CN102257140A

CN102257140A - Treating hepatitis c virus infection with over-expression of microrna-196

Info

Publication number: CN102257140A
Application number: CN2009801451621A
Authority: CN
Inventors: 赫伯特·L·帮科夫斯基; 侯卫红
Original assignee: Charlotte Mecklenburg Hospital
Current assignee: Charlotte Mecklenburg Hospital
Priority date: 2008-10-08
Filing date: 2009-10-08
Publication date: 2011-11-23
Also published as: AU2009302344A2; KR20110065568A; CA2739948A1; WO2010042683A1; AU2009302344A1; AR073800A1; BRPI0914060A2; MX2011003813A; JP2012505233A; EP2352829A1

Abstract

The present invention is directed to methods of treating cells infected with HCV and mammals suffering from HCV infection by transfecting the infected cells with miRNA-196 mimic. miRNA-196 mimic significantly down-regulates Bach1 protein and HCV gene expression, while also up-regulating HMOX1 gene expression. miRNA-196 binds with the 3'-UTR of Bach1 mRNA to reduce the expression of Bach1. As such, miRNA-196 can play an important role in the regulation of HCV replication and HMOX1/Bach1 expression in hepatocytes. The present invention also provides a formulation for the treatment of cells expressing HCV comprising a therapeutically effective amount of miRNA-196 such that Bach1 and HCV gene expression are down- regulated while HMOX1 expression is increased. The formulations are adapted to enable the transfection of miRNA-196 mimic into hepatocytes expressing HCV proteins.

Description

Mistake expression treatment infection with hepatitis C virus with microRNA-196

The research and development of federal funding

The present invention obtains United States Government's support and finishes under the RO1-DK38825 that NIH/NIDDK authorized.United States Government enjoys certain right in the present invention.

Technical field

The present invention relates generally to the method and formulation that is used for the treatment of hepatitis C infection.More specifically, the present invention relates to Bach1 and/or HCV.Can make Bach1 and the downward modulation of HCV NS5A albumen by miR-196, HMOX1 is raised.

Background technology

Hepatitis C virus (HCV) is a kind of small volume (being of a size of 50nm) in the flaviviridae, the sense single stranded rna virus of tool coating.Though hepatitis A virus, hepatitis B virus and hepatitis C virus have similar title because of it all causes the liver inflammation, in heredity with clinically every kind all be unique and different virus.HCV causes haematogenous (promptly propagating with contacting of blood by blood) transmissible disease, i.e. hepatitis C.This infection often is asymptomatic, but in a single day sets up, and chronic infection can cause liver inflammation (chronic hepatitis).This illness can be made progress and is liver scar (fibrosis) and scar in late period (liver cirrhosis).In some cases, the suffering from liver cirrhosis person can continue to develop liver failure or other liver cirrhosis complication, comprises liver cancer.

Acute hepatitis C is meant preceding 6 months after the HCV infection.Infected people symptom can not occur in acute phase about 60%～70%.In the sub-fraction patient of experience acute stage symptom, these symptoms normally slight and nonspecific, and can cause particular diagnosis hardly to hepatitis C.The symptom of acute hepatitis C infection comprises appetite reduction, fatigue, stomachache, jaundice, itches and influenza-like symptom.After infection, in 1～3 week, in blood, can detect HCV usually, and in 3～12 weeks, can detect usually at this viral antibody.As shown in normalization method in the hepatic function test (LTF) (for example alanine aminotransferase (ALT) and aspartate aminotransferase (AST) normalization method) and blood plasma HCV-RNA removing (spontaneous virus sweep), about 15%～40% can dispose virus in acute phase from its health in the people of HCV infection.Remaining 60%～85% patient who infected by HCV can develop into chronic hepatitis C (CHC).

Chronic hepatitis C is defined as continuing to surpass 6 months HCV infection.The nature process of chronic hepatitis C can be obviously different between different people.Basically all people that infected by HCV demonstrate the inflammation sign on liver biopsy.Yet the advance rate of liver scar (fibrosis) can demonstrate significant mutability between individuality.Nearest data be illustrated among the not subject patient about 1/3 can be in less than 20 years progress be liver cirrhosis.Other 1/3 can progress be liver cirrhosis in 30 years.Before substantial liver scar occurs, there is not to indicate specifically the symptom of hepatic diseases usually.Yet hepatitis C is a systemic disease, and before developing into the hepatic diseases in late period, the patient may experience the various clinical symptoms form from asymptomatic to the more obvious patient's condition of symptom.The general S﹠S relevant with chronic hepatitis C comprises fatigue, significantly loses weight, influenza-like symptom, myalgia, arthrodynia, intermittent slight fever, itch, somnopathy, stomachache (especially at right upper quadrant of the abdomen), appetite change, feel sick, diarrhoea, maldigestion, cognitive change, depression, headache and anxious state of mind.

Be liver cirrhosis in case chronic hepatitis C has made progress, the S﹠S that causes by decreased liver function or liver circulation high pressure (a kind of illness that is called as portal hypertension) usually may occur.Possible liver cirrhosis S﹠S comprises ascites (accumulation of liquid in the abdomen), contusion and bleeding tendency, ostalgia, varix (venectasia is especially in stomach and esophagus), fatty stool (steatorrhea), jaundice and a kind of cognitive impairment syndrome that is called as hepatogenic encephalopathy.

Chronic hepatitis C is diagnosed more than other form hepatitis, its reason is the outer performance form of the liver relevant with the existence of HCV, for example has especially membranoproliferative glomerulo nephritis (MPGN) of hyperthyroidism or hypothyroid thyroiditis (Tiroidina inflammation), porphyria cutanea tarda, cryoglobulinemia (a kind of little blood vessel vasculitis form) and glomerulonephritis (kidney inflammation).Hepatitis C is also relevant with sjogren syndrome, thrombopenia, lichen planus, diabetes and B cell lymphocytic hyperplasia sexual disorder.

Infection with hepatitis C virus is worldwide health problem, and its vaccine does not still exist at present.The present standard treatment of chronic hepatitis C (CHC) is to be used in combination Peg-Intron (IFN) and ribavirin, but only has 50% patient that this treatment is had response.In addition, this treatment price height, use duration, and with multiple undesirable side effect.The whole world has 1.5～200,000,000 people to infect hepatitis C according to estimates.

Therefore, still need antiviral therapy and the program finding and develop at HCV's and be used for the treatment of whole hepatitis C forms.

Summary of the invention

The invention provides by making Bach1 protein expression level in the human liver tumor cell of expressing hepatitis C virus protein reduce to treat and suffer from cell or the mammiferous method that HCV infects, thereby satisfy at least some aforementioned need.The reduction of Bach1 protein expression level can realize by following manner: come transfectional cell with the miRNA-196 stand-in, thereby make miRNA-196 combine the expression that reduces Bach1 with 3 '-UTR of Bach1 mRNA." seed zone " that this miRNA only need comprise matching comes effectively 3 '-UTR in conjunction with Bach1 mRNA.Preferably make this miRNA rise or cross the reduction amplitude that increases the Bach1 expression level of expressing.By synthetic miRNA stand-in the level of miRNA-196 is raised, these synthetic miRNA stand-in enter the miRNA approach and serve as ripe miRNA-196.

Can also suffer from the Mammals that HCV infects by transferring to treat in the HMOX1 genetic expression in the cell that makes the expression of HCV Nonstructural Protein.In a plurality of embodiments, the rise of HMOX1 genetic expression is with the downward modulation of Bach1 genetic expression in the cell.Therefore, miRNA-196 combines by the Bach1 with negative regulation HMOX1 HMOX1 is raised.Regulation and control to each can both be by realizing with miRNA-196 stand-in transfectional cell.In addition, reduce by make the expression of HCV in infected cell with miRNA-196 stand-in transfectional cell, thereby can treat the cell that infected by HCV, for example liver cell.

On the other hand, the invention provides pharmaceutical preparation, said preparation is suitable for the stand-in to administration miRNA-196, thus the cell that the transfection of the enough miRNA stand-in of energy is infected by HCV.Using these preparations can check the Bach1 expression in the translating phase, HMOX1 rise and regulation and control HCV are duplicated in liver cell.

Importantly, the present invention has showed the functional miRNA-196 binding site that is arranged in 3 ' of Bach1-UTR, and this site causes the downward modulation of Bach1 genetic expression, the downward modulation of going up mediation HCV genetic expression of HMOX1 genetic expression.The treatment that is applied as of miRNA-196 is subjected to the cell that HCV infects and suffers from the Mammals of hepatitis C (for example chronic HCV infection) and be that the other diseases of feature provides new extra therapy for treatment with the oxidative stress rising also perhaps.

Description of drawings

After the present invention having been carried out above-mentioned general the description, now address accompanying drawing, described accompanying drawing is not necessarily drawn in proportion, wherein:

Figure 1A has shown that fixed first of miR-196 and institute's target infer the synoptic diagram of first seed zone coupling between Bach13 '-UTR site;

Figure 1B has shown miR-196 and the fixed synoptic diagram of inferring second seed zone coupling between Bach13 '-UTR site of institute's target;

Fig. 2 A has illustrated the Bach1 protein level of the downward modulation relevant with using the transfection of miRNA-196 stand-in;

Fig. 2 B has illustrated the Bach1 protein level of the rise relevant with using the transfection of miRNA-196 inhibitor;

Fig. 2 C has shown that the transfection of miRNA-196 stand-in does not change the level of Bach1 mRNA;

Fig. 3 A has illustrated the HMOX1 mRNA level relevant with the miRNA-196 stand-in and has raised;

Fig. 3 B has shown the level that does not change Cullin 3 mRNA with the transfection of miRNA-196 stand-in;

Fig. 4 A has illustrated and has reduced with the relevant HCV NS5A protein level of miRNA-196 stand-in transfection;

Fig. 4 B has illustrated and has raised with the relevant HCV NS5A protein level of miRNA-196 inhibitor transfection;

Fig. 4 C has illustrated in Con 1 (hypotype 1b) total length replicon cell by the HCV NS5A level that causes with the transfection of miRNA-196 stand-in and the downward modulation of core mRNA level;

Fig. 5 A has shown two seed coupling sites at miRNA-196 of Bach13 '-UTR;

Fig. 5 B has illustrated the synoptic diagram of pGL3-Bach1, and pGL3-Bach1 is used from Photinus pyralis LUC (f-luc) the reporter gene construct that pair cell carries out cotransfection by Lipofectamine 2000 and pGL3-Bach1, pRL-TK (sea pansy) and miR-196 stand-in or inhibitor one;

Fig. 5 C has shown that the miRNA-196 stand-in have suppressed the f-luc activity of pGL3-Bach1 reporter gene carrier;

Fig. 5 D has shown that the miRNA-196 inhibitor raises the f-luc activity of pGL3-Bach1 reporter gene carrier slightly;

Fig. 6 A has illustrated the displacement to four Nucleotide in two seed coupling sites of Bach13 '-UTR;

Fig. 6 B has illustrated in the cell of sudden change pGL3-Bach1 or pGL3-Bach1, pRL-TK (sea pansy) and miR-196 stand-in cotransfection that the miRNA-196 stand-in make pGL3-Bach1-WT but not the f-luc of pGL-Bach1-Mut reporter gene is active reduces;

Fig. 6 C has illustrated active reduction of f-luc that in the cell of sudden change pGL3-Bach1 or pGL3-Bach1, pRL-TK (sea pansy) and miR-155 stand-in cotransfection miR-155 stand-in make pGL3-Bach1-WT and pGL-Bach1-Mut reporter gene;

Fig. 7 A has illustrated four coding mutations that are introduced into miRNA-196 seed coupling site;

Fig. 7 B has shown the uciferase activity in the cell of the stand-in negative control that progressively raises with pGL3-Bach1, pRL-TK and concentration, miR-196 stand-in or sudden change miR-196 cotransfection;

Fig. 8 A has illustrated the recovery of the seed coupling between sudden change miRNA-196 and sudden change Bach13 '-UTR;

Fig. 8 B has shown with the uciferase activity of measuring in sudden change reporter gene (pGL3-Bach1-Mut), pRL-TK and sudden change miRNA-196 or 48 hours the cell of wild-type miRNA-196 cotransfection;

Fig. 9 A has shown in transfection has in the Huh-7.5 cell of J6/JFH1 RNA the miRNA-196 stand-in to the downward modulation effect of HCVJ6/JFH1 rna level;

Fig. 9 B has shown in transfection the Huh-7.5 cell that has been secreted into the J6/JFH1 hepatitis C virus in the culture supernatants; With

Fig. 9 C has shown in transfection has in the Huh-7.5 cell that has been secreted into the J6/JFH1 hepatitis C virus in the culture supernatants miRNA-196 stand-in to the downward modulation effect of HCV J6/JFH1 protein level.

Embodiment

Now hereinafter the present invention is described more fully with reference to the accompanying drawings, has shown some but non-all embodiments of the present invention in the accompanying drawings.In fact, these inventions can be implemented and it should be read as and be subject to embodiment as herein described with multiple different form; And these embodiments are provided is in order to make the disclosure satisfy the legal requirements that is suitable for.

Admitted that hepatitis C virus (HCV) directly brings out oxidative stress in liver cell.Same approved is that heme oxygenase 1 (HMOX1) is to have anti-oxidant and key cells protective enzyme anti-inflammatory activity.Therefore, the existence of HMOX1 or application can be considered as being used for eliminating or alleviate the method for the oxidative stress that HCV brings out.Yet, Bach1, a kind of alkaline leucine zipper (bZip) Mammals transcription repressor can negative regulation HMOX1.Therefore, only the reduction of Bach1 level or its are followed the rising of HMOX1 level, can prevent or improve hepatitis C potentially.

Bach1 is the gene of the encoding transcription factor, and the described factor belongs to cap ' n ' collar type basic region leucine zipper factor family (CNC-bZip).Coded albumen contains Broad bunch of (broad complex), Tramtrack, bric-a-brac/ poxvirus and zinc and refers to (BTB/POZ) structural domain, and this is atypical for the CNC-bZip family member.These BTB/POZ structural domains promote the formation of protein-protein interphase interaction and homology and oligomeric body.When this encoded protein and MafK formed heterodimer, it served as the repressor of Maf recognition component (MARE) and makes to transcribe and is checked.More outstanding is, Bach1 is mammiferous HMOX1 transcription repressor, and negative regulation is carried out in its genetic expression to HMOX1.The relevant oncogene with Maf of Bach1 family forms the antagonism heterodimer.These heterodimers combine with Maf recognition component (MARE), and suppress expression that the heterodimer that contains Maf and other positive transcription factors are produced the gene (for example, HMOX1 and NQO1) of response.

MiRNA is small molecules non-coding RNA (about 22nt), it has been generally acknowledged that it is the important regulon of genetic expression.Before the present invention, Bach1 or also the unknown substantially of HCV are regulated and control and how to be regulated and control to microRNA whether.The present invention recognizes that first miRNA-196 directly acts on 3 ' of Bach1mRNA-UTR, and checks this proteic expression and HMOX1 is raised in the translating phase.In addition, miR-196 also suppresses the proteic expression of HCV NS5A.Perhaps or even keying action so miRNA-196 plays an important role in the HCV in the liver cell being duplicated the regulation and control of expressing with HMOX1/Bach1.Therefore, thus by using the miRNA-196 stand-in with the infected cell of miRNA-196 stand-in transfection, be subjected to cell that HCV infects or the Mammals that suffers from hepatitis thereby can treat.Preferably, miRNA-196's cross to express and can advantageously provide prevention or improve the method for hepatitis C infection to the transfection of infected cell.In one embodiment, the level of miRNA-196 raises by synthetic miRNA stand-in, and these miRNA stand-in enter the miRNA approach and serve as ripe miRNA-196.

As mentioned above, miRNA is small molecules non-coding RNA (about 22nt), it has been generally acknowledged that it is the important regulon of genetic expression.More specifically, miRNA is that length is the single stranded RNA molecule of about 21～23 Nucleotide, and it mainly comes genetic expression is regulated and control by translation repression.MiRNA is by transcribing from DNA but do not translate into proteic gene (non-coding RNA) coding; Opposite miRNA through being processed into short loop-stem structure (being called pre-miRNA), and finally becomes functional miRNA from primary transcript (being called pri-miRNA).The part of ripe miRNA molecule and one or more messenger RNA(mRNA)s (mRNA) is complementary, and its major function is to make down regulation of gene expression.MiRNA appears in and brings into play function in the gene regulating.For this purpose, the part of miRNA and one or more messenger RNA(mRNA)s (mRNA) is complementary.Animal miRNA usually with 3 ' UTR in the site complementation.MiRNA can translate by arrestin subsequently to the annealing of mRNA, but promotes the shearing of mRNA sometimes.In this case, the double-stranded RNA formation meeting that combination caused by miRNA by be similar to the degraded that process that RNA disturbs (RNAi) triggers the mRNA transcript.Yet, in other cases, it is believed that the miRNA complex body can not cause blocks protein translating mechanism or prevention protein translation under the situation that mRNA degrades.At present, target gene reduces really that cutter system it be unclear that.

For purposes of the present invention, microRNA stand-in (for example, miRNA-196 stand-in) are to use

Carry out chemically modified to increase its stability and to improve its active double-stranded RNA oligonucleotides.The microRNA stand-in are simulated the endogenous precursor miRNA and are entered the miRNA approach and serve as sophisticated miRNA species.

As if determined the specificity of miRNA-target RNA interphase interaction in the sequence complementarity of 6～8 base pairs " seed zone " of miRNA-mRNA heteroduplex end.Recognize that the earliest miR-196 and homology frame (HOX) bunch (to the vital correlated transcription factor gene group of multiple development program in the animal) have quite big degree and complementarity that evolve and upward guard, and the regulation and control hox gene is expressed.

The present invention proposes the miRNA-196 target and decides the HCV genome and by the 3 ' UTR that target is decided Bach1 mRNA the HMOX1 gene is raised.Therefore, can treat this infected cell by the cell that infected by HCV.By the cell that infected by HCV is carried out transfection, can when improving the HMOX1 level, reduce the expression level of Bach1.Advantageously, the expression that the downward modulation of the Bach1 of negative regulation HMOX1 is helped to improve HMOX1.As mentioned above, HMOX1 provides antioxidant effect to alleviate the oxidative stress that HCV brings out.In addition, can treat the rise of inducing miRNA-196 with IFN β.It is this that inducing of miRNA-196 is found in the human hepatocytes oncocyte is in Huh-7 and the former generation mouse liver cell.

The quantity of miRNA continues to increase, and its other mRNA that regulate and control and candidate gene continue to obtain to identify.With regard to liver, miRNA-122 expresses the highest miRNA of abundance in being accredited as liver cell, and it has material impact to some cholesterol metabolic enzymes according to the show.In addition according to the show, miRNA-122 is that the HCV expression is necessary.Usually, the environment and the position of its homology kind subsequence binding site depended in the influence of miRNA-122, and mainly relevant with the rise of expressing in the site in 5 ' district, and mainly relevant with checking of expression in the site in 3 ' the untranslated district.Embodiments of the present invention comprise uses the miRNA-196 stand-in to be used as the downward modulation regulon of HCV NS5A protein expression.This albumen is absolutely necessary for the expressed intact of HCV and normal expression.Therefore, embodiments of the present invention comprise with the miRNA-196 stand-in of treatment significant quantity and treat infected cell or suffer from the mammiferous method of hepatitis C.The treatment significant quantity can provide by known route of administration.For example, a plurality of embodiments according to the present invention can be by the topical application approach, use the therapeutical agent that comprises the miRNA-196 stand-in through intestines route of administration or parenteral route of administration.

Now generally accept, HCV infection meeting causes oxidative stress to raise in infected liver cell, and may also be like this in other infected cells.An important mediators of the oxidative stress of this rising is the HCV core protein.Received equally is that HMOX1 helps to protect various kinds of cell and the potential damage effect of organizing the oxidative stress of avoiding too high.As mentioned above, HMOX1 has anti-oxidant and key cells protective enzyme anti-inflammatory activity, and it can eliminate or alleviate the oxidative stress that HCV brings out.More specifically, HMOX1 is that catalysis hemachrome degradation and produce has ferrous, the carbon monoxide of anti-oxidant and anti-inflammatory activity and the key cells protective enzyme of uteroverdine in vivo.The favourable character of these of HMOX1 is based on the following ability of HMOX1: it can reduce " dissociating " or the loose bonded protoheme that can be used as powerful prooxidant, and improves carbon monoxide, uteroverdine and the bilirubinic output have powerful antioxidant and anti-inflammatory and to resist into fiber effects.

Regulation and control to HMOX1 genetic expression are complicated.Yet, the present invention shows, in the untranslated district of the HMOX of a plurality of species gene 5 ', the important site that is used to regulate and control HMOX1 comprises the AP-1 site of a series of expansions (also being called anti-oxidant response element), Maf albumen response element [MARE] and metalloporphyrin response element [MPRE].Bach1 is the zinc leucine zipper protein member of cap ' n ' collar family.It plays a crucial role in that the enhancing of HMOX1 genetic expression is checked in (tonic repression).It is by forming heterodimer with little Maf albumen and blocking this gene transcription and activate this effect of bringing into play.Bach1 comprises some total binding sites (comprising the CP motif), these positions are in conjunction with protoheme the time, can cause this proteic conformation to change and make it and the proteic avidity of Maf obviously reduces and restraining effect is subsequently reduced, and increase the HMOX1 activity of gene expression.A kind of main pungency Maf albumen is Nrf2.The rise of Nrf2 is relevant with the rising of HMOX1 genetic expression.

In view of mentioned above, the HMOX1 activity may raise in HCV infects.Yet clinical study has identified HMOX1 expression reduction in the chronic hepatitis C situation.Therefore, patient with the inherited genetic factors that causes lower level HMOX1 genetic expression or other factors is developed chronic hepatitis C infection behind acute contact HCV risk may raise, and/or to infect the risk of hepatic diseases of developing faster speed progress higher because of HCV.

The HMOX1 genetic expression of lower level may be relevant with chronic hepatitis C infection, and Bach1 negative regulation HMOX1.Therefore, in the mode that reduces the Bach1 level in the infected cell cell that is subjected to the HCV infection or the Mammals that suffers from hepatitis C are treated the influence that can advantageously alleviate HCV.Therefore, an embodiment of the invention comprise the mammiferous method of suffering from hepatitis C infection for the treatment of, described method is undertaken by following manner: with miRNA-196 stand-in transfectional cell, thereby make miRNA-196 combine the expression that reduces Bach1 with 3 '-UTR of Bach1 mRNA and make the HMOX1 rise.Preferably make this miRNA-196 cross expression, thereby provide more miRNA-196 to be used for and HMOX1 is raised in conjunction with Bach1.

In an embodiment of the invention, suffers from the Mammals that HCV infects (for example chronic hepatitis C) with the miRNA-196 treatment.In particular, with making the miRNA-196 stand-in of Bach1 down-regulated expression come the infected cell of transfection.In a specific implementations, the Bach1 protein expression level can reduce by following manner in the infected cell of expression of HCV Nonstructural Protein: with miRNA-196 stand-in transfectional cell, thereby make miRNA-196 combine the expression that reduces Bach1 with 3 '-UTR of Bach1 mRNA.In a preferable methods, crossed with 3 '-UTR bonded miRNA-196 of Bach1 mRNA and to be expressed.In a specific implementations, also miRNA-196 is raised by using interferon beta.In another embodiment, using separately or miRNA-196 raised also by the miRNA-196 stand-in with using jointly of interferon beta.

To single infected cell or can cause the remarkable reduction of Bach1 protein expression level when the miRNA-196 of the administering therapeutic significant quantity stand-in to the mammiferous treatment of carrying this type of infected cell." treatment significant quantity " be meant be used for effectively treating and/or prevent one or more targets illness at infected cell or have the amount of the mammiferous miRNA-196 stand-in of hepatitis.Instruct as generality, therapeutic dose every day that is used for the treatment of the miRNA-196 stand-in of HCV infection generally can be 0.5 μ mol/kg body weight～5 μ mol/kg body weight, or 1.0 μ mol/kg body weight～4 μ mol/kg body weight, or 2 μ mol/kg body weight～3 μ mol/kg body weight.In substituting embodiment, the therapeutic dose of miRNA-196 stand-in can be 0.1 μ mol/kg body weight～1 μ mol/kg body weight, or 0.25 μ mol/kg body weight～1 μ mol/kg body weight, or 0.25 μ mol/kg body weight～0.75 μ mol/kg body weight.

In a plurality of embodiments, that the Bach1 protein expression level can reduce is about 10%～and about 75%, or about 25%～about 65%.In other embodiments, after the transfection 24 hours Bach1 protein expression level can reduce about 25%～about 75%, or about 50%～about 60%.In other embodiment, after the transfection 48 hours Bach1 protein expression level can reduce about 40%～about 80%, or about 60%～about 70%.

As discussed above, improve the HMOX1 gene expression dose and can help eliminating or alleviating the oxidative stress that HCV brings out.After carrying out transfection, the HMOX1 genetic expression in the cell of expression of HCV Nonstructural Protein is raise with miRNA-196.Therefore, an embodiment of the invention provide by making in the cell of expression of HCV Nonstructural Protein and transfer to treat the cell of HCV infection in the HMOX1 genetic expression or suffer from the mammiferous method that HCV infects.By the infected cell of miRNA-196 stand-in transfection HMOX1 genetic expression is raised with the treatment significant quantity.In one embodiment, in cell the rise of HMOX1 with the downward modulation of Bach1 genetic expression.In a preferred implementation, aforesaid method comprises that miRNA-196 rise or mistake are expressed treats liver cell.In a plurality of embodiments, with respect to the HMOX1 expression level of not using in the miRNA-196 stand-in cells transfected, the expression of HMOX1 has improved about 2 times～about 3 times.

Except checking the Bach1 expression, in the human hepatocytes of expression of HCV Nonstructural Protein, the expression of HCV Nonstructural Protein 5a (HCV NS5A) also has been subjected to checking.Therefore, embodiments of the present invention comprise the cell that infected by HCV with the treatment of miRNA stand-in.Can come the infected cell of transfection with the miRNA-196 stand-in, thereby make the down-regulated expression of HCVNS5A.Preferably make described miRNA-196 cross expression.

Some embodiment of the present invention comprises: express by the HCV NS5A in the cell that makes the expression of HCV Nonstructural Protein with miRNA-196 stand-in transfectional cell and reduce, thereby treatment is subjected to the cell of HCV infection or suffers from the Mammals that HCV infects.That is to say that in some embodiments, by making HCV replicon cell with miRNA stand-in transfectional cell and reduced by HCV rna expression and/or protein expression in the cell that HCV infects, thereby treatment suffers from the Mammals that HCV infects.In such embodiment, the proteic expression level of HCV NS5A has reduced by 10%～60%, or 20%～50%.In another embodiment, after the transfection 24 hours HCV NS5A protein expression level reduced about 45%～about 55%, and 48 hours HCVNS5A protein expression level has reduced about 35%～about 45% after the transfection.

Used term " transfection " is used for describing and uses virus vector or other transfer methods that foreign matter is imported in the eukaryotic cell among the application.The transfection of zooblast is usually directed to get through hole or " hole " of of short duration existence in cytoplasmic membrane, thereby allows the picked-up material.Can transfection genetic material (for example super spirial plasmid DNA or siRNA construct) even protein (for example antibody).Except electroporation, can also carry out transfection by positively charged ion lipid is mixed with described material to produce liposome, described liposome and cytoplasmic membrane merge and its load are stored in inside.

A feasible method of transfection can be utilized calcium phosphate.The HEPES buffer salt solution (HeBS) that will contain phosphate ion remains the calcium chloride solution of transfection material and mixes with containing.When the two mixes, will form the calcium of positively charged and the tiny throw out of electronegative phosphoric acid, and the material that remains transfection in its surface bonding.Subsequently should sedimentary suspension add to and treat cells transfected.Though do not understand this process as yet fully, cell can absorb some throw outs and follow picked-up to treat the transfection material.

Additive method uses highly branched organic compound (for example dendrimer) to come in conjunction with genetic material (for example DNA, RNA or miRNA) and makes it enter cell.Another kind method is that the genetic material that will treat transfection covers in the liposome, promptly has the corpusculum of membrane boundary, thereby it is similar to cellularstructure in some aspects and in fact also can merge with plasma membrane genetic material is discharged in the cell.For eukaryotic cell, the more usually transfection of fat-cationic is because cell can be responsive more.

Other method is to use cationic polymers, for example DEAE-dextran or poly-thanomin.Electronegative genetic material is in conjunction with polycation, and this mixture is absorbed by endocytosis by cell subsequently.

The direct method of transfection is a particle gun, wherein makes the nanoparticle coupling of genetic material and inert solid (being generally gold), and subsequently its direct " penetrating " is gone in the nucleus of target cell.Can also be with virus as vehicle with in the genetic material transfered cell.In this case, this technology is called viral transduction, and claims that described cell is transduceed.Other transfection methods comprise that consideration convey dyes (nucleofection), electroporation, heat-shocked, magnetic transfection and special-purpose transfection reagent (for example Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect).

Described use positively charged ion parents molecule for the 5th, 942, No. 634 and promote biological activity (therapeutic) molecule to be transported in the cell by quoting the United States Patent (USP) of incorporating this paper into.United States Patent (USP) the 5th, 942 has also instructed how to make the therapeutic composition that contains this therapeutic molecules by therapeutic molecules is contacted with the dispersion of one or more positively charged ions parents molecule No. 634.The therapeutic molecules that can be delivered in the cell comprises DNA, RNA and polypeptide.This based composition can be used to provide gene therapy and antisense polynucleotides or biologically active polypeptides are delivered in the cell.

Aspect another, the invention provides be used for treating suffer from hepatitis C, the cell or the mammiferous pharmaceutical preparation of preferred chronic hepatitis C.Preparation of the present invention should comprise the miRNA-196 stand-in for the treatment of significant quantity, thereby makes Bach1 and/or HCV NS5A downward modulation when HMOX1 expresses rising.Embodiments of the present invention are adapted such that can be with the transfection of miRNA-196 stand-in to the liver cell of expression of HCV.Preparation of the present invention can comprise or use but be not limited to be used for any preceding method of genetic material transfection to the target cell.

According to a plurality of embodiments, preparation of the present invention can provide through the form that intestines are used being used for.For example, can provide the preparation of embodiment of the present invention to be used for Orally administered tablet, capsule or liquid preparation form.Yet more preferably, described preparation is provided as the intravenous injection liquid preparation.

In other embodiments, can provide described preparation with the form that injection or infusion are used.For example, various preparation intravenouslys of the present invention can be used (entering vein), intra-arterial uses (entering artery), intramuscular and uses and use (entering spinal canal) in (entering muscle), subcutaneous administration (subcutaneous), intradermal administration (entering skin itself), the sheath or intraperitoneal is used (infusion or be injected to intraperitoneal).

When the miRNA-196 of administering therapeutic significant quantity stand-in, can cause the remarkable reduction of Bach1 and HCVNS5A protein expression level and the rising of HMOX1 level to single infected cell or to the treatment that the Mammals that contains this type of infected cell carries out with the preparation of embodiment of the present invention." treatment significant quantity " be meant the illness that is used for effectively treating and/or preventing one or more targets at infected cell or suffer from the amount of the mammiferous miRNA-196 stand-in of hepatitis.Instruct as generality, therapeutic dose every day that is used for the treatment of the miRNA-196 stand-in of HCV infection generally can be 0.5 μ mol/kg body weight～5 μ mol/kg body weight, or 1.0 μ mol/kg body weight～4 μ mol/kg body weight, or 2 μ mol/kg body weight～3 μ mol/kg body weight.In substituting embodiment, the therapeutic dose of miRNA-196 stand-in can be 0.1 μ mol/kg body weight～1 μ mol/kg body weight, or 0.25 μ mol/kg body weight～1 μ mol/kg body weight, or 0.25 μ mol/kg body weight～0.75 μ mol/kg body weight.

Embodiment

I. material and experiment

A. reagent and antibody

(Rockford IL) obtains BCA protein determination reagent from Pierce Biotechnology.Da Erbaikeshi MEM (DMEM) and foetal calf serum (FBS) from HyClone (Logan, UT).

The luciferase assay system from Promega (Madison, WI).TRIzol available from Invitrogen (Carlsbad, CA), Geneticin (G-418) from Gibco (Grand Island, NY).(Coralville IA) synthesizes primer by Integrated DNA Technologies.The SDS-PAGE gel of 4%～15% gradient and ImmunBlot pvdf membrane available from Bio-Rad (Hercules, CA).Mouse-anti HCV NS5A and mouse-anti HCV NS3 monoclonal antibody available from Virogen (Watertown, MA).Anti-people Bach1 of goat and GAPDH polyclonal antibody from Santa CruzBiotechnology (Santa Cruz, CA).ECL-Plus from Amersham (Piscataway, NJ).

B. cell cultures

The 9-13 cell is provided by Ruprecht-Karls-Universitat Heidelberg (Heidelberg, Germany).9-13 clone is human hepatocytes knurl Huh-7 cell mass, contains the non-structural area of HCV and the stably express HCV Nonstructural Protein NS3 to NS5B of replicability.Cell is maintained in the DMEM that is supplemented with 10% (volume/volume) FBS, 100u/ml penicillin, 100 μ g/ml Streptomycin sulphates and 500 μ g/ml G-148.Con 1 (hypotype 1b) total length replicon Huh-7.5 cell (Con1 cell) from Rockefeller university (New York, NY).Con1 clone is the Huh-7.5 cell mass that contains total length HCV genotype 1b replicon, has wherein carried out the replacement from the Serine to the Isoleucine of high degree of adaptability on the 2204th amino acids of polypeptide.The maintenance of Con1 cell is being supplemented with 10% (volume/volume) FBS and the nonessential amino acid of 0.1mM, 100 units/ml penicillin, 100 μ g/ml Streptomycin sulphates and is selecting among the DMEM of antibody 750 μ g/ml G148.With atmosphere (95% room air and the 5%CO of cell maintenance at 37 ℃ humidification ₂) in.

C. microRNA and construct

(Lafayette CO) obtains from Dharmacon to be used for the sudden change has-miRNA-196 of has-miRNA-196, has-miRNA-16, customization and the miRIDIAN microRNA stand-in of miRNA stand-in negative control (MMNC).The microRNA stand-in are to use

Thereby it has been carried out chemically modified improved its stability and active double-stranded RNA oligonucleotides.The microRNA stand-in are simulated endogenous precursor miRNA and are entered the miRNA approach and serve as sophisticated miRNA species.In addition, following miRNA inhibitor is also from Dharmacon:has-miR-196 and miRNA inhibitor negative control (MINC).MiRIDIAN microRNA hair fastener inhibitor is the synthetic oligonucleotide inhibitor of latest generation, and it is designed to suppress natural miRNA activity.The pRL-TK carrier is from Promega.The cDNA (Rluc) that pRL-TK reporter gene carrier comprises the renilla luciferase of encoding is used as confidential reference items contrast reporter gene.(Gainesville FL) provides by the University of Florida to comprise the segmental pGL3-Bach1 luciferase reporter gene of the Bach1 3 '-UTR construct of 1837bp.PGL3-Bach1 is by GENEWIZ in sudden change, and (South Plainfield NJ) makes Inc..Construct is confirmed by restriction enzyme digestion and order-checking.

D. transfection and uciferase activity are measured

Use Lipofectamine 2000 (Invitrogen) to carry out transfection according to the working specification of manufacturers.In brief, the miRNA with pGL3-Bach1 or sudden change pGL3-Bach, pRL-TK and test comes cotransfection 9-13 cell.After the transfection 48 hours, collecting cell also made its cracking.Use

The activity of luciferase reporter gene is measured by the luciferase assay system.The Photinus pyralis LUC activity is normalized to the renilla luciferase activity, and uses the BCA protein determination kit to determine total protein.For the ease of relatively, there is the value of the cell of pGL3-Bach1 and pRL-TK to be made as transfection and equals 1.

E. in-vitro transcription, transfection and infection

HCV infections clone body pJ6/JFH1 is by C.M.Rice (Rockefeller university, New York, NY) generosity is gifted, and this clone body is a total length mosaic gene group, wherein core NS2 district is from infective J6 (genotype 2a), and NS3～NS5B district is from infective JFH1 (genotype 2b).The previous report by preparation based on the HCV (HCVcc) of the cell cultures deposits yields of J6/JFH1.In brief, make pJ6/JFH1 plasmid line styleization, carry out purifying by ethanol sedimentation subsequently, cut, and carry out phenol-chloroform extraction with the Proteinase K enzyme with XbaI.Use the template of this linear plasmid as in-vitro transcription, (Ambion, Austin TX) carry out described in-vitro transcription with MEGAscript T7 test kit.In order to carry out HCV RNA transfection, transfection the day before yesterday with Hub-7.5 cell plating to 24 orifice plate in, and be to carry out transfection at 70%～80% o'clock in coverage (confluence).Thereby the Lipofectamine 2000 in HCV RNA by using 2 μ g/ holes and 4 μ l/ holes with 1: 2 RNA/Lipofectamine recently pair cell carry out 48 hours transfection.In order to infect initial Hub-7.5 cell, collected personal HCV RNA transfection 48 hours the cell culture supernatant liquid of cell filters by 0.20 μ m strainer it, and is added in the culture of initial Huh-7.5 cell.

F. western blotting

Carry out western blotting as previously mentioned.In brief, on the SDS-PAGE gel, separate total protein (30 μ g～50 μ g).After electrophoretic transfer is to the ImmunBlot pvdf membrane, in the PBS that contains 5% skim-milk and 0.1%Tween-20,, and educates with a temperature resistance in 4 ℃ subsequently and spend the night membrane closure 1 hour.As follows to a dilution that resists: for anti-Bach1 antibody is 1: 1000, is 1: 2000 for anti-HCV NS5A and anti-GAPDH antibody.Make film and two anti-(1: 10, the 000 dilution) incubation that is conjugated with horseradish peroxidase 1 hour subsequently.At last, the working specification according to manufacturers makes bonded antibody visual with the ECL-Plus chemiluminescence system.(Rochester NY) comes the relative optical density of each particular bands of carrying out being obtained behind the western blotting is measured to use Kodak 1DV3.6 computer based imaging system.

G. quantitative RT-PCR

From tested cell, extract total RNA, synthetic as previously mentioned cDNA.Used primer is as follows: Bach-1 specificity sense primer 5 '-GGACACTCCTTGCCAAATGCAG-3 ' (22bp), antisense primer 5 '-TGACCTGGTTCTGGGCTCTCAC-3 ' is (22bp); HMOX1 specificity sense primer 5 '-CGGGCCAGCAACAAAGTG-3 ' (18bp), antisense primer 5 '-AGTGTAAGGACCCATCGGAGAA-3 ' is (22bp); Cul3 specificity sense primer 5 '-GTGCTCACGACAGGATA-3 ' (17bp), antisense primer 5 '-GTTTGGCTAAGTAGAACCTTC-3 ' 21bp); GAPDH specificity sense primer 5 '-TTGTTGCCATCAATGACCC-3 ' (19bp), antisense primer 5 '-CTTCCCGTTCTCAGCCTTG-3 ' is (19bp).Use CFX96 ^TMPCR in real time detection system (Bio-Rad) and iQ ^TMSYBR Green Supermix PCR in real time test kit (Bio-Rad) is carried out real-time quantitative RT-PCR.The sample that has comprised no template and no reversed transcriptive enzyme is used as negative control, and it has produced insignificant signal (Ct value＞35) as expection.After carrying out normalization method, analyze by Ct relatively and to calculate the multiple changing value at the GAPDH in same sample amount.Initial experiment shows that some processing and operation that pair cell carries out do not influence the GAPDH expression of gene.

H. statistical analysis

The initial analysis display result is normal distribution.Therefore, used the parameter statistical method.Use student t check (being used for the comparison of two kinds of situations) or variance analysis (being used for comparison) to come the difference between analytic sample more than two kinds of situations.To think though statistically significant less than 0.05 P value.Experiment has repeated three times at least, and its result is similar.All experiments to each treatment group all comprise the sample of trisection at least.Show the representative result of single experiment.Use that (Cary, JMP 4.0.4 software NC) carries out statistical analysis from SAS Institute.

II. result

A. computer for analysis dopes two seed zone couplings of miR-196 and Bach1 mRNA 3 '-UTR

Use bioinformatics method to identify potential miRNA target.On-line search to TargetScan 4.0 databases has shown that comprising at least two miRNA-196 seeds of inferring in 3 '-UTR of Bach1 mRNA mates sites.Shown in Figure 1A and 1B, (2280～2286nt) is conservative at people, mouse, rat, chicken and dog camber, and (2161～2166nt) conservative propertys between different plant species are relatively poor and another infers the position for one of binding site of being predicted.In Nrf2 and HMOX1 gene, find the miRNA-196 binding site predicted, in the coding region of Bach1 gene, find the miRNA-196 binding site (data not shown goes out) of inferring.

B. miRNA-196 reduces transcription repressor Bach1 and raises HMOX1 in the 9-13 of expression of HCV Nonstructural Protein cell

For the miR-196 binding site that confirms to infer in experiment has function, with miRNA-196 specificity stand-in or inhibitor transfection the 9-13 cell.Bach1 albumen and mRNA level are assessed by western blotting and qRT-PCR respectively.Compare with miRNA stand-in negative control (MMNC), the 9-13 cell that transfection has miRNA-196 stand-in demonstrates the remarkable reduction (having reduced about 55%) of Bach1 protein expression level in transfection after 24 hours, and has reduced about 64% after 48 hours in transfection.Dye (mock) with idle running and compare, in transfection has the cell of miRNA stand-in negative control, do not detect influence (Fig. 2 A) the Bach1 protein level.Different with miRNA inhibitor negative control is, the miRNA-196 specific inhibitor makes Bach1 protein level significantly raise (Fig. 2 B) to the downward modulation effect of miRNA-196.Yet not observing miRNA-196 in the 9-13 cell has remarkably influenced to Bach1 mRNA level.The transfection of miRNA-196 stand-in does not change the level (Fig. 2 C) of Bach1 mRNA.These results show that in human stem cell knurl 9-13 cell miRNA-196 may appear at translation skill to the regulation and control of Bach1.

Because Bach1 is the HMOX1 gene transcription repressor of generally acknowledging, next the present invention has determined whether proteic downward modulation effect can make HMOX1 genetic expression raise to miRNA-196 to Bach1.So the 9-13 cell has been carried out 48 hours transfection with miRNA-196 stand-in or miRNA stand-in negative control.After 48 hours, the mRNA level of HMOX1 and Cullin 3 (Cul 3, non-specific genetic contrast) has been carried out quantitatively by qRT-PCR.As shown in Figure 3A, compare with the miRNA stand-in negative control of equivalent, the mRNA-196 stand-in make the mRNA level of HMOX1 significantly raise 2.4 times.Shown in Fig. 3 B, the mRNA level of Cul 3 does not raise.

C. miRNA-196 checks the proteic expression of the non-structure NS5A of HCV in the 9-13 of expression of HCV Nonstructural Protein cell

Before the present invention, do not know still whether miRNA-196 can make the proteic level of the non-structure NS5A of HCV change in the human hepatocytes oncocyte of expression of HCV Nonstructural Protein.In order to determine whether miRNA-196 can make the proteic level of the non-structure NS5A of HCV change in the human hepatocytes oncocyte of expression of HCV Nonstructural Protein, with miRNA-196 stand-in or inhibitor transfection the 9-13 cell.After transfection 24 hours and 48 hours, carry out western blotting and analyze NS5A and the proteic level of GAPDH.Shown in Fig. 4 A, compare with the miRNA stand-in negative control of equivalent, transfection after 24 hours 50nM miRNA-196 simulation to cause the NS5A protein expression level significantly to reduce about 52%, and reduced about 37% after 48 hours in transfection.By contrast, shown in Fig. 4 B, the miRNA-196 specific inhibitor makes the proteic level of NS5A significantly raise 2.2 times to the downward modulation effect of miRNA-196.Whether can suppress HCV and duplicate in order to assess miRNA-196, Con1 total length HCV replicon Huh-7.5 cell carried out 48 hours transfection with negative control or miRNA-196 stand-in.Shown in Fig. 4 C, compare with miRNA stand-in negative control (MMNC), miRNA-196 causes the viral RNA level significantly to reduce.

D. miR-196 suppresses the HCV expression in based on the cell culture system of HCV JFH1

HCV J6/JFH1 RNA transfection Huh-7.5 cell by Lipofectamine 2000 usefulness 2 μ g/ holes.After 48 hours, with miRNA-196 stand-in or miRNA stand-in negative control (MMNC) transfectional cell.Infect in order to carry out HCV, as previously mentioned, have the 1ml cell culture supernatant liquid of the cell of J6/JFH1 to cultivate with gathering from transfection initial Huh-7.5.Be exposed to this supernatant liquor after 48 hours, use miRNA-196 stand-in or miRNA stand-in negative control (MMNC) transfectional cell 48 hours.Collecting cell extracts total RNA and albumen.By qRT-PCR HCV RNA has been carried out quantitatively, and measured HCV NS3 and GAPDH protein level by western blotting.

In the coding region of the genomic HCVNS5A gene of HCV JFH1, found the splendid coupling of miRNA-196.In HCV J6/JFH1 cell culture system, observed the downward modulation effect that miRNA-196 expresses HCV.Have in the Huh-7.5 cell of J6/JFH1 in transfection, 50nM miRNA-196 causes HCV J6/JFH1 RNA significantly to reduce almost 70% (shown in Fig. 9 A), in infection has the Huh-7.5 cell of J6/JFH1, reduced about 50% (shown in Fig. 9 B), and in infection has the Huh-7.5 cell of J6/JFH1, made HCV NS3 albumen reduce about 60% (shown in Fig. 9 C).These results observed unanimity in the JFH1 cell culture system.

E. 3 '-UTR direct interaction of miRNA-196 and Bach1 mRNA in the 9-13 of expression of HCV Nonstructural Protein cell

In order further to determine that the miRNA-196 target decide 3 '-UTR of Bach1 mRNA, shown in Fig. 5 A, 3 ' of Bach1mRNA-UTR comprises two miR-196 seeds of being predicted and mates sites.Used the reporter gene construct that is known as pGL3-Bach1, rather than carried out indirect and the lower regulation and control of specificity, Bach13 ' in this construct-UTR is positioned at Photinus pyralis LUC (f-luc) open reading frame downstream (Fig. 5 B).Come cotransfection 9-13 cell with pGL-Bach1 (f-luc), pRL-TK (sea pansy is used for carrying out normalization method by transfection efficiency) and miRNA negative control and miRNA-196 stand-in or inhibitor or miRNA-196 (in 3 '-UTR of Bach1 mRNA, do not have prediction binding site " feminine gender " miR).After the transfection 48 hours, test luciferase reporter gene activity.It is about 53% that the transfection of miRNA-196 stand-in has significantly reduced reporter gene activity, and miRNA stand-in negative control and miRNA-196 stand-in do not have an influence to luciferase reporter gene is active.In addition, not observing reporter gene activity in transfection has the cell of miRNA-196 inhibitor has significant change.Shown in Fig. 5 C, the miRNA-196 stand-in have suppressed the f-luc activity of pGL3-Bach1 reporter gene, and the miRNA-196 inhibitor raises the f-luc activity of pGL3-Bach1 reporter gene slightly.

Next, use Luc reporter gene construct (being called pGL3-Bach1-Mut) (Fig. 6 A) and pRL-TK, miRNA-196 stand-in or miRNA-155 stand-in (not " feminine gender " miR that the miR-155 binding site of being predicted among pGL3-Bach1-WT and the pGL3-Bach1-Mut is changed) the cotransfection 9-13 cell of the Bach1 3 '-UTR that contains four coding mutations.After the transfection 48 hours, use the plain enzymatic determination system of double fluorescent (from Promega) to measure the luciferase reporter gene activity, the Photinus pyralis LUC activity is normalized to renilla luciferase activity and total protein.Shown in Fig. 6 B, the miRNA-196 stand-in make the active reduction of the f-luc of pGL3-Bach1-WT, but do not make active reduction of f-luc of pGL-Bach1-Mut reporter gene carrier.The transfection of miRNA-196 stand-in significantly reduces uciferase activity in the dose-dependently mode, and miRNA-196 changes reporter gene activity in the cell of the reporter gene construct that comprises the miRNA-196 binding site that has suddenlyd change and have in transfection.Shown in Fig. 6 C, miRNA-155 descends the reporter gene activity among pGL3-Bach1-WT and the pGL3-Bach1-Mut slightly.These results show miRNA-196 direct regulation and control Bach1 genetic expression, and miRNA-196 mediates the downward modulation of Bach1 by 3 ' of Bach1mRNA-UTR.

In order further to determine the direct interaction between miRNA-196 and the Bach1-3 '-UTR, shown in Fig. 7 A, import the tetranucleotide mutation-ure and produce the miRNA-196 of sudden change, wherein deleted the seed coupling site of 3 '-UTR of Bach1 mRNA.The miRNA-196 stand-in of miRNA stand-in negative control, miRNA-196 stand-in or the sudden change that progressively raises with pGL3-Bach1-WT, pRL-TK, concentration come the cotransfection cell, test luciferase reporter gene activity.Shown in Fig. 7 B, miRNA-196 significantly reduces uciferase activity in the dose-dependently mode, and this is consistent with the observation herein; Yet the miR-196 of miRNA negative control and sudden change does not influence uciferase activity, has further shown the direct interaction between 3 '-UTR of miR-196 and Bach1 mRNA.

Since comprise with the sudden change miRNA-196 (miR-196-Mut) of sudden change pGL3-Bach1 (pGL3-Bach1-Mut) complementary base and sudden change reporter gene (Bach1-3 '-UTR-Mut) form splendid coupling (shown in Fig. 8 A) at its seed zone again, so miR-196-Mut should recover it to the active influence of Bach1-3 '-UTR-Mut.Have in transfection in the cell of sudden change pGL3-Bach1 (it " agrees with " with the miRNA-196 that suddenlys change through sudden change), sudden change miRNA-196 stand-in have obviously suppressed uciferase activity.On the other hand, do not observe wild-type miRNA-196 to the sudden change reporter gene (pGL3-Bach1-Mut) the luciferase gene activity remarkably influenced is arranged.So shown in Fig. 8 B, this has further confirmed the direct interaction between 3 '-UTR of miR-196 and Bach1 mRNA.

Above-mentioned cut-and-try work explanation, the transfection of miRNA-196 stand-in is significantly reduced Bach1 and the non-structure NS5A protein level of HCV, and HMOX1 genetic expression is raised.Therefore, in the HCV in the liver cell being duplicated the regulation and control that expression is carried out with HMOX1/Bach1, miRNA-196 can play an important role or even keying action.So the rise of miR-196 can provide extra new treatment approach for chronic hepatitis C perhaps also has the therapy of other hepatopathy diseases.

Have benefited from the instruction that presented in above-mentioned specification sheets and the accompanying drawing, those skilled in the art in the invention will expect multiple modification and other embodiments of invention as herein described.Therefore, it should be understood that the present invention will be not limited to disclosed embodiment, and be intended to modification and other embodiments are comprised within the scope of the appended claims.Though this paper has adopted specific term, to its use only on general and descriptive meaning and be not for restricted purpose.

Claims

1. a treatment suffers from the mammiferous method that HCV infects, and described method comprises that the Bach1 protein expression level in the infected cell that makes expression of HCV reduces.

2. the method for claim 1, wherein, described Bach1 protein expression level in the cell of expression of HCV Nonstructural Protein is reduced to be comprised: with the described cell of miRNA-196 stand-in transfection, thereby make miRNA-196 combine the expression that reduces Bach1 with 3 ' of Bach1mRNA-UTR.

3. method as claimed in claim 2, described method also comprise makes miRNA-196 or miRNA-196 stand-in raise or cross and express.

4. method as claimed in claim 2 wherein, raises miRNA-196 by using interferon beta.

5. method as claimed in claim 2, wherein, described Bach1 protein expression level has reduced about 10%～about 75%.

6. method as claimed in claim 2, wherein, transfection after 24 hours described Bach1 protein expression level reduced about 50%～about 60%.

7. method as claimed in claim 2, wherein, transfection after 48 hours described Bach1 protein expression level reduced about 60%～about 70%.

8. a treatment suffers from the mammiferous method that HCV infects, and described method comprises raises the HMOX1 genetic expression that is subjected in the cell that HCV infects.

9. method as claimed in claim 8, described method also comprise the Bach1 down regulation of gene expression that makes in the described cell.

10. method as claimed in claim 9, wherein, by making the Bach1 down regulation of gene expression with the described cell of miRNA-196 stand-in transfection.

Make the miRNA-196 rise or cross expression 11. method as claimed in claim 10, described method also comprise.

12. method as claimed in claim 8, wherein said cell comprises liver cell.

13. method as claimed in claim 11, wherein, with respect to the HMOX1 expression level of not using in the miRNA-196 stand-in cells transfected, the expression of HMOX1 has improved about 2 times～about 3 times.

14. a treatment suffers from the mammiferous method that HCV infects, described method comprises: by with miRNA stand-in transfection HCV replicon cell and be subjected to the cell of HCV infection that HCV rna expression and protein expression in the described cell are reduced.

Make the miRNA-196 rise or cross expression 15. method as claimed in claim 14, described method also comprise.

16. method as claimed in claim 15, wherein, HCV NS5A protein expression level has reduced by 10%～60%.

17. method as claimed in claim 15, wherein, 24 hours described HCV NS5A protein expression levels have reduced about 45%～about 55% after the transfection.

18. method as claimed in claim 15, wherein, 48 hours described HCV NS5A protein expression levels have reduced about 35%～about 45% after the transfection.

19. mammiferous preparation that is used for the treatment of the cell of expression of HCV Nonstructural Protein and suffers from hepatitis C, described preparation comprises the miRNA-196 stand-in for the treatment of significant quantity, thereby makes Bach1 and the downward modulation of HCV NS5A expression level when the HMOX1 expression is raise; Wherein, described preparation be suitable for making the miRNA-196 stand-in can transfection to the proteic liver cell of expression of HCV.

20. preparation as claimed in claim 19, wherein, described miRNA-196 stand-in are contained in the liposome, thereby described miRNA-196 stand-in can be released in the proteic cell of expression of HCV.

21. preparation as claimed in claim 20, wherein, described preparation comprises cationic lipid.

22. preparation as claimed in claim 19, described preparation also comprise positively charged ion parents molecule, thereby described miRNA-196 stand-in can be released in the proteic cell of expression of HCV.