CN113817823B - Long non-coding RNA LETN as tumor marker and therapeutic target - Google Patents

Abstract

The present application relates to a long non-coding RNA (LETN) useful as a diagnostic target and therapeutic target for cancer. In particular, the present application discloses that lncRNA RP11-196G18.22 (LETN) is overexpressed in cancer cells, which is capable of promoting proliferation of cancer cells and is associated with short prognosis survival in cancer patients. Decreasing the lncRNA expression leads to inhibition of cancer cell growth, and thus inhibition of the lncRNA expression represents a new strategy for cancer treatment.

Description

Long non-coding RNA LETN as tumor marker and therapeutic target

The application is a divisional application of Chinese patent application No.201911265749.8 with the application date of 2019, 12 months and 11 days and the name of 'long non-coding RNA LETN as a tumor marker and a treatment target'.

Technical Field

The present application relates to the field of cancer. More particularly, the application relates to the use of long non-coding RNA LETN as a tumor marker and therapeutic target.

Background

With the rapid development of sequencing technology in recent years, research has found that a large number of non-coding regions on the human genome transcribe long-chain non-coding RNAs (lncrnas) without translational activity, and the number is far greater than that of proteins. Hundreds of lncRNA have been found to be associated with various diseases or physiological functions in humans. In particular in cancer, there is growing evidence that lncRNA plays a synergistic role in tumor suppression or tumorigenesis [1]. However, for the vast majority of lncRNA, little is known about its molecular and biological functions.

Liver cancer is the fifth most common cancer worldwide, with 841000 new cases and 782000 deaths each year [2] among which male morbidity and mortality are 2 to 3 times higher, and the second among male deaths. China belongs to a high-risk country with liver cancer incidence, and is mainly caused by chronic HBV infection and aflatoxin contact. However, despite the increasing incidence of liver cancer, surprisingly, few treatments are available. In addition to physical treatments such as radiation, transplantation and surgery, there is only one approved drug for the treatment of advanced hepatocellular carcinoma, sorafenib, but it is very expensive and only extends the lifetime of 2.8 months on average, and causes various side effects such as diarrhea and nausea [3].

Nucleolus is a non-membrane structured subnuclear chamber located in the nucleus and is an important organelle for the basic processes of rDNA transcription and ribosome biosynthesis. NPM1 (also called B23) is a protein expressed in nucleolus in abundance, its protein sequence contains three distinct domains, the N-terminal domain can affect its biological function by regulating NPM1 oligomerization and interactions with other proteins, and numerous studies have shown that NPM1 functions by forming pentamers. The central region of NPM1 is an intrinsically disordered region marked by the presence of highly acidic regions, involved in binding to histones. The C-terminal region provides a sufficient platform to allow binding to nucleic acids [4]. NPM1 is an important cellular protein that has been shown to be involved in a range of biological processes including ribosomal biogenesis, chromatin remodeling and centrosome replication. Abnormal expression or mutation causes embryo dysplasia and tumor development.

Disclosure of Invention

The inventor performs comprehensive analysis and exploration on lncRNA which possibly has potential functions in the cancer background through a multi-group data mining tool by combining theoretical research and experimental means. In Hepatocellular Carcinoma (LIHC), the present inventors have functionally investigated lncRNA using data of cancer genomic map (TCGA) by means of algorithms designed in the present laboratory, found a previously unexplored lncRNA RP11-196G18.22 (we named LETN), predicted that its regulatory 191 has a broad and powerful regulatory potential for transcription factors and target genes in the development and progression of liver cancer, and verified through experiments.

The present application thus relates to a long non-coding RNA (lncRNA RP11-196G18.22, herein designated LETN) useful as a diagnostic and therapeutic target for cancer that is overexpressed in cancer cells, which is capable of promoting proliferation of cancer cells and is associated with a short prognostic survival in cancer patients, and thus can be used as a tumor marker and diagnostic marker. Decreasing the lncRNA expression leads to inhibition of cancer cell growth, and thus inhibition of the lncRNA expression represents a new strategy for cancer treatment. The present application also investigated the mechanism of action of LETN, and found that LETN functions by binding to NPM1, and promotes the occurrence and development of cancers (e.g., liver cancer) by affecting the production of rRNA and the assembly function of nucleosomes by binding to NPM 1.

According to one aspect of the present application there is provided the use of a reagent for detecting the expression level of lncRNA RP11-196G18.22 (LETN) in the preparation of a diagnostic agent or diagnostic kit for cancer. Detecting the expression level of lncRNA RP11-196G18.22 (LETN) may be detecting the DNA or RNA level thereof.

In one embodiment, the application provides a method of diagnosing cancer, the method comprising detecting the expression level of lncRNA RP11-196G18.22 (LETN) in a sample of a subject, wherein overexpression of LETN in the sample of the subject relative to a control (healthy or normal sample) is indicative of the subject having a (high) risk of having cancer or having cancer.

In one embodiment, the application provides an agent for detecting the expression level of lncRNA RP11-196G18.22 (LETN) for use in diagnosing cancer.

In one embodiment, the agent is a lncRNA RP11-196G18.22 specific probe, a gene chip, or a PCR primer.

In another embodiment, the cancer is a solid tumor, preferably selected from the group consisting of: liver cancer, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, stomach cancer, soft tissue cancer, gall bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.

In another preferred embodiment, the nucleotide sequence of lncRNA RP11-196G18.22 is set forth in SEQ ID NO. 1 (Ensembl accession No. ENST 00000564237.1).

According to another aspect of the application there is provided the use of an agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) in the manufacture of a medicament for the treatment of cancer.

In one embodiment, the application provides an agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) for use as a medicament, in particular for use as a medicament for treating cancer.

In another embodiment, the application provides a method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of an agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN).

The nature of the agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) is not critical to the application, as long as it reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN).

According to a preferred embodiment, the agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) is selected from the group consisting of: gapmer, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamer, TALEN, CRISPR, and zinc finger nucleases. In a particularly preferred embodiment, the specific sequences for antisense RNA, siRNA, shRNA and CRISPR are those used in the examples of the present description.

In another embodiment, the cancer is a solid tumor, preferably selected from the group consisting of: liver cancer, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, stomach cancer, soft tissue cancer, gall bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.

According to a preferred embodiment, the nucleotide sequence of lncRNA RP11-196G18.22 is shown in SEQ ID NO. 1 (Ensembl accession No. ENST 00000564237.1).

In another embodiment, the medicament further comprises an additional anticancer agent such as a chemotherapeutic agent, for example an agent for reducing or inhibiting NPM1 expression or mutation or an agent that inhibits the binding of LETN to NPM 1. Or the agent is used in combination with a method for reducing or inhibiting NPM1 expression or mutation or a method for inhibiting the binding of LETN to NPM 1. Even though inhibition of lncRNA RP11-196G18.22 (LETN) is sufficient to achieve therapeutic effects for cancer, it is expected that a stronger or even synergistic anti-cancer effect may be obtained when agents that reduce or inhibit lncRNA RP11-196G18.22 (LETN) expression are combined with other anti-cancer agents, such as chemotherapeutic agents. Since the present application has found that LETN functions by binding to NPM1, the occurrence and progression of cancer (e.g., liver cancer) is promoted by affecting the production of rRNA and the assembly function of nucleosomes by binding to NPM1, this is particularly true for anticancer drugs or chemotherapeutic agents having reduced or suppressed NPM1 expression or mutation.

According to another aspect of the present application, there is provided a method of screening for an anticancer drug, the method comprising the steps of:

1) Determining the expression level of lncRNA RP11-196G18.22 (LETN) in cells that overexpress lncRNA RP11-196G18.22 (LETN);

2) Contacting the candidate compound with the cells of step 1);

3) Determining the expression level of lncRNA RP11-196G18.22 (LETN) in the cell after step 2); and

4) Comparing the expression levels of lncRNA RP11-196G18.22 (LETN) determined in step 1) and step 3), wherein a decrease in the expression level of lncRNA RP11-196G18.22 (LETN) is indicative of the candidate compound having anti-cancer potential, preferably the cell is a cancer cell.

According to another aspect of the present application there is provided a method of identifying whether a tumour is suitable for treatment with an inhibitor of LETN expression, the method comprising the steps of:

1) Determining whether expression of LETN in a tumor or tumor cell sample is increased relative to a control (normal or healthy tissue/cells);

2) Determining whether the tumor is suitable for treatment, wherein increased expression indicates suitability for treatment with an inhibitor of LETN expression.

According to another aspect of the present application there is provided a method of evaluating the effect of an agent in the treatment and/or prophylaxis of cancer, wherein the method comprises testing whether the agent is capable of reducing the expression of LETN in a tumour or tumour cell sample and if so, indicating that the agent is suitable for the treatment and/or prophylaxis of cancer. In a preferred embodiment, the cancer is a solid tumor, preferably selected from the group consisting of: liver cancer, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, stomach cancer, soft tissue cancer, gall bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer. According to a preferred embodiment, the nucleotide sequence of LETN is shown in SEQ ID NO. 1 (Ensembl accession ENST 00000564237.1).

Drawings

The above features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

the results shown in fig. 1 a to I show that the LETN can promote the development of liver cancer.

A shows the expression of LETN in TCGA databases for various cancers and related paracancers (CHOL, LIHC, LUAD, lung adenocarcinoma, KIRC, clear cell carcinoma of the kidney, BLCA, bladder, BRCA, PRAD, prostate, READ, LUSC, lung squamous cell carcinoma, UCEC, endometrial, PAAD, pancreatic, HNSC, head and neck squamous cell carcinoma, KIRP, COAD, colon carcinoma, STAD, gastric cancer, SARC, soft tissue carcinoma, ESCA, esophageal carcinoma, THCA, THYM, thymus, KICH, renal suspicion cell carcinoma, PCPG, CESC, cervical squamous cell carcinoma), and it was observed that LETN expression in most human cancer tissues (solid tumors) was higher than that of the corresponding paracancerous tissues; b shows CRISPR-Cas9 knockdown of LETN in HUH7 cell line, observing that the proliferation rate of the LETN knockdown group (sgLETN) cells is much lower than the control (sgEV); c shows that the LETN is knocked down in liver cancer cell lines HUH7, SMMC-7721, lung cancer cell lines HCC827, prostate cancer cell lines PC3 and DU145 respectively, and the proliferation rate of LETN knockdown group cells is far lower than that of control group cells (siNC and siLMNA are two different negative controls; to prevent off-target effect, two siRNA knockdown are designed for the LETN: siLETN-1 and siLETN-2); d, E shows that the cell clonogenic capacity of LETN is significantly impaired after stable knockdown/knockdown in HUH7 and HCC827 cells; the F is that LETN is over-expressed in liver cancer cell lines HUH7 and SMMC-7721, and the proliferation rate of the LETN over-expression group cells is far higher than that of the control group cells; g shows that the cell clone forming ability of LETN is obviously enhanced after the stable overexpression in HUH7 and SMMC-7721 cells; h shows that the subcutaneous oncologic capacity of the LETN knockdown group is significantly reduced after stable knockdown of the LETN in the liver cancer cell line HUH 7; i shows that the LETN over-expression group is observed to have significantly enhanced subcutaneous tumorigenicity after stable over-expression of LETN in liver cancer cell line HUH7 (LETN-OE group is over-expression LETN group, EV group is control group).

The results shown in fig. 2a through E demonstrate that the LETN functions by binding to NPM 1.

Specifically, a shows that the LETN is found to be predominantly located in the cell and in the clusters by in situ hybridization experiments; b shows that LETN is mostly located in the nucleus through a nucleoplasm separation experiment, and GAPDH and LaminA/C are markers of cytoplasm and the nucleus respectively; c shows that NPM1 is the protein with the strongest binding capacity in two experiments through mass spectrometry analysis of LETN interacted proteins; d shows that the LETN and NPM1 are bound to each other and localized in the nucleolus as further demonstrated by a cell fluorescence co-localization experiment; e shows that NPM1 was able to pull down (pull down) RNA in formaldehyde crosslinked or uncrosslinked state, and NPM1 was found to actually pull down lncRNALETN (MALAT 1 in the figure indicates a negative control).

The results shown in FIGS. 3A to C demonstrate that LETN promotes the development of liver cancer by affecting the production of rRNA and the assembly function of nucleosomes by binding to NPM1 (detection antibody: murine anti-human NPM1 antibody (ab 10530, abcam), histone H2A (EPR 17470, ab177308, abcam), histone H2B (EP 957Y, ab52599, abcam), histone H3 (17168-1-AP, proteontech), histone H4 (16047-1-AP, proteontech)).

Specifically, A shows that the expression of various rRNAs can be obviously reduced by knocking down LETN or NPM1 in HUH7 and HCC827 cell lines respectively, and the functions of the LETN and the NPM1 are consistent; b shows that the knocking down of LETN can weaken the binding capacity of NPM1 and histone, thereby affecting the assembly of nucleosomes; c shows that the prognosis survival time of the patients which are singly divided into LETN or NPM1 high expression groups is shorter than that of the patients in the low expression groups for the clinical data analysis survival time analysis of the liver cancer patients in the TCGA database; when the four groups NPM1-low+LETN-low, NPM1-low+LETN-high, NPM1-high+LETN-low and NPM1-high+LETN-high are further subdivided, the survival time of the patients with high expression of NPM1 and LETN is far lower than that of the patients with low expression of NPM1 and LETN.

The results shown in figures 4 a through D demonstrate various knockdown and overexpression efficiencies of LETN.

A shows LETN knockdown by siRNA on five cell lines, and knockdown efficiency is detected by RT-qPCR; b shows LETN knockdown is carried out on two cell lines through lentivirus shRNA, and the knockdown efficiency is detected through RT-qPCR; c shows that LETN is knocked out by CRISPR-Cas9 technology, and the knockdown efficiency is detected by RT-qPCR; d shows LETN overexpression by lentiviral overexpression system for both cell lines, and the overexpression efficiency was detected by RT-qPCR.

FIG. 5 shows the effect on proliferation of the liver cancer cell line HUH7 after knocking down the corresponding lncRNA.

Detailed Description

Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art. For definitions and terms in the art, reference is made to Sambrook et al, molecular Cloning: a Laboratory Manual, version 2, cold Spring Harbor Press, plainsview, new York (1989); and Ausubel et al Current Protocols in Molecular Biology (supply 47), john Wiley & Sons, new York (1999).

The term "long non-coding RNAs (in English: long non-coding RNAs, abbreviated as lncRNA)" refers to transcripts longer than 200 nucleotides of non-coding proteins.

The term "lncRNA RP11-196G18.22" or "LETN" refers to the gene of Ensembl accession number ENST00000564237.1 and mRNA transcribed from the gene. Since it is a non-protein encoding gene, no protein product is present. In the present application, the sequence thereof can be represented by SEQ ID NO. 1 (shown below). The application also contemplates variations in non-coding exons that may be present in the above genes, which variations are considered to belong to the corresponding transcripts, i.e., unless otherwise indicated, the term "lncRNA RP11-196G18.22" or "LETN" encompasses different isoforms.

By expression of lncRNA RP11-196G18.22 (LETN), it is meant expression at two levels thereof: one is expression at the DNA level; and secondly, expression at the RNA level.

The term "overexpression" refers to the fact that a gene may not be properly "turned off" or transcribed at a high rate when the strict control of gene expression (transcription) is disturbed. High-speed transcription results in the production of large amounts of mRNA. By "lncRNA RP11-196G18.22" or "LETN" in the present application is meant that the DNA or RNA expression level is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher than the control (normal or healthy tissue/cell), or even 4, 5, 6, 7, 8, 9, 10 times or more the expression level of the LETN in the control.

Techniques and reagents for detecting gene expression levels are well known to those skilled in the art. In the present application, it is preferred that the reagent is selected from the group consisting of a specific probe (preferably a nucleic acid probe, carrying a detection label, usually complementary to the gene of interest) of lncRNA RP11-196G18.22, a gene chip or PCR primers for a PCR specific amplification reaction.

The term "reducing or inhibiting lncRNA RP11-196G18.22 (LETN) expression" refers to reducing the expression level of lncRNA RP11-196G18.22 (LETN) to 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, or 10% or less, for example 5% or less, 2% or less, 1% or less, or even 0% of the original expression level. In one embodiment, expression of lncRNA RP11-196G18.22 (LETN) can be reduced or inhibited by gene knockout or knockdown.

The term "knockout" refers to a genetic engineering technique in which an endogenous normal homologous gene is replaced by homologous recombination using an exogenous mutated gene, thereby inactivating the endogenous gene to express the mutant's trait.

The term "knockdown" refers to the effect of preventing gene expression by degrading mRNA of a target gene having a homologous sequence. The double-stranded small RNA is utilized to efficiently and specifically degrade homologous mRNA in cells, so that the expression of target genes in the body is blocked, and the cells have a target gene deletion phenotype. It is different from gene knockout to make the target gene permanently expressed and silenced, but can be used for preventing gene expression by degrading mRNA of target gene with homologous sequence.

Techniques for gene knockout or knockdown are well known in the art and include, but are not limited to, retroviral gene transfer, resulting in mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations. Another way in which the gene can be knocked out is by using zinc finger nucleases. Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target DNA sequences of interest, which can target zinc finger nucleases to unique sequences in complex genomes. Other genomic customization techniques that can be used to knock out genes are TAL effector nucleases (TALENs). Another technique is the genome editing technique CRISPR/Cas system, which can be used to achieve RNA-guided genome engineering.

Techniques for achieving "reducing or inhibiting lncRNA RP11-196G18.22 (LETN) expression" may also include the use of gapmers, antisense RNA, siRNA, esiRNA, shRNA, miRNA, or RNA aptamers.

"antisense RNA" refers to RNA molecules that are complementary to mRNA, and also includes RNA molecules that are complementary to other RNAs. Since ribosomes are unable to translate double-stranded RNA, specific complementary binding of antisense RNA to mRNA inhibits translation of the mRNA. The antisense construct can be delivered, for example, as an expression plasmid that, when expressed in a cell, produces RNA that is complementary to at least one unique portion of cellular lncRNA RP11-196G18.22 (LETN).

Another particular form of antisense RNA strategy is gapmer. Gapmer is a chimeric antisense oligonucleotide comprising a central segment (central block) of deoxynucleotide monomers of sufficient length to induce cleavage by RNase H. The design and synthesis of gapmers is well known to those skilled in the art and can be accomplished by commercial companies (e.g., exiqon, isis pharmaceuticals).

"Small interfering RNAs (siRNAs), sometimes referred to as short interfering RNAs or silencing RNAs, are a class of double-stranded RNA molecules that are about 20-25 base pairs in length and function via an RNA interference (RNAi) pathway. It interferes with post-transcriptional degraded mRNA of a specific gene expressing a nucleotide sequence complementary to it, thereby preventing translation. The siRNAs of the present application can target any segment of about 19 to 25 contiguous nucleotides in the lncRNA RP11-196G18.22 (LETN) target sequence, examples of which are provided herein. Techniques for selecting target sequences for siRNA are well known in the art.

"short hairpin RNA" (short hairpin RNA, abbreviated shRNA) is an RNA sequence comprising two short inverted repeats that can silence gene expression via RNA interference (RNAi).

The full name of "esiRNA" is endonucleonucleotide-prepared siRNAs, which is an siRNA mixture generated by cutting long double-stranded RNA (dsRNA) by RNase III (a ribonuclease) of escherichia coli, has a length of 18-25bp, and can be used for efficiently knocking out the expression level of a target gene.

The present application is based on the following unexpected findings: i.e., lncRNA RP11-196G18.22 (LETN) can be used as a tumor marker and therapeutic target. Accordingly, the present application provides the use of a reagent for detecting the expression level of lncRNA RP11-196G18.22 (LETN) in the preparation of a diagnostic agent or diagnostic kit for cancer. The application also provides the use of an agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) in the manufacture of a medicament for treating cancer. In addition, the present application provides a method of screening for an anticancer agent comprising the step of determining whether a candidate compound is capable of reducing or inhibiting expression of lncRNA RP11-196G18.22 (LETN).

The application is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present application. The chemicals used in the following reactions are all commercially available products unless otherwise indicated.

Unpaired student's t-test was used as a statistical analysis in the present application. Statistical calculations were performed using Microsoft Excel. When P < 0.05, the P value is significant.

Example 1 screening of lncRNA RP11-196G18.22 (LETN)

In Hepatocellular Carcinoma (LIHC), we utilized data of cancer genomic map (TCGA)https:// www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) The function of the lncRNA is investigated, and the lncRNA with the functions of regulating and controlling the transcription factors and target genes thereof is predicted through an algorithm designed in the laboratory. We finally selected 22 lncRNAs (UBE 2SP2, BMS1P8, RP11-443P15.2, LINC 0196/DUXAP 10, LL22NC03-N14H11.1, RP11-284F21.10,DUXAP8, CRNDE, CTD-2227E11.1, LINC00853, LINC00665, RP11-196G18.22, GOLGA2P7, RP11-14N7.2, PVT1, LINC00511, RP11-396C23.2, MIR4435-2HG, AL450992.2, HCG25, PCAT6, LINC 00152) by comprehensive screening for factors that regulate how much, whether cancer differs from cancer to cancer and genomic copy number variation. Some of the non-specific lncRNA (whose sequence is entirely within the exons of a gene, for which no specific siRNA can be designed for functional verification) was removed, leaving 16 lncRNA, which we then experimentally phenotyped. Specifically, siRNA specific for its transcript was designed (see Table 1 below) by liposome Lipofectamine2000 The effect of (Thermo Fisher, 11668019) transfected liver cancer cell line HUH7 (Shanghai cell bank of the national institute) on cell proliferation was examined, and the result (see fig. 5) was found that only 7 lncRNA (DUXAP 8, PCAT6, LINC00511, LINC00152, RP11-198g18.22, pvt1, crnde, 7 lncRNA on the right in fig. 5) could inhibit proliferation of HUH7 cells, whereas only 1 out of these 7 lncRNA was not studied, i.e. lncRNA RP11-196G18.22 (we named LETN), which was the lncRNA that was found in the algorithm prediction before to regulate the transcription factor and the maximum number of target genes, suggesting that it may have a broad and powerful regulatory potential in the development of liver cancer. Based on the above findings, we analyzed the expression levels of LETN in liver cancer and paracancerous tissues in the TCGA database, and found that the expression levels of LETN in liver cancer tissues were far higher than those in paracancerous tissues; further, it was found that the expression level of LETN was higher in most cancers than in other cancer tissues (FIG. 1, A).

TABLE 1

EXAMPLE 2 cellular level study of lncRNA RP11-196G18.22 (LETN)

Two liver cancer cell lines, HUH7 (Shanghai cell bank in the Chinese sciences) and SMMC-7721 (Shanghai cell bank in the Chinese sciences), were selected, and by siRNA knockdown (siRNA primer shown below) of LETN (cells were inoculated in 35mm dishes and transfected after cell attachment, 250ml of medium was used to dilute 2ul of Lipofectamine 2000 (Thermo Fisher, 11668019) and 20nM siRNA, respectively, and then mixed evenly, and left to stand for 20min, and then slowly dropped into dishes containing cells, after 48 hours, the knockdown was detected by RT-qPCR (primer pair sequences see Table 2 below), the knockdown efficiency was shown in FIG. 4, A, and as a result, it was found that knockdown LETN significantly reduced proliferation of cells (FIG. 1, C) (siNC and siLMNA were two different negative controls).

siLETN-1Sense 5'-3'GCUGUCUCCAUGUCUUCUU(SEQ ID NO:34)

Antisense 5'-3'AAGAAGACAUGGAGACAGC(SEQ ID NO:35)

siLETN-2Sense 5'-3'GCUCUCUGCUCAAGUAUUA(SEQ ID NO:36)

Antisense 5'-3'UAAUACUUGAGCAGAGAGC(SEQ ID NO:37)

siNC(negative control)Sense 5'-3'ACGUGACACGUUCGGAGAA(SEQ ID NO:38)

Antisense 5'-3'UUCUCCGAACGUGUCACGU(SEQ ID NO:39)

siLMNA Sense 5'-3'AUCUCAUCCUGAAGUUGCUUC(SEQ ID NO: 40)

Antisense 5'-3'GAAGCAACUUCAGGAUGAGAU(SEQ ID NO:41)

TABLE 2

Because it takes two weeks to stay knocked down to detect colony formation, we constructed a LETN knockdown stable screen cell line (see example 3 for details). Knockdown levels were detected by RT-qPCR (primer pair sequences see table 3 below), knockdown efficiencies are shown in fig. 4, b, and as a result, knockdown of LETN was found to significantly reduce cell clonality (fig. 1, d, e). shNC is a negative control group.

TABLE 3 Table 3

/>

In addition, we also knocked out LETN by CRISPR-Cas9 technique (cells were inoculated in 35mm dishes and transfected after cell attachment, 250ml of medium was used to dilute 2ul of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 2ug of px458-sgRNA plasmid (the sgLETN sequence was added on the basis of px458 (adedge, category No. 48138) plasmid, specific methods were reference [5 ]), then mixed well, incubated for 20min at rest, then slowly dropped into the dishes containing cells, and the knockdown level was detected by RT-qPCR after 48 hours, knockdown efficiency was seen in FIG. 4, C. It was found that proliferation of cells was significantly inhibited (FIG. 1, B).

sgLETN-1Sense 5'-3'TCAAATTTCAGTCGGAACTC(SEQ ID NO：70)

sgLETN-2Sense 5'-3'GAGACGATATGCTACGGGTG(SEQ ID NO:71)

sgEV-1Sense 5'-3'GAACGTTGGCACTACTTCAC(SEQ ID NO:72)

sgEV-2Sense 5'-3'GCGCCTTAAGAGTACTCATC(SEQ ID NO:73)

Otherwise, LETN over-expression was performed (cells were inoculated in 35mm dishes and transfected after the cells were attached to the walls), 250ml of medium was used to dilute 2ul of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 2ug of LETN over-expression plasmid plv-LETN (constructed by Cellless blue Biotechnology Co., ltd.) respectively, which was then mixed uniformly, incubated for 20min at rest, and then slowly dropped into the dishes containing cells, and after 48 hours, the over-expression efficiency was examined by RT-qPCR (see FIG. 4, D.) it was found that proliferation and clone formation of the cells were promoted (FIG. 1, F, G).

Subsequently we selected the lung cancer cell line HCC 827%CRL-2868) and the prostate cancer cell line DU145 (++>HTB-81)、PC3(/>CRL-1435), a similar effect was also found when the proliferation rate of cells was significantly reduced and the clonogenic capacity was also destroyed after knocking down LETN with siRNA (the procedure was as previously performed in the liver cancer cell line HUH7, see FIG. 4, A).

EXAMPLE 3 animal level study of lncRNA RP11-196G18.22 (LETN)

We further explored the effect of LETN on the tumorigenicity of tumor cells. Firstly, constructing a LETN knockdown stable sieve cell strain by using shRNA packaged by viruses (shRNA sequences are shown in a table 3. Firstly, packaging viruses, then, slowly dripping 293T cells (Shanghai cell bank of China) into a 100mm dish, carrying out transfection on the next day, taking 1ml of culture medium to dilute 12ul liposome Lipofectamine 2000 (Thermo Fisher, 11668019), taking 1ml of culture medium to dilute packaging vectors 7.1ug delta 8.9 (Qinghai university library platform) and 3.55ug VSVG (Qinghai university library platform), and expression vectors 3ug plv-LETN, then, uniformly mixing, standing and incubating for 20min, then, slowly dripping the 293T cells (Shanghai cell bank of China) into a culture dish containing cells, supplementing the culture medium to 10ml, centrifuging at 3000rpm for 10min, namely virus liquid, and sub-packaging to 80 ℃ for standby. Tumor removal measures the weight and volume, and as a result, the LETN knockdown cells were found to form significantly smaller tumor masses than the control group, and the tumor volumes and weights were much smaller than the control group (FIG. 1, H). Correspondingly, we selected HUH7 to construct a stable screen cell strain with over-expressed LETN, injected the same procedure was performed under the skin of the above athymic nude mice, and after 5 weeks of incubation, it was found that the cells with over-expressed LETN formed a significantly higher tumor mass than the control group, and the tumor volume and weight were also significantly larger than those of the control group (fig. 1, i). In a word, through detailed experimental verification of tumor indexes such as cell proliferation, clone formation, subcutaneous tumor formation and the like, the lncRNA LETN is considered to have obvious function of inhibiting occurrence and development of liver cancer, and is a potential therapeutic target.

EXAMPLE 4 investigation of the mechanism of action of LETN

LETN was first identified as being predominantly localized in the nucleus and distributed in clusters by RNA in situ hybridization [6] and Nuclear cytoplasmic separation techniques (Nuclear/Cytosol Fractionation Kit (Biovision, K266-25)). Indicating that LETN is likely to function through binding proteins. And carrying out mass spectrometry on the pulled protein through RNA pull down to find out the protein interacted with LETN, and finally finding out that NPM1 is a functional protein combined with the protein. Subsequently we performed pull down by RNA pull down technique [7] using the NPM1 antibody Anti-NPM1 (Abcam, ab 10530), and also found that NPM1 was indeed able to pull down lncRNA LETN. Cell fluorescence co-localization experiments also further demonstrated that LETN and NPM1 bound to each other and were localized in nucleoli.

NPM1 is a very important nucleolar function protein, which can bind to rDNA promoter to promote rDNA transcription; participating in rRNA shearing and maturation; binding to histones, involved in nucleosome assembly, and the like. We found that LETN did also participate in these functions, and that expression of various rRNAs was significantly reduced after knockdown of LETN, and overexpression of LETN promoted expression of rRNAs. In addition, knocking down LETN can also decrease the binding capacity of NPM1 to histone, thereby affecting nucleosome assembly. The kernel is composed of three basic structural components, namely a fiber center, a dense fiber component and a particle component from inside to outside. NPM1 is mainly located in the outermost layer and is the most important component of the particle component, and studies have shown that when NPM1 is knocked down, the morphology of nucleolus can be destroyed.

We have found that the regularly dense spheres of the nucleolus become irregularly scattered when the LETN is knocked down, suggesting that the LETN also affects the nucleolus structure. According to the clinical data analysis survival analysis of liver cancer patients in the TCGA database by using an algorithm, the clinical data analysis survival analysis is independently divided into an LETN high expression group and a LETN low expression group or an NPM1 high expression group and an NPM1 low expression group, and the prognosis survival time of the NPM1 or LETN high expression group patients is shorter than that of the low expression group patients; when the four groups NPM1-low+LETN-low, NPM1-low+LETN-high, NPM1-high+LETN-low and NPM1-high+LETN-high are further subdivided, the survival time of the patients with high expression of NPM1 and LETN is far lower than that of the patients with low expression of NPM1 and LETN.

It will be appreciated by persons skilled in the art that although the application has been specifically described with reference to the above embodiments, the application is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present application, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present application, and the equivalent embodiments thus obtained are within the scope of the present application.

Reference to the literature

1.Schmitt AM,Chang HY:Long Noncoding RNAs in Cancer Pathways.Cancer Cell 2016,29(4):452-463.

2.Bray F,Ferlay J,Soerjomataram I,Siegel RL,Torre LA,Jemal A:Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185countries.CA:A Cancer Journal for Clinicians 2018, 68(6):394-424.

3.Scudellari M:Drug development:Try and try again.Nature 2014,516:S4.

4.Box JK,Paquet N,Adams MN,Boucher D,Bolderson E,O'Byrne KJ,Richard DJ: Nucleophosmin:from structure and function to disease development.BMC Mol Biol 2016,17(1):19.

5.Ran FA,Hsu PD,Wright J,Agarwala V,Scott DA,Zhang F:Genome engineering using the CRISPR-Cas9 system.Nat Protoc 2013,8(11):2281-2308.

6.Kawaguchi T,Tanigawa A,Naganuma T,Ohkawa Y,Souquere S,Pierron G,Hirose T:SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies.Proceedings of the National Academy of Sciences of the United States of America 2015,112(14):4304-4309.

7.Li X,Wang X,Song W,Xu H,Huang R,Wang Y,Zhao W,Xiao Z,Yang X: Oncogenic Properties of NEAT1 in Prostate Cancer Cells Depend on the CDC5L-AGRN Transcriptional Regulation Circuit.Cancer Res 2018, 78(15):4138-4149。

Claims (4)

1. Use of an agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) in the manufacture of a medicament for treating cancer, wherein

1) The cancer is liver cancer and the agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) is selected from the group consisting of: gapmer, TALEN and zinc finger nucleases, or

2) The cancer is selected from lung cancer and prostate cancer, and the agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) is selected from the group consisting of: antisense RNA, shRNA, TALEN, CRISPR and zinc finger nucleases.

2. The use of claim 1, wherein the cancer is selected from lung cancer and prostate cancer and the agent that reduces or inhibits expression of lncRNA RP11-196G18.22 (LETN) is gapmer.

3. The use according to claim 1, wherein the nucleotide sequence of lncRNA RP11-196G18.22 is shown in SEQ ID No. 1.

4. The use of any one of claims 1 to 3, wherein the medicament further comprises an additional anti-cancer agent, wherein the additional anti-cancer agent is a chemotherapeutic agent, wherein the chemotherapeutic agent is an agent for reducing or inhibiting NPM1 expression or mutation or an agent that inhibits the binding of LETN to NPM 1.