterwards for 24 hours.

(6) after cultivating 72 hours, luciferase expression situation is observed.For the cell of slow growth, observing time can be delayed, in Interchangeable liquid is on the way to guarantee cell state.If cell is overstocked, can be passed on.

(7) concentration needed for determining the virus infection liquid of target cell and infection number of days.

2, formal test

Concentration needed for target cell infection virus liquid has been determined by preliminary experiment and the number of target cell and infection day Number, final to determine, target cell number is 2 × 10⁵A, when MOI value=100, virus liquid is best to the infectious effect of target cell.

(1) day before transfection is by HeLa and A549 cell inoculation in the culture dish of 60mm.Every ware inoculation 2 × 10⁵It is a thin 5mL culture medium is added in born of the same parents, until second day about grows to 60-70%, and keeps cell growth state good.

(2) it takes out virus liquid from -80 DEG C of refrigerators and helps and turn agent Polybrene, melt on ice.

(3) according to the MOI (MOI=100) determined in preliminary experiment, virus liquid volume is calculated according to formula.Then it carries out Virus infection, according to Polybrene when infecting: the condition addition of nutrient solution volume ratio 1:1000, which helps, turns agent Polybrene, to mention High infect efficiency.

(4) cell state is observed after 12h, observation cell state and untransfected slow virus group have no significant difference, if without bright Significant difference is different, then continues to cultivate, and is changed to fresh complete culture solution afterwards for 24 hours.

(5) after cultivating 72h, under fluorescence microscope, GFP green fluorescence expression is observed.And it is struck with RT-qPCR The verifying of inefficient fruit.

Primer sequence for detecting β-actin (internal reference) is as follows:

F:5 '-GAATCAATGCAAGTTCGGTTCC-3 '；

R:5 '-TCATCTCCGCTATTAGCTCCG-3 '.

Primer sequence for detecting the coding DNA of LncRNA RET is as follows:

F:5 '-GGGACAGTTGACACCCATCT-3 '；

R:5 '-AACATTTGGGCCACTATTGC-3 '.

(6) points for attention: during operation slow virus, ultra-clean Fans is closed, gloves and mask are worn, properly protects and arranges It applies.It carries out disinfection after the completion of operation to super-clean bench, in bromogeramine or other disinfectants and waste liquid.Experimental waste liquid is by laboratory Centralized processing.

As shown in A in Fig. 7, shNC and shRET transfection HeLa and A549 cell after, the expression of fluorescence.Luciferase expression Account for about 60%-80%, indicates that transfection is preferable.Using LncRNA in the method detection HeLa and A549 cell of RT-qPCR The expression of RET, as shown in B in Fig. 7.Under shRET group LncRNA RET expression is significant in HeLa and A549 cell It adjusts (P < 0.05).Should be the result shows that LncRNA RET expression be significantly lowered after cell infection LncRNA RET shRET, card It is preferable that bright slow virus knocks out effect.

Seven, LncRNA RET, which is knocked out, inhibits radiation to cause DNA of tumor cell injury repair

It is above-mentioned experiments have shown that knocking out LncRNA RET after, the radiosensitivity of tumour cell increases, and DNA damage reparation lacks Mistake is a key factor of cellular radiosensitivity, so the present invention, which has detected, knocks out LncRNA RET to radiation injury mark The expression of γ-H2AX.

As shown in figure 8,⁶⁰After Co gamma-rays irradiates (dosage 4Gy), in two kinds of cells (recombinant lentiviral disease of step 1 preparation Malicious shNC and shRNA infects HeLa and A549 cell respectively) in, the content of control group shNC γ-H2AX is with the time after irradiation Increase first increase and reduce afterwards, content highest when 4h after irradiation, the content of shRET group γ-H2AX is also with the time after irradiation Increase the trend for showing reduction after first increasing, 4h reaches highest after irradiation, then also gradually decreases.By Western Blot As a result (A and C in Fig. 8) is as can be seen that the content of the γ-H2AX of shRET group is obviously higher than shNC group.Statistical result such as Fig. 8 Shown in middle B and D, difference has statistical significance (P < 0.01 * P < 0.05, * *).As a result prompt: LncRNA RET, which is knocked out, inhibits spoke It penetrates and causes DNA of tumor cell injury repair.

Eight, LncRNA RET knocks out the generation for the tumour cell EMT for inhibiting radiation-induced

In recent years evidence show tumour cell Epithelial and stromal conversion (EMT) it is related with tumour cell radioresistance increase, The experimental data display of front knocks out the radiosensitivity that LncRNA RET increases tumour cell, and existing document report spoke Penetrate the generation of induction EMT.After recombinant slow virus shNC and shRNA (step 1 preparation) infect HeLa and A549 cell respectively, into Row⁶⁰Co gamma-rays irradiates (dosage is set as 6Gy), and 48h is with Western Blot and immunofluorescence experiment to different items after irradiation The expression of EMT GAP-associated protein GAP is detected under part.It tests while undosed control group is set.

As shown in figure 9, control group is compared with irradiation group, epithelium marker E-cadherin is reduced, interstitial marker N- Cadherin and Vimentin is reduced, it was demonstrated that the generation of radiation-induced EMT.And under irradiation condition, lower LncRNA RET's Compared with shNC group, E-cadherin expression increases for expression, and N-cadherin and vimentin expression are reduced.As a result it prompts: spoke The generation of induction EMT is penetrated, and knocks out the generation that LncRNA RET inhibits radiation-induced tumour cell EMT.

Nine, LncRNA RET passes through the radiosensitivity of target protein PTEN modulate tumor cell

For previous experiments to LncRNA RET under radiation pressure, the detection of radiosensitivity and its influence factor finds spoke LncRNA RET is knocked out under injection pressure can increase the radiosensitivity of cell.But the target gene that LncRNA RET is specifically acted on And its molecular mechanism is still not clear, and in order to more accurately illustrate LncRNA RET under radiation pressure to the influence of cell, seeks The target protein and molecular mechanism of LncRNA RET are looked for, it is most important.The present invention passes through bioinformatic analysis and literature survey, The target protein PTEN (amino acid sequence is as shown in SEQ ID No.4) for having interaction with LncRNA RET is searched out, and preliminary Inquire into the mechanism of action of LncRNA RET.

1, radiation-induced LncRNA RET expression and PTEN are negatively correlated

The present invention has detected after irradiation that the content of PTEN changes with time in HeLa and A549 cell, RT-qPCR detection ?⁶⁰Co gamma-rays irradiation after 0,0.5,1,2,4,8, for 24 hours when PTEN variation.

Primer sequence for detecting β-actin (internal reference) is as follows:

F:5 '-GAATCAATGCAAGTTCGGTTCC-3 '；

R:5 '-TCATCTCCGCTATTAGCTCCG-3 '.

Primer sequence for detecting PTEN is as follows:

PTEN_F:5 '-AGAACTTATCAAACCCTT-3 '；

PTEN_R:5 '-GTCCTTACTTCCCCAT-3 '.

As shown in Figure 10, downward trend is totally presented in the content for irradiating the intracellular PTEN of latter two, after irradiation when 4h most Low, difference has statistical significance (P < 0001 * P < 005, * *).

(3) expression of LncRNA RET and PTEN are negatively correlated

According to the experiment of front, present invention discover that the expression of radiation-induced LncRNA RET, and radiating leads to PTEN content It reduces, there are certain interactions by guess LncRNA RET and PTEN, so the present invention, which has detected, strikes low LncRNA RET (ginseng See step 6) influence to PTEN, influence of the LncRNA RET to PTEN is knocked out in mRNA level in-site and protein level detection respectively.

As shown in A in fig. 11, RT-qPCR is the results show that the content for knocking out PTEN after LncRNA RET increases, B in Figure 11 Shown, after Western Blot result also shows knockout LncRNA RET, the content of PTEN is increased.As a result prompt, no matter MRNA level in-site is still in protein level, and after knocking out LncRNA RET, PTEN expression contents are increased, it was demonstrated that LncRNA RET is adjusted The variation of PTEN, and be negatively correlated.Difference has statistical significance (P < 0.05 *).

2, it knocks out PTEN and reverses influence of the LncRNA RET silencing to growth of tumour cell under radiation pressure

SiRET-1:5 '-GAGGAGACGGGAGAAACAUTT-3 ' (SEQ ID No.2)；

SiPTEN:5 '-GUAUGACAACAGCCUCAAGTT-3 ' (SEQ ID No.5).

According to transfection siNC, siRET or siRET+siPTEN into HeLa and A549 cell as follows:

1) by HeLa on the day before transfection siRNA sequence, for A549 cell inoculation in 60mm culture dish, every ware is inoculated with 6x105 A cell, until second day long to 60% or so.

2) 30min-1h discards old culture solution before transfecting, and is changed to culture solution (double no cultures without antibiotic and serum Liquid).

3) take the 1.5mL Ep pipe of 4 sterilizings (by taking transfection HeLa cell as an example).1. EP pipe 1 plus the bis- no culture solutions of 50 μ l, Add 15 μ l siNC, mixes well.2. EP pipe 2 plus the bis- no culture solutions of 100 μ l, add 10 μ l lipofectmine 2000, gently mix It is even.3. EP pipe 1 plus the bis- no culture solutions of 50 μ l, add 15 μ l siRET, mix well.4. EP pipe 2 plus the bis- no culture solutions of 100 μ l, add 10 μ l lipofectmine 2000, mix gently.5. EP pipe 3 plus the bis- no culture solutions of 100 μ l, add 15 μ l siRET+15 μ l SiPTEN is mixed well.6. EP pipe 4 plus the bis- no culture solutions of 200 μ l, add 20 μ l lipofectmine 2000, mix gently.It will It is above-mentioned 1. and 2., 3. and 4., 5. and 6. mix, be stored at room temperature 5min, no more than 20min.

4) mixed liquor for having added 2000 solution of lipofectmine is added in the mixed liquor that siRNA is added, is gently mixed It is even, make sure to keep in mind to blow and beat.Room temperature condition stands 20min.

5) suspension is added drop-wise in cell culture fluid, front and back gentle agitation, is uniformly mixed, is put into incubator and continues to train It supports.

6) complete culture solution is changed to after cultivating 6h.

7) after transfecting 48h, the content of RT-qPCR detection PTEN is carried out, strikes inefficient fruit (Figure 12) to determine three groups.

With the mtt assay life to siNC, siRET and siRET+siPTEN group of HeLa and A549 cell under radiation pressure respectively Long situation is detected.As a result as shown in figure 13, under 4Gy irradiation pressure (⁶⁰Co gamma-rays irradiation), siRET+siPTEN group and SiRET group is compared, while knocking out LncRNA RET and PTEN, and cell growth curve is delayed compared with knocking out LncRNA RET Solution, and with the increase of number of days, gap is more and more obvious.Difference has statistical significance (P < 0.05 *).As a result it prompts: knocking out PTEN can reverse influence of the LncRNA RET silencing to the growth of tumour cell under radiation pressure.

3, it knocks out PTEN and reverses influence of the LncRNA RET silencing to radiosensitivity for tumor cell

SiRNA infects HeLa and A549 cell and (referring to after step 2), knocks out PTEN pairs with colony formation detection Influence of the LncRNA RET silencing to radiosensitivity for tumor cell.By siNC, siRET and siRET+siPTEN group cell into Row⁶⁰Cell is inoculated with by the irradiation of Co gamma-rays, exposure dose 0,2,4,6,8Gy after irradiation according to designed cell number.2 weeks Afterwards, number of cell clones is counted, cell survival rate is calculated.

As shown in figure 14, LncRNA RET is knocked out as the result is shown, cell survival rate reduces, and cellular radiosensitivity increases, And in the case where LncRNA RET silencing, PTEN is knocked out, the radiosensitivity of cell obviously weakens, and difference is anticipated with statistics Adopted (P < 0.05 *).Prompt: the radiosensitivity as caused by LncRNA RET silencing can be reversed by knocking out PTEN.

4, it knocks out PTEN and reverses influence of the LncRNA RET silencing to radiation-induced apoptosis of tumor cells

SiRNA, which infects HeLa and A549 cell, (referring to after step 2), to carry out⁶⁰Co gamma-rays irradiates (dosage 4Gy), so The Apoptosis situation of siNC, siRET and siRET+siPTEN group of two kinds of cells is divided respectively with flow cytometry afterwards Analysis.

Flow cytometry apoptosis result is as shown in figure 15.It is learnt through statistical analysis: thin in HeLa cell and A549 In born of the same parents, compared with siNC group, siRET group percentage of cerebral apoptosis is significantly raised, and siRET+siPTEN group is compared with siRET, Apoptosis rate reduces, and difference has statistical significance (P < 0.05 *).Prompt: knocking out PTEN can alleviate by LncRNA RET Radiation-induced Apoptosis caused by silencing.

5, it knocks out PTEN and reverses influence of the LncRNA RET silencing to radiation-induced tumour cell EMT

SiRNA, which infects HeLa and A549 cell, (referring to after step 2), to carry out⁶⁰Co gamma-rays irradiates (dosage 4Gy), so Western blot experimental verification knocks out influence of the PTEN to radiation-induced EMT caused by LncRNA RET silencing afterwards.

As shown in figure 16, in A549 cell under the conditions of different disposal knock out LncRNA RET after EMT marker protein variation, From result it can be seen that knocking out LncRNA RET and PTEN under radiation parameter simultaneously compared with only knocking out LncRNA RET, upper leather mark Will object E-cadherin expression is reduced, and interstitial marker N-cadherin and Vimentin expression increase, transcription factor twist Content also increase.It proves that knocking out LncRNA RET under radiation pressure inhibits the generation of EMT, and is knocked out when LncRNA RET silencing PTEN but promotes the generation of EMT.Prompt: under radiation pressure, the EMT inhibited by LncRNA RET silencing can be reversed by knocking out PTEN Effect.

<110>PLA Academy of Military Sciences's military medical research institute

<120>application of LncRNA RET modulate tumor cellular radiosensitivity

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Claims (10)

1. Application of the 1.LncRNA RET relevant biological material in following (A1) or (A2):

   (A1) preparation is used for the product of modulate tumor cellular radiosensitivity；

   (A2) modulate tumor cellular radiosensitivity；

   The LncRNA RET relevant biological material is the LncRNA RET, or the LncRNA RET can be promoted to express Substance, or be able to suppress the substance of LncRNA RET expression.
2. 2. application according to claim 1, it is characterised in that: the modulate tumor cellular radiosensitivity is by target egg The radiosensitivity of white PTEN modulate tumor cell.
3. 3. being able to suppress application of the substance of LncRNA RET expression in following (a)-(e) is at least one:

   (a) product for inhibiting the growth of tumour cell under radiation pressure is prepared, or inhibits tumour cell under radiation pressure Growth；

   (b) product for increasing radiosensitivity for tumor cell is prepared, or increases radiosensitivity for tumor cell；

   (c) product for increasing radiation-induced apoptosis of tumor cells is prepared, or increases radiation-induced apoptosis of tumor cells；

   (d) preparation is for inhibiting radiation to cause the product of tumor cell damage reparation, or inhibition radiation that DNA of tumor cell damage is caused to repair It is multiple；

   (e) product or reverse radiation induction of the preparation for the generation of the tumour cell Epithelial and stromal conversion of reverse radiation induction Tumour cell Epithelial and stromal conversion generation.
4. 4. application according to claim 1 to 3, it is characterised in that: described to be able to suppress what LncRNA RET was expressed Substance is siRNA or shRNA；The sequence of the siRNA is as shown in SEQ ID No.2；The sequence of the shRNA is by SEQ ID T in No.3 replaces with resulting sequence after U.
5. 5.LncRNA RET or the substance that LncRNA RET can be promoted to express answering in following (a ')-(e ') is at least one With:

   (a ') prepares the product for promoting the growth of tumour cell under radiation pressure, or promotes tumour cell under radiation pressure Growth；

   (b ') prepares the product for mitigating radiosensitivity for tumor cell, or mitigates radiosensitivity for tumor cell；

   (c ') prepares the product for reducing radiation-induced apoptosis of tumor cells, or reduces radiation-induced tumour cell and wither It dies；

   (d ') preparation is for promoting radiation that the product of DNA of tumor cell injury repair, or promotion radiation is caused to cause DNA of tumor cell damage Wound is repaired；

   The product of generation of (the e ') preparation for promoting radiation-induced tumour cell Epithelial and stromal to convert, or promote radiation-induced Tumour cell Epithelial and stromal conversion generation.
6. 6. any application in -5 according to claim 1, it is characterised in that: described that LncRNA RET can be promoted to express Substance is following any: can be transcribed into the DNA of the LncRNA RET, expression cassette, recombinant vector containing the DNA or again Group cell.
7. 7. being able to suppress application of the substance of pten protein expression in the product that preparation has at least one of following function:

   (A) feelings that growth of tumour cell is suppressed under radiation pressure caused by due to inhibiting LncRNA RET expression are reversed Condition；

   (B) radiosensitivity for tumor cell caused by due to inhibiting the phenomenon that LncRNA RET expression is reversed to enhance；

   (C) radiation-induced resulted tumour Apoptosis caused by due to inhibiting the phenomenon that LncRNA RET expression is reversed to increase；

   (D) reverse due to inhibit LncRNA RET expression caused by radiation-induced resulted tumour cell epithelia mesenchymal transformation by The case where inhibition.
8. 8. application according to claim 7, it is characterised in that: the amino acid sequence of the pten protein such as SEQ ID Shown in No.4；And/or

   The substance for being able to suppress pten protein expression is siRNA shown in SEQ ID No.5.
9. 9. any application in -8 according to claim 1, it is characterised in that: the LncRNA RET is following any:

   (a1) RNA shown in SEQ ID No.1；

   (a2) nucleotide sequence shown in SEQ ID No.1 is passed through to the substitution and/or missing of one or several nucleotide residues And/or addition and RNA with the same function；

   (a3) have 99% or more, 95% or more, 90% or more, 85% or more with nucleotide sequence defined by (a1) or (a2) Or 80% or more homology and RNA with the same function.
10. 10. any application in -9 according to claim 1, it is characterised in that: the tumour cell be cervical cancer cell or Lung adenocarcinoma cell；

    Specifically, the cervical cancer cell is HeLa cell；The lung adenocarcinoma cell is A549 cell.