Presentasi 3 Oktober
Identification of the Role of MirNa 3145
- Slide 1Good Morning
Today I want to give a presentation about the Identification of the role of micro RNA 3145 in hbv life cycle. - Slide 2Before we go further, I need to give a brief description about hepatitis B virus.
Hepatitis B is a viral infection that attack the liver and can cause acute and chronic disease. Hepatitis B infection is caused by the Hepatitis B virus. This virus is passed from person to person through blood, semen, or other body fluids.
According to world health organization, there are 2 57 million of people got infected by HBV virus.
In 2015, 887 thousand of people died because of this virus.
From this map, whe can see the variation of population of people with HBV pver the world. There are six region with different color. Japan is included in the western pacific region, (brown color). According to WHO, there are 6,2 % of adult population in this brown area got infected by HBV. Even thought, HBV has high impact, Until now, there is still no effective medication can cure this disease. - Slide 3Hepatitis B Virus is included in Family of Hepadnaviridae. The disease associated with this family is liver disease such as hepatitis, hepatocellular carcinoma, and cirrochis.
Hepadnaviridae consist of two genus.
First genus, is Avihepadnavirus. It infect birds.
Second genus is Orthohepadnavirus. It infect human and several mammals. Hepatitis B virus is included in orthohepadnavirus.
From this picture, we can see the picture of hbv virus. It is usually named as Dane Particle. Its size is about 42 nm. When hbv infect hepatocyte, besides it induces the replication of intact virus like this. It also induce the formation of subviral particle such as filament which is actually not contagious. The function of this subviral particle is to evade the host immunity.
The middle picture is the schematic image of Dane particle.
The most outer part is the envelope, consist of Hepatitis B surface antigen or usually called as HbsAg. There are three conformation of HbsAg. Small, medium, and long protein. Small protein is the most component of HbsAg.
Between the core and surface, there is protein HBeAg. The detection of HbeAg in the blood of patient is indicator to know whether the status of virus replication is active or not.
This is the core particle of the virus, consist of protein HbcAg. It covers the DNA of the virus.
The DNA itself constitute partiall double stranded DNA. There is DNA polymerase attached to the DNA that will be used in the process of replication inside the host.
The lowest part is the schematic genome of the HBV DNA.
There are four open reading frame of the genome. S, P, C, and X. We can seen here, the model of this genome.
S will transcribe HbsAg.
C will transcribe HbeAg and HbcAg
P will transcribe DNA polymerase
and X will transcribe Hbx Ag
According to Baltimore classification system, There are 7 type of virus. Hepatitis B virus is included into Group VII which is for the category of virus that has double stranded DNA but the process of replication need reverse trancriptase from RNA. - Slide 4I will explain a little about the life cycle of HBV virus.
This is the picture of the life cycle of the Hepatitis B Virus. And also the target therapeutic of the drug. The black lines are the life cycle pathway. The red line are the target therapeutic.
Virus will get into the hepatocyte cell through NTCP receptor.
Inside the cytoplasm, it will uncoat the surface of the body.
Then the DNA will get into the nucleus.
In nucleus, it will become cccDNA.
cccDNA will trancribe two genomic transcript and subgenomic trancripts.
Genomic transcript consist of DNA polymerase, precore and core protein
Subgenomic transcript consist of protein S and protein X.
There will be two pathway.
Process 1 is the formation of envelope. The formation of envelope is performed through protein S in this pathway.
Process 2 is the formation of core and process of encapsidation of RNA HBV. It is performed in this pathway. In this encapsidation pathway, the RNA will become DNA by reverse trancriptase system.
After complete, the core will get assembly with the envelope from the other pathway.
After that, the virus can get recycling process or it can secreted to the outside of the cell and it will attact the other hepatocyte.
From this picture we can also see the target therapeutic. There are several target to kill the virus.
- inhibit the entry
- degradation of cccDNA
- inhibit the transcription process
- inhibit the translation process
- inhibit the encapsidation
- inhibit the reverse trancriptase
- inhibit the assembly process - Slide 5Now the medicine that are available for HBV are interferon and NRTI.
We can see interferon can inhibit encapsidation, translation, degrading cccDNA, and also reverse transcriptase
But NRTI only work in one way which inhibiting Reverse trancriptase.
Now there are several potential drug which can inhibit the entry of HBV, inhibit the formation cccDNA,inhibit transcription inhibit encapsidation, inhibit the assembly of the virus. I will explain about it later.
Back to the interferon and NRTI.
The problem in NRTI is the rate of drug resistance is very high due to DNA mutation of the Hepatitis B virus. For example for lamivudine drug. It has been reported so many resistance for this drug.
The problem of interferon in treatment is that so many side effect happened after taking
Therefore, it is very necessary to figure out more effective medicine for hbv. There are several potential drug under research. - Slide 6There are several potential drug under predlinical stage.
They work as :
Direct acting antiviral
Host trageting agents and
Immune modulatory agents.
In this first type. The drug target dna polymerase which is inhibit reverse transcriptase. Inhibit the capsid formation, and by working as RNA interference.
In this second type. The drug target the host by inhibit the entry process of the hbv, and induce the apoptosis of infected cell
in this third type. it will strengthen the immune system to detect and kill HBV virus by being agonist of TLR 7 or block PD 1. - Slide 7miRNA is one of the potential drug to use in HBV therapy.
But before we talk about that, I will give a brief explanation about what micro RNA is.
A microRNA is a small non-coding RNA
molecule (containing about 22 nucleotides) found in plants, animals and
some viruses. Human genome encode over 1000 miRNA.
The functions of microRNA are in RNA silencing and post-transcriptional
regulation of gene expression. So microRNA will make complement to the mRNA so, the translation will be inhibited.
This is the picture of biogenesis of microRNA.
so, Inside the nucleus, gene will transcribe pri-miRNA and then become pre microRNA.
microRNA - Slide 8In this project we will use microRNA 3145. We want to know how is the role of this microRNA to Hepatitis B virus.
Why we choose this microRNA?
First,
From the next generation sequencing, the researcher has made several databases about the thousand microRNA sequencing.
From that database, we can predict which microRNA can have complementary sequence with the target we want.
Second
To confirm the computational analysis, we made preliminary test. - Slide 9We can see in this picture. The target gene has been confirmed have the function that related with the expression of HBV.
We found that from several database or several sofwatere, mirRNA 3145, 3p and 5 p has been confirmed to complement with this target gene. - Slide 10
- Slide 11This is the preliminary experiment.
We compare the Relative expression of several mRNA. Y3, Y4, SNORA2B, and primiR3145 in HBV positive cell .
We can see that in HBV positive cell the relative expression they were increased compared with control.
PrimiR3145 Relative expression is increased highest than other mRNA. - Slide 12This is another preliminary experiment.
We measure the relative expression of several gene. USP10, KAT6B, ADAMTS4, PPP2R5E, STRN4, TOB1, and DICER1.
When we compare with the control, the Relative expression is higher in HBV positive cell.
From the computational method and preliminary test, we have a hypothesis that this microRNA 3145 has a potential role to be one of therapy of HBV in the future. We will figure out in this project, what the specific role will be. - Slide 13This is the picture of pre miR 3145 sequence
It is the 5 part
It is the 3 part
According to website mirDB.org, 5p can make complementary to 450 target of mRNA
3p can make complementary to 293 target.
From other paper, this miR-3145 can inhibit influenza A virus, by targeting and silencing viral PB1 gene.
In this project we hipotesize that this microRNA will also have an effect to HBV virus. - Slide 14
- Slide 15I need to talk about the previous paper that related with this project.
This is written by Hironori Nishitsuji. Novel Reporter system ...
the point of this paper is, they have been success to establish the new reporter system to monitor HBV in vitro.
They use recombinant virus which contain NL luciferase gene. Nanoluc luciferase gene is similar with firefly luciferase, but the size is very small. NL gene is the shortest luciferase gene comercially available.
They inserted NL gene into to the HBV genome. We can see here. Compared with wildtype, the the genome is not complete. These recombinant itself can not produce all protein they need to live. So, paralellly, HBV- delta which produced all protein inserted. This HBV D is also not prefect, because there is a mutation in this part.
After transfecting of the plasmid, If the virus exist, These gene will express Nanoluc protein that can be detected by luciferase machine.
The advantage of this method is :
1. It is very safe, because the recombinant virus has change the genome. It has not complete gene. So, it can not replicate.
2. NL luciferase is 100 fold brighter compared with other - Slide 16The step of this project, I divided into to big steps.
Preparation and main steps.
In preparation or optimization,
The first thing we did is to confirm if the we can do the process of infection in this laboratory. We did the nishitsuji protocol to know, Does it really work or not to make the infection of HBV happened.
After that, we will measure the viral titer by infecting the virus in different solution from 1 times to 100 times dilution.
After that will find which is the best condition, for example what is the medium better to use, to do in this experiment.
After getting the best condition, we will go through the main step.
The first thing is . We want to to check the effect of overexpression or lowexpression of this microRna to HBV.
IF we have already get the relationship, we will figure out, in what way, the microrna affect the HBV.
We also will confirm conversely the effect of HBV existence to the expression of miRNA 3145 itself. - Slide 17In this first step, we will confirm the HBV infection.
There are two step.
First HBV production
Second, HBV infection.
This all step based on Nishitsuji protocol, that I will explain in next slide. - Slide 18
- Slide 19In original protocol, they use different medium which is DMEM from Gibco. We use DMEM-F12 from Wako. Gibco DMEM contain more nutrition than DMEM-F12. I will show the difference in other slide, later.
- Slide 20In this protocol, we use two combination of recombinant HBV plasmid.
It is the genome of wild type HBV.
HBV/NL is the main plasmid. But it can not produce all protein that virus need to live.
So, it is parallelly added with HBV-delta. HBV delta produced all kind of HBV protein.
The virus obtained from this plasmid can not replicate due to incomplete genome.
Komentar
Posting Komentar