LONG NON-CODING RNA LETN SERVING AS TUMOR MARKER AND THERAPEUTIC TARGET POINT
20230129013 · 2023-04-27
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
C12N15/1135
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
A61K31/713
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a long non-coding RNA (LETN) useful as a diagnostic and therapeutic target for cancer. In particular, the invention discloses that lncRNA RP11-196G18.22 (LETN) is overexpressed in cancer cells, and such overexpression can promote the proliferation of cancer cells and are associated with short prognostic survival time in cancer patients. Reducing the expression of this lncRNA results in the inhibition of cancer cell growth, and thus inhibiting the expression of this lncRNA represents a new strategy for cancer therapy
Claims
1. A method for diagnosing cancer in a subject, the method comprising: detecting the expression level of lncRNA RP11-196G18.22 (LETN) in a sample from a subject, wherein relative to a control (healthy or normal sample), the overexpression of LETN in the subject's sample is indicative of the subject suffering from a (high) risk of cancer or suffering from cancer.
2. The method according to claim 1, wherein a probe, gene chip, or PCR primer specific for lncRNA RP11-196G18.22 is used for detecting the expression level of lncRNA RP11-196G18.22 (LETN).
3. The method according to claim 1, wherein the cancer is a solid tumor, preferably selected from the group consisting of liver hepatocellular carcinoma, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, gastric cancer, soft tissue cancer, biliary cancer, bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.
4. The method according to claim 1, wherein the lncRNA RP11-196G18.22 has a nucleotide sequence as shown in SEQ ID NO: 1 (Ensembl accession number: ENST00000564237.1).
5. A method for treating cancer, the method comprising administrating to a subject in need thereof an effective amount of an agent for reducing or inhibiting the expression of lncRNA RP11-196G18.22 (LETN).
6. The method according to claim 5, wherein the agent for reducing or inhibiting the 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 nuclease.
7. The method according to claim 5, wherein the cancer is a solid tumor, preferably selected from the group consisting of liver hepatocellular carcinoma, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, gastric cancer, soft tissue cancer, biliary cancer, bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.
8. The method according to claim 5, wherein the lncRNA RP11-196G18.22 has a nucleotide sequence as shown in SEQ ID NO: 1 (Ensembl accession number: ENST00000564237.1).
9. The method according to claim 5, wherein the method further comprises administrating to the subject an additional anticancer agent such as chemotherapeutic agent, for example, an agent for reducing or inhibiting the expression or mutation of NPM1 or an agent for inhibiting the binding of LETN to NPM1.
10. A method for screening anticancer drugs, comprising the following steps: 1) determining the expression level of lncRNA RP11-196G18.22 (LETN) in cells overexpressing lncRNA RP11-196G18.22 (LETN); 2) contacting a candidate compound with the cells of step 1); 3) determining the expression level of lncRNA RP11-196G18.22 (LETN) in the cells after step 2); and 4) comparing the expression levels of lncRNA RP11-196G18.22 (LETN) determined in step 1) and step 3), wherein a reduced expression level of lncRNA RP11-196G18.22 (LETN) is indicative of the candidate compound having anticancer potential, preferably the cells are cancer cells.
11. A method of identifying whether a tumor is susceptible to treatment with an inhibitor of LETN expression, comprising the following steps: 1) determining whether the expression of LETN in a tumor or tumor cell sample is increased relative to a control (normal or healthy tissue/cell); 2) determining whether the tumor is susceptible to treatment, wherein the increased expression is indicative of being susceptible to treatment with an inhibitor of LETN expression.
12. The method according to claim 11, wherein the cancer is a solid tumor, preferably selected from the group consisting of liver hepatocellular carcinoma, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, gastric cancer, soft tissue cancer, biliary cancer, bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.
13. The method according to claim 11, wherein the lncRNA RP11-196G18.22 has a nucleotide sequence as shown in SEQ ID NO: 1 (Ensembl accession number: ENST00000564237.1).
14. A method of evaluating the effect of an agent in the treatment and/or prevention of cancer, wherein the method includes testing whether the agent can reduce the expression of LETN in a tumor or tumor cell sample, and if so, the agent is suitable for the treatment and/or prevention of cancer.
15. The method according to claim 14, wherein the cancer is a solid tumor, preferably selected from the group consisting of liver hepatocellular carcinoma, lung cancer, prostate cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, gastric cancer, soft tissue cancer, biliary cancer, bladder cancer, rectal cancer, endometrial cancer, head and neck cancer, colon cancer, esophageal cancer, and thyroid cancer.
Description
DESCRIPTION OF THE DRAWINGS
[0030] The above-mentioned features and advantages of the invention will become more apparent from the detailed descriptions below in conjunction with the accompanying drawings, wherein:
[0031] The results shown in A to J in
[0032] A shows the expression status of LETN in various cancers and related paracancerous tissues in the TCGA database (CHOL: biliary cancer; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; KIRC: renal clear cell carcinoma; BLCA: bladder cancer; BRCA: breast cancer; PRAD: prostate cancer; READ: rectal cancer; LUSC: lung squamous cell carcinoma; UCEC: endometrial cancer; PAAD: pancreatic cancer; HNSC: head and neck squamous cancer; KIRP: papillary renal cell carcinoma; COAD: colon cancer; STAD: gastric cancer; SARC: soft tissue cancer; ESCA: esophageal cancer; THCA: thyroid cancer; THYM: thymic cancer; KICH: renal chromophobe cell carcinoma; PCPG: adrenal carcinoma; CESC: cervical squamous cell carcinoma), and it is observed that the expression of LETN in most cancer tissues (solid tumors) in humans is higher than that in the corresponding paracancerous tissues; B shows the CRISPR-Cas9 knockout of LETN in the HUH7 cell line, and it is observed that the proliferation rate of cells in the LETN knockout group (sgLETN) is much lower than the control (sgEV); C shows the knockdown of LETN in liver hepatocellular carcinoma cell lines HUH7 and SMMC-7721, lung cancer cell line HCC827, and prostate cancer cell lines PC3 and DU145, respectively; and it is detected that the proliferation rate of cells in the LETN knockdown group is much lower than that of cells in the control group (siNC and siLMNA are two different negative controls; to prevent off-target effects, two siRNAs are designed for LETN knockdown: siLETN-1 and siLETN-2); D and E show that after the stable knockdown/knockout of LETN in HUH7 and HCC827 cells, the colony formation ability of cells is significantly destroyed; F shows the overexpression of LETN in liver hepatocellular carcinoma cell lines HUH7 and SA/MC-7721, and it is detected that the proliferation rate of cells in the LETN overexpression group is much higher than that of cells in the control group; G shows that after the stable overexpression of LETN in HUH7 and SMMC-7721 cells, the colony formation ability of cells is significantly enhanced; H shows that after the stable knockdown of LETN in the liver hepatocellular carcinoma cell line HUH7, it is observed that the subcutaneous tumorigenic ability of the LETN knockdown group is significantly reduced; I showed that after the stable overexpression of LETN in the liver hepatocellular carcinoma cell line HUH7, it is observed that the subcutaneous tumorigenic ability of the LETN overexpression group is significantly enhanced (LETN-OE group is the LETN overexpression group, and EV group is the control group).
[0033] The results shown in A to E in
[0034] Specifically, A shows that it is found in the in situ hybridization experiment that LETN is mainly located in the nucleus and appears in clusters; B shows that it is demonstrated in the nucleocytoplasmic separation experiment that most of LETN is located in the nucleus, and GAPDH and LaminA/C are markers for cytoplasm and nucleus, respectively; C shows that through the mass spectrometry analysis of proteins interacted with LETN, it is found that NPM1 is the protein with the strongest binding ability in both experiments; D shows that it is further confirmed by the cellular fluorescence co-localization experiment that LETN binds to NPM1 and is localized in the nucleolus; E shows the pull down of RNA by NPM1 in the formaldehyde crosslinked or uncrosslinked state, and it is also found that NPM1 can indeed pull down the lncRNA LETN (MALAT1 in the figure represents the negative control).
[0035] The results shown in A to C in
[0036] Specifically, A shows that the respective knockdown of LETN or NPM1 in HUH7 and HCC827 cell lines can significantly reduce the expressions of various rRNAs, and the functions of LETN and NPM1 are consistent; B shows that the knockdown of LETN can attenuate the binding ability of NPM1 to histones, thereby affecting the assembly of nucleosome; C shows the survival time analysis of the clinical data of liver hepatocellular carcinoma patients in the TCGA database, wherein the prognostic survival time of patients individually divided in LETN or NPM1 high expression group is shorter than that of patients in low expression group; and when further subdivided into four groups: NPM1-low+LETN-low, NPM1-low+LETN-high, NPM1-high+LETN-low, and NPM1-high+LETN-high, it is found that the survival time of patients with high expressions of both NPM1 and LETN is much shorter than that of patients with low expressions of both NPM1 and LETN.
[0037] The results shown in A to D in
[0038] A shows the knockdown of LETN by siRNA in five cell lines, wherein the knockdown efficiency is detected by RT-qPCR; B shows the knockdown of LETN by lentiviral shRNA in two cell lines, wherein the knockdown efficiency is detected by RT-qPCR; C shows the knockout of LETN by CRISPR-Cas9 technology, wherein the knockdown efficiency is detected by RT-qPCR; D shows the overexpression of LETN by the lentiviral overexpression system in two cell lines, wherein the overexpression efficiency is detected by RT-qPCR.
[0039]
DESCRIPTION OF EMBODIMENTS
[0040] Unless otherwise indicated, the terms used herein have ordinary technical meanings as understood by those skilled in the art. For definitions and terms in the art, the skilled artisan is specifically referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).
[0041] The term “long non-coding RNA” (abbreviated as lncRNA) refers to the transcripts longer than 200 nucleotides that do not encode proteins.
[0042] The term “lncRNA RP11-196G18.22” or “LETN” refers to the gene with Ensembl accession number: ENST00000564237.1 and mRNAs transcribed from this gene. Since it is a non-protein-coding gene, there is no protein product. In present invention, its sequence can be represented by SEQ ID NO: 1 (shown below). The invention also contemplates variations in the non-coding exons that may exist in the above-mentioned gene, which variations are considered to belong to the corresponding transcripts; that is, unless otherwise specified, the term “lncRNA RP11-196G18.22” or “LETN” encompasses different isotypes.
TABLE-US-00001 (SEQ ID NO: 1) AGAGTTCCGACTGAAATTTGAGAAGCTCTTTGCTATTCAAGTGGATATGT GCAGTTGACAGTTTGAGAGATGCATCTAGGGTTCAGTAAAGACAACACAA GCCTGTCTTTAGGGTCTACCTGTGAACTGTGAACACAGCAATGAGAATGA TGGACATCACCTTTAAGTATTTTTCTAGACTTTATTACTCATGTGTTTGT CATGAGGTGTAACTTAGTAGTTCATAGTCCTATAATGTATGTTATTGACT AGGTAGCATTTATTTTTCTAATTGTTTCTGTTATAGTGCTGCCACATGTG TTTCCCAGAAACGCATTTTACCCACAGTTCTTAGGGTTGGCCTGATTAGT TTAATTGCTGTCTGAACCTGCTTCTTACTGTGATTAGTTCAGGAATCTAG ATCAAACTCATTGGCATTTAACATTTCAGGAAGTGAACTGAGTAACAACT AACTCAGCAGGGGAGTGTAGTATGCTATTATCTTTTGGGAAAGCAGCTTA TTTGCTTTCAAGAGGCAGCAGGAGGATGGACTGTCTTTAATGGAGTTCAG GTATGAAGGCAAGAATGATTATAGACAATATGCAGAGGAGGACACAGTGT GGGAGAATCAGGGAACTGAGCCACTCGCCAACCTTGGATCCCTGCTGCAC CTTTGGACTTCCAGTTAAGCCAATTTGTCTGACATATTTACTTATACCAG TTTGAATCTTGAAATATTTCAGGAATAATAATTTCCTAGATAAAAGGAAA GACCTTTCATGAAAGGTCTCAAGTCAAATAGGGTCAATTAGGACAGAGTT GCTCCAATTACATATTTGGAACAGATGTCCAAATGTTAATACTTGACTAA GGCTAAAGACTAATATTACCATCACAGGAAAAATGTCCAGGGTTTTTTTT CAGATGTGAAATTTTATTTAAAAATTTTAAATAAACTAAATCAAAAAATT TTAGTAGTTGTACTAATTTCCTGGGGCTGTCAAAGTACCACAAACTGTAT GGCGTAAAACAACACAAAGTTATTCTTTCATGGTTTTAGAGGCTAGAAGT GTTGAAATCAACGTGTTGGTAGGGCCATCTCTCTCCAAACCCACTAGGGG AAGACTCCTGTCTTTCAGTGTCTGGTAGCCCCACTTGTTCTTTGGTTTCT GGCAGCATAACTGTAATCTCTACCTCAGTTTTTTCATGTATGTCTCCATG TTTTTTTACTTTCTTTCTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCT GGAGTGCAGTGGCATGATCTTGGCTTACTGCAACCTCTGTGCCCCGGGTT CAAGCAATTTTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGCATG CGTCAGCACGCCCGGCTGATTTTGTATTTTTGGTAGAGATGGAGTTTCAT CATGTTAGTCAGGCTGGCCTCGAACTGACCTCAGGTGATCCACCTGCCTT GGCCTCCCAAAGTGCTGGGATTACAGATGTGAGCCACTGCACCCGGCTGT CTCCATGTCTTCTTATAAGGGTATCAGTCATACTGGATTAGGGCCCACCC TAAAGACCTCATTTTAACTTGATTACCTCTGTAAAGACCCTGTTTCCAAA GAAGGCAAAATTCTAAGCAACTAGGGGTTAGACTTCAACATATCTTTCGG GGGGACACAACTCAACCCATAACAGTAGTCAATGGCTGTGGCAGGCTAAA TGTGGCTCCCAAATATGTCCATATCCTAATCCCTACAGCCTGTGAATATT ACCTTATATAGCCAAGAGGATTTTGCAGATGTGATTCTGAGATTGAGAGA TTATGCCAGATTATCCAGGTAGGCCCCAAATGTAATCACCACAGTCCTTA TAGGAGAGGCAAGAAAGTCAAGTGTAGAAGGAGGCGATAGAAGGAGAGAG GGATTTGAAGATTAATAGGCTGCTTGCTTTGAAGACAGAGGGAAGGGACC ATAAACCAGAAATAAACCTCTAGAAGCTGGAAAAGGCATGGAAATAGACC CTCCCTTAAGGTCTCTGGAGGGAGTGCAGCCTTGATTTCTACCGAGTAAA ATTGATTTTGTACTTCAGACCTCCAAAACTGTAAGAGAATGACTGTTGTT TTAAAACCATTGAGTTTGTAGTAATTTGTTGCAGCAGCCACAAGAAACTA ATACAACATCTATATAGAATTTTTTCAATAATTGGAGAAATTTGAATATG GATTGCATATTAATATTACTGAATCAGCATTAAATTTGTTAGGTGTAATA ATGTGATTGTAGCTATTTAGGAGAATATCCTATTTTTAAGAGACATGCCA CCATATTTAGGGAGAAGTGCCAACATATTTGCAGTTTATTTTCAAATGGT TCAGAGGCTGTCTGTGTACATGAGAAGACAAAGATAAGGCAAATGCAGCA AAATTGTAATAATTGGTGAATCCAGGTGAAGGGACTATGGCTGGTCTTTG TACTTTTTTTTCCAACTTTTCTGTAGGTTTAAAATTTTCAAAATAAAAAA ATGGGAAATACTTTAAAAATTGTAATCAAAGACATTAGTACAGAAACTTT CATAATGTATTTTATTTTTACAGTAAAATTAATTTATGTAAATTGATAGA ATTTTACTAATTTCACTCCCAAGTTACATTAAAAGGCTTACATATGTTTG ATAATAGCATATGTAAACTAGAACTCTGAATGATATCCATTGGTCATAAT ACGTACTATGTAGCGGTAATGGTGACTTTTGTGATTGCACAAGTCTAGAG ATGCCCCAAATGACATTGACTTAGACATCTGGTTATTCTAAGGCTGAAAC TGAAGTTGAATAGAAGGTTTTAGTCAAATACTGAGATGAAAACTGAGGCA GTCCTGGCGGGGGGGAGTGAGTGTGTGTGTATATATACACACATAGACAT CATGCTTCTAAACATTTACAGAAAGAAAGGGTAGATTATCTACAAAAAAA TAAGAATCAGACTGATATGAGATCTTACAAACCTAACCCCCTTCTCTTTC CTAAACTCCAGATTCTCATATTTCTGACTTCCTATTTGATATTTACACTT CGATATTTACCAGGAGTCTTCAACATTTTGTTCAAAACAGTACTCTTGGT TTTCTTCCTCCAAGACTACTCCTTACTCATATCAGCAAATAGCAGCTCTT TTCAAGTGCTCAGTGTAAAAACCTACAATTAATCCTTGATTTCTCTTTCA GTCAGCCTATACTAAATCAATTTCATTTAAAATATCTCGGCTACTACTCT GCATCTCCACTGCTACCATCGGCCTCTCCAGTCACATTCTCCAAGAGCAC TCTATCTCATTTAAAAGACAAAATCTCTGCAGTGGCCTGTGATGCTCCTT AATGGCCTACATAATCCAGCCCTCAAGCACCTCCGTGATCTCTGTAAAAC TTTCCCTTGGTCACTGTGCTTCAGCCACATTAACCAGCTTGCATATTTCT CACATTCACCAAGCTTGTTCCTGCCTTGGGGCCTTTGTACTTACCATGTT CTGTTCTGAGAATACTCTGCCTCAAGATATCCTACAACTATCTTACTGTA TTCAGCTCTCTGCTCAAGTATTAACTGATGAAACCTGTCATCCCTACTCC ACTCCATGTTCTGCTTTACTTAACAGCAATTGCACATATGGCCCCCTGAA TAATATACATTTAGTCACTTATTTTTACTTATCTGCTAATTAAAATGTAG ACTTTTTCTATTCTGTTTACTGCTGTATTCCCAGCATGTTTTATCCGAAT GTGCAGTGGTTTCTTTTCTTCTCCCTTATCGTGGGAAGTGATGTGCACAA ATACACATAATGGAGCCTGAATGTCATATTGCTTTCATACCTGTGTGAAT TTTGGTAAGAAAGGAAAAGTAGCGATTGACAGGTAATATAATTACATTAA GTCACTCTCATAGTTAGCTGTTTATTGCTTTCCTGCTCTTATTCTCAGTC CCCAGGACCAAATGTTGACCACTACCTTCCCCCACATATAATTAGGTTAT TTACCGAACGCCATGCAGGTGGCTGTTAAAAGGAAGATATATACTTACCT TATAAACTCAACTTTTCCCTGTTGTCTTTCTGTCTCACCCCTACCTCCAT GCTTTAAATTAACTTTTCAGGCTTAGGCCTTATCTCTCAGTAGAGCCATA TAAGGTATGTGTAAAAGCAGGAAAATGTTTCCTGGGGATGAAGCTTTGAA AAGCTTTTTTTTTTTTTTCTTTTGGCAATAAAATAAGGTAGATTCAGCAC AATACCTAATAACTAAAAAATCTGTTTTTAATTGGGTGGGGCAGACAGCA AGTGTGTCATCCTGGAAGATACTATTTGGGATTTTATGTAGGTACATAAG AGAAAAAAGTGAACAAAAGCAAGGGGCTACCAGGACGCCGCAGTATGCTT AACATGTATTTTCTAAGTTTGTATTATGCCTTTATCTTGGTACTTTTATC TTCTGTTCTCACTTGATCTTTTTGAAATGTATTTTAAATCCTAATAAAAA TATATAAAGTCTGGAATTAATAAAGGA
[0043] Regarding the expression of lncRNA RP11-196G18.22 (LETN), it means expressions at two levels: one involves the expression at the DNA level; and the other involves the expression at the RNA level.
[0044] The term “overexpression” refers to when the strict control of gene expression (transcription) is disrupted, a gene may be inappropriately “turned off”, or be transcribed at a high rate. The transcription at a high rate results in the production of a large amount of mRNA. For the overexpression of “lncRNA RP11-196G18.22” or “LETN” of the invention, it means that its DNA or RNA expression level is higher than that of the control (normal or healthy tissue/cell) by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or 300%, or even 4, 5, 6, 7, 8, 9, 10 times or more higher than the expression level of LETN in the control.
[0045] The techniques and reagents for detecting the gene expression level are well known to those skilled in the art. In the invention, the reagent is preferably selected from a specific probe (preferably a nucleic acid probe with a detection label, usually complementary to the target gene), gene chip, or PCR primer used in the PCR specific amplification reaction for lncRNA RP11-196G18.22.
[0046] The term “reducing or inhibiting the expression of lncRNA RP11-196G18.22 (LETN)” 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, e.g., 5% or less, 2% or less, 1% or less, or even 0% of the original expression level. In one embodiment, the expression of lncRNA RP11-196G18.22 (LETN) can be reduced or inhibited by gene knockout or knockdown.
[0047] The term “knock out” refers to a genetic engineering technique in which an exogenous mutated gene is used to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene and exhibiting the performances of mutant.
[0048] The term “knock down” refers to the effect of preventing the gene expression by degrading the mRNA of a target gene with a homologous sequence. It efficiently and specifically degrades the homologous mRNA in cells using a double-stranded small RNA, thereby blocking the expression of the target gene in vivo and presenting the cell phenotype with target gene deletion. It prevents the gene expression by degrading the mRNA of the target gene with homologous sequence, different from the gene knockout which permanently silences the expression of the target gene.
[0049] The techniques for gene knockout or knockdown are well known in the art, and include but are not limited to the gene transfer by retrovirus and the generation of mutation, such as point mutation, insertion, deletion, frame shift, or missense mutation. Another means for gene knockout is by the use of zinc finger nuclease. The zinc finger nuclease (ZFN) is an artificial restriction enzyme produced by fusing the zinc finger DNA binding domain with the DNA cleavage domain. The zinc finger domain can be engineered to target the DNA sequence of interest, allowing the zinc finger nuclease to target the unique sequence in the complex genome. Other genome customization techniques useful for the gene knockout include TAL effector nucleases (TALENs). Another technique involves CRISPR/Cas system for genome editing, which can be used to achieve the RNA-guided genome engineering.
[0050] The techniques to achieve “reducing or inhibiting the expression of lncRNA RP11-196G18.22 (LETN)” may also include the use of gapmer, antisense RNA, siRNA, esiRNA, shRNA, miRNA, or RNA aptamer.
[0051] “Antisense RNA” refers to an RNA molecule complementary to mRNA, and also includes RNA molecules complementary to other RNAs. Since the double-stranded RNA cannot be translated in the ribosome, the specific complementary binding of antisense RNA to mRNA inhibits the translation of the mRNA. An antisense construct can be delivered, for example, as an expression plasmid that, when expressed in a cell, produces RNA complementary to at least a unique portion of cellular lncRNA RP11-196G18.22 (LETN).
[0052] Another specific form in the antisense RNA strategy is the gapmer. The gapmer is a chimeric antisense oligonucleotide containing a central block of deoxynucleotide monomer with a sufficient length to induce the cleavage by RNase H. The design and synthesis of gapmer are well known to those skilled in the art and can be accomplished by commercial companies (e.g., Exiqon, Isis pharmaceuticals).
[0053] “Small interfering RNA (siRNA),” sometimes referred to as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, with approximately 20-25 base pairs in length, that function by the way of RNA interference (RNAi). It interferes with the expression of mRNA post-transcriptionally degraded for the specific gene with complementary nucleotide sequence, thereby preventing translation. The siRNA of the invention can target any segment of about 19 to 25 continuous nucleotides in the lncRNA RP11-196G18.22 (LETN) target sequence, examples of which are provided in this application. The techniques for selecting the target sequence for siRNAs are well known in the art.
[0054] “Short hairpin RNA” (abbreviated as shRNA) is an RNA sequence comprising two short inverted repeat sequences that can silence the gene expression via RNA interference (RNAi).
[0055] The full English name of “esiRNA” is endoribonuclease-prepared siRNA. It is a mixture of siRNAs generated by cleaving long double-stranded RNAs (dsRNAs) by RNase III (ribonuclease) of E. coli, with a length of 18-25 bp, and can be used to efficiently knock down the expression level of the target gene.
[0056] The invention is based on the unexpected finding that lncRNA RP11-196G18.22 (LETN) can be used as a tumor marker and a therapeutic target. Thus, the invention provides the use of an agent for detecting the expression level of lncRNA RP11-196G18.22 (LETN) in the manufacture of a diagnostic agent or a diagnostic kit for cancer. The invention also provides the use of an agent for reducing or inhibiting the expression of lncRNA RP11-196G18.22 (LETN) in the manufacture of a medicament for treating cancer. In addition, the invention also provides a method for screening anticancer drugs, comprising the step of determining whether the candidate compound can reduce or inhibit the expression of lncRNA RP11-196G18.22 (LETN).
[0057] The invention is further illustrated in Examples below. These Examples are for illustrative purposes only and are not intended to limit the scope of the invention. The chemicals used in the reactions below are all commercially available products, unless otherwise indicated.
[0058] The unpaired student's t-test is used for statistical analysis in the invention. Statistical calculations are performed using Microsoft Excel. When P<0.05, P value is significant.
Example 1 Screening of lncRNA RP11-196G18.22 (LETN)
[0059] For the liver hepatocellular carcinoma (LIHC), lncRNA functions were investigated using the data of the Cancer Genome Atlas (TCGA) (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga), to predict lncRNAs with the functions of regulating transcription factors and their target genes through an algorithm designed in the laboratory. Through the comprehensive screening based on three factors: the regulation logarithm, the presence or absence of a difference in expression between cancer and paracancerous tissue, and the variation in genome copy number, 22 lncRNAs were finally selected (UBE2SP2, BMS1P8, RP11-443P15.2, LINC01296/DUXAP10, 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, LINC00152). After removing several lncRNAs with non-specific sequences (the sequences are completely located in the exon of a gene, and specific siRNAs cannot be designed for functional verification), there remained 16 lncRNAs, and then the phenotypes of these lncRNAs were verified through experiments. Specifically, siRNAs specific for its transcript were designed (see Table 1 below), and the liver hepatocellular carcinoma cell line HUH7 (Shanghai Cell Bank, Chinese Academy of Sciences) was transfected by liposome Lipofectamine 2000 (Thermo Fisher, 11668019), to detect the effect of the knockdown of corresponding lncRNA on the cell proliferation. It was found that (see
TABLE-US-00002 TABLE 1 siRNA Sense 5′-3′ Antisense 5′-3′ siRP11- CCAGUUCUCCUAAA AAGCAUUUAGGAGAACUGG 284F21.10 UGCUU (SEQ ID NO: 3) (SEQ ID NO: 2) siLL22NC03- GAGCCUUUCCUGCC AAGCGGGCAGGAAAGGCUC N14H11.1 CGCUU (SEQ ID NO: 5) (SEQ ID NO: 4) siLINC00853 GCUCUCAUCAAUCU AGCGUAGAUUGAUGAGAGC ACGCU (SEQ ID NO: 7) (SEQ ID NO: 6) siCTD- GUGUCUCUGGGAAG AAGGGCUUCCCAGAGACAC 2227E11.1 CCCUU (SEQ ID NO: 9) (SEQ ID NO: 8) siAL450992.2 GGAGUCAUGUGUUG AUCUCCAACACAUGACUCC GAGAU (SEQ ID NO: 11) (SEQ ID NO: 10) siRP11- CUCCACAUAUGAAAC AAGGGUUUCAUAUGUGGAG 14N7.2 CCUU (SEQ ID NO: 13) (SEQ ID NO: 12) siLINC00665 CAGAUGAAGGUAUAU AAUGAUAUACCUUCAUCUG CAUU (SEQ ID NO: 15) (SEQ ID NO: 14) siRP11- GCCGUCGCCGCAGCU AAGGAGCUGCGGCGACGGC 396C23.2 CCUU (SEQ ID NO: 17) (SEQ ID NO: 16) siHCG25 CCACUCUCAGCCUCA UUCCUGAGGCUGAGAGUGG GGAA (SEQ ID NO: 19) (SEQ ID NO: 18) siDUXAP8 CCUGGUAAGGCUUGA AACUUCAAGCCUUACCAGG AGUU (SEQ ID NO: 21) (SEQ ID NO: 20) siPCAT6 CUCCAAUUCAAUGCC UGAUGGCAUUGAAUUGGAG AUCA (SEQ ID NO: 23) (SEQ ID NO: 22) siLINC00511 CUCCCAGAAUGCCAG AAUCCUGGCAUUCUGGGAG GAUU (SEQ ID NO: 25) (SEQ ID NO: 24) siLINC00152 CCGUCUGCAUCCCUC AUUCGAGGGAUGCAGACGG GAAU (SEQ ID NO: 27) (SEQ ID NO: 26) siRP11- GCUGUCUCCAUGUCU AAGAAGACAUGGAGACAGC 196G18.22 UCUU (SEQ ID NO: 29) (SEQ ID NO: 28) siPVT1 GGACUUGAGAACUGU AAGGACAGUUCUCAAGUCC CCUU (SEQ ID NO: 31) (SEQ ID NO: 30) siCRNDE UGUUGAAAUGAAAAA AAUAUUUUUCAUUUCAACA UAUU (SEQ ID NO: 33) (SEQ ID NO: 32)
Example 2 Study of lncRNA RP11-196G18.22 (LETN) on the Cellular Level
[0060] Two liver hepatocellular carcinoma cell lines were selected, namely HUH7 (Shanghai Cell Bank, Chinese Academy of Sciences) and SMMC-7721 (Shanghai Cell Bank, Chinese Academy of Sciences), and LETN was knocked down by siRNA (siRNA primers are shown below). The cells were seeded in a 35 mm dish for transfection after cell adherence. 2 μl of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 20 nM of siRNA were diluted with 250 ml of medium, respectively. After mixing well, the mixture was incubated for 20 min on standing, then slowly dropped into the dish containing the cells, and the knockdown level was detected by RT-qPCR after 48 hours (see Table 2 below for the sequence of primer pairs). The knockdown efficiency is shown in
TABLE-US-00003 siLETN-1 Sense (SEQ ID NO: 34) 5′-3′ GCUGUCUCCAUGUCUUCUU Antisense (SEQ ID NO: 35) 5′-3′ AAGAAGACAUGGAGACAGC siLETN-2 Sense (SEQ ID NO: 36) 5′-3′ GCUCUCUGCUCAAGUAUUA Antisense (SEQ ID NO: 37) 5′-3′ UAAUACUUGAGCAGAGAGC siNC(negative control) Sense (SEQ ID NO: 38) 5′-3′ ACGUGACACGUUCGGAGAA Antisense (SEQ ID NO: 39) 5′-3′ UUCUCCGAACGUGUCACGU siLMNA Sense (SEQ ID NO: 40) 5′-3′ AUCUCAUCCUGAAGUUGCUUC Antisense (SEQ ID NO: 41) 5′-3′ GAAGCAACUUCAGGAUGAGAU
TABLE-US-00004 TABLE 2 Gene forward primer reverse primer ID 5′-3′ 5′-3′ LETN- GGGTCTACCTGTGAACTG GGAAACACATGTGGCAGCAC 1 TGA (SEQ ID NO: 43) (SEQ ID NO: 42) LETN- TGGTTTCTGGCAGCATAA AGCCTGGGCAACAAGAGTGA 2 CT (SEQ ID NO: 45) (SEQ ID NO: 44) NPM1- TTCGGTTGTGAACTAAAG CAAGGGAAACCGTTGGCTGT N GC (SEQ ID NO: 47) (SEQ ID NO: 46) NPM1- TCTGTAGAAGACATTAAA AATAGCCTCTTGGTCAGTCAT C GCAAA (SEQ ID NO: 49) (SEQ ID NO: 48) Pre- GCCTTCTCTAGCGATCTG CCATAACGGAGGCAGAGACA rRNA AGAG (SEQ ID NO: 51) (SEQ ID NO: 50) 18S TCCTTTGGTCGCTCGCTC GATCTGATAAATGCACGCATC rRNA CT CC (SEQ ID NO: 52) (SEQ ID NO: 53) 5.8S ACTCGGCTCGTGCGTC GCGACGCTCAGACAGG rRNA (SEQ ID NO: 54) (SEQ ID NO: 55) 28S GCGGGTAAACGGCGGGAG TTGGCTGTGGTTTCGCTGGAT rRNA TA (SEQ ID NO: 57) (SEQ ID NO: 56) GAPDH GGTCACCAGGGCTGCTTT TTCCCGTTCTCAGCCTTGAC TA (SEQ ID NO: 59) (SEQ ID NO: 58) β- TGGACATCCGCAAAGACC CCGATCCACACGGAGTACTT actin TG (SEQ ID NO: 61) (SEQ ID NO: 60) MALAT1 GCTCTGTGGTGTGGGATT GTGGCAAAATGGCGGACTTT GA (SEQ ID NO: 63) (SEQ ID NO: 62)
[0061] Since two weeks of continuous knockdown was required to detect the colony formation, a stably screened cell line with LETN knockdown was constructed (see Example 3 for details). The knockdown level was detected by RT-qPCR (see Table 3 below for the sequence of primer pairs). The knockdown efficiency was shown in
TABLE-US-00005 TABLE 3 shRNA Sense 5′-3′ Antisense 5′-3′ shLETN- GGACTTCCAGTTAAGC AATTGGCTTAACTGGA 1 CAATT AGTCC (SEQ ID NO: 64) (SEQ ID NO: 65) shLETN- GGCTGTCTCCATGTCT TAAGAAGACATGGAGA 2 TCTTA CAGCC (SEQ ID NO: 66) (SEQ ID NO: 67) shNC ACGTGACACGTTCGGA TTTCTCCGAACGTGTC GAAA ACGT (SEQ ID NO: 68) (SEQ ID NO: 69)
[0062] In addition, LETN was knocked out by CRISPR-Cas9. The cells were seeded in a 35 mm dish for transfection after cell adherence. 2 μl of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 2 μg of px458-sgRNA plasmid (sgLETN sequence was added on the basis of px458 (Addgene, catalog no. 48138), and the specific method is shown in the reference [5]) were diluted with 250 ml of medium, respectively. After mixing well, the mixture was incubated for 20 min on standing, then slowly dropped into the dish containing the cells, and the knockdown level was detected by RT-qPCR after 48 hours. The knockdown efficiency is shown in
TABLE-US-00006 sgLETN-1 Sense (SEQ ID NO: 70) 5′-3′ TCAAATTTCAGTCGGAACTC sgLETN-2 Sense (SEQ ID NO: 71) 5′-3′ GAGACGATATGCTACGGGTG sgEV-1 Sense (SEQ ID NO: 72) 5′-3′ GAACGTTGGCACTACTTCAC sgEV-2 Sense (SEQ ID NO: 73) 5′-3′ GCGCCTTAAGAGTACTCATC
[0063] On the other hand, LETN overexpression was performed. The cells were seeded in a 35 mm dish for transfection after cell adherence. 2 μl of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 2 μg of LETN overexpression plasmid plv-LETN (constructed by Wuxi Qinglan Biotechnology Co., Ltd., the expression plasmid: plv-mCherry, catalog no. 36084) were diluted with 250 ml of medium, respectively. After mixing well, the mixture was incubated for 20 min on standing, then slowly dropped into the dish containing the cells, and the overexpression efficiency was detected by RT-qPCR after 48 hours (see
[0064] Subsequently, the lung cancer cell line HCC827 (ATCCRCRL-2868) as well as the prostate cancer cell lines DU145 (ATCCRHTB-81) and PC3 (ATCCRCRL-1435) were selected, and similar effects were found. When LETN was knocked down by siRNA (the method is identical to that previously manipulated in the liver hepatocellular carcinoma cell line HUH7, and the knockdown efficiency is shown in
Example 3 Study of lncRNA RP11-196G18.22 (LETN) on Animal Level
[0065] The effect of LETN on the tumorigenic ability of tumor cells was further investigated. Firstly, a stably screened cell line with LETN knockdown was constructed using the virus packaging shRNA (shRNA sequences are shown in Table 3). First, for virus packaging, 293T cells (Shanghai Cell Bank, Chinese Academy of Sciences) were seeded in a 100 mm plate, and transfected on the next day. 12 μl of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) was diluted with 1 ml of medium, and the packaging vector 7.1 μl 48.9 (Tsinghua University library platform), 3.55 μg VSVG (Tsinghua University library platform), and the expression vector 3 μg plv-LETN were diluted with 1 ml of medium. After mixing well, the mixture was incubated for 20 min on standing, then slowly dropped into the dish containing the cells, and the medium was supplemented to 10 ml. After 48 hours, the supernatant was collected and centrifuged at 3000 rpm for 10 min. This supernatant as the virus solution was aliquoted, and stored at −80° C. until use. For the construction of the stably screened cell line with LETN knockdown, first, the cells were seeded in a 35 mm dish, 500 μl of virus solution was added on the next day, and the medium was supplemented to 2 ml. After 48 hours, puromycin was added once every three days to remove cells without viral expression, to obtain the stably screened cell line as desired). The HUH7 cell line with LETN knockdown (the knockdown efficiency was detected by RT-qPCR, and shown in
Example 4 Exploration of Action Mechanism of LETN
[0066] First, it was identified by RNA in situ hybrization[6] and nucleocytoplasmic separation (Nuclear/Cytosol Fractionation Kit (Biovision, K266-25)) that LETN was mainly localized in the nucleus and distributed in clusters. This indicated that LETN was likely to function through binding to proteins. The proteins pulled down through RNA pull down were analyzed by mass spectrometry to find the proteins that interact with LETN, and finally NPM1 was found to be the functional protein that binds thereto. Then NPM1 antibody Anti-NPM1 (Abcam, ab10530) was used for pulling down through RNA pull down technology[7], and it was also found that NPM1 could indeed pull down lncRNA LETN. It was further confirmed by the cellular fluorescence co-localization experiment that LETN was localized in the nucleolus.
[0067] NPM1 is a very crucial functional protein in the nucleolus, which can bind to rDNA promoter to promote rDNA transcription; participate in rRNA splicing and maturation; bind to histones and participate in nucleosome assembly; and the like. It was found that LETN was indeed involved in these functions. It was found that the knockdown of LETN could significantly reduce the expressions of various rRNAs, and the overexpression of LETN could promote the expression of rRNA. In addition, the knockdown of LETN could also attenuate the binding ability of NPM1 to histones, thereby affecting the nucleosome assembly. The nucleolus is composed of three basic structural components, namely fibrillar center, dense fibrillar component, and granular component (from the inside to the outside). NPM1 is mainly located in the outermost layer and is the most important constituent of the granular component. Studies have shown that the knockdown of NPM1 can destroy the morphology of the nucleolus.
[0068] Our study found that when LETN was knocked down, the nucleolus became irregular and scattered from the regular and dense spherical shape, indicating that LETN also affected the structure of the nucleolus. Through our algorithm, the survival time was analyzed based on the clinical data of liver hepatocellular carcinoma patients in the TCGA database, wherein these groups were individually divided: LETN high expression group and low expression group, or NPM1 high expression group and low expression group. It was found that the prognostic survival time of patients in NPM1 or LETN high expression group was shorter than that of patients in low expression group; and when further subdivided into four groups: NPM1-low+LETN-low, NPM1-low+LETN-high, NPM1-high+LETN-low, and NPM1-high+LETN-high, it was found that the survival time of patients with high expressions of both NPM1 and LETN was much shorter than that of patients with low expressions of both NPM1 and LETN.
[0069] Those skilled in the art should understand that, although the invention is described in details with reference to the above Examples, the invention is not limited to these specific Examples. Based on the methods and technical solutions taught by the invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the invention, and the equivalent embodiments thus obtained are all within the scope of the invention.
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