WO2016124002A1 - 一种竞争性消耗致癌性microRNAs的LncRNA、溶瘤腺病毒及其用途 - Google Patents

一种竞争性消耗致癌性microRNAs的LncRNA、溶瘤腺病毒及其用途 Download PDF

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WO2016124002A1
WO2016124002A1 PCT/CN2015/091892 CN2015091892W WO2016124002A1 WO 2016124002 A1 WO2016124002 A1 WO 2016124002A1 CN 2015091892 W CN2015091892 W CN 2015091892W WO 2016124002 A1 WO2016124002 A1 WO 2016124002A1
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lncrna
mir
sequence
tumor
oncolytic adenovirus
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French (fr)
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苏长青
苏英晗
李晓雅
季卫丹
刘扣勇
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中国人民解放军第二军医大学
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Priority to US15/523,422 priority Critical patent/US10323244B2/en
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Definitions

  • the present invention relates to the field of genetic engineering and virology, and in particular to a LncRNA, an oncolytic adenovirus which competitively consumes oncogenic microRNAs and uses thereof.
  • miRNAs are small, numerous, and widely used. It can even be said that miRNAs are ubiquitous in cells and in humans. miRNAs play a key role in a variety of biological processes, including regulation of early cell development, cell proliferation, stem cell differentiation, and apoptosis. The regulation of miRNAs on tumorigenesis and its important role in tumor diagnosis and treatment have attracted attention. Studies have shown that changes in the expression profile of miRNAs are involved in the whole process of tumorigenesis and development, including the origin, invasion and metastasis of tumor cells. Therefore, the value of miRNAs for tumor-targeted therapy is immeasurable.
  • miRNAs are a class of small, non-coding RNAs of 18-22 nucleotides in length that are highly conserved and widely present in eukaryotic cells.
  • the miRNAs are miRNAs transcripts (pri-miRNAs) in the nucleus by RNA transcriptase, and then pri-miRNAs are cleaved under the action of RNA polymerase III to generate miRNAs precursors of about 70 nucleotides in length (pre - miRNAs). Thereafter, the pre-miRNA is transported from the nucleus into the cytoplasm and processed by the DICER enzyme to generate a single-stranded RNA molecule of approximately 18-22 nucleotides, ie, mature miRNAs.
  • miRNAs selectively bind to an RNA-induced silencing complex (RISC) to form a RISC complex that functions as a biological function.
  • RISC RNA-induced silencing complex
  • the mature miRNAs can form a RISC complex together with other proteins, and bind to the target gene mRNAs by complementary nucleic acid sequences, thereby inhibiting the translation of the target gene mRNA or degrading the target gene mRNA, thereby exerting its post-transcriptional regulation effect on the target gene.
  • miRNAs are widely involved in many important processes in the life process of organisms, including individual development, organ formation, and cell proliferation, differentiation, and apoptosis. They are also involved in tumor development, invasion, and metastasis.
  • miRNAs may act as oncogenic miRNAs (oncogenic microRNAs, OncomiRs) or tumor suppressor miRNAs. miRNAs may participate in the development of tumors by causing changes in the expression of certain apoptosis-related factors, affecting the activity of various intracellular and extracellular signaling pathways, and regulating the activity of transcriptional regulators.
  • miR-17-92 clusters (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92-1) A marker for poor prognosis in multiple myeloma and can promote the malignant development of B-cell lymphoma. Down-regulation or even loss of expression of certain miRNAs in tumor cells may also lead to tumors, and such miRNAs may be considered as tumor suppressor genes.
  • miR-15 and miR-16 are associated with chronic lymphocytic leukemia, miR-26a, miR-129, miR-143 and miR-145 in breast, prostate, and cervical cancer
  • Low expression in lymphoid tumors and colorectal cancer, and decreased expression of miR-122 may mediate the pathogenesis of primary liver cancer.
  • miR-34 including miR-34a, miR-34b, and miR-34c, exhibits abnormally low expression in a variety of tumors. Changes in the expression levels of these miRNAs are closely related to the development of cancer cells.
  • the miRNAs highly expressed in liver cancer were screened by miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR-106b, miR-151-. 5p, miR-155, miR-181a/181b, miR-184, and miR-1 and miR-449a were also detected in hepatitis B virus-associated liver cancer.
  • miRNAs in invasive and non-invasive liver cancer 20 miRNAs were also found to be associated with liver cancer metastasis and postoperative recurrence.
  • miR-185, miR-219-1, miR-207, miR-338 were up-regulated.
  • miR-21 inhibiting PTEN is Activate the PI3K/AKT pathway to promote the proliferation and metastasis of cancer cells.
  • miR-221/222 acts on many key tumor suppressor factors, including Bmf, p27Kip1, p57Kip2l6, PTEN, tissue inhibitor of metalloproteinase-3, TIMP-3, and DNA damage-inducible transcript 4 (DDIT4), which mediates tumor Development has taken place.
  • liver cancer miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR106b, miR-151-5p, miR-155, miR-181a/181b, miR-184 Expression can promote cancer cell proliferation, invasion and metastasis; high expression of miR-221/222, miR-224, miR-10b and miR-155 can inhibit cancer cell apoptosis; miR-21, miR-221/222, miR-143 High expression of miR-1 and miR-449a can promote the proliferation of hepatitis B virus, induce cell carcinogenesis and enhance the proliferation activity of cancer cells.
  • miRNAs Due to the large number of target genes acting on miRNAs, the regulatory mechanism is complex. One miRNA can target multiple target genes, and one target gene may also be regulated by multiple miRNAs. The development of tumors must involve many miRNAs and regulate a wide range of genes. A pan-target gene affects many signaling pathways. Therefore, the intervention of single miRNA expression has a limited inhibitory effect on tumors, and cancer cells can easily regain proliferation activity through the bypass signaling pathway.
  • Tumor-specific proliferating adenovirus can specifically proliferate and replicate in tumor cells, dissolve and destroy tumor cells, release progeny virus, and infect more tumor cells, so it is also called Oncolytic adenovirus.
  • Oncolytic adenovirus By using the high diffusivity of tumor-specific proliferating adenovirus, high transfection ability, and the ability to specifically proliferate and replicate in tumor cells, the number of anti-cancer gene copies carried by the tumor-proliferating adenovirus increases with virus proliferation and is high in tumor cells. Transfection efficiency, high copy number and highly expressed anti-cancer factors enhance the anti-cancer effect on tumors. Because adenovirus is a common and safe viral vector for tumor gene therapy, it has been widely used in human gene therapy programs.
  • the genome is completely clear and easy to operate; the human cells that can be infected are widely and can be efficiently transferred. Different types of human tissue cells, including quiescent cells; high virus titer, high titer of recombinant virus production in cell culture; large genome, which can insert large fragments of foreign genes; Integration into the host cell genome is therefore safer and less carcinogenic.
  • SFDA National Food and Drug Administration
  • LncRNA-mediated artificially designed LncRNA specifically propagates in cancer cells, and replicates with adenovirus to achieve high-copy, high-efficiency expression of LncRNA and enhance competition for binding to miRNAs.
  • the effect is to fully protect the target genes of various OncomiRs and ultimately exert a more effective anti-cancer effect.
  • the object of the present invention is to provide a LncRNA which can be used for competitive treatment of OncomiRs for various tumor treatments such as liver cancer and realizes joint intervention of various miRNAs, and an oncolytic adenovirus which can specifically propagate and replicate in cancer cells and express the LncRNA. .
  • the present invention provides a LncRNA competitively consuming OncomiRs, the sequence of which is n copies of the sequence shown in SEQ ID NO. 4, wherein n is an integer greater than or equal to 1.
  • the n is equal to 6.
  • the present invention provides a coding sequence encoding the LncRNA, wherein the coding sequence of the LncRNA is n copies of the sequence shown in SEQ ID NO. 1, wherein n is an integer greater than or equal to 1.
  • the n is equal to 6.
  • the present invention provides an oncolytic adenovirus in which the expression cassette of the LncRNA is integrated into the genome of the oncolytic adenovirus.
  • the expression cassette of the LncRNA comprises the coding sequence of the LncRNA, and a promoter which regulates expression of the coding sequence of the LncRNA, and the promoter which regulates expression of the coding sequence of the LncRNA is inserted in the The transcription initiation site of the coding sequence of LncRNA is described before.
  • the genome of the oncolytic adenovirus contains a virus-proliferating essential gene and a tumor-specific promoter that regulates expression of an essential gene for viral proliferation.
  • the oncolytic adenovirus is constructed based on human type 5 adenovirus; the promoter sequence for regulating expression of the coding sequence of LncRNA is as shown in SEQ ID NO.
  • the virus increase The sequence of the essential gene is shown in SEQ ID NO. 8, and the tumor-specific promoter sequence is shown in SEQ ID NO.
  • the whole genome sequence of the oncolytic adenovirus is as shown in SEQ ID NO.
  • the invention provides the LncRNA, the coding sequence of the LncRNA or the use of the oncolytic adenovirus in the preparation of a medicament for treating a tumor.
  • the tumor has miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR106b, miR-151-5p, miR-155, miR -181a/181b, miR-184, miR-1 and/or miR-449a are highly expressed.
  • the tumor is liver cancer, particularly primary hepatocellular carcinoma.
  • the present invention provides the LncRNA, the coding sequence of the LncRNA or the use of the oncolytic adenovirus in a preparation reagent for studying a molecular mechanism of action or/and a liver cancer in a liver cancer cell. treatment.
  • the invention has the advantages of providing a LncRNA, recombinant oncolytic adenovirus and its application for competitive consumption of OncomiRs, and has the following important meanings:
  • the oncolytic adenovirus constructed under the effective control of the tumor-specific Survivin gene promoter can specifically proliferate and replicate in tumor cells, dissolve and destroy tumor cells, and release the hormone. Generation of virus infection destroys more tumor cells; 2) By continuously expressing artificially designed LncRNA in cancer cells, the LncRNA also contains a sequence capable of complementary binding to multiple OncomiRs, and can compete with OncomiRs for all target gene mRNAs of OncomiRs, Thereby, the high level of OncomiRs in the cells is consumed, and the anti-cancer gene in the cell is protected, and the anticancer effect is exerted.
  • This treatment strategy can effectively block multiple OncomiRs with different mechanisms of action or complementary mechanisms, and widely inhibit multiple signaling pathways related to OncomiRs, which is more effective in treating tumors and overcomes the effect of single miRNA intervention on tumor suppression.
  • cancer cells can easily regain the proliferative viability through the bypass signaling pathway; 3) use tumor-specific proliferation oncolytic adenovirus as a vector to mediate the expression of artificially designed LncRNA, with the specificity of the virus in the tumor Proliferation and replication, the copy number and expression of LncRNA are geometrically multiplied, and high-copy and high-efficiency expression of LncRNA can consume OncomiRs in large quantities, showing a good protective effect on intracellular tumor suppressor genes, thereby inhibiting tumor development and even completely disappearing. .
  • the Survivin promoter is a cis-acting element, inserted between the transcription initiation site of the adenovirus-proliferating essential gene E1a and the translation initiation site ATG, and regulates E1a transcriptional activation.
  • E1a can only be specifically expressed in Survivin-positive tumor cells, but has no effect or minimal effect on normal cells, and its regulation has better reliability and safety;
  • Synthetic LncRNA contains a group of The complementary binding sequence of OncomiRs, which is highly expressed in cancer cells and capable of promoting tumorigenesis through various mechanisms, whose goal is to consume or interfere with OncomiRs, and does not contribute to other tumor suppressor miRNAs in cancer cells.
  • Normal cells do not function because of OncomiRs.
  • the expression level is extremely low and will not be affected, thus improving the anti-tumor effect and improving the safety; 3) introducing a translation stop codon at the initiation and termination positions of the LncRNA coding sequence to prevent its translation into any protein or polypeptide. molecule.
  • the anti-tumor system combining LncRNA and adenovirus vector provided by the invention is adapted to most humans
  • the treatment of malignant tumors has not seen similar treatments at home and abroad. Based on this, a variety of anti-tumor biological treatment products can be constructed, and a reliable technical platform for tumor treatment has been established.
  • FIG. 1 Comparison of AdSVPE1a-lncR-mediated expression levels of LncRNA in different cell lines.
  • AdSVPE1a-lncR mediates the effect of LncRNA expression on cell viability.
  • AdSVPE1a-lncR mediates the effect of LncRNA expression on cell migration invasiveness.
  • AdSVPE1a-lncR mediates the effect of LncRNA expression on gene expression profiles of hepatoma cells.
  • the inventors of the present application utilize a tumor-specific Survivin gene promoter to regulate the oncolytic adenovirus-proliferating essential gene E1a, so that the virus can specifically proliferate and replicate in cancer cells; and the oncolytic adenovirus is used as a vector to express a synthetic synthesis.
  • the LncRNA coding sequence comprising a set of complementary binding sequences of a seed sequence of OncomiRs capable of promoting the development of cancer cells by various mechanisms; achieving specific high-copy replication of adenovirus in cancer cells, enabling LncRNA in cancer cells Large-scale expression and high-copy accumulation, by competing with OncomiRs target gene and consuming OncomiRs, thereby protecting a large number of tumor suppressor genes in cells from interference and inhibition by OncomiRs, and achieving targeted intervention therapy for cancer cells.
  • Such a strategy has improved anti-tumor efficacy and improved safety due to its tumor-targeted specific proliferation of viral vectors and specific interventions against OncomiRs carcinogenesis.
  • the "LncRNA coding sequence” is a DNA sequence encoding LncRNA.
  • the coding sequence of LncRNA is shown in SEQ ID NO.
  • the coding sequence of LncRNA is shown in SEQ ID NO. 2 and in order to enable the coding sequence of LncRNA to obtain relatively strong transcriptional activity and prevent translation thereof, as shown in SEQ ID NO.
  • the 5'-end of the sequence was added with CACCATGC, and the AG was added at the 3'-end.
  • the increased LncRNA coding sequence is shown in SEQ ID NO. The above sequence was introduced to increase the translation stop codon at the start and end positions of the LncRNA coding sequence, which may be TAG, TGA or TAA.
  • the LncRNA coding sequence of the present invention is preferably obtained by a synthetic method.
  • the LncRNA coding sequence comprises a complementary binding sequence of a seed sequence of OncomiRs, and the group of OncomiRs is expressed at a high level in liver cancer cells, and can promote the development of liver cancer cells through various mechanisms.
  • This group of OncomiRs includes miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR106b, miR-151-5p, miR-155, miR-181a/181b, miR-184, miR-1, miR-449a. They play different roles in different genes and signaling pathways in the development of liver cancer.
  • miR-21 in liver cancer can inhibit the expression of the tumor suppressor gene PTEN, which is An important inhibitory protein of the phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT) pathway. Therefore, the consequence of miR-21 inhibiting PTEN is to activate PI3K/ AKT pathway promotes proliferation and metastasis of cancer cells; miR-221/222 acts on many key tumor suppressor factors, including Bmf, p27Kip1, p57Kip2l6, PTEN, tissue inhibitor of metalloproteinase-3, TIMP-3, and DNA damage-induced transcription Sub 4 (DDIT4), which mediates the development of tumors.
  • PTEN phosphoinositide 3-kinase
  • AKT threonine kinase
  • DDIT4 DNA damage-induced transcription Sub 4
  • miRNAs are classified by function, miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR106b, miR-151-5p, miR-155, miR-181a/181b in liver cancer.
  • High expression of miR-184 can promote cancer cell proliferation, invasion and metastasis;
  • high expression of miR-221/222, miR-224, miR-10b and miR-155 can inhibit cancer cell apoptosis;
  • miR-21, miR-221 /222, miR-143, miR-1, miR-449a high expression can promote hepatitis B virus proliferation, induce cell carcinogenesis and enhance cancer cell proliferation activity. Inhibition of the expression and function of these carcinogenic and cancer-promoting miRNAs can inhibit the development of liver cancer through multiple pathways and multiple effects.
  • the coding sequence of the LncRNA may further comprise a complementary binding sequence of one or a group of OncomiRs seed sequences, and the OncomiRs may be one or a group of high expression in tumors other than liver cancer, and OncomiRs that can promote the development of tumors other than liver cancer through various mechanisms, but also for liver cancer except miR-21, miR-221/222, miR-224, miR-17-5p, miR-10b, miR-151- One or a group of OncomiRs other than 5p, miR-155, miR-181a/181b, miR-184, miR-1, miR-449a, miR106b.
  • LncRNA Long non-coding RNA
  • the LncRNA of the present invention is obtained by encoding a DNA sequence comprising a set of complementary sequences of a seed sequence of OncomiRs capable of promoting the development of liver cancer cells by various mechanisms.
  • the encoded LncRNA sequence is shown in SEQ ID NO.
  • the coding sequence of the above LncRNA is repeated 6-fold in the genome of the virus, and the encoded LncRNA sequence is shown in SEQ ID NO. 5 to enhance the effect of competitive binding to the corresponding OncomiRs.
  • the "promoter of the LncRNA coding sequence” is used to initiate transcription of the LncRNA coding sequence, which includes other cis-acting elements having the same function.
  • the LncRNA coding sequence of the present invention may be regulated by any of the following promoters or cis elements in the genome of the virus: (a) carcinoembryonic antigen promoter; (b) alpha-fetoprotein promoter; (c) human epidermal growth Receptor tyrosine kinases (including EGFR, Her-2, Her-3, and Her-4) promoters of factor receptor family (EGFRs); (d) breast cancer-associated antigen DF3/MUC1 promoter; (e) blood vessels Promoter of endothelial growth factor (VEGF) receptor KDR; (f) herpes simplex virus promoter; (g) E2F promoter; (h) prostaglandin-specific antigen promoter.
  • VEGF endothelial growth factor
  • the LncRNA coding sequence is under the control of a murine cytomegalovirus (CMV) promoter, and the cis-acting element is inserted into the transcription initiation site of the LncRNA coding sequence to ensure that the LncRNA is infected by a virus.
  • the tumor is highly expressed inside and exerts biological activity.
  • the sequence of the CMV promoter is preferably as shown in SEQ ID NO.
  • the tumor-specific promoter is a tumor-specific regulatory sequence. Since many tumors have their own specific tumor markers whose expression is regulated by the tumor-specific cis-acting elements or trans-acting factors, the viral proliferative genes are placed in the regulation of such tumor-specific regulatory sequences. Underneath, the virus can be selectively propagated in the corresponding tissue cells without affecting other tissue cells.
  • the tumor-specific promoter of the present invention may be composed of any of the following promoters and enhancers: (a) carcinoembryonic antigen promoter, enhancer and mutant sequences thereof; (b) alpha-fetoprotein promoter, enhancement And its mutant sequences; (c) receptor tyrosine kinases (including EGFR, Her-2, Her-3 and Her-4) promoters and enhancers of the human epidermal growth factor receptor family (EGFRs) Mutant sequence; (d) breast cancer-associated antigen DF3/MUC1 promoter, enhancer and its mutant sequence; (e) vascular endothelial growth factor (VEGF) receptor KDR promoter, enhancer and its mutant sequence; (f) L-plastin promoter, enhancer and its mutant sequences; (g) promoters, enhancers and their mutant sequences of members of the inhibitor of apoptosis inhibitory protein (IAP); (h) prostaglandin-specific antigens Promoter, enhancer and its mutant sequences; (i)
  • Survivin a member of the apoptosis inhibitory protein factor (IAP) family, is involved in the regulation of various aspects of embryonic cell physiological development and cell cycle regulation, inhibiting cell apoptosis through various pathways, and promoting cell proliferation and cell cycle progression.
  • Survivin is expressed in embryonic developmental tissues and most tumor tissues, but not in normal adult tissues.
  • the expression of Survivin in malignant tumors is highly selective and is highly expressed in most of the most common tumor tissues in humans such as lung cancer, colon cancer, pancreatic cancer, prostate cancer and breast cancer, and non-Hodgkin's lymphoma.
  • viruses regulated by the Survivin promoter are expected to achieve a broad-spectrum anticancer effect against most human tumors.
  • the core sequence of the Survivin promoter is a cis-acting element, inserted between the transcription initiation site of the adenovirus-proliferating essential gene E1a and the translation initiation site ATG, and regulates E1a transcriptional activation, thereby making E1a It can only be specifically expressed in Survivin-positive tumor cells, but has no effect on normal cells or has very weak effects, and its regulation has better reliability and safety.
  • the sequence of the tumor-specific Survivin gene promoter used in the present invention is shown in SEQ ID NO.
  • the virus-producing essential gene provides the protein necessary for the proliferative replication of the virus.
  • Adenoviral proliferation essential genes include E1a, and also include Elb-55kDa, Elb-19kDa, E3 or E4.
  • Elb-55kDa is a protein essential for adenovirus to reproduce in normal cell proliferation and is not essential in tumor cells.
  • the selective deletion of Elb-55kDa encoding gene can enable adenovirus to maintain proliferation and replication in tumor cells, while in normal cells. Lost ability to copy.
  • Elb-55kDa can inactivate and degrade P53 protein, and the selective deletion of Elb-55kDa coding gene is beneficial to the cells to maintain the anti-tumor activity of P53 protein and improve the targeting of viral vectors.
  • the adenoviral Elb-19kDa gene is homologous to the apoptosis-inhibiting gene Bcl-2, and the encoded Elb-19kDa binds to Bax or/and Bak to initiate downstream apoptosis inhibition. Elb-19kDa also disrupts Fas-mediated apoptosis. The process protects infected cells from TNF- ⁇ mediated killing.
  • the viral growth essential gene E1a sequence is shown in SEQ ID NO.
  • the present invention uses a recombinant viral vector to mediate the specific expression of the LncRNA coding sequence in tumor cells to exert an anticancer effect.
  • the recombinant viral vector can be constructed from an existing viral vector, a tumor-specific promoter and an expression cassette of LncRNA, and the expression cassette of the LncRNA comprises a LncRNA coding sequence and a promoter of the LncRNA coding sequence.
  • Human oncolytic adenoviruses have six different subgenus A, B, C, D, E, and F, which have different affinities, tumorigenicity, and disease history to host cells.
  • the invention uses Form 5 of the subgenus C of the oncolytic adenovirus.
  • the recombinant oncolytic adenovirus is constructed from an expression cassette of human type 5 adenovirus, tumor-specific promoter and LncRNA.
  • the recombinant oncolytic adenovirus was produced by site-directed recombination of the framework plasmid pBHGloxdeltaE1, 3Cre containing the type 5 adenovirus genome and an adenoviral shuttle plasmid carrying the LncRNA coding sequence in a 293 cell by a Cre-loxP recombinase cleavage system.
  • sequence other than the recombinant region of the recombinant oncolytic adenovirus is identical to the sequence other than the recombinant region of the pBHGloxdeltaE1,3Cre adenoviral vector, and the sequence in the recombinant region of the recombinant oncolytic adenovirus is as shown in SEQ ID NO. .
  • the whole genome sequence of the recombinant oncolytic adenovirus is as shown in SEQ ID NO. 10, and the whole genome sequence of the recombinant oncolytic adenovirus finally constructed in the examples is based on SEQ ID NO.
  • Example 1 Tumor-specific Survivin promoter controls the construction of E1a adenovirus plasmid
  • the first step artificially synthesize the tumor-specific Survivin promoter, introduce the XbaI site at the 5'-end, introduce the EcoRI site at the 3'-end, insert the XbaI+EcoRI site of the plasmid pDC315, and construct the plasmid pDC315 containing the Survivin promoter.
  • SVP. pDC315 is a product of Microbix Biosystems, Canada (catalog number: PD-01-27).
  • the synthetic Survivin promoter was designed to be 1002 bp in length, and the sequence was introduced into the XbaI restriction site (TCTAGA) at the 5'-end of SEQ ID NO. 7, and the EcoRI restriction site (GAATTC) was introduced at the 3'-end.
  • the second step cloned the adenovirus E1a sequence (containing PolyA tailing sequence), introduced the EcoRI site and ACC at the 5'-end, introduced the BamHI site at the 3'-end, and inserted the EcoRI+BamHI site of pDC315-SVP to construct
  • the plasmid pDC315-SVPE1a, which controls the expression of E1a, is a tumor-specific Survivin promoter.
  • E1a (containing PolyA tailing sequence) is 1311 bp in length, of which, 1-6 bp: EcoRI cleavage site (GAATTC); 7-9 bp: ACC; 10-95 bp: E1a gene cDNA sequence (SEQ ID NO. 8) ); 996-1305 bp: SV40PolyA tailing sequence (SEQ ID NO. 11); 1306-1311 bp: BamHI restriction site (GGATCC).
  • the first step the mouse cytomegalovirus (CMV) promoter sequence was cloned, the BamHI site was introduced at the 5'-end, the SalI site was introduced at the 3'-end, and the BamHI+SalI site of the plasmid pDC315 was inserted to construct a mCMV-containing site.
  • the mCMV sequence is 543 bp in length, wherein the 1-6 bp: BamHI restriction site (GGATCC); 7-537 bp: murine cytomegalovirus (CMV) promoter sequence (SEQ ID NO. 6); 538-543 bp: SalI cleavage site (GTCGAC).
  • the second step artificially synthesize 6 copies of the DNA sequence encoding the LncRNA (including PolyA tailing sequence), and introduce the SalI site at the 5'-end and 3'-end, insert the SalI site of pDC315-mCMV, and construct it into a The expression plasmid pDC315-mCMVLncR of the LncRNA coding sequence.
  • the DNA sequence encoding the LncRNA (containing the PolyA tailing sequence) is 1348 bp in length, wherein the 1-6 bp: SalI cleavage site (GTCGAC); the 7-13 bp: transcription start site (CACCATG); 14-1192 bp: LncRNA coding sequence (SEQ ID NO. 12) containing 6 replicate copies; 1193-1352 bp: LncRNA tailing sequence (SEQ ID NO. 13); 1353-1358 bp: SalI cleavage site (GTCGAC).
  • the third step: pDC315-mCMVLncR was digested with BamHI+SalI, and the fragment containing the complete expression cassette of LncRNA was recovered and inserted into the BamHI+SalI site of pDC315-SVPE1a plasmid to construct the tumor-specific proliferation adenovirus packaging plasmid pDC315 carrying LncRNA coding sequence.
  • -SVPE1a-mCMVLncR The LncRNA complete expression cassette is 1895 bp in length, wherein the 1-6 bp: BamHI restriction site; 7-537 bp: murine cytomegalovirus (CMV) promoter sequence (SEQ ID NO.
  • bp SalI Enzyme cleavage site
  • 544-550 bp: transcription initiation site (CACCATG); 55111-29 bp: LncRNA coding sequence containing 6 repeated copies (SEQ ID NO. 12); 1730-1889 bp: LncRNA tailing sequence (SEQ ID NO. 13); 1890-1895 bp: SalI cleavage site.
  • First step The constructed adenovirus type 5 left arm packaging plasmid pDC315-SVPE1a-mCMVLncR and the type 5 adenovirus right arm packaging plasmid pBHGloxdelE13cre were co-transfected into HEK293 cells by LipoFectamine2000.
  • LipoFectamine2000 For specific method steps for transfection, see Invitrogen's LipoFectamine 2000 kit operating instructions.
  • the plasmids pBHGloxdelE13cre and HEK293 cell lines are products of Microbix Biosystems, Canada.
  • pBHGloxdelE13cre contains the right arm of adenovirus type 5, which lacks the E1 and E3 regions, and its recombinase system Cre/LoxP can ensure efficient recombination of the virus.
  • HEK293 cells are transformed from spliced adenovirus type 5 DNA, containing the E1 region of adenovirus type 5, which has high transfection efficiency and can promote the recombination and packaging of adenovirus.
  • the complete adenoviral AdSVPE1a-lncR genomic sequence is set forth in SEQ ID NO. Among them: (1) 1-85bp: type I adenovirus ITR sequence; (2) 86-437bp: type 5 adenovirus genome sequence; (3) 438-443bp: XbaI restriction site; (4)444-1433bp: Survivin promoter sequence; (5) 1434-1439bp: EcoRI restriction site; (6) 1440-2428bp: E1a gene cDNA sequence; (7) 2429-2738bp: SV40PolyA tail sequence; (8) 2739-2744bp: BamHI Enzyme cleavage site; (9) 2745-3275 bp: murine cytomegalovirus (CMV) promoter sequence; (10) 3276-3281 bp: SalI cleavage site; (11) 3282-3288 bp: LncRNA transcription initiation site; (12) 3289-4467bp: LncRNA coding sequence
  • the second step Adenovirus AdSVPE1a-lncR was multiplied in HEK293 cells, and the adenovirus was purified by cesium chloride gradient centrifugation (for details, see GeneTransfer and Expression Protocols, edited by Murray EJ, Humana Press, Clifton, New Jersey).
  • AdSVPE1a-lncR The insert of the recombinant adenovirus AdSVPE1a-lncR was confirmed by sequencing and PCR.
  • AdSVPE1a-lncR is an adenovirus type 5, and the insertion sequence includes 6 copies of the LncRNA coding sequence, the mCMV promoter, the Survivin promoter, and the E1a sequence.
  • the other DNA sequences are identical to the type 5 adenovirus.
  • the PCR primers used for the identification are shown in Table 1:
  • PCR with the above primer sequences could amplify the same target band with the same theoretical length and the sequencing was correct.
  • AdSVPE1a-lncR has strong ability to specifically proliferate and replicate in HepG2, Hep3B, MHCC97H, Huh-7, PLC/PRF/5, and can reach thousands to tens of thousands of times after 72h, up to 68465.66 times (Huh- 7), in the SMMC-7721, MHCC97L up to hundreds of times, while in normal cells L02, WRL-68 proliferation is not obvious, below 10 times (Figure 1).
  • the second step the specific and efficient expression of LncRNA.
  • the LncRNA-specific PCR primers were LncR-F (5'-CTGCACTGTCAGCACTTTA-3') and LncR. -R(5'-ACATTCATTGCTGTCGGTG-3').
  • AdSVPeGFP-lncR-mediated LncRNA expression The highest level was found in Huh-7, Hep3B and HepG2, followed by PLC/PRF/5, MHCC97H, BEL-7402, MHCC97L and SMMC-7721, which were extremely low in normal liver cells L02 and WRL-68 (Fig. 2).
  • Example 6 Effect of recombinant adenovirus AdSVPE1a-lncR on biological behavior of hepatoma cells
  • the first step the effect of LncRNA expression on cell proliferation activity.
  • the effect of viral AdSVPE1a-lncR on tumor cells and normal cells was examined by a tetrazolium salt colorimetric assay (MTT).
  • MTT tetrazolium salt colorimetric assay
  • Cell Proliferation Kit I was purchased from Roche Diagnostics GmbH. The cells in the logarithmic growth phase were collected, counted, and the cells were diluted with 10% serum culture solution, 1 ⁇ 10 4 cells/100 ⁇ l/well, and transferred to a 96-well plate; after the cells were attached, the virus was diluted with serum-free medium, and pressed.
  • the second step the effect of LncRNA expression on cell migration and invasion ability.
  • ER1 group tumor cells
  • Ad2VPeGFP-lncR positive virus control
  • Ad5-eGFP Ad5-eGFP as negative virus control.
  • CR1 group non-proliferating virus not expressing LncRNA
  • virus-free cells were simultaneously cultured as a blank control (CR2 group).
  • the Transwell chamber was placed in a 24-well plate, cells were added to the upper chamber of the chamber, 4 x 10 5 cells/200 ⁇ l were added to each chamber, and 500 ⁇ l of medium containing 10% fetal bovine serum was added to the lower chamber.
  • the polycarbonate membrane matrix glue was added to the upper chamber, and the matrix migration was observed without adding matrigel.
  • the supernatant cells were removed, and the cells were stained with 0.1% crystal violet for 20 min.
  • Three fields of view ( ⁇ 200 times) were randomly taken under an optical microscope to count and photograph, and the experiment was repeated three times. The results showed that AdSVPE1a-lncR could significantly inhibit the migration and invasion of hepatoma cells.
  • AdSVPeGFP-lncR also inhibited the migration and invasion of hepatoma cells, but the activity was weaker than that of AdSVPE1a-lncR.
  • AdSVPE1a-lncR and AdSVPeGFP-lncR had no significant effect on normal hepatocytes (Fig. 4).
  • Example 7 Effect of recombinant adenovirus AdSVPE1a-lncR on gene expression profile of hepatoma cells
  • 100 ⁇ l of AdSVPE1a-lncR virus was added, and Ad5-eGFP was used as a negative virus control, and cells without virus were cultured as a blank control.
  • the cells were cultured for 2 h in the incubator; the serum culture medium was changed to 100 ⁇ l/well, and after 48 hours of culture, the cells were collected, the total protein was extracted, and the expression of the protein was detected by a gene expression profile chip.
  • the blank control group was marked with green by the cy3 fluorescent signal, and the experimental group was marked with red by the cy5 fluorescent signal.
  • the fluorescence signals of the two groups were superimposed, and the green signal was green when the cy3 signal was strong. The difference was lower than 0.5 times, indicating that the gene expression was down-regulated.
  • the cy5 signal was strong, it was red, and the difference was 2 times, indicating that the gene expression was up-regulated.
  • cy3 Similar to the cy5 signal intensity, it shows yellow, and the trend is between 0.5 and 2 times. The results showed that the gene expression profile of Huh-7 was significantly infected after infection with AdSVPE1a-lncR.
  • 708 genes were detected, including 13 protooncogenes and tumor suppressor genes, 11 ion channels and transport proteins, 12 cyclins, 13 cytoskeletal and motor proteins, and apoptosis. 5 related proteins, 6 DNA synthesis and recombinant proteins, 30 DNA binding and transcription factors, 10 cell receptors, 27 immune-related proteins, 74 cell signaling and transduction proteins, metabolic molecules There were 61 protein translation synthesis factors, 9 differentiation and development related classes, and 389 other molecules; 628 genes were detected, including 12 protooncogenes and tumor suppressor genes, ion channels and transport proteins.
  • FIG. 5 shows changes in the expression levels of some representative genes, of which PTEN, p27 kip1 , TIMP3, and RECK are more prominent, and p38/MAPK, Survivin, CDK4, and c-myc are more prominent in down-regulation (Fig. 5).
  • Example 8 Inhibition of recombinant adenovirus AdSVPE1a-lncR on transplanted tumor of liver cancer in nude mice
  • the virus treatment group was given multiple injections in the corresponding adenovirus tumors, each dose of 2 ⁇ 10 8 pfu/100 ⁇ l, once every other day, a total of 5 times; the blank control group was injected synchronously with physiological saline, each time 100 ⁇ l.
  • the tumor size was measured weekly, and the tumor volume was calculated by the formula of "maximum diameter x minimum diameter 2 ⁇ 0.5", and a growth curve was drawn.
  • the tumor growth rate of the AdSVPE1a-lncR treatment group was significantly lower than that of the blank control group, and the tumor volume decreased on the 21st day; the AdSVPeGFP-lncR group also showed a certain degree of growth inhibition at 28 days after treatment.
  • the difference was significant compared to the blank control group, but the tumor continued to grow; the Ad5-eGFP group showed no growth inhibition from start to finish (Table 2).
  • the tumor volume of the AdSVPE1a-lncR group was significantly lower than that before treatment (Fig. 6). The observation was terminated and the nude mice were euthanized.

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Abstract

提供了一种LncRNA、溶瘤腺病毒及其用途。以溶瘤腺病毒为载体表达所述LncRNA，实现该LncRNA在癌细胞内表达，通过与OncomiRs的靶基因竞争性结合并消耗OncomiRs，从而保护抑癌基因免受OncomiRs的干扰和抑制，实现对癌细胞的靶向干预治疗。

Description

一种竞争性消耗致癌性microRNAs的LncRNA、溶瘤腺病毒及其用途 技术领域
本发明涉及基因工程和病毒学技术领域，具体地说，涉及一种竞争性消耗致癌性microRNAs的LncRNA、溶瘤腺病毒及其用途。
背景技术
恶性肿瘤是以细胞异常增殖及转移为特点的一大类疾病，在我国其发病率及死亡率一直呈上升趋势，严重威胁国民的健康和生命。目前对恶性肿瘤的治疗仍以常规的手术、放疗、化疗为主，这种常规治疗对绝大多数肿瘤来说，难以收到预期的疗效，很重要的原因是没有特异的、敏感的早期诊断指标以及缺乏有效的治疗性分子靶标，这成为改善肿瘤患者临床疗效和预后的重要障碍。近年来的生物信息学分析表明，人类全部基因的三分之一都受到microRNAs(miRNAs)的调控，表明miRNAs实际上是基因调控网络中的核心成分。miRNAs分子小、数量多、作用广，甚至可以说miRNAs在细胞内、在人体内无处不在。miRNAs在多种生物进程中起到关键作用，包括调节细胞早期发育、细胞增殖、干细胞分化和凋亡。关于miRNAs对肿瘤发生发展的调控作用及其在肿瘤诊断治疗中的重要作用受到人们的关注。研究表明，miRNAs表达谱的改变参与肿瘤发生、发展的整个过程，包括肿瘤细胞的起源、侵袭及转移。因此，miRNAs对肿瘤靶向治疗的价值不可估量。
miRNAs是一类长度为18-22个核苷酸、高度保守的小分子非编码RNA，广泛存在于真核细胞中。miRNAs基因在细胞核内由RNA转录酶作用先生成miRNAs转录前体(pri-miRNAs)，然后pri-miRNAs在RNA聚合酶Ⅲ作用下，剪切生成约70个核苷酸长度的miRNAs前体(pre-miRNAs)。之后，pre-miRNA被转运由细胞核进入细胞质，经过DICER酶加工，生成约18-22个核苷酸的单链RNA分子，即成熟的miRNAs。miRNAs选择性地与RNA介导的沉默复合体(RNA-induced silencing complex，RISC)特异结合，形成RISC复合物，发挥生物学功能。成熟的miRNAs可与其他蛋白质一起组成RISC复合体，通过核酸序列互补结合于靶基因mRNAs，发挥抑制靶基因mRNA翻译或降解靶基因mRNA的作用，从而发挥其对靶基因的转录后调节效应。miRNAs广泛参与生物体生命过程中的许多重要进程，包括个体发育、器官形成以及细胞增殖、分化、凋亡等，同时也参与了肿瘤的发生发展和侵袭转移。越来越多的证据表明，miRNAs的异常表达与多种恶性肿瘤密切相关。有超过一半的miRNAs位于肿瘤相关基因组区域或脆性位点、杂合性丢失区和扩增区，提示miRNAs可能作为致癌性miRNAs(致癌性microRNAs，OncomiRs)或抑癌性miRNAs而发挥作用。miRNAs有可能通过引起某些凋亡相关因子表达的变化、影响各种细胞内外信号通路的活性、调控基因转录调节因子的活性，参与肿瘤的发生发展过程中。不同人类肿瘤的miRNAs表达谱，在乳腺癌、肝癌、肺癌、大 肠癌、脑瘤、白血病等多种肿瘤中均发现特异性miRNAs的高表达，这些miRNAs被视作一类OncomiRs。miR-155在慢性淋巴细胞性白血病、何杰金氏淋巴瘤、B细胞淋巴瘤、乳腺癌、肺癌、结肠癌和甲状腺癌中的表达上调，预示这些疾病患者治疗后难以缓解。miR-17-92家簇(miR-17-5p、miR-17-3p、miR-18a、miR-19a、miR-20a、miR-19b-1、miR-92-1)的高表达，可作为多发性骨髓瘤预后不良的标志物，并能够促进B细胞淋巴瘤的恶性发展。肿瘤细胞中某些miRNAs表达下调甚至缺失，也可能导致肿瘤，这类miRNAs可视作抑癌基因。如let-7表达下调与肿瘤发生有关，miR-15和miR-16与慢性淋巴细胞性白血病相关，miR-26a、miR-129、miR-143和miR-145在乳腺癌、前列腺癌、宫颈癌、淋巴系统肿瘤及大肠癌中低表达，miR-122表达下降可能介导原发性肝癌的发病过程。miR-34包括miR-34a、miR-34b和miR-34c等，在多种肿瘤中都呈现非正常的低表达。这些miRNAs表达水平的改变，与癌细胞发生发展密切相关。
以肝癌为例，通过文献检索，筛选出在肝癌中高表达的miRNAs有miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR-106b、miR-151-5p、miR-155、miR-181a/181b、miR-184，此外在乙肝病毒相关的肝癌中还可检出miR-1和miR-449a。通过比较侵袭性和非侵袭性肝癌的miRNAs表达谱，还发现20种miRNAs与肝癌转移、术后复发相关，其中上调表达的有miR-185、miR-219-1、miR-207、miR-338；下调表达的有Let-7g、miR-1-2、miR-122、miR-124a-2、miR-125b-2、miR-126、miR-148a、miR-148b、miR-15a、miR-194、miR-19a、miR-30a、miR-30c-1、miR-30e、miR-34a、miR-9-2，它们在肝癌的发生发展中分别起不同的作用。肝癌中miR-21高表达可抑制抑癌基因PTEN的表达。PTEN蛋白是3-磷酸肌醇激酶(phosphoinositide 3-kinase，PI3K)/丝/苏氨酸蛋白激酶(serine/threonine kinase，AKT)通路的重要抑制蛋白，因此，miR-21抑制PTEN的后果即是激活PI3K/AKT通路，促进癌细胞的增殖、转移。miR-221/222作用于许多关键的肿瘤抑制因子，包括Bmf、p27Kip1、p57Kip2l6、PTEN、金属蛋白酶-3的组织抑制因子TIMP-3以及DNA损伤诱导转录子4(DDIT4)，从而介导肿瘤的发生发展。总之，肝癌中miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR106b、miR-151-5p、miR-155、miR-181a/181b、miR-184高表达能够促进癌细胞增殖、侵袭、转移；miR-221/222、miR-224、miR-10b、miR-155高表达能够抑制癌细胞凋亡；miR-21、miR-221/222、miR-143、miR-1、miR-449a高表达能够促进乙肝病毒增殖，诱导细胞癌变和增强癌细胞增殖活性。
肿瘤细胞与其起源的相应正常细胞之间miRNA表达谱的差异，不同肿瘤之间miRNA表达谱的特异性，以及miRNAs在肿瘤发生、发展、转移、复发过程中起到的作用，为肿瘤的生物治疗提供了有用的分子靶标，可通过调控miRNAs的表达来治疗肿瘤。近来，以miRNAs为靶点的肿瘤治疗方案有很多报道，给肿瘤的治疗带来了希望。对于在肿瘤细胞中高表达的OncomiRs，已有研究采用miRNAs抑制剂或反义序列封闭miRNAs的表达，抑制肿瘤生长。但是，现有的治疗性研究大多针对单一miRNA或者miRNAs家族。由于miRNAs作用的靶基因众多，调控机制复杂，一个miRNA可以靶向多个靶基因，一个靶基因也可能受多种miRNAs的调控，而肿瘤的发生发展一定涉及很多miRNAs分子，调控广 泛的靶基因或者影响很多信号途径。因此，单一miRNA表达的干预对肿瘤的抑制效果有限，癌细胞很容易通过旁路信号途径重新获得增殖活力。
肿瘤特异性增殖腺病毒能够特异性地在肿瘤细胞内大量增殖复制，溶解破坏肿瘤细胞，释放子代病毒，感染更多的肿瘤细胞，故又称之为溶瘤腺病毒(Oncolytic adenovirus)。利用肿瘤特异性增殖腺病毒的高弥散力、高转染力以及在肿瘤细胞内特异性增殖复制的能力，可使其携带的抗癌基因拷贝数随病毒增殖而增加，在肿瘤细胞中获得高转染率、高拷贝数及高效表达的抗癌因子，提高对肿瘤的抗癌效应。由于腺病毒是目前肿瘤基因治疗的常见的、安全的病毒载体，已广泛地应用于人体基因治疗方案中，具有以下优点：基因组完全清楚，易于操作；能感染的人类细胞广泛，可高效地转导不同类型的人组织细胞，包括静止期细胞；病毒滴度较高，在细胞培养物中有高滴度的重组病毒产量；基因组大，因而可插入大片段外源基因；进入细胞内并不整合到宿主细胞基因组，因而安全性较高，无致癌性。中国国家食品药品监督管理局(SFDA)2004年和2005年已批准了2个腺病毒产品上市，临床应用至今未发现明显的毒副作用。
目前，还未见报道采用肿瘤特异性增殖腺病毒介导人工设计的LncRNA在癌细胞中特异性增殖，随腺病毒的增殖复制，以实现高拷贝、高效率表达LncRNA，提高其竞争结合miRNAs的效果，全面保护多种OncomiRs的靶基因，最终发挥更有效的抗癌效应。
发明内容
本发明的目的在于提供一种可用于肝癌等多种肿瘤治疗的竞争性消耗OncomiRs并实现多种miRNAs联合干预的LncRNA、可在癌细胞内特异性增殖复制并表达所述LncRNA的溶瘤腺病毒。
本发明提供了一种竞争性消耗OncomiRs的LncRNA，所述的LncRNA的序列为SEQ ID NO.4所示序列的n个拷贝，其中，n为大于等于1的整数。
作为本发明的一个优选例，所述的n等于6。
本发明提供了编码所述的LncRNA的编码序列，所述的LncRNA的编码序列为SEQ ID NO.1所示序列的n个拷贝，其中，n为大于等于1的整数。
作为本发明的一个优选例，所述的n等于6。
本发明提供了一种溶瘤腺病毒，所述的溶瘤腺病毒的基因组中整合有所述的LncRNA的表达盒。
作为本发明的一个优选例，所述的LncRNA的表达盒包含所述的LncRNA的编码序列，以及调控LncRNA的编码序列表达的启动子，所述的调控LncRNA的编码序列表达的启动子插入在所述LncRNA的编码序列的转录起始位点之前。
作为本发明的一个优选例，所述的溶瘤腺病毒的基因组中含有病毒增殖必需基因和调控所述病毒增殖必需基因表达的肿瘤特异性启动子。
作为本发明的一个优选例，所述的溶瘤腺病毒是以人类5型腺病毒为基础构建而成；所述的调控LncRNA的编码序列表达的启动子序列如SEQ ID NO.6所示，所述的病毒增 殖必需基因序列如SEQ ID NO.8所示，所述的肿瘤特异性启动子序列如SEQ ID NO.7所示。
作为本发明的一个优选例，所述的溶瘤腺病毒的全基因组序列如SEQ ID NO.10所示。
本发明提供了所述的LncRNA、所述的LncRNA的编码序列或所述的溶瘤腺病毒在制备药物中的用途，所述的药物用于治疗肿瘤。
作为本发明的一个优选例，所述的肿瘤中miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR106b、miR-151-5p、miR-155、miR-181a/181b、miR-184、miR-1和/或miR-449a高表达。
作为本发明的一个优选例，所述的肿瘤是肝癌，特别是原发性肝细胞肝癌。
本发明提供了所述的LncRNA、所述的LncRNA的编码序列或所述的溶瘤腺病毒在制备试剂中的用途，所述的试剂用于研究肝癌细胞中的分子作用机制或/和肝癌的治疗。
本发明的优点在于提供了一种竞争性消耗OncomiRs的LncRNA、重组溶瘤腺病毒及其应用，具有以下重要意义：
1、具有多重有效的抗肿瘤机制：1)所构建的溶瘤腺病毒在肿瘤特异性Survivin基因启动子的有效控制下，能在肿瘤细胞内特异性大量增殖复制，溶解破坏肿瘤细胞，释放子代病毒感染破坏更多的肿瘤细胞；2)通过在癌细胞内持续表达人工设计的LncRNA，该LncRNA同时包含能与多个OncomiRs互补结合的序列，能够与OncomiRs的所有靶基因mRNAs竞争结合OncomiRs，从而消耗细胞内高水平的OncomiRs，对细胞内的抑癌基因起保护作用，发挥抗癌效应。这种治疗策略，可以同时有效封闭多个作用机制不同、或者作用机制互补的OncomiRs，广泛抑制OncomiRs相关的多条信号传导途径，对肿瘤的治疗更为有效，克服了单一miRNA干预对肿瘤抑制效果有限、癌细胞很容易通过旁路信号途径重新获得增殖活力的缺陷；3)以肿瘤特异性增殖溶瘤腺病毒作为载体，介导人工设计的LncRNA的表达，随着病毒在肿瘤内的特异性增殖复制，LncRNA的拷贝数和表达量呈几何倍数增长，高拷贝、高效率表达LncRNA，可大量消耗OncomiRs，对细胞内的抑癌基因显现良好的保护效果，从而抑制肿瘤发生发展乃至完全消褪。
2、具有多重有效的靶向安全机制：1)以Survivin启动子为顺式作用元件，插入腺病毒增殖必需基因E1a的转录起始位点与翻译起始位点ATG之间，调控E1a转录激活，使E1a只能特异性地在Survivin阳性的肿瘤细胞内表达，而对正常细胞没有影响或者影响极弱，其调控具有更好的可靠性和安全性；2)人工合成的LncRNA包含一组在癌细胞内高表达、能够通过多种机制促进肿瘤发生发展的OncomiRs的互补结合序列，其目标是消耗或干预OncomiRs，对癌细胞内其他的抑癌性miRNAs不起作用，正常细胞因为其OncomiRs不表达或水平极低而不会受到影响，因而使其抗肿瘤疗效得以提高，安全性得以改善；3)在LncRNA编码序列起始和终止位置引入翻译终止密码子，阻止其翻译成为任何蛋白或多肽分子。
3、本发明提供的LncRNA和腺病毒载体相结合的抗肿瘤体系适应于人类绝大多数 类型恶性肿瘤的治疗，目前国内外未见类似治疗方案，可据此构建多种抗肿瘤生物治疗产品，为肿瘤治疗建立了一个疗效可靠的技术平台。
附图说明
图1.AdSVPE1a-lncR在不同细胞系中的增殖倍数比较。
图2.AdSVPE1a-lncR介导LncRNA在不同细胞系中表达水平的比较。
图3.AdSVPE1a-lncR介导LncRNA表达对细胞存活率的影响。
图4.AdSVPE1a-lncR介导LncRNA表达对细胞迁移侵袭力的影响。
图5.AdSVPE1a-lncR介导LncRNA表达对肝癌细胞基因表达谱的影响。
图6.AdSVPE1a-lncR对Huh-7肝癌细胞裸鼠移植瘤的抑瘤效果。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
本申请的发明人利用肿瘤特异性的Survivin基因启动子调控溶瘤腺病毒增殖必需基因E1a，使病毒能在癌细胞内特异性增殖复制；以此溶瘤腺病毒为载体，表达一种人工合成的LncRNA编码序列，该LncRNA编码序列包含一组能够通过多种机制促进癌细胞发生发展的OncomiRs的种子序列的互补结合序列；随腺病毒在癌细胞内特异性高拷贝复制，实现LncRNA在癌细胞内大量表达和高拷贝积累，通过与OncomiRs的靶基因竞争性结合并消耗OncomiRs，从而保护细胞内大量的抑癌基因免受OncomiRs的干扰和抑制，实现对癌细胞的靶向干预治疗。这样的策略由于其病毒载体的肿瘤靶向特异性增殖和针对OncomiRs致癌作用的特异性干预，因而使其抗肿瘤疗效得以提高，安全性得以改善。
LncRNA编码序列
所述的“LncRNA编码序列”即编码LncRNA的DNA序列。作为本发明的一个优选例，LncRNA的编码序列如SEQ ID NO.1所示。作为本发明的一种具体实施方式，LncRNA的编码序列如SEQ ID NO.2所示，且为了使LncRNA的编码序列能够得到比较强的转录活性，防止其翻译，在SEQ ID NO.2所示序列的5’-端加入了CACCATGC，在3’-端加入AG，增加后的LncRNA编码序列如SEQ ID NO.3所示。以上序列的引入是为了在LncRNA编码序列的起始和终止位置增加翻译终止密码子，该终止密码子可以是TAG、TGA或TAA。
本发明的LncRNA编码序列优选为人工合成的方法获得。
作为本发明的一种具体实施方式，所述的LncRNA编码序列包含一组OncomiRs的种子序列的互补结合序列，该组OncomiRs在肝癌细胞中高水平表达，能够通过多种机制促进肝癌细胞发生发展。该组OncomiRs包括miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR106b、miR-151-5p、miR-155、miR-181a/181b、miR-184、miR-1、miR-449a。它们在肝癌的发生发展中分别作用于不同的靶基因和信号传导途径，分别起不同的作用。如：肝癌中miR-21高表达可抑制抑癌基因PTEN的表达，PTEN蛋白是 3-磷酸肌醇激酶(phosphoinositide 3-kinase，PI3K)/丝/苏氨酸蛋白激酶(serine/threonine kinase，AKT)通路的重要抑制蛋白，因此，miR-21抑制PTEN的后果即是激活PI3K/AKT通路，促进癌细胞的增殖、转移；miR-221/222作用于许多关键的肿瘤抑制因子，包括Bmf、p27Kip1、p57Kip2l6、PTEN、金属蛋白酶-3的组织抑制因子TIMP-3以及DNA损伤诱导转录子4(DDIT4)，从而介导肿瘤的发生发展。将这些miRNAs按功能分类，肝癌中miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR106b、miR-151-5p、miR-155、miR-181a/181b、miR-184高表达则能够促进癌细胞增殖、侵袭、转移；miR-221/222、miR-224、miR-10b、miR-155高表达能够抑制癌细胞凋亡；miR-21、miR-221/222、miR-143、miR-1、miR-449a高表达能够促进乙肝病毒增殖，诱导细胞癌变和增强癌细胞增殖活性。抑制这些有致癌、促癌活性的miRNAs的表达及功能，可通过多种途径多种效应抑制肝癌的发生发展。
本发明不仅限于此，所述的LncRNA的编码序列还可以包含一个或一组OncomiRs的种子序列的互补结合序列，所述的OncomiRs既可以是一个或一组在肝癌之外其他肿瘤中高表达、并能够通过多种机制促进肝癌之外其他肿瘤发生发展的OncomiRs，也可以是针对肝癌除miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR-151-5p、miR-155、miR-181a/181b、miR-184、miR-1、miR-449a、miR106b之外的一个或一组OncomiRs。
LncRNA
长链非编码RNA(Long non-coding RNA，LncRNA)是长度在200-100000nt之间的RNA分子，不编码蛋白，参与细胞内多种过程调控。
本发明的LncRNA由包含一组能够通过多种机制促进肝癌细胞发生发展的OncomiRs的种子序列互补结合序列的DNA序列编码获得。作为本发明的一个优选例，编码得到的LncRNA序列如SEQ ID NO.4所示。作为本发明的一种具体实施方式，以上LncRNA的编码序列在病毒的基因组中重复6倍，编码得到的LncRNA序列如SEQ ID NO.5所示，以提高竞争性结合相应OncomiRs的效果。
LncRNA编码序列的启动子
所述的“LncRNA编码序列的启动子”用于启动LncRNA编码序列的转录，其包含其它的具备相同功能的顺式作用元件。本发明的LncRNA编码序列在病毒的基因组中可以受以下任意一种启动子或顺式元件的调控：(a)癌胚抗原启动子；(b)甲胎蛋白启动子；(c)人表皮生长因子受体家族(EGFRs)的受体酪氨酸激酶(包括EGFR、Her-2、Her-3和Her-4)启动子；(d)乳腺癌相关抗原DF3/MUC1启动子；(e)血管内皮生长因子(VEGF)受体KDR的启动子；(f)单纯疱疹病毒启动子；(g)E2F启动子；(h)前列腺素特异性抗原的启动子。作为本发明的一个优选例，所述的LncRNA编码序列受鼠巨细胞病毒(CMV)启动子的控制，该顺式作用元件插入LncRNA编码序列的转录起始位点之前，保证LncRNA在病毒感染的肿瘤内部高效表达，发挥生物学活性。所述的CMV启动子的序列优选如SEQ ID NO.6所示。
肿瘤特异性启动子
所述的肿瘤特异性启动子为一种肿瘤特异性的调控序列。由于许多肿瘤都有其自身特异性的肿瘤标志物，其表达受该肿瘤特异性顺式作用元件或反式作用因子的调控，因此，将病毒增殖基因置于此类肿瘤特异性调控序列的调控之下，可使病毒选择性地在相应的组织细胞中增殖，而不影响其它组织细胞。
本发明所述的肿瘤特异性启动子可以由以下任何一组启动子和增强子构成：(a)癌胚抗原启动子、增强子及其突变体序列；(b)甲胎蛋白启动子、增强子及其突变体序列；(c)人表皮生长因子受体家族(EGFRs)的受体酪氨酸激酶(包括EGFR、Her-2、Her-3和Her-4)启动子、增强子及其突变体序列；(d)乳腺癌相关抗原DF3/MUC1启动子、增强子及其突变体序列；(e)血管内皮生长因子(VEGF)受体KDR的启动子、增强子及其突变体序列；(f)L-plastin启动子、增强子及其突变体序列；(g)凋亡抑制蛋白家族(IAP)成员的启动子、增强子及其突变体序列；(h)前列腺素特异性抗原的启动子、增强子及其突变体序列；(i)缺氧诱导因子-1(HIF-1)调控的缺氧反应元件保守序列；(j)转录因子E2F启动子、增强子及其突变体序列。
凋亡抑制因子Survivin属于凋亡抑制蛋白因子(IAP)家族成员，参与调节胚胎细胞生理发育和细胞周期调控的各个方面，通过多种途径抑制细胞凋亡，促进细胞增殖和周期进展。Survivin表达于胚胎发育组织和大多数肿瘤组织，但在正常成人组织不表达。Survivin在恶性肿瘤中的表达具有高度选择性，在人类最常见的绝大多数肿瘤组织如肺癌、结肠癌、胰腺癌、前列腺癌和乳腺癌以及非霍奇金淋巴瘤中都呈高表达，并且与肿瘤的复发转移以及患者的预后不佳有一定关系，使其成为一种广谱的肿瘤诊断标志物。因此，以Survivin启动子调控的病毒，有望实现针对大多数人体肿瘤的广谱的抗癌效应。
作为本发明的一个优选例，以Survivin启动子核心序列为顺式作用元件，插入腺病毒增殖必需基因E1a的转录起始位点与翻译起始位点ATG之间，调控E1a转录激活，使E1a只能特异性地在Survivin阳性的肿瘤细胞内表达，而对正常细胞没有影响或者影响极弱，其调控具有更好的可靠性和安全性。本发明采用的肿瘤特异性Survivin基因启动子的序列如SEQ ID NO.7所示。
病毒增殖必需基因
病毒增殖必需基因为病毒的增殖复制提供所必需的蛋白。腺病毒增殖必需基因包括E1a，还包括Elb-55kDa、Elb-19kDa、E3或E4。Elb-55kDa是腺病毒在正常细胞增殖复制必需的而在肿瘤细胞中不必需的蛋白，Elb-55kDa编码基因的选择性缺失可以使腺病毒在肿瘤细胞内保持增殖复制的能力，而在正常细胞中失去复制能力。Elb-55kDa能够灭活并降解P53蛋白，Elb-55kDa编码基因的选择性缺失有利于细胞保持P53蛋白的抗肿瘤活性，同时提高病毒载体的靶向性。腺病毒Elb-19kDa编码基因与凋亡抑制基因Bcl-2同源，编码的Elb-19kDa能够结合Bax或/和Bak启动下游的凋亡抑制程序，Elb-19kDa还可以破坏Fas介导的凋亡过程，保护感染的细胞免受TNF-α介导的杀伤作用。
作为本发明的一个优选例，所述的病毒增殖必需基因E1a序列如SEQ ID NO.8所示。
重组溶瘤腺病毒
本发明使用重组病毒载体介导LncRNA编码序列在肿瘤细胞的特异性表达，以发挥抗癌作用。所述的重组病毒载体可由现有的病毒载体、肿瘤特异性启动子和LncRNA的表达盒构建而成，所述的LncRNA的表达盒包含LncRNA编码序列及该LncRNA编码序列的启动子。
人类溶瘤腺病毒有A、B、C、D、E、F 6个不同亚属，它们对宿主细胞的亲嗜性、致瘤性及疾病史各不相同。本发明优选使用溶瘤腺病毒C亚属中的5型。
作为本发明的一个优选例，所述的重组溶瘤腺病毒由人类5型腺病毒、肿瘤特异性启动子和LncRNA的表达盒构建而成。该重组溶瘤腺病毒由含5型腺病毒基因组的骨架质粒pBHGloxdeltaE1,3Cre与携带LncRNA编码序列的腺病毒穿梭质粒在293细胞内通过Cre-loxP重组酶切割系统定点重组而产生。所述的重组溶瘤腺病毒的重组区以外的序列与pBHGloxdeltaE1,3Cre腺病毒载体重组区以外的序列一致，所述的重组溶瘤腺病毒的重组区内的序列如SEQ ID NO.9所示。
其中：(1)1-6bp：XbaI酶切位点；(2)7-996bp：Survivin启动子序列；(3)997-1002bp：EcoRI酶切位点；(4)1003-1991bp：E1a基因cDNA序列；(5)1992-2301bp：SV40PolyA加尾序列；(6)2302-2307bp：BamHI酶切位点；(7)2308-2838bp：鼠巨细胞病毒(CMV)启动子序列；(8)2339-2844bp：SalI酶切位点；(9)2845-2851bp：LncRNA转录起始位点；(10)2852-4030bp：6个重复拷贝的LncRNA编码序列；(11)4031-4190bp：LncRNA加尾序列；(12)4191-4196bp：SalI酶切位点。
所述的重组溶瘤腺病毒的全基因组序列如SEQ ID NO.10所示，且实施例中最终构建的重组溶瘤腺病毒的全基因组序列以SEQ ID NO.10为准。
实施例1肿瘤特异性Survivin启动子控制E1a的腺病毒质粒的构建
第一步：人工合成肿瘤特异性Survivin启动子，5’-端引入XbaI位点，3’-端引入EcoRI位点，插入质粒pDC315的XbaI+EcoRI位点，构建含Survivin启动子的质粒pDC315-SVP。pDC315为加拿大Microbix Biosystems公司产品(产品目录号：PD-01-27)。设计合成的Survivin启动子全长1002bp，序列是在SEQ ID NO.7的5’-端引入XbaI酶切位点(TCTAGA)；在3’-端引入EcoRI酶切位点(GAATTC)。
第二步：克隆获得腺病毒E1a序列(含PolyA加尾序列)，5’-端引入EcoRI位点和ACC，3’-端引入BamHI位点，插入pDC315-SVP的EcoRI+BamHI位点，构建成肿瘤特异性Survivin启动子控制E1a表达的质粒pDC315-SVPE1a。E1a(含PolyA加尾序列)全长1311bp，其中，第1-6bp：EcoRI酶切位点(GAATTC)；第7-9bp：ACC；第10-995bp：E1a基因cDNA序列(SEQ ID NO.8)；第996-1305bp：SV40PolyA加尾序列(SEQ ID NO.11)；第1306-1311bp：BamHI酶切位点(GGATCC)。
实施例2表达人工合成的长链非编码RNA(LncRNA)的腺病毒包装质粒的构建
第一步：克隆获得鼠巨细胞病毒(CMV)启动子序列，5’-端引入BamHI位点，3’-端引入SalI位点，插入质粒pDC315的BamHI+SalI位点，构建成含mCMV启动子的质粒pDC315-mCMV。mCMV序列长543bp，其中，第1-6bp：BamHI酶切位点(GGATCC)； 第7-537bp：鼠巨细胞病毒(CMV)启动子序列(SEQ ID NO.6)；第538-543bp：SalI酶切位点(GTCGAC)。
第二步：人工合成编码LncRNA的6个拷贝的DNA序列(含PolyA加尾序列)，5’-端和3’-端均引入SalI位点，插入pDC315-mCMV的SalI位点，构建成含LncRNA编码序列的表达质粒pDC315-mCMVLncR。编码LncRNA的DNA序列(含PolyA加尾序列)长1348bp，其中，第1-6bp：SalI酶切位点(GTCGAC)；第7-13bp：转录起始位点(CACCATG)；第14-1192bp：含有6个重复拷贝的LncRNA编码序列(SEQ ID NO.12)；第1193-1352bp：LncRNA加尾序列(SEQ ID NO.13)；第1353-1358bp：SalI酶切位点(GTCGAC)。
第三步：pDC315-mCMVLncR经BamHI+SalI酶切，回收含有LncRNA完整表达框的片段，插入pDC315-SVPE1a质粒的BamHI+SalI位点，构建携带LncRNA编码序列的肿瘤特异性增殖腺病毒包装质粒pDC315-SVPE1a-mCMVLncR。LncRNA完整表达框全长1895bp，其中，第1-6bp：BamHI酶切位点；第7-537bp：鼠巨细胞病毒(CMV)启动子序列(SEQ ID NO.6)；第538-543bp：SalI酶切位点；第544-550bp：转录起始位点(CACCATG)；第551-1729bp：含有6个重复拷贝的LncRNA编码序列(SEQ ID NO.12)；第1730-1889bp：LncRNA加尾序列(SEQ ID NO.13)；第1890-1895bp：SalI酶切位点。
实施例3携带LncRNA编码序列的肿瘤特异性增殖腺病毒的重组、扩增和纯化
第一步：将构建好的5型腺病毒左臂包装质粒pDC315-SVPE1a-mCMVLncR与5型腺病毒右臂包装质粒pBHGloxdelE13cre通过LipoFectamine2000共转染HEK293细胞。转染具体方法步骤参见Invitrogen公司的LipoFectamine2000试剂盒操作说明书。质粒pBHGloxdelE13cre和HEK293细胞株为加拿大Microbix Biosystems公司产品。pBHGloxdelE13cre含有5型腺病毒右臂，缺失E1、E3区，其重组酶系统Cre/LoxP可以保证病毒的高效重组。HEK293细胞由剪切的5型腺病毒DNA转化，含5型腺病毒E1区，腺病毒DNA对其具有高转染效率，能够促进腺病毒的重组和包装。在载体共转染后的14天出现病毒空斑，经过三次病毒空斑纯化(具体方法参见GeneTransfer and Expression Protocols，Murray EJ主编，Humana Press,Clifton,New Jersey)，重组出腺病毒AdSVPE1a-lncR。
完整的腺病毒AdSVPE1a-lncR基因组序列如SEQ ID NO.10所示。其中：(1)1-85bp：5型腺病毒ITR序列；(2)86-437bp：5型腺病毒基因组序列；(3)438-443bp：XbaI酶切位点；(4)444-1433bp：Survivin启动子序列；(5)1434-1439bp：EcoRI酶切位点；(6)1440-2428bp：E1a基因cDNA序列；(7)2429-2738bp：SV40PolyA加尾序列；(8)2739-2744bp：BamHI酶切位点；(9)2745-3275bp：鼠巨细胞病毒(CMV)启动子序列；(10)3276-3281bp：SalI酶切位点；(11)3282-3288bp：LncRNA转录起始位点；(12)3289-4467bp：含有6个重复拷贝的LncRNA编码序列；(13)4468-4627bp：LncRNA加尾序列；(14)4628-4633bp：SalI酶切位点；(14)4634-4668bp：LoxP 序列；(15)4669-34339bp：5型腺病毒基因组序列；(16)34340-34441bp：5型腺病毒ITR序列。
第二步：腺病毒AdSVPE1a-lncR在HEK293细胞中大量繁殖，应用氯化铯梯度离心的方法大量纯化腺病毒(具体方法参见GeneTransfer and Expression Protocols，Murray EJ主编，Humana Press,Clifton,New Jersey)。
实施例4重组腺病毒AdSVPE1a-lncR的鉴定
重组腺病毒AdSVPE1a-lncR的插入序列通过测序和PCR证实。AdSVPE1a-lncR为5型腺病毒，插入序列包括6个拷贝的LncRNA编码序列、mCMV启动子、Survivin启动子、E1a序列，其他DNA序列与5型腺病毒相同。鉴定所用PCR引物如表1所示：
表1.重组腺病毒AdSVPE1a-lncR鉴定的PCR引物
Figure PCTCN2015091892-appb-000001
结果：以上述引物序列进行PCR，均能扩增出与理论长度相同的目的条带，且测序正确。
实施例5重组腺病毒AdSVPE1a-lncR在肝癌细胞中的增殖及基因表达实验
第一步：重组腺病毒AdSVPE1a-lncR的特异性增殖。收集对数生长期的肝癌细胞(HepG2、Hep3B、SMMC-7721、MHCC97H、MHCC97L、Huh-7、PLC/PRF/5)和正常肝细胞(L02、WRL-68)，计数，铺96孔板，1×104/孔，细胞贴壁后，换用无血清培养液；以MOI＝1加入AdSVPE1a-lncR。病毒感染2h后调节至5％血清培养液，继续培养12h、24h、48h、72h，分别在这三个时间段收集细胞，TCID50方法检测病毒滴度。结果显示：AdSVPE1a-lncR在HepG2、Hep3B、MHCC97H、Huh-7、PLC/PRF/5中特异性增殖复制的能力非常强，72h后可达数千至数万倍，最高达68465.66倍(Huh-7)，在SMMC-7721、MHCC97L中达数百倍，而在正常细胞中L02、WRL-68增殖不明显，在10倍以下(图1)。
第二步：LncRNA的特异性高效表达。上述细胞以MOI＝1感染实验腺病毒AdSVPE1a-lncR，培养48h后收集细胞，实时定量RT-PCR检测LncRNA的表达，LncRNA特异性PCR引物为LncR-F(5’-CTGCACTGTCAGCACTTTA-3’)和LncR-R(5’-ACATTCATTGCTGTCGGTG-3’)。结果显示：AdSVPeGFP-lncR介导的LncRNA表 达水平，在Huh-7、Hep3B、HepG2中最高，其次为PLC/PRF/5、MHCC97H、BEL-7402、MHCC97L、SMMC-7721，在正常肝细胞L02和WRL-68中表达量极低(图2)。
实施例6重组腺病毒AdSVPE1a-lncR对肝癌细胞生物学行为的影响
第一步：LncRNA表达对细胞增殖活性的影响。通过四唑盐比色实验(MTT)检测病毒AdSVPE1a-lncR对肿瘤细胞和正常细胞的作用。Cell Proliferation Kit I(MTT)购于Roche Diagnostics GmbH。收集对数生长期的细胞，计数，用10％血清培养液稀释细胞，1×104细胞数/100μl/孔，传至96孔板；细胞贴壁后，以无血清培养液稀释病毒，按MOI＝1～100pfu/cell的感染强度加入相应病毒100μl，对应于每个病毒设8个复孔，孵箱内培养2h；换血清培养液100μl/孔，培养48h后，弃培养液，加入0.1mol/L PBS溶液100μl/孔，再加MTT labeling reagent 10μl/孔至终浓度0.5mg/ml，置孵箱内4h；加Solubilization solution(10％SDS in 0.01mol/L HCl)100μl/孔，置孵箱内过夜；采用Model 550Microplate Reader(BIO-RAD)测定570nm波长光吸收值，校正波长为655nm；绘制曲线。结果显示：AdSVPE1a-lncR对Hep3B和Huh-7杀伤活性最强，Hep3B存活率在MOI＝2pfu/cell时已下降到10％以下，在MOI＝0.5pfu/cell时已下降到50％以下；Huh-7存活率在MOI＝100pfu/cell时已下降到10％以下，在MOI＝1pfu/cell时已下降到50％以下。AdSVPE1a-lncR对HepG2和MHCC97L的杀伤，在MOI＝20pfu/cell时存活率下降到50％以下；AdSVPE1a-lncR对PLC/PRF/5的杀伤，在MOI＝50pfu/cell时存活率下降到50％以下；AdSVPE1a-lncR对MHCC97H和SMMC-7721的杀伤，在MOI＝100pfu/cell时存活率下降到50％以下；AdSVPE1a-lncR对BEL-7402的杀伤活性较弱，在MOI＝200pfu/cell时存活率才下降到50％以下。AdSVPE1a-lncR对正常肝细胞无明显影响，在MOI＝500以下细胞存活率一直维持在80％以上。
第二步：LncRNA表达对细胞迁移侵袭能力的影响。实验组为以MOI＝10pfu/cell感染AdSVPE1a-lncR病毒的肿瘤细胞(ER1组)，以AdSVPeGFP-lncR为阳性病毒对照(ER2组，表达LncRNA的非增殖病毒)，以Ad5-eGFP为阴性病毒对照(CR1组，不表达LncRNA的非增殖病毒)，另设不加病毒的细胞同步培养作为空白对照(CR2组)。将Transwell小室置于24孔板中，在小室的上室加入细胞，每小室加入4×105细胞数/200μl，下室中加入500μl含10％胎牛血清的培养基。观察细胞侵袭力时上室加聚碳酸酯膜基质胶，观察细胞迁移力时不加基质胶。细胞培养24h后取出，擦去小室上层细胞，用0.1％结晶紫染色20min，光学显微镜下随机取3个视野(×200倍)进行计数并拍照，实验重复3次。结果显示：AdSVPE1a-lncR能够明显抑制肝癌细胞的迁移侵袭能力，AdSVPeGFP-lncR也有抑制肝癌细胞迁移侵袭力的活性，但活性弱于AdSVPE1a-lncR。AdSVPE1a-lncR和AdSVPeGFP-lncR对正常肝细胞无明显作用(图4)。
实施例7重组腺病毒AdSVPE1a-lncR对肝癌细胞基因表达谱的影响
收集对数生长期的Huh-7细胞，1×106细胞数/100μl/孔，传至24孔板；细胞贴壁后，以无血清培养液稀释病毒，按MOI＝10pfu/cell的感染强度加入AdSVPE1a-lncR病毒100μl，设Ad5-eGFP为阴性病毒对照，不加病毒的细胞同步培养作为空白对照。孵箱内 培养2h；换血清培养液100μl/孔，培养48h后，收集细胞，提取细胞总蛋白，基因表达谱芯片检测蛋白表达的变化。空白对照组以cy3荧光信号标记为绿色，实验组以cy5荧光信号标记为红色。两组荧光信号叠加，在cy3信号较强时显绿色，差值低于0.5倍提示基因表达呈下调趋势；在cy5信号较强时显红色，差值达2倍提示基因表达呈上调趋势；cy3和cy5信号强度相似，即显示黄色，变化趋势在0.5倍至2倍之间。实验结果显示：Huh-7感染AdSVPE1a-lncR后，基因表达谱变化明显。检出升高表达者708个基因，其中原癌基因和抑癌基因类13个，离子通道和运输蛋白类11个，细胞周期蛋白类12个，细胞骨架及运动蛋白类13个，细胞凋亡相关蛋白类5个，DNA合成修复及重组蛋白类6个，DNA结合及转录因子类30个，细胞受体类10个，免疫相关蛋白类27个，细胞信号及传导蛋白类74个，代谢分子61个，蛋白翻译合成因子类48个，分化发育相关类9个，其他类分子389个；检出降低表达者628个基因，其中原癌基因和抑癌基因类12个，离子通道和运输蛋白类14个，细胞周期蛋白类21个，细胞骨架及运动蛋白类32个，细胞凋亡相关蛋白类5个，DNA合成修复及重组蛋白类7个，DNA结合及转录因子类17个，细胞受体类12个，免疫相关蛋白类31个，细胞信号及传导蛋白类59个，代谢分子63个，蛋白翻译合成因子类48个，分化发育相关类8个，其他类分子299个。图5显示一些代表性基因表达水平的变化，其中上调比较明显的是PTEN、p27kip1、TIMP3、RECK，下调比较明显的是p38/MAPK、Survivin、CDK4、c-myc(图5)。
实施例8重组腺病毒AdSVPE1a-lncR对肝癌裸鼠移植瘤的抑制作用
健康纯种BALB/C裸鼠40只，5周龄，雄性，中科院上海斯莱克实验动物中心提供，合格证号SCXK(沪)2012-0002。取对数生长期Huh-7细胞悬液注射于裸鼠右腋皮下，5×106细胞数/100μl/只。接种后10天，成瘤率100％，移植瘤直径大约0.8-1.0cm。随机分为4组(AdSVPE1a-lncR、AdSVPeGFP-lncR、Ad5-eGFP、空白对照组)。病毒治疗组给予相应腺病毒瘤内多点注射，每次每只剂量2×108pfu/100μl，隔天一次，共5次；空白对照组同步注射生理盐水，每次每只100μl。治疗后，每周测量瘤体大小，以“最大径×最小径2×0.5”公式计算瘤体体积，绘制生长曲线。
至治疗后第14天，AdSVPE1a-lncR治疗组肿瘤生长速度已明显低于空白对照组，至第21天，肿瘤体积出现下降；AdSVPeGFP-lncR组在治疗后28天也出现一定程度的生长抑制，与空白对照组相比，差别具有显著意义，但瘤体仍在持续增长中；Ad5-eGFP组自始至终未出现生长抑制(表2)。观察至治疗后42天，AdSVPE1a-lncR组瘤体体积已明显低于治疗前(图6)。终止观察，无痛处死裸鼠。
表2.AdSVPE1a-lncR对Huh-7肝癌细胞裸鼠移植瘤的抑瘤效果
Figure PCTCN2015091892-appb-000002
注：*与同一时间点的对照组相比，T检验。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Figure PCTCN2015091892-appb-000003
Figure PCTCN2015091892-appb-000004
Figure PCTCN2015091892-appb-000005
Figure PCTCN2015091892-appb-000006
Figure PCTCN2015091892-appb-000007
Figure PCTCN2015091892-appb-000008
Figure PCTCN2015091892-appb-000009
Figure PCTCN2015091892-appb-000010
Figure PCTCN2015091892-appb-000011
Figure PCTCN2015091892-appb-000012
Figure PCTCN2015091892-appb-000013
Figure PCTCN2015091892-appb-000014
Figure PCTCN2015091892-appb-000015
Figure PCTCN2015091892-appb-000016
Figure PCTCN2015091892-appb-000017
Figure PCTCN2015091892-appb-000018
Figure PCTCN2015091892-appb-000019

Claims (10)

  1. 一种竞争性消耗致癌性microRNAs的LncRNA，其特征在于，所述的LncRNA的序列为SEQ ID NO.4所示序列的n个拷贝，其中，n为大于等于1的整数。
  2. 编码权利要求1所述的LncRNA的编码序列，其特征在于，所述的编码序列为SEQ ID NO.1所示序列的n个拷贝，其中，n为大于等于1的整数。
  3. 一种溶瘤腺病毒，其特征在于，所述的溶瘤腺病毒的基因组中整合有权利要求1所述的LncRNA的表达盒。
  4. 根据权利要求3所述的溶瘤腺病毒，其特征在于，所述的LncRNA的表达盒包含权利要求2所述的LncRNA的编码序列，以及调控LncRNA的编码序列表达的启动子，所述的调控LncRNA的编码序列表达的启动子插入在所述LncRNA的编码序列的转录起始位点之前。
  5. 根据权利要求4所述的溶瘤腺病毒，其特征在于，所述的溶瘤腺病毒的基因组中含有病毒增殖必需基因和调控所述病毒增殖必需基因表达的肿瘤特异性启动子。
  6. 根据权利要求5所述的溶瘤腺病毒，其特征在于，所述的溶瘤腺病毒是以人类5型腺病毒为基础构建而成；所述的调控LncRNA的编码序列表达的启动子序列如SEQ ID NO.6所示，所述的病毒增殖必需基因序列如SEQ ID NO.8所示，所述的肿瘤特异性启动子序列如SEQ ID NO.7所示。
  7. 根据权利要求3所述的溶瘤腺病毒，其特征在于，所述的溶瘤腺病毒的全基因组序列如SEQ ID NO.10所示。
  8. 权利要求1所述的LncRNA、权利要求2所述的LncRNA的编码序列或权利要求3-7任一所述的溶瘤腺病毒在制备药物中的用途，其特征在于，所述的药物用于治疗肿瘤。
  9. 根据权利要求8所述的用途，其特征在于，所述的肿瘤中miR-21、miR-221/222、miR-224、miR-17-5p、miR-10b、miR106b、miR-151-5p、miR-155、miR-181a/181b、miR-184、miR-1和/或miR-449a高表达。
  10. 权利要求1所述的LncRNA、权利要求2所述的LncRNA的编码序列或权利要求3-7任一所述的溶瘤腺病毒在制备试剂中的用途，其特征在于，所述的试剂用于研究肝癌细胞中的分子作用机制或/和肝癌的治疗。
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