CN108653315B

CN108653315B - Application of long non-coding RNA linc00637

Info

Publication number: CN108653315B
Application number: CN201810429626.2A
Authority: CN
Inventors: 胡颖; 王星文; 赵坤明
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2018-05-08
Filing date: 2018-05-08
Publication date: 2020-04-24
Anticipated expiration: 2038-05-08
Also published as: CN108653315A

Abstract

The invention provides application of long non-coding RNA linc00637 and provides novel application of the long non-coding RNA linc 00637. The long non-coding RNA linc00637 is applied to preparation of anti-tumor targeted drugs. linc00637 can inhibit HIF-1a expression, thereby inhibiting tumorigenesis, angiogenesis and tumor metastasis. The volume, tumor weight, number of vessels and vessel lumen size of one to four weeks of tumors were measured in the HCT116 control group and the over-expressed linc00637 xenograft model, with 93% less tumor volume, 86% less weight, 57% reduction in vessel number and 68% reduction in vessel lumen size in the over-expressed linc00637 xenograft model. The invention is applied to the field of tumor targeted therapy.

Description

Application of long non-coding RNA linc00637

Technical Field

The invention relates to a new application of linc 00637.

Background

Hypoxia is one of the important characteristics of the tumor microenvironment, nuclear transcription factor HIF-1a is the core signal molecule of the tumor cells in hypoxia tolerance reaction,

HIF-1a activates the expression of target genes by combining hypoxia response elements located in promoter regions of genes, resulting in biological effects such as adaptive survival of tumors and angiogenesis of tumors. Studies have shown that hypoxia/HIF-1 a is closely associated with tumor development and progression. The results suggest that HIF-1a is a potential target for tumor therapy.

Although a large number of in vitro experiments and preclinical experimental data show that the inhibition of HIF-1a expression or activity can produce obvious tumor inhibition effect, at present, no high-efficiency and safe HIF-1a inhibitor exists clinically. The discovery of highly potent, safe HIF-1a inhibitors relies on an in-depth understanding of the HIF-1a regulatory mechanisms. However, the mechanisms that regulate HIF-1a degradation are well understood, but the molecular mechanisms that regulate HIF-1a at both the transcriptional and translational levels are poorly understood. Undoubtedly, the research of new HIF-1a regulation mechanism can provide a brand new target for the treatment of tumors in hypoxic environment.

Disclosure of Invention

The invention provides application of long non-coding RNA linc 00637.

The invention discloses application of long non-coding RNA linc00637 in preparation of anti-tumor targeted drugs.

Based on the expression of the hypoxia inducible gene HIF-1a in tumors, the invention analyzes whether linc00637 can inhibit the generation and angiogenesis of tumors by reducing the expression condition of the HIF-1 a. The research finds that linc00637 can inhibit HIF-1a mRNA expression by combining with PRC2 complex, further inhibit the combination of YB-1 and HIF-1a 5' -UTR region by combining with YB-1, inhibit the regulation function of HIF-1a translation activity, and the linc00637 can efficiently and specifically inhibit HIF-1a expression by combining with the two. In vivo experiments in nude mice, it was further confirmed that linc00637 can inhibit the expression of HIF-1a, thereby inhibiting tumorigenesis, angiogenesis and tumor metastasis. The volume, tumor weight, number of vessels and vessel lumen size of 1 to four weeks of tumors were measured in the HCT116 control group and the over-expressed linc00637 xenograft model, with 93% less tumor volume, 86% lighter weight, 57% reduction in vessel number and 68% reduction in vessel lumen size in the over-expressed linc00637 xenograft model.

The invention discovers for the first time that the gene linc00637 with unknown function has an important inhibition effect on the biosynthesis and transcription level of HIF-1 a. linc00637 can be a potential high-efficiency and safe HIF-1a inhibitor, and the research result provides a new approach for targeting HIF-1 a.

Drawings

FIG. 1 is a characteristic analysis of the linc00637 gene;

FIG. 2 is an analysis of the expression of linc00637 in 46 pairs of colon cancer, 6 pairs of cervical cancer and 17 pairs of kidney cancer tissues;

FIG. 3 is a graph of 3 'and 5' RACE experiments;

FIG. 4 is a graph showing the length and expression of linc00637 in normal large intestine tissue and large intestine cancer tissue;

FIG. 5 is a graph showing the length and expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in normal large intestine tissue and large intestine cancer tissue;

FIG. 6 is a diagram showing the separation experiment of linc00637 nucleoplasm in HCT116 cells; wherein a is the nucleus and b is the cytoplasm;

FIG. 7 is a diagram showing the separation experiment of linc00637 nucleoplasm in Hela cells; wherein a is the nucleus and b is the cytoplasm;

FIG. 8 is a diagram of an in vitro transcription and translation experiment;

FIG. 9 is a graph that shows the expression of HIF-1a and-2 a protein in HCT116 cells under hypoxic conditions at 0, 12, and 24 hours;

FIG. 10 is a graph showing protein expression profiles of HIF-1a and HIF-2a in Hela cells under hypoxic conditions at 0, 12, and 24 hours;

FIG. 11 is a graph of linc00637 expression assay in HCT116 cells under hypoxic conditions of 0, 12 and 24 hours;

FIG. 12 is a graph showing the expression of linc00637 in Hela cells measured at 0, 12 and 24 hours under hypoxic conditions;

FIG. 13 is a graph that shows the expression of HIF-1a and-2 a in HCT116 cells at oxygen concentrations of 20, 5, and 2 percent;

FIG. 14 is a graph showing the expression of HIF-1a and-2 a in Hela cells at oxygen concentrations of 20, 5, and 2 percent;

FIG. 15 is a graph showing the expression of linc00637 in HCT116 cells at oxygen concentrations of 20, 5 and 2 percent;

FIG. 16 is a graph showing the expression of linc00637 in Hela cells at oxygen concentrations of 20, 5 and 2 percent;

FIG. 17 is a graph of the change in HIF-1a mRNA levels following overexpression of HCT116 under hypoxic conditions (control) and linc 00637; wherein c is a control and d is linc 00637;

FIG. 18 is a graph of the change in HIF-1a mRNA levels by Hela after overexpression of empty vector (control) and linc00637 under hypoxic conditions; wherein c is a control and d is linc 00637;

FIG. 19 is a graph of HIF-1a protein levels altered by HCT116 and Hela after overexpression of linc00637 under hypoxic conditions;

FIG. 20 shows transcriptional activity of HIF-1a downstream target gene HRE following HCT116 overexpression under hypoxic conditions (control), linc00637 and cotransforming linc00637 with HIF-1 a; wherein c is control, d is line 00637, e is line 00637+ HIF-1 a;

FIG. 21 shows HRE transcriptional activity of HIF-1a downstream target genes following Hela overexpression of empty (control), linc00637 and cotransforming linc00637 under hypoxic conditions with HIF-1 a; wherein c is control, d is line 00637, e is line 00637+ HIF-1 a;

FIG. 22 is the mRNA expression levels of the HIF-1a downstream target genes VEGF, AGN1 and MMP2 after HCT116 over-expressed unloaded (control) and linc00637 under hypoxic conditions; wherein c is a control and d is linc 00637;

FIG. 23 is the mRNA expression levels of the HIF-1a downstream target genes VEGF, AGN1 and MMP2 after Hela overexpresses empty (control) and linc00637 under hypoxic conditions; wherein c is a control and d is linc 00637;

FIG. 24 shows the HIF-1a promoter luciferase activity following HCT116 overexpression under hypoxic conditions with no-load (control) and linc00637, wherein c is control and d is linc 00637;

FIG. 25 is a graph showing the luciferase activity of the HIF-1a promoter after Hela overexpresses empty vector (control) and linc00637 under hypoxic conditions, wherein c is control and d is linc 00637;

FIG. 26 is a graph of HIF-1a and EZH2 protein levels following downregulation of EZH2 following overexpression of empty vector (control) and linc00637 by HCT116 and Hela under hypoxic conditions;

FIG. 27 is a graph of HIF-1a mRNA levels after HCT116 overexpression of empty vector (control) and linc00637 downregulation of EZH2 under hypoxic conditions; wherein c is a control, d is linc00637, f is linc00637+ siEZH 2;

FIG. 28 is a graph of HIF-1a mRNA levels after Hela overexpresses empty (control) and linc00637 downregulated after EZH2 under hypoxic conditions; wherein c is a control, d is linc00637, f is linc00637+ siEZH 2;

FIG. 29 is the binding of the HIF-1a promoter to EZH2 and IgG after HCT116 overexpression under hypoxic conditions, with empty vector (control) and linc 00637; wherein g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 30 shows the binding of the HIF-1a promoter to EZH2 and IgG after Hela overexpresses empty vector (control) and linc00637 under hypoxic conditions; wherein g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 31 is the binding of the HIF-1a promoter to H3K27me3 and IgG following overexpression of empty vector (control) and linc00637 by HCT116 under hypoxic conditions; wherein g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 32 is a graph showing the binding of the HIF-1a promoter to H3K27me3 and IgG by Hela after overexpression of empty vector (control) and linc00637 under hypoxic conditions; wherein g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 33 is an RNA in vitro co-immunoprecipitation assay;

FIG. 34 is a graph of linc00637 binding to EZH2 and SUZ12 in vivo following HCT116 overexpression of empty (control) and linc00637 under hypoxic conditions, wherein c is control and d is linc 00637;

FIG. 35 is a graph of linc00637 binding to EZH2 and SUZ12 in vivo after Hela overexpresses empty (control) and linc00637 under hypoxic conditions, wherein c is control and d is linc 00637;

FIG. 36 shows that HCT116 and Hela add ActD under hypoxic conditions, overexpress linc00637 or down-regulate YB-1 to detect HIF-1a and YB-1 protein expression levels;

FIG. 37 shows the binding of YB-1 to linc00637 and HIF-1a 5' -UTR in vivo following overexpression of empty (control) and linc00637 by HCT116 under hypoxic conditions; wherein g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 38 is the binding of YB-1 to linc00637 and HIF-1a 5' -UTR in vivo following Hela overexpression under hypoxic conditions (control) and linc 00637; medium g is normoxia, h is hypoxia, i is hypoxia + linc 00637;

FIG. 39 shows HIF-1a 5' -UTR luciferase activity in HCT116 under hypoxic conditions by overexpression of linc00637, downregulation of YB-1, or both; wherein c is a control and d is linc 00637; j is si-YB-1; k is si-YB-1+ line 00637;

FIG. 40 shows HIF-1a 5' -UTR luciferase activity by Hela under hypoxic conditions by overexpressing linc00637, downregulating YB-1, or both; wherein c is a control and d is linc 00637; j is si-YB-1; k is si-YB-1+ line 00637;

FIG. 41 is the volume of one to four weeks of tumors in the HCT116 control group and the line 00637 overexpressing xenogenic model; wherein c is a control and d is linc 00637;

FIG. 42 is a graph of the weight of one to four weeks of tumors in the HCT116 control group and the line 00637 overexpressing xenogenic model; wherein c is a control and d is linc 00637;

FIG. 43 is a photograph of a tumor after dissection;

FIG. 44 is an analysis of CD31 expression in the HCT116 control group and the line 00637 overexpressing xenogenic model;

FIG. 45 is an analysis of ki-67 expression in HCT116 control and the overexpression of linc00637 heterogeneous model;

FIG. 46 is the number of vessels in the HCT116 control group and the line 00637 overexpressing xenogenic model; wherein c is a control and d is linc 00637;

FIG. 47 is the vessel lumen size in the HCT116 control group and the line 00637 overexpressing xenogenic model; wherein c is a control and d is linc 00637;

FIG. 48 is the ki-67 relative value, p 0.04, in the HCT116 control and the overexpressed linc00637 xenogeneic model; wherein c is a control and d is linc 00637;

FIG. 49 is the expression level of linc00637 after overexpression of empty (control) and linc00637 in three pairs of xenogenic model specimens (T1, T2 and T3); wherein c is a control and d is linc 00637;

FIG. 50 is a graph showing the detection of HIF-1a mRNA expression levels in three pairs of xenogenic model specimens (T1, T2, and T3) overexpressing empty (control) and linc 00637; wherein c is a control and d is linc 00637;

FIG. 51 is a graph showing the detection of VEGF mRNA expression levels in three pairs of xenogenic model specimens (T1, T2 and T3) over-expressed empty cells (control) and linc 00637; wherein c is a control and d is linc 00637;

FIG. 52 is a graph of HIF-1a and VEGF protein expression levels in three xenogenic model specimens (T1, T2, and T3).

Detailed Description

The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments.

The first embodiment is as follows: the long noncoding RNA linc00637 of the embodiment is applied to preparation of anti-tumor targeted drugs.

From the viewpoint of expression of the hypoxia inducible gene HIF-1a in tumors, the present embodiment was conducted to examine whether linc00637 could inhibit tumor development and angiogenesis by decreasing the expression of HIF-1 a. The research finds that linc00637 can inhibit HIF-1a mRNA expression by combining with PRC2 complex, further inhibit the combination of YB-1 and HIF-1a 5' -UTR region by combining with YB-1, inhibit the regulation function of HIF-1a translation activity, and the linc00637 can efficiently and specifically inhibit HIF-1a expression by combining with the two. In vivo experiments in nude mice, it was further confirmed that linc00637 can inhibit the expression of HIF-1a, thereby inhibiting tumorigenesis, angiogenesis and tumor metastasis. The volume, tumor weight, number of vessels and vessel lumen size of one to four weeks of tumors were measured in the HCT116 control group and the over-expressed linc00637 xenograft model, with 93% less tumor volume, 86% less weight, 57% reduction in vessel number and 68% reduction in vessel lumen size in the over-expressed linc00637 xenograft model.

The embodiment discovers for the first time that the function unknown gene linc00637 has an important inhibition effect on the HIF-1a biosynthesis and transcription level. linc00637 can be a potential high-efficiency and safe HIF-1a inhibitor, and the research result provides a new approach for targeting HIF-1 a.

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the anti-tumor is used for inhibiting tumor growth, tumor angiogenesis and tumor metastasis. The others are the same as in the first or second embodiment.

The third concrete implementation mode: the present embodiment differs from the first or second embodiment in that: inhibiting tumor growth is the inhibition of the volume and weight of the tumor. The same as in the first or second embodiment.

The fourth concrete implementation mode: the difference between this embodiment mode and one of the first to third embodiment modes is: the tumor is carcinoma of large intestine, renal carcinoma or cervical carcinoma. The rest is the same as one of the first to third embodiments.

The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: long noncoding RNArinc 00637 inhibits HIF-la expression. The rest is the same as one of the first to fourth embodiments.

The effect of the invention was verified by the following experiments:

example 1:

firstly, molecular characteristic analysis of linc 00637: the molecular characteristics of linc00637 were analyzed by searching UCSC website, and as a result, as shown in FIG. 1, linc00637 is located on chromosome 14q32 (FIG. 1), and the gene contains 3 exons, has a full length of 2056nt, and is distributed in cytoplasm and nucleus. FIG. 3 is a3 'and 5' RACE experiment from which the 3 'and 5' bands of the corresponding linc00637 can be derived. The 3 'and 5' sequences of linc00637 were determined. FIGS. 4 and 5 are Northern blot experiments to examine the length and expression of linc00637 and GAPDH in normal and large intestine tissues. It can be seen that a band appears at 2000bp in linc00637, and the expression of linc00637 is reduced in colorectal cancer tissues, and the length of linc00637 can be obtained from the figure, and RACE and Northern blot experiments jointly determine that the length of the linc00637 is 2050, and the sequence on the website UCSC is lack of the first six bases.

The Northern blot experiment method comprises the following steps:

1ug of linearized plasmid DNA was used for the synthesis of digoxigenin-labeled RNA probes, which were synthesized according to the experimental procedure provided by the Roche kit DIGRNALabeling Mix. Trizol method is used for extracting the tissue RNA of the patient with colorectal cancer. According to the procedure provided in the Northern Max Kit of the Kit, 30ug of RNA sample was electrophoresed in agarose gel containing one hundredth of formaldehyde at 80V, after electrophoresis, the RNA was transferred to a nylon membrane, and the UV-crosslinked nylon membrane was incubated with digoxigenin-labeled RNA probe overnight at 65 ℃. The next day, the target bands were detected using the Roche Anti-Digoxigenin-AP Fabfragments and NBT/BCIP Stock Solution kit. Construction of antisense linc00637 sequence into PCDNA3.1 vector

The RACE experimental method comprises the following steps:

linc 006373 'and 5' RACE experiments Using test Marathon-Ready cDNA and FirstChoice^TMRLM-RACE Kit with Manual Kit. Experimental procedures refer to the kit.

Linc00637-F and Linc00637-R are used as primers, real-time quantitative PCR is adopted to analyze the expression conditions of the Linc00637 in tissues of 46 pairs of colorectal cancers, 6 pairs of cervical cancers and 17 pairs of kidney cancers, the result is shown in figure 2, and the expression of the Linc00637 in the colorectal cancers, the kidney cancers and the cervical cancers is obviously reduced as shown in figure 2.

FIGS. 6 and 7 show the Linc00637 nucleoplasm separation experiments with Linc00637-F and Linc00637-R as primers and GAPDH as a cytoplasmic marker; MALAT1-F and MALAT1-R are used as primers, MALAT1 is used as a nuclear marker, and it can be seen from the figure that linc00637 is distributed in HCT116 (colon cancer cell) and Hela (cervical cancer cell) cells in the nucleus and cytoplasm.

FIG. 8 shows an in vitro transcription and translation experiment with iASPP as a positive control, and it can be seen that there is no band in the linc00637 group, indicating that linc00637 does not have the ability to encode a protein.

Secondly, the research of the linc00637 regulation HIF-1a expression and activity:

the protein expression of HIF-1a (hypoxia inducible factor 1a) and HIF-2a (hypoxia inducible factor 2a) under the hypoxia condition of 0, 12 and 24 hours is detected by using a Western blot method, and as can be seen from FIGS. 9 and 10, HIF-1a and HIF-2a can be detected under the hypoxia condition, which indicates that the hypoxia condition is established.

Linc00637-F and Linc00637-R are used as primers, and real-time quantitative PCR is adopted to detect the expression conditions of the Linc00637 under the anoxic conditions of 0 hour, 12 hours and 24 hours. From FIGS. 11 and 12, it can be concluded that linc00637 is down-regulated under hypoxic conditions and gradually down-regulated as the time of hypoxia increases.

And detecting the protein expression of the HIF-1a and the HIF-2a under the condition that the oxygen concentration is 20 percent, 5 percent and 2 percent by using a Western blot method. From FIGS. 13 and 14, it can be seen that HIF-1a and HIF-2a are detectable under hypoxic conditions, indicating that hypoxic conditions are established.

Linc00637-F and Linc00637-R are used as primers, and real-time quantitative PCR is adopted to detect the expression condition of the Linc00637 under the condition that the oxygen concentration is 20 percent, 5 percent and 2 percent. From FIGS. 15 and 16, it can be concluded that linc00637 is down-regulated in hypoxic conditions, and gradually down-regulated as the concentration of hypoxia decreases. As can be seen in FIGS. 9-16, linc00637 was down-regulated under hypoxic conditions.

After overexpression of no-load (control) and linc00637 under the hypoxia condition of HCT116 and Hela, a linc00637 sequence is constructed into a PLNC vector, HIF-1 α -F and HIF-1 α -R are used as primers, and real-time quantitative PCR is adopted to detect the change condition of HIF-1amRNA level, so that the mRNA level of HIF-1a under the hypoxia condition is increased as shown in FIGS. 17 and 18, and the mRNA expression level of HIF-1a is remarkably reduced after overexpression of linc00637 under the hypoxia condition, which indicates that the HIF-1a mRNA expression level can be reduced by linc 00637.

The conclusion that HIF-1a is increased in protein level under the anoxic condition is that HIF-1a is increased in protein level under the anoxic condition, and the protein expression level of HIF-1a is remarkably reduced after linc00637 is over-expressed under the anoxic condition is shown in the figure 19, which shows that linc00637 can reduce the protein expression of HIF-1a, and α -tublin is an internal reference.

The method comprises the steps of constructing a linc00637 sequence into a PLNC vector, after HCT116 and Hela overexpress no-load (control), linc00637 and cotransforming linc00637 and HIF-1a under the anoxic condition, constructing an HRE sequence into a PGL3 vector, and detecting HRE transcription activity of a downstream target gene of the HIF-1a by adopting a luciferase reporter gene method. From FIGS. 20 and 21, it can be seen that HRE transcription activity of the HIF-1a downstream target gene is increased under the anoxic condition, and is reduced after the linc00637 is over-expressed, and the influence of the linc00637 on the HRE transcription activity of the HIF-1a downstream target gene is eliminated after the linc00637 and the HIF-1a are co-transformed, which indicates that the linc00637 can regulate the transcription of the HIF-1a downstream target gene.

After HCT116 and Hela over-express no-load (control) and linc00637 under the anoxic condition, a linc00637 sequence is constructed into a PLNC vector, VEGF-F, VEGF-R, AGN1-F, AGN1-R, MMP2-F and MMP2-R are used as primers, and the mRNA expression levels of HIF-1a downstream target genes VEGF (vascular growth factor), AGN1 (angiopoietin 1) and MMP2 (matrix metalloproteinase 2) are detected by real-time quantitative PCR. 22 and 23 in the figure, the mRNA expression of VEGF, AGN1 and MMP2 is increased under the anoxic condition, and the mRNA expression of VEGF, AGN1 and MMP2 is obviously reduced after the linc00637 is over-expressed under the anoxic condition, which shows that the linc00637 can inhibit the expression of HIF-1a downstream target genes.

Restoration of linc00637 expression completely abolished hypoxia-induced HIF-1a mRNA and protein (FIGS. 17-19), suggesting that a decrease in linc00637 levels is an essential factor in hypoxia-induced HIF-1a expression and activation. In addition, with the change of HIF-1a expression level, linc00637 effectively regulates the transcriptional activity of HIF-1a and the expression of its downstream target genes (FIGS. 20-23).

Thirdly, the molecular mechanism of the linc00637 for regulating the expression and the activity of HIF-1a is as follows:

when the linc00637 sequence is constructed into a PLNC vector, HCT116 and Hela express no-load (control) and linc00637 under the anoxic condition, the luciferase activity of the HIF-1a promoter is detected by adopting a luciferase reporter gene method, and the conclusion can be drawn from the graphs in FIGS. 24 and 25 that the luciferase activity of the HIF-1a promoter is increased under the anoxic condition and the fluorescence activity is reduced after the linc00637 is over-expressed under the anoxic condition, which indicates that the linc00637 can influence the HIF-1a expression by the transcription level. The HIF-1a promoter luciferase vector was purchased from Addgene.

The linc00637 sequence is constructed into a PLNC vector, HCT116 and Hela are used for down-regulating EZH2(Zeste gene enhancer homolog 2) after over-expressing no-load (control) and linc00637 under the anoxic condition, and the protein levels of HIF-1a and EZH2 are detected by a Western blot method. From FIG. 26, it can be concluded that HIF-1a protein expression decreased and EZH2 protein expression did not change after line 00637 was overexpressed under hypoxic conditions, and HIF-1a expression was restored after EZH2 was downregulated after line 00637 was overexpressed, indicating that HIF-1a was regulated at transcriptional level by line 00737 via EZH 2.

The sequences of linc00637 are constructed into a PLNC vector, HCT116 and Hela express no-load (control) under the anoxic condition and down-regulate EZH2 after linc00637, HIF-1 α -F and HIF-1 α -R are used as primers, real-time quantitative PCR is adopted to detect the mRNA level of HIF-1a, and the conclusion can be drawn from FIGS. 27 and 28 that the expression level of HIF-1a mRNA is reduced after the linc00637 is over-expressed under the anoxic condition, and the expression level of HIF-1a is recovered after the EZH2 is down-regulated after the linc00637 is over-expressed, which indicates that the HIF-1a is regulated by the linc00637 at the transcription level through EZH 2.

The sequences of linc00637 were constructed into PLNC vectors, HCT116 and Hela over-expressed the no-load (control) and linc00637 under hypoxic conditions, and HIF-1 α promoter-F and HIF-1 α promoter-R were used as primers to detect the binding of HIF-1a promoter to EZH2 and IgG by real-time quantitative PCR it can be concluded from FIGS. 29 and 30 that the binding of EZH2 to HIF-1a promoter was reduced under hypoxic conditions, that the binding of EZH2 to HIF-1a promoter was increased after over-expression of linc00637 under hypoxic conditions, and that no IgG was bound, indicating that linc00637 promoted the binding of EZH2 to HIF-1a promoter under hypoxic conditions.

The linc00637 sequence was constructed into PLNC vector, HCT116 and Hela over-expressed no-load (control) and linc00637 under hypoxic conditions, and HIF-1 α promoter-F and HIF-1 α promoter-R were used as primers to detect the binding of HIF-1a promoter to H3K27me3 (trimethylation at lysine 27 of histone H3) and IgG using real-time quantitative PCR the conclusion from FIGS. 31 and 32 is that H3K27me3 binds to HIF-1a promoter less strongly under hypoxic conditions, H3K27me3 binds to HIF-1a promoter more strongly after over-expressing linc00637 and does not bind to IgG, indicating that linc00637 promotes H3K27me3 binds to HIF-1a promoter more strongly under hypoxic conditions.

An in vitro co-immunoprecipitation assay was performed to detect binding of both sense and antisense linc00637 to EZH2 and SUZ12(Zeste gene suppressor 12) in vitro, and it can be concluded from fig. 33 that linc00637 binds EZH2 and SUZ12 in vitro.

The Linc00637 sequence was constructed into PLNC vector, HCT116 and Hela over-expressed no-load (control) and Linc00637 under hypoxic conditions, Linc00637-F and Linc00637-R, GAPDH-F and GAPDH-R were used as primers to detect the binding of Linc00637 to EZH2 and SUZ12 in vivo by real-time quantitative PCR, and it can be concluded from FIGS. 34 and 35 that the binding of Linc00637 to EZH2 is reduced under hypoxic conditions, the binding is increased after over-expression of Linc00637, and the binding to SUZ12 is weak.

A linc00637 sequence is constructed into a PLNC vector, HCT116 and Hela are added with ActD under the anoxic condition to overexpress linc00637 or to down-regulate YB-1 to detect the expression level of HIF-1a and YB-1(Y box binding protein 1) proteins. Using the Westernblot approach for detection, it can be concluded from FIG. 36 that HIF-1a protein expression is reduced and YB-1 protein expression is unchanged after overexpression of linc00637, HIF-1a protein expression is reduced after YB-1 is down-regulated, HIF-1a protein expression is consistent with the independent down-regulation of YB-1 after overexpression of linc00637, indicating that linc00637 regulates HIF-1a protein translation level through YB-1.

A Linc00637 sequence is constructed into a PLNC vector, HCT116 and Hela overexpress no-load (control) and Linc00637 under an anoxic condition, Linc00637-F, Linc00637-R, GAPDH-F and GAPDH-R are used as primers, and the binding condition of YB-1, Linc00637 and HIF-1a 5' -UTR is detected in vivo by adopting real-time quantitative PCR. From FIGS. 37 and 38, it can be seen that YB-1 binds to HIF-1a mRNA under hypoxic conditions, and is less bound to linc00637, and that YB-1 binds to HIF-1a mRNA and is more bound to linc00637 after overexpression of linc00637 under hypoxia. GAPDH was a negative control. Indicating that HIF-1a competes with lin 00637 for binding to YB-1.

The sequences of linc00637 are constructed into PLNC vectors, HCT116 and Hela are used for detecting the luciferase activity of HIF-1a5 '-UTR by a luciferase reporter gene method under the condition of overexpression of linc00637, down-regulation of YB-1 or the combined treatment of the linc00637 and the YB-1 a under the anoxic condition, wherein the luciferase vector is formed by constructing HIF-1 α Wild Type (WT) 5' -UTR (1-256), 5 '-UTR (1-138, MT1) and 5' -UTR (139-256, MT2) into PGL3 vectors with CMV promoters, and the conclusion that the fluorescence activity of MT1 and MT2 is enhanced under the anoxic condition and the fluorescence activity of WT 2 is weakened under the condition of overexpression of the linc00637, down-regulation of the YB-1 or the combined treatment of the linc MT 00637 and the MT2 under the anoxic condition can be obtained from the conclusion that the fluorescence activity of the WT 1 is not changed, thereby indicating that the MT2 is the main region influenced by the YB-1 and the linc 00637.

As can be seen from FIGS. 24-35, linc00637 could bind to PRC2 and exert inhibitory effect on HIF-1a mRNA transcription, which has certain regulatory effect on HIF-1a protein level (FIG. 26). As can be seen from FIGS. 37 and 38, linc00637 can bind to YB-1, thereby inhibiting the binding of YB-1 to the HIF-1a 5' -UTR region and inhibiting the regulation of the expression level of HIF-1a by YB-1 (FIGS. 36 to 40).

And fourthly, the inhibition effect of the expression of linc00637 on the growth of the tumor:

in the HCT116 control group and the overexpressed linc00637 xenogeneic model, the volume of the tumor was measured from 1 to four weeks in a real-time measurement manner, and the tumor weight was measured after dissection, and a photograph of the tumor after dissection is shown below. From FIGS. 41-43, it can be concluded that the tumor volume and weight are significantly reduced after overexpression of linc00637, indicating that linc00637 inhibits tumor growth in vivo. In the overexpression linc00637 heterogeneous model, a linc00637 sequence is constructed into a PLNC vector.

In the HCT116 control group and the overexpressed linc00637 xenogeneic model, CD31 and ki-67 were detected and the number of vessels and the size of the vessel lumen were counted. The conclusions that can be drawn from FIGS. 44-48 are that CD31 and ki-67 expression decreased and the number of vessels and vessel lumen size decreased after overexpression of linc00637, suggesting that linc00637 inhibits tumor angiogenesis. In the overexpression linc00637 heterogeneous model, a linc00637 sequence is constructed into a PLNC vector.

The Linc00637 sequence was constructed into PLNC vector, no-load (control) and Linc00637 were overexpressed in three pairs of heterogeneous model specimens (T1, T2 and T3), Linc00637-F and Linc00637-R, HIF-1 α -F and HIF-1 α -R were used as primers, VEGF-F and VEGF-R were used as primers, and real-time quantitative PCR was used to detect the expression levels of Linc00637, HIF-1a and VEGF mRNA, respectively, it can be concluded from FIG. 49 that Linc00637 overexpression occurred, and HIF-1a (FIG. 50) and VEGF mRNA (FIG. 51) decreased in the Linc00637 overexpression group.

The linc00637 sequence was constructed into PLNC vector, and in three pairs of heterogeneous model specimens (T1, T2 and T3), "+" indicates overexpression of no-load (control) or linc00637, and expression levels of HIF-1a and VEGF protein were detected by Western blot method. From FIGS. 49-51, it can be concluded that the linc00637 overexpression group HIF-1a and VEGFmRNA expression was decreased.

In three pairs of heterogeneous model specimens (T1, T2 and T3), "+" indicates overexpression of null-load (control) or linc00637, and HIF-1a and VEGF protein expression levels were detected, respectively. From FIG. 52, it can be concluded that the expression of HIF-1a and VEGF proteins in the linc00637 overexpression group was reduced.

FIGS. 41-52 show that linc00637 overexpression inhibited tumor tumorigenicity volume and angiogenesis in nude mice, and that this effect was accompanied by changes in HIF-1a mRNA and protein levels in the tumor, as well as changes in angiogenesis. It was suggested that linc00637 has tumor growth and angiogenesis inhibitory effects, and that the effects are exerted through the HIF-1a signaling pathway.

The real-time quantitative PCR method described in this example is: RNA was extracted from the tissues and cells by Trizol, and cDNA was synthesized using the RNA as a template, with a total amount of 1. mu.g of RNA. cDNA was synthesized using the reverse transcription kit of TAKARA, and the reagents in the kit were placed on ice and allowed to melt. After thawing, the system configuration is given by a kit. TAKARA fluorescent quantitative PCR kit was used for full quantitative PCR, 10. mu.l of each reaction system. The cDNA concentration is not less than 100 ng.

The Western blot method comprises the following steps:

after the concentration of the protein is measured, the concentration of each histone is leveled and boiled for five minutes in a metal bath at 100 ℃. Preparing SDS-PAGE protein gel according to the requirement, wherein the SDS-PAGE protein gel comprises an upper layer gel and a lower layer gel, the upper layer is a concentrated gel, and the lower layer is a separation gel, and preparing the protein gel according to the specified proportion. And 5% milk is prepared and sealed for 1 hour after the film transfer is finished. Once at 4 ℃ overnight. The following day, after removing the PVDF membrane of the primary antibody hybridization, washing with TBST for 5 minutes was repeated three times, and development was carried out after hybridization of the secondary antibody for 1 hour.

The luciferase reporter gene method comprises the following steps:

3×10⁴cells were seeded in 24-well plates, and Linpfectamin 2000 was used to transfect Renila and the corresponding Luciferase Reporter vector, 48 hours after transfection, cells were collected and the fluorescence activity was detected using Dual Luciferase Reporter Assay system. The activity of the corresponding luciferase reporter gene was normalized to that of Renilla.

The experimental method of RNA in vitro co-immunoprecipitation comprises the following steps:

after the Biotin-labeled RNA probe was transcribed in vitro using the Roche Biotin RNA Labeling Mix kit, the RNA secondary structure was restored after incubation for 3 minutes at 95 ℃ for 2 minutes on ice. RNA probes that restored secondary structure were mixed with streptavidin agarose beads overnight. After the cell lysate is mixed with the RNA-column conjugate for 1 hour at 4 degrees, the beads with the mixture are washed 5 times, SDS loading buffer is added and boiled for 5 minutes, and finally the protein band bound to the RNA is detected by the Westernblot method.

The sequences of the primers used in this example are shown in Table 1:

TABLE 1

As is clear from this example, linc00637 inhibits tumorigenesis and angiogenesis by decreasing HIF-1a expression. linc00637 can inhibit HIF-1a mRNA expression by combining PRC2 complex, inhibit YB-1 from being combined with HIF-1a 5' -UTR region by combining YB-1, inhibit regulation of HIF-1a translation activity, and can efficiently and specifically inhibit HIF-1a expression by combining linc00637 with the two. In vivo experiments in nude mice, it was further confirmed that linc00637 can inhibit the expression of HIF-1a, thereby inhibiting tumorigenesis, angiogenesis and tumor metastasis.

Claims

1. The application of the long non-coding RNA linc00637 in the preparation of anti-tumor targeted drugs is characterized in that the tumor is colorectal cancer, renal cancer or cervical cancer.

2. The use of the long noncoding RNA linc00637 according to claim 1 in the preparation of a targeted drug against tumors, wherein said tumors are the inhibition of tumor growth, tumor angiogenesis and tumor metastasis.

3. The use of the long non-coding RNA linc00637 in the preparation of a targeted drug against tumors according to claim 1, wherein the inhibition of tumor growth is the inhibition of tumor volume and weight.

4. The use of the long non-coding RNA linc00637 in the preparation of a targeted drug against tumors as claimed in claim 1, wherein the long non-coding RNA linc00637 inhibits the expression of HIF-la.