阅读说明：本技术 长链非编码rna xloc_102237及其在人脐静脉内皮细胞中的应用 (Long-chain non-coding RNA XLOC _102237 and application thereof in human umbilical vein endothelial cells ) 是由 袁晓龙 王希龙 张豪 潘金春 李加琪 龚宝勇 张哲� 高洪彬 唐龙龙 谭伟江 李 于 2019-11-07 设计创作，主要内容包括：本发明公开一种长链非编码RNA XLOC_102237及其在人脐静脉内皮细胞中的应用。本发明检通过基因工程技术外源合成lncRNA XLOC_102237的超表达载体和沉默试剂lncRNA XLOC_102237 Silencer,检测超表达/沉默lncRNA XLOC_102237后人脐静脉内皮细胞的增殖、凋亡情况,结果显示lncRNA XLOC_102237能够参与抑制人脐静脉内皮细胞的增殖、促进人脐静脉内皮细胞的凋亡,并抑制内皮细胞体外小管的形成,进一步说明lncRNA XLOC_102237可能通过抑制人脐静脉内皮细胞的增殖而抑制血管新生。(The invention discloses a long-chain non-coding RNA XLOC _102237 and application thereof in human umbilical vein endothelial cells. The invention detects the overexpression vector and silencing reagent lncRNA XLOC _102237Silencer which exogenously synthesize lncRNA XLOC _102237 by genetic engineering technology, detects the proliferation and apoptosis conditions of human umbilical vein endothelial cells after overexpression/silencing lncRNA XLOC _102237, and the result shows that lncRNA XLOC _102237 can participate in inhibiting the proliferation of human umbilical vein endothelial cells, promoting the apoptosis of human umbilical vein endothelial cells and inhibiting the formation of endothelial cell external tubules, further shows that lncRNA XLOC _102237 can inhibit angiogenesis by inhibiting the proliferation of human umbilical vein endothelial cells.)

1. A long non-coding RNA XLOC _102237, characterized by: the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. A silencing agent that inhibits the function of said long non-coding RNA XLOC _102237, wherein: the silencing reagent is lncRNA XLOC _102237Silencer, and the nucleotide sequence of the silencing reagent is shown in SEQ ID NO: 2, respectively.

3. A recombinant expression vector comprising the long non-coding RNA XLOC _102237 of claim 1.

4. A recombinant cell, wherein: to transfect the recombinant expression vector of claim 3 or the silencing agent of claim 2 into human umbilical vein endothelial cells.

5. The use of the long non-coding RNA XLOC _102237 of claim 1 or the silencing agent of claim 2 that inhibits the function of long non-coding RNA XLOC _102237 in human umbilical vein endothelial cells, wherein:

the environment of the application is an in vitro environment;

the lncRNA XLOC _102237 inhibits the proliferation of human umbilical vein endothelial cells, promotes the apoptosis of the human umbilical vein endothelial cells and inhibits the human umbilical vein endothelial cells from forming external tubules;

the silencing reagent for inhibiting the function of the long-chain non-coding RNA XLOC _102237 promotes the proliferation of human umbilical vein endothelial cells, inhibits the apoptosis of the human umbilical vein endothelial cells and promotes the human umbilical vein endothelial cells to form in-vitro tubules.

6. Use according to claim 5, characterized in that:

the inhibition of the proliferation of human umbilical vein endothelial cells, the promotion of the apoptosis of the human umbilical vein endothelial cells and the inhibition of the formation of external tubules of the human umbilical vein endothelial cells are realized by adding exogenous long-chain non-coding RNA XLOC _ 102237;

the promotion of the proliferation of the human umbilical vein endothelial cells, the inhibition of the apoptosis of the human umbilical vein endothelial cells and the promotion of the human umbilical vein endothelial cells to form in vitro tubules is realized by transferring exogenous lncRNA XLOC-102237 Silenecer.

7. The use of the long non-coding RNA XLOC _102237 of claim 1 in the manufacture of a medicament for inhibiting proliferation of human umbilical vein endothelial cells, promoting apoptosis of human umbilical vein endothelial cells, and/or inhibiting formation of tubules in vitro by human umbilical vein endothelial cells.

8. Use according to claim 7, characterized in that:

the medicine contains a sequence of long-chain non-coding RNA XLOC _102237 and/or contains a recombinant expression vector;

the recombinant expression vector contains a sequence of long-chain non-coding RNA XLOC _ 102237.

9. The use of the silencing agent of claim 2 for inhibiting the function of long-chain non-coding RNA XLOC _102237 in the preparation of a medicament for promoting proliferation of human umbilical vein endothelial cells, inhibiting apoptosis of human umbilical vein endothelial cells, and/or promoting formation of small tubes in vitro by human umbilical vein endothelial cells.

10. Use according to claim 9, characterized in that:

the medicine contains the sequence of lncRNA XLOC-102237 Silencer.

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to long-chain non-coding RNA XLOC _102237 and application thereof in human umbilical vein endothelial cells.

Background

Angiogenesis (Angiogenesis) refers to the development of new blood vessels from existing capillaries or postcapillary veins, and mainly comprises: the degradation of blood vessel basement membrane during activation, the activation, proliferation and migration of blood vessel endothelial cells and the reconstruction of new blood vessels and blood vessel networks are complex processes involving various molecules of various cells, which depend on the delicate regulation of gene expression and also require various factors to participate together. Angiogenesis is a complex process of the coordination of a pro-angiogenic factor and an inhibitory factor, and normally, the two are in equilibrium, and once the equilibrium is broken, the vascular system is activated, so that the angiogenesis is excessive or the vascular system is inhibited to degenerate the blood vessel.

Human Umbilical Vein Endothelial Cells (HUVECs) are specialized epithelial Cells consisting of a layer of flattened Cells that form the inner wall of a blood vessel. Endothelial cells have a variety of biological functions including angiogenesis, arteriosclerosis, control of vasoconstriction and dilation, and the like. The endothelial cells have the potential of stem cells, can be transmitted for 50-60 generations theoretically, and have very important relation with angiogenesis, namely the self-proliferation capacity of the endothelial cells can directly influence the angiogenesis, so the endothelial cells are ideal cells for researching the formation of blood vessels.

Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length of more than 200 nt. Research shows that lncRNA is widely involved in biological processes, can be involved in gene expression regulation from an apparent modification level, a transcription level and a post-transcription level, and further plays an important role in the processes of proliferation, differentiation, survival and transfer of cells. However, no report about the regulation of the expression change of lncRNA XLOC _102237 on the function of human umbilical vein endothelial cells is available at home and abroad at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a long-chain non-coding RNAXLOC _ 102237.

Another object of the invention is to provide a silencing agent that inhibits the function of lncRNA XLOC _ 102237.

The invention further aims to provide application of the long-chain non-coding RNA XLOC _102237 or the silencing agent for inhibiting the function of lncRNAAXLOC _102237 in human umbilical vein endothelial cells. The expression level of lncRNA XLOC _102237 in human umbilical vein endothelial cells is changed by a genetic engineering technology, and the application of the lncRNA XLOC _102237 in regulating and controlling the proliferation, apoptosis and angiogenesis of the human umbilical vein endothelial cells is determined.

The purpose of the invention is realized by the following technical scheme:

a long non-coding RNA XLOC _102237(lncRNA XLOC _102237) having a nucleotide sequence set forth in SEQ ID NO: 1 is shown.

A silencing agent for inhibiting the function of the long non-coding RNA XLOC _102237 is lncRNA XLOC _102237Silencer, and the nucleotide sequence of the silencing agent is shown as follows (SEQ ID NO: 2):

TAATCTCATCCTAGGAATTTGCCATCAAAGTTCAAAGTCTAGGTCATAAGGATGAAAAGGAATTACGTGCATGCACACTTATTCTCCGTGAAGCTCATCCCTTTCAGGATGGATTTA。

a recombinant expression vector comprising said long non-coding RNA XLOC _ 102237.

A recombinant cell, wherein the recombinant expression vector or the silencing agent for inhibiting the function of the long-chain non-coding RNA XLOC _102237 is transfected into a human umbilical vein endothelial cell.

The application of the long-chain non-coding RNA XLOC _102237 or the silencing agent for inhibiting the function of the long-chain non-coding RNA XLOC _102237 in human umbilical vein endothelial cells is in an in vitro environment.

The lncRNA XLOC _102237 inhibits the proliferation of human umbilical vein endothelial cells, promotes the apoptosis of the human umbilical vein endothelial cells, and inhibits the human umbilical vein endothelial cells from forming external tubules, and the lncRNA XLOC _102237 participates in the angiogenesis process by regulating the functions of the human umbilical vein endothelial cells.

The inhibition of the proliferation of human umbilical vein endothelial cells, the promotion of the apoptosis of the human umbilical vein endothelial cells and the inhibition of the formation of external tubules of the human umbilical vein endothelial cells are realized by adding exogenous long-chain non-coding RNA XLOC _ 102237.

The addition of exogenous long-chain non-coding RNA XLOC _102237 is realized by the following steps: the long-chain non-coding RNA XLOC _102237 is connected to pcDNA3.1(-) vector to construct a super-expression vector of the long-chain non-coding RNA XLOC _102237, and then the super-expression vector is transfected into human umbilical vein endothelial cells.

The overexpression vector of the long-chain non-coding RNA XLOC _102237 is preferably constructed by the following method: carrying out PCR amplification by taking human umbilical vein endothelial cell cDNA as a template and taking lncRNA-F and lncRNA-R as primers to obtain lncRNA XLOC _ 102237; then XbaI and XhoI enzyme are used for double enzyme digestion, and the mixture is recycled and connected to a pcDNA3.1(-) vector to obtain a super expression vector of the long-chain non-coding RNA XLOC _ 102237; wherein the nucleotide sequences of lncRNA-F and lncRNA-R are shown as follows:

lncRNA-F：CTAGTCTAGACTAGCGGTTTGCAATTAGGAGATGTTAG；

lncRNA-R：CCGCTCGAGCGGCTCATTTATCTTCACTGGAGGTAAG。

the silencing reagent for inhibiting the function of the long-chain non-coding RNA XLOC _102237 promotes the proliferation of human umbilical vein endothelial cells, inhibits the apoptosis of the human umbilical vein endothelial cells and promotes the human umbilical vein endothelial cells to form in-vitro tubules.

The promotion of the proliferation of the human umbilical vein endothelial cells, the inhibition of the apoptosis of the human umbilical vein endothelial cells and the promotion of the formation of the external tubules of the human umbilical vein endothelial cells are realized by transferring exogenous lncRNA XLOC _102237Silencer, namely, lncRNA XLOC _102237Silencer is directly transfected into the human umbilical vein endothelial cells.

The long-chain non-coding RNA XLOC _102237 is applied to the preparation of medicines for inhibiting the proliferation of human umbilical vein endothelial cells, promoting the apoptosis of the human umbilical vein endothelial cells and/or inhibiting the formation of external tubules of the human umbilical vein endothelial cells.

The medicine contains a sequence of long-chain non-coding RNA XLOC _102237 and/or contains a recombinant expression vector.

The recombinant expression vector contains a sequence of long-chain non-coding RNA XLOC _102237, and can express long-chain non-coding RNA XLOC _102237 in human umbilical vein endothelial cells; preferably, the construction is carried out by the following method: carrying out PCR amplification by taking human umbilical vein endothelial cell cDNA as a template and taking lncRNA-F and lncRNA-R as primers to obtain lncRNA XLOC _ 102237; then XbaI and XhoI enzymes are used for double enzyme digestion, and the product is recovered and connected to a pcDNA3.1(-) vector to obtain a recombinant expression vector; wherein the nucleotide sequences of lncRNA-F and lncRNA-R are shown as follows:

lncRNA-F：CTAGTCTAGACTAGCGGTTTGCAATTAGGAGATGTTAG；

lncRNA-R：CCGCTCGAGCGGCTCATTTATCTTCACTGGAGGTAAG。

the silencing reagent for inhibiting the function of the long-chain non-coding RNA XLOC _102237 is applied to the preparation of medicaments for promoting the proliferation of human umbilical vein endothelial cells, inhibiting the apoptosis of the human umbilical vein endothelial cells and/or promoting the human umbilical vein endothelial cells to form in-vitro tubules.

The medicine contains a sequence of lncRNA XLOC _102237Silencer, and can inhibit the expression of long-chain non-coding RNA XLOC _102237 in human umbilical vein endothelial cells.

The verification results of the invention are as follows:

1. according to the invention, the proliferation condition of the human umbilical vein endothelial cells is detected by an EdU method, and the proliferation rate of the human umbilical vein endothelial cells is remarkably reduced (P <0.01) compared with a control group (pcDNA3.1) after the lncRNAXOCO _102237 is over-expressed (figure 1). After silencing lncRNA XLOC _102237, the proliferation rate of human umbilical vein endothelial cells was very significantly increased (P <0.01) compared to the control (NC) (fig. 2).

2. The apoptosis condition of the human umbilical vein endothelial cells is detected by an Annexin V-FITC/PI technology, and the apoptosis rate of the human umbilical vein endothelial cells is remarkably increased (P is less than 0.01) compared with a control group (pcDNA3.1) after the lncRNA XLOC _102237 is over-expressed (figure 3). After silencing lncRNA XLOC _102237, the proliferation rate of human umbilical vein endothelial cells was significantly decreased (P <0.05) compared to the control group (NC) (fig. 4).

3. In the invention, Matrigel is used as a carrier to simulate in vitro tubule formation, and the tube formation number of human umbilical vein endothelial cells is remarkably reduced (P <0.01) compared with that of a control group (Blank) after the lncrnax LOC-102237 is over-expressed (FIG. 5). After silencing lncRNA XLOC _102237, the tube formation number of human umbilical vein endothelial cells was very significantly increased (P <0.01) compared to the Blank control (Blank) (fig. 6).

The invention takes lncRNA XLOC _102237(SEQ ID NO: 1) as a research object, adopts a molecular cell biology method to research the application of the lncRNA XLOC _102237 in human umbilical vein endothelial cells, and proves the effect of lncRNA XLOC _102237 on the human umbilical vein endothelial cells for the first time. The lncRNA XLOC _102237 is proved to be capable of participating in inhibiting the proliferation of human umbilical vein endothelial cells, promoting the apoptosis of the human umbilical vein endothelial cells and inhibiting the formation of endothelial cell in vitro tubules by over-expressing and silencing lncRNA XLOC _102237, and further shows that lncRNA XLOC _102237 can inhibit angiogenesis by inhibiting the proliferation of the human umbilical vein endothelial cells.

Compared with the prior art, the invention has the following advantages and effects:

the invention detects the overexpression vector and the silencing reagent which are exogenously synthesized into lncRNA XLOC _102237 by the genetic engineering technology, detects the proliferation and apoptosis conditions of human umbilical vein endothelial cells after overexpression/silencing of lncRNA XLOC _102237, and is a molecular mechanism accumulation material for lncRNA to regulate and control the human umbilical vein endothelial cells. Meanwhile, the results of the invention show that lncRNAXLOC _102237 inhibits the formation of endothelial cells in vitro tubules, and further shows that lncRNA XLOC _102237 can inhibit the neogenesis of blood vessels by inhibiting the proliferation of endothelial cells in human umbilical veins.

Drawings

FIG. 1 is a graph showing the result of EdU method detecting the regulation of human umbilical vein endothelial cell proliferation after overexpression of lncRNA XLOC _ 102237.

FIG. 2 is a graph showing the result of EdU method detecting the regulation of human umbilical vein endothelial cell proliferation after silencing lncRNA XLOC _ 102237.

FIG. 3 is a graph showing the results of the Annexin V-FITC method for detecting the apoptosis of human umbilical vein endothelial cells after overexpression of lncRNA XLOC _ 102237.

FIG. 4 is a graph showing the results of the Annexin V-FITC method for detecting the regulation of human umbilical vein endothelial cell apoptosis after the silencing of IncRNA XLOC _ 102237.

FIG. 5 is a graph of matrigel vascularization assay results of human umbilical vein endothelial cell vascularization after overexpression of lncRNA XLOC _ 102237.

FIG. 6 is a graph of matrigel vascularization assay results of in vitro vascularization of human umbilical vein endothelial cells following silencing of lncRNA XLOC _ 102237.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results. The examples are all reagents and process steps conventional in the art, except where specifically indicated.

The base sequence related by the invention is shown as SEQ ID NO: the long-chain non-coding RNA shown in 1 is an lncRNA which is discovered for the first time by constructing a heart stalk model of a miniature pig, performing high-throughput sequencing on RAN and analyzing biological information, wherein a human genome also contains the long-chain non-coding RNA, the full-length sequence is 1018bp, and the long-chain non-coding RNA is named as lncRNA XLOC _102237, and the sequence is shown as follows:

lncRNA XLOC_102237(SEQ ID NO：1)：

GGGAACTCCAAAACTTTCTATTCTTATGTTTTAAGAAAGATCACACGGTTTGCAATTAGGAGATGTTAGTTTAATTTCTTTTCTACCCCTGGTCACTGTGATAGTGGAGAGATCACTAACCAATAACTCTAGGACCCATCTGCTAAATAAGGATAATCTCATCCTAGGAATTACGTGCATGCACACAGAGGTACCTTGAGTATTTGGGAAGGATGGTGCCATCAAAGTTCAAAGTATATTGATTATAATTTGCTTTCTTATTCTCCGTGAAGCTCAGAGCCTATTATAGGCATTTTATACACAGTTTCCCTCTCTTGTGTGGTTGGCAAGTCTGGCCATAAAGCATCTGGAGGAAAAACCCAGAGATGCTGAGGGCCGTGAGTCTCAGTTCCACCTCTTTTTACTCCTTAGCTTCTTCAAGAGTCAGTTGCGTTTTGGCATTCCCATTGTGGCACAGTGGAAACGAATCTGACTAGGAACCATGAGGTTTCAGGTTCGATCCCTGATCTTGCTTGGTAGGTTAAGGATCCTGCATTGCCATGCTCTATGATGTAGGTCTCAGATGTGGCTTGGATCCAGCATTGCTGTGGCTGTGGTGTAGACCAGCGAGCGCTACAGCTCCAATTCGACTCCTAGCCAGGGAACCTCCATGTGCTGCGGGTACAGTCCTTAAAAAAAAAAAAAAAAAAAAAAAAGAGTCAGTTGTGCTTCTTTGGTACGGGGGAGGAGTGCCATTCTAATTAGAAGAAAATGATGTCCTTTGGAAAATTACCTACATGCTAAACAGCAGTAATGAGGAACCCTGTTCCAGCCTCCCTTTCAGGATGGATTTATTGGTTTGAGGCCTTCCTAGGTCATAAGGATGAAAAGGCCGCTCAGGAACAGCACAGGTGATTTAGCTTAAGTTGGTGCCCAGGAACTTAGAGGCTGCTCTCTGTCTTACCTCCAGTGAAGATAAATGAGGTAAAGGAGTAGACATATTGAATTCTGAAATAGAAAAAGGAAGAGGAGTACAGGG。