CN111139239A - Application of brand-new PANC754 in preparation of tumor-inhibiting drugs - Google Patents

Abstract

The invention belongs to the technical field of medicines, and through whole genome big data analysis, the ENSG00000213754.2 is found to be a brand-new tumor inhibition ncRNA, and the invention is named as PANC754 (a)PAn‑cancerNon‑Coding RNA754Pan-cancer non-coding RNA 754); the invention provides application of brand-new PANC754 in preparation of a tumor-inhibiting medicament. The embodiment of the invention also provides a brand-new verification method for preparing the tumor-inhibiting medicament by the PANC754, which comprises the steps that the PANC754 presents a wide-range low-expression mode in 23 cancers; patients with low expression of PANC754 were confirmed to have significantly lower survival rates in 23 cancers than PANC754 high-expression patients; the invention focuses on the discovery of cancer suppressor elements in pan-cancerous ncRNAThe method removes the limitation of traditional tumor suppressor gene screening, discovers a brand-new tumor suppressor ncRNA-PANC 754, and has the function of widely suppressing tumors.

Description

Application of brand-new PANC754 in preparation of tumor-inhibiting drugs

Technical Field

The invention belongs to the technical field of medicines, and relates to a method for discovering that ENSG00000213754.2 is a brand-new tumor inhibition ncRNA through whole genome big data analysis, and can have a wide tumor inhibition effect; this is named PANC754 (in the present invention)PAn-cancerNon-Coding RNA754Pan-cancer non-coding RNA 754); in particular to a brand-new PANC754 for preparing a tumor-inhibiting medicamentApplication of the aspect.

Background

In the past decade, although the incidence and mortality of cancer has been reduced, cancer remains the second leading killer of human health, cancers represented by liver cancer, stomach cancer, colorectal cancer are also the second leading cause of threat to Chinese health, oncogenes and Tumor Suppressor Genes (TSGs) are the two major gene types involved in tumorigenesis, oncogenes lead to uncontrolled growth of cells, while TSGs generally act as negative regulators of oncogenes, cell cycle checkpoints or gene products, inhibiting the development of cancer, classical tumor suppressor genes are RB (retinoblastoma gene) and TP53 (tumor protein P53), subsequently discovered tumor suppressor genes include the telangiectasia mutated gene, BRCA1 (breast cancer gene 1), BRCA2, BRIP1(BRCA1 interoperable protein carboxy-terminal helicase 1), PALB1(BRCA1 partner and localizer genes), NF1 (neurofibromatosis type protein), buldii 2 (BRCA 3626), bust 2 (bms3 h 2), TGF β 2 receptor homolog, TGF β 9B 2 homolog, TGF β -kinase, TGF β -.

Non-coding RNA (ncRNA) is a class of RNA molecules that are not translated into protein, including transfer RNA (tRNAs), ribosomal RNA (rRNAs), microRNAs, siRNAs, piRNAs, snorRNAs, exRNAs, snRNAs, scarNAs, circRNAs, and lncRNAs. Early studies thought it to be "garbage" of the genome, but with increasing evidence that the ncRNA gene is deeply involved in the development of disease, it is increasingly important to study its role in disease pathogenesis. Especially, the function and mechanism of ncRNAs in tumor inhibition are important. Traditional tumor suppressor gene screening has been largely limited to protein-coding genes, and extension of the screening for tumor suppressor elements to include non-protein-coding genes/regions allows for the discovery of new TSGs.

Traditional tumor suppressor gene screening is mainly limited to protein coding genes, and finding new tumor suppressor genes based on coding regions is increasingly difficult along with the development of exon sequencing technology and the like. RNA in the human genome can be used as an information carrier and can also perform a catalytic function, and is considered to be the oldest molecule in life origin. Among them, the non-coding rna (ncRNA) gene, which is a dark substance of genome, accounts for about 98% of genome, and more researches have found that ncRNA is widely involved in various links of life phenomena, such as growth, differentiation, development, immunity, and even has an important regulation and control function in tumor formation.

Disclosure of Invention

The invention aims to solve the technical problem of providing a brand-new application of PANC754 in preparing a tumor-inhibiting medicament.

In order to solve the above technical problems, the embodiment of the present invention provides an application of a brand-new PANC754 in the preparation of a tumor-inhibiting drug.

Further, the tumor inhibiting medicine at least comprises PANC 754.

Wherein the PANC754 is ENSG 00000213754.2.

Wherein the PANC754 is analyzed by genome-wide big data analysis and pan-cancer analysis as research tools.

The embodiment of the invention also provides a brand-new PANC754, which is characterized in that the PANC754 is ENSG 00000213754.2.

The embodiment of the invention also provides a brand-new verification method for preparing the tumor-inhibiting medicine by the PANC754, which is characterized by comprising the following steps:

(1) PANC754 exhibits a broadly low expression pattern in 23 cancers; low expression of PANC754 was confirmed in colorectal and gastric cancer patient tissues;

(2) through whole genome level meta-analysis and Cox regression analysis, patients with low expression of PANC754 were confirmed to have significantly lower survival rates in 23 cancers than those with high expression of PANC 754;

(3) through in vitro experiments, the PANC754 gene cDNA is overexpressed in six cancer cell lines of liver cancer, gastric cancer, esophageal cancer and colorectal cancer, and the PANC754 can obviously inhibit the proliferation, clone formation, invasion and metastasis of tumor cells;

(4) according to the characteristic that PANC754 is antisense ncRNA and based on the sequence complementation principle that the antisense ncRNA plays a role, the whole genome blast analysis finds that the PANC754 sequence similarity genes are KPNA4 and MTMR8, and the PANC754 is verified to interact with target genes KPNA4 and MTMR8 through an immunoblotting experiment;

(5) bioinformatics discovers the correlation between the expression of PANC754 and the expression of whole genome genes, and then a fluorescent quantitative PCR experiment verifies that the PANC754 can directly regulate the mRNA expression of CDK2, DNMT1 and NRAS genes in a coexpression mode, wherein CDK2 has direct correlation, R is-0.05, and P is 4.9 multiplied by 10^-9This suggests that the compound exerts a cancer suppressing effect through KPNA4-CDK2 signaling pathway.

The technical scheme of the invention has the following beneficial effects: the present invention focuses on a novel cancer suppressor, non-coding RNA, to discover a novel cancer therapeutic target; the invention focuses on the discovery of cancer suppressor elements in pan-cancer ncRNA, removes the limitation of traditional tumor suppressor gene screening, and further discovers a brand-new tumor suppressor ncRNA-PANC 754, wherein the PANC754 has the function of widely suppressing tumors.

Drawings

FIG. 1 is a diagram of the validation of the whole genome RNA analysis screening of PANC754 and the validation of its under-expression pattern in the example of the present invention;

FIG. 2 is a validation graph of the fact that the Meta-analysis found a low expression pattern of PANC754 from 23 cancers in the examples of the present invention;

FIG. 3 is a graph of the overall survival of whole genome level meta analysis and Cox regression analysis PANC754 in an embodiment of the present invention;

FIG. 4 is a validation graph of the significant inhibition of tumor cell proliferation and colony formation by PANC754 in accordance with the present invention;

FIG. 5 is a verification that PANC754 significantly inhibits tumor cell invasion and metastasis in accordance with an embodiment of the present invention;

FIG. 6 is a validation graph of the finding of a typical PANC754 sequence similarity gene by whole genome blast analysis in an embodiment of the present invention;

FIG. 7 is a validation graph of the finding that PANC754 can interact with its target genes KPNA4 and MTMR8 in the immunoblotting experiment in the examples of the present invention;

FIG. 8 is a graph showing the results of the fluorescent quantitative PCR experiment conducted by PANC754 according to the present invention in regulating the expression of COPS7B, PMVK, SMIM12, CDK2, DNMT1 and SRSF genes.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

According to the invention, through genome-wide big data analysis, the ENSG00000213754.2 is a brand-new tumor inhibition ncRNA, and can have a wide tumor inhibition effect. This is named PANC754 (in the present invention)PAn-cancerNon-CodingRNA754Pan-cancer non-coding RNA754) and associated application protection.

An application of brand-new PANC754 in preparing tumor-inhibiting medicine.

In a further embodiment, at least PANC754 is included in the tumor-inhibiting drug. Wherein the PANC754 is ENSG 00000213754.2. Wherein the PANC754 is analyzed by genome-wide big data analysis and pan-cancer analysis as research tools.

The embodiment of the invention also provides a brand-new PANC754, and the PANC754 is ENSG 00000213754.2.

The embodiment of the invention also provides a brand-new verification method for preparing the tumor-inhibiting medicine by the PANC754, which comprises the following steps:

(1) PANC754 showed a broadly low expression pattern in 23 cancers (whole genome screen results are shown in fig. 1A and fig. 2); low expression of PANC754 was confirmed in colorectal and gastric cancer patient tissues (clinical specimen validation results are shown in fig. 1B and fig. 1C). FIG. 1A, Manhattan plot, shows the screening of whole genome RNA sequencing data from 23 cancers in the TCGA database to yield PANC 754; FIG. 1B, quantitative PCR results for 30 colorectal cancer tissues and 26 paracancer control tissues show that, relative to the paracancer control tissues,PANC754 is low expressed in nodular carcinoma tissue (P ═ 0.0269); FIG. 1C, quantitative PCR results for 12 cases of gastric cancer tissues and corresponding paracancer control tissues show that PANC754 is less expressed in gastric cancer tissues (P) than in paracancer control tissues<0.001). FIG. 2, forest plot of regression analysis by meta analysis of 10490 gene expression data in 23 cancers combined with a fixed effect model (fixed effect model) and a random effect model (random effect model) shows that PANC754 is universally low expressed in cancer tissue (SMD ═ 1.2), where the fixed effect model P is<1.0x10^-264(ii) a Random effect model P1.78 x10^-29。

(2) Through genome-wide-level meta-analysis and Cox regression analysis, it was found that patients with low expression of PANC754 were significantly lower in survival than those with high expression of PANC754, and the results are shown in fig. 3, and as shown by the forest plot of the meta-analysis of 8074 survival data in 23 cancers combined with regression analysis of fixed effect model (fixed effect model) and random effect model (randomeffect model), PANC754 is a protective factor for cancer, where the fixed effect model risk coefficient HR is 0.84, 95% CI:0.76-0.94, and P is 0.0024; random effect model HR 0.86, 95% CI 0.78-0.94, P9.0 x10^-4。

(3) Through in vitro experiments, PANC754 gene cDNA is over-expressed in six cancer cell lines of liver cancer, gastric cancer, esophageal cancer and colorectal cancer, and PANC754 can obviously inhibit tumor cell proliferation, clone formation, invasion and metastasis (verification results are shown in figures 4 and 5). Fig. 4A, CCK8 experiment found that PANC754 was overexpressed in the gastric cancer cell line MGC803, with a significantly lower cell proliferation rate than the empty and untransfected plasmid groups (P < 0.001); fig. 4B, CCK8 found that PANC754 was overexpressed in the esophageal cancer cell line Eca109, and the cell proliferation rate was significantly lower than that of the empty and untransfected plasmid groups (P < 0.001); fig. 4C, flow cytometry examination found that the apoptosis rate was significantly increased in the PANC754 over-expressed group in colorectal cancer cell lines relative to the empty and untransfected plasmid groups (P ═ 0.0012). Fig. 5A, cell scratch experiments show that the PANC754 group over-expressed in the hepatoma cell line HepG2 has significantly lower cell migration capacity than the empty plasmid group and the untransfected plasmid group; FIG. 5B, statistical histogram showing cell scratch experiments for hepatoma cell lines, over-expressed PANC754 VS empty plasmid group and untransfected plasmid group (P < 0.001); fig. 5C, cell invasion experiments show that the cell invasion capacity of the liver cancer cell line Bel-7402 overexpression PANC754 group is significantly lower than that of the empty plasmid group and untransfected plasmid group; fig. 5D, statistical histogram, shows cell invasion experiments for liver cancer cell lines, over-expression PANC754 VS empty plasmid group and untransfected plasmid group (P ═ 0.0003).

(4) According to the characteristic that PANC754 is antisense ncRNA, based on the principle of sequence complementation that antisense ncRNA plays a role, the PANC754 sequence similarity genes are KPNA4 and MTMR8 through whole genome blast analysis, as shown in FIG. 6; by immunoblotting experiments, it was verified that PANC754 can interact with its target genes KPNA4 and MTMR8, as shown in fig. 7. In FIG. 6A, the sequence alignment analysis of PANC754 and 55270388 non-redundant sequences from GenBank, EMBL, DDBJ, PDB and RefSeq databases using the NCBI blastn suite tool and default algorithm parameters found that the PANC754 has very high homology with the KPNA4 and MTMR8 genes (score > 1200); FIG. 6B, a dendrogram showing that PANC754 has very high homology with KPNA4 and MTMR8 gene sequences; FIG. 6C, based on the theory of sequence complementarity, using a heat map of co-expression analysis, the antisense ncRNA PANC754 was found to target the KPNA4 and MTMR8 genes. FIG. 7A, immunoblot experiments found that the levels of KPNA4 and MTMR8 were significantly reduced in the group of liver cancer cell line Bel-7402 overexpressing PANC754, relative to the empty and untransfected plasmid groups; fig. 7B, immunoblot experiments found that colorectal cancer cell line DLD1 overexpressed PANC754 panel with significantly reduced levels of KPNA4 and MTMR8 protein relative to the empty and untransfected plasmid panels; fig. 7C, immunoblot experiments found that esophageal cancer cell line Eca109 over-expressed PANC754 panel with significantly reduced levels of KPNA4 and MTMR8 protein relative to the empty and untransfected plasmid panels; fig. 7D, immunoblot experiments found that the levels of KPNA4 and MTMR8 protein were significantly reduced in the group of hepatocellular carcinoma cell line HepG2 overexpressing PANC754 relative to the empty and untransfected plasmid groups; fig. 7E, immunoblot experiments found that the levels of KPNA4 and MTMR8 protein were significantly reduced in the gastric cancer cell line MGC803 over-expressing PANC754 group relative to the empty and untransfected plasmid groups; FIG. 7F, immunoblot assay finding that colorectal cancer cell line SW480 overexpresses PANC754 panels with slightly reduced KPNA4 and MTMR8 protein levels relative to the empty and untransfected plasmid panels; figure 7G, a statistical histogram, shows that six cell lines overexpress PANC754 group with significantly reduced levels of KPNA4 protein (P <0.05) relative to empty and untransfected plasmid groups; fig. 7H, a statistical histogram, shows that the six cell lines over-expressed PANC754 group with significantly reduced MTMR8 protein levels (P <0.05) relative to the empty and untransfected plasmid groups.

(5) Bioinformatics excavates the correlation between the expression of PANC754 and the expression of whole genome genes, and then a fluorescent quantitative PCR experiment verifies that the expression of CDK2, DNMT1 and NRAS gene mRNA can be directly regulated and controlled by the PANC754 through a co-expression form, as shown in figure 8, the fluorescent quantitative PCR experiment finds that the expression of COPS7B, PMVK, SMIM12, CDK2, DNMT1 and SRSF genes is regulated and controlled by the PANC 754; of these, CDK2 has direct relevance, R ═ 0.05, P ═ 4.9 × 10^-9This suggests that the compound exerts a cancer suppressing effect through KPNA4-CDK2 signaling pathway. FIG. 8A shows that, based on the sequence similarity analysis in FIG. 6C, COPS7B, which is one of the genes with the most significant P-value co-expression similarity, was selected, and quantitative PCR detection revealed that the levels of COPS7B mRNA were significantly reduced (P7B mRNA levels) in the Panc754 group over-expressed in esophageal cancer, gastric cancer, liver cancer, and colorectal cancer cell lines, compared to the empty plasmid group and the untransfected plasmid group (P7C)<0.05); FIG. 8B shows that PMVK, one of the genes with the most significant co-expression similarity P value, was selected based on the sequence similarity analysis of FIG. 6C, and quantitative PCR detection revealed that compared to the empty plasmid group and the untransfected plasmid group, the PMVK mRNA level was significantly reduced in the over-expressed PANC754 group among esophageal cancer, gastric cancer, liver cancer and colorectal cancer cell lines (P is a significant reduction in the mRNA level of PMVK (P is<0.05); FIG. 8A shows that SMIM12, one of the genes with the most significant co-expression similarity P value, was selected based on the sequence similarity analysis of FIG. 6C, and quantitative PCR detection revealed that SMIM12 mRNA levels were significantly reduced (P12 mRNA levels were significantly reduced) compared to empty and untransfected plasmid groups, in the Panc754 group of esophageal cancer, gastric cancer, liver cancer, colorectal cancer cell lines<0.05); FIG. 8D shows that one of the most significant oncogenes CDK2 with co-expression correlation P value was selected and quantitative PCR revealed esophageal cancer, gastric cancer, liver cancer, and colorectal cancer, relative to the empty plasmid group and untransfected plasmid groupThe cancer cell line over-expression PANC754 group has obviously reduced CDK2 mRNA level (P)<0.05); FIG. 8E shows that DNMT1, one of the most significant oncogenes with the co-expression correlation P value, was selected, and quantitative PCR revealed that DNMT1 mRNA levels were significantly reduced in the over-expressed PANC754 group in esophageal cancer, gastric cancer, liver cancer and colorectal cancer cell lines (P) compared to the empty plasmid group and the untransfected plasmid group (P value is shown in FIG. 8E)<0.05); FIG. 8F shows that SRSF2, one of the most significant oncogenes with co-expression correlation P value, was selected, and quantitative PCR revealed that the SRSF2 mRNA levels were significantly reduced (P is a significant decrease) in the over-expression PANC754 group in the esophageal, gastric, liver, and colorectal cancer cell lines, relative to the empty and untransfected plasmid groups (P is a significant decrease in the P-level of the mRNA in the cell lines of esophageal, gastric, liver, and colorectal cancers) (P is a significant decrease<0.05)。

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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<120> application of brand-new PANC754 in preparation of tumor inhibition drugs

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Claims (6)

1. An application of brand-new PANC754 in preparing tumor-inhibiting medicine.

2. The use of the PANC754 in the preparation of a tumor suppressor according to claim 1, wherein the tumor suppressor comprises at least PANC 754.

3. The use of a novel PANC754 in the manufacture of a medicament for the inhibition of tumors as in claim 1 or 2, wherein said PANC754 is ENSG 00000213754.2.

4. The use of a novel PANC754 for the preparation of a tumor suppressor according to claim 1 or 2, wherein the PANC754 is analyzed by genome-wide big data analysis with pan-cancer analysis as a research tool.

5. A completely new PANC754, characterized in that said PANC754 is ENSG 00000213754.2.

6. The method for verifying the preparation of the anti-tumor drug according to claim 1, comprising the following steps:

(1) PANC754 exhibits a broadly low expression pattern in 23 cancers; low expression of PANC754 was confirmed in colorectal and gastric cancer patient tissues;

(2) through whole genome level meta-analysis and Cox regression analysis, patients with low expression of PANC754 were confirmed to have significantly lower survival rates in 23 cancers than those with high expression of PANC 754;

(3) through in vitro experiments, the PANC754 gene cDNA is overexpressed in six cancer cell lines of liver cancer, gastric cancer, esophageal cancer and colorectal cancer, and the PANC754 can obviously inhibit the proliferation, clone formation, invasion and metastasis of tumor cells;

(4) according to the characteristic that PANC754 is antisense ncRNA and based on the sequence complementation principle that the antisense ncRNA plays a role, the whole genome blast analysis finds that the PANC754 sequence similarity genes are KPNA4 and MTMR8, and the PANC754 is verified to interact with target genes KPNA4 and MTMR8 through an immunoblotting experiment;

(5) bioinformatics discovers the correlation between the expression of PANC754 and the expression of whole genome genes, and then a fluorescent quantitative PCR experiment verifies that the PANC754 can directly regulate the mRNA expression of CDK2, DNMT1 and NRAS genes in a coexpression mode, wherein CDK2 has direct correlation, R is-0.05, and P is 4.9 multiplied by 10^-9This suggests that the compound exerts a cancer suppressing effect through KPNA4-CDK2 signaling pathway.

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