TWI642787B - The use of let-7g for anti-hepatitis c virus - Google Patents

Abstract

The purpose of this case is to investigate the regulation and correlation of Let-7g in hepatitis C virus infection. Let-7g can effectively reduce the hepatitis C virus NS5B gene, core protein and viral load, and Let-7g combined with interferon / ribavirin also synergistically inhibited the expression level of hepatitis C virus. Let-7g binds directly to the hepatitis C virus gene to inhibit viral expression, and the effect of inhibiting the virus is also related to interferon/rebavirin. It was found in the samples that the expression level of Let-7g in the serum or liver tissues of the patients was significantly lower in the non-viral sustained reaction group before the treatment than in the patients in the virus continuous reaction group. This case proposes the pathogenesis of Let-7g in the hepatitis C virus infection, which can make Let-7g develop into a potential target for hepatitis C virus therapy.

Description

Use of a small ribonucleic acid Let-7g for anti-hepatitis C virus

The present invention primarily discloses the use of a small ribonucleic acid (miRNA) Let-7g, particularly in the application of hepatitis C.

Approximately 1300-170 million people worldwide (about 2% to 3% of the world's population) are chronically infected with hepatitis C virus (HCV). Hepatitis C is one of the main causes of liver disease and liver cancer. Among the developed countries, the prevalence of hepatitis C virus antibodies is about 1-2%, and in some areas where hepatitis C is highly prevalent, it can even exceed 20%. At present, the clinical treatment is mainly based on a combination of long-acting interferon (IFN-α) and ribavirin (Ribavirin). In addition, clinically, according to the genotype of the virus or host, and the combination of interferon / ribavirin, in the treatment of chronic hepatitis C patients with viral sustained response (SVR), the ratio can reach 50% to 85%. About 3% to 4% of people in China are infected with hepatitis C. However, the effect of interferon combined with ribavirin therapy is only about 70%, and the course of treatment is not only as long as 6-12 months, but also has many side effects, which seriously affects daily life.

Because the expression level of microribonucleic acid is related to disease, it is possible to identify the role of the disease as a target for new drug development by quantification of microribonucleic acid. Previous studies have found that when normal liver cells are infected with hepatitis C virus, miR-122 (a tiny ribonucleic acid in the vertebrate) binds to two locations in the hepatitis C virus gene pool to cause a virus. A lot of copying. Therefore, if the specificity of binding to miR-122 is used to block viral replication, it may improve the hepatitis C virus infection. At present, a pharmaceutical company has developed a drug that can inhibit the activity of miR-122, and the drug has entered the human trial stage.

Some studies have shown that certain miRNAs upregulated by β-interferon (IFN-β), including: miR-196, miR-296, miR-351, miR-431 and miR-448, will be associated with the hepatitis C virus genome. Predicted sequence binding, which in turn reduces viral replication and infection. In addition, among lung cancer, hepatocellular carcinoma (HCC), liver cancer cell lines, and metastatic liver cancer, the expression level of the Let-7 family members was found to be decreased.

Cheng JC et al., Cell Mol Life Sci. 2012 Aug; 69(15): 2621-33 has suggested that Let-7b, a member of the small ribonucleic acid Let-7 family, can reduce the hepatitis C virus NS5A gene and Core protein expression level, and mention that in Hepatitis C virus Replicon cells, the expression level of Let-7b is decreased, and interferon can cooperate with viral luciferase activity. Inhibition. However, Cheng did not discuss whether Let-7b affects Interferon Stimulated Genes (ISGs); in addition, these results are from in vitro experiments and do not present actual in vivo tests. The experiment of the body is used as evidence.

In this case, it is proposed that Let-7g will bind to one position in the hepatitis C virus gene pool to inhibit viral replication. Under the cell model of hepatitis C virus infection: Let-7g can not only inhibit the expression level of core protein, but also reduce the hepatitis C virus load (cell) secreted by cells. In addition, whether in serum or liver tissue before treatment, the expression level of Let-7g is significantly lower in patients with non-SVR than in patients with viral persistent response (SVR), ie non-viral The expression level of Let-7g is low in patients with persistent response, so Let-7g can be used to predict the efficacy of interferon; the pathogenesis of Let-7g in chronic hepatitis C virus infection and Therapeutic response may be related, and Let-7g has the potential to develop the target of treatment for hepatitis C virus. Let-7g can be used as a therapeutic drug for hepatitis C virus and can be used together with interferon/rapavir.

The main object of the present invention is to provide a method for pre-evaluating the efficacy of Interferon/Ribavirin in the treatment of hepatitis C, comprising the steps of: providing a sample; and detecting the sample with a microRNA; a primer pair of miRNA) Let-7g and a polymerase chain reaction (PCR) reagent are mixed; and the expression level of the microribonucleic acid Let-7g of the sample detected by the PCR reagent can be pre-evaluated for the test The efficacy of interferon/rebavirin in the treatment of hepatitis C.

Another object of the present invention is to provide a kit for pre-evaluating the efficacy of Interferon/Ribavirin in the treatment of hepatitis C, comprising: a primer pair of a small ribonucleic acid Let-7g, Mixing with a sample; a polymerase chain reaction (PCR) reagent for mixing with the primer pair of the microribonucleic acid Let-7g and the sample, wherein the sample is detected according to the PCR reagent The expression level of the small ribonucleic acid Let-7g can be pre-evaluated for the efficacy of the interferon/rebavirin in the treatment of hepatitis C for the sample.

Another object of the present invention is to provide a use of a small ribonucleic acid Let-7g, which is mainly used for down-regulating at least one of a NS5B gene expression level, a core protein expression and a viral load in a hepatitis C virus. .

Another object of the present invention is to provide a nucleic acid binding protein lin28 for use in downregulating the expression level of a small ribonucleic acid, Let-7g, in a sample which replicates or infects hepatitis C virus.

HCV‧‧‧C hepatitis virus

Let-7g‧‧‧a tiny ribonucleic acid

IFN‧‧ interferon

RBV‧‧‧Rebavirin

AVA5‧‧‧ cell line carrying the hepatitis C virus gene pool

JFH1‧‧‧In vitro hepatitis C virus infection cells

Huh7, Huh7.5.1‧‧‧ liver cancer cell line

NC‧‧‧negative control group

siRNA‧‧‧ small interfering nucleic acid

Figure 1 (A) ~ Figure 1 (C) shows that Let-7g may be associated with hepatitis C among AVA5 cells Correlation experiments on the combination of the 5' untranslated region of the viral gene bank; Figure 2 (A) ~ Figure 2 (D) shows the expression level of Let-7g in Huh7, hepatitis C virus replicon and infected cells; Figure 3 ( A) ~ Figure 3 (D) shows the cell model of Let-7g in vitro infection with hepatitis C virus JFH1 virus, in which cells transfected with miR-122 inhibitor served as a positive control group; Figure 4 (A) ~ Figure 4 (F) shows the results of related experiments on whether there is synergy between Let-7g and interferon/rapavirin; Figure 5 (A) shows the expression of Let-7g in serum before drug treatment of patients with hepatitis C virus infection Level 5; Figure 5 (B) shows the expression level of Let-7g in liver tissue before drug treatment in patients with hepatitis C virus infection; Figure 6 (A) ~ Figure 6 (F) shows nucleic acid binding protein lin28, lin28 small interfering nucleic acid (siRNA) can regulate the expression of Let-7g; Figure 7 shows an example of a method for pre-evaluating the efficacy of interferon/rebavirin in the treatment of hepatitis C; Figure 8 shows the evaluation of interferon/rebavirin treatment type C An embodiment of a method of treating hepatitis.

The invention will be described in detail by the following preferred examples and their associated experimental results.

[First Embodiment]

In this case, the sequence of AVA5 cells (Gene Bank No. AJ242654.1) was used to predict, and it was found that Let-7g may bind to hepatitis C in the hepatitis C virus genome sequence. The position of the nucleotide of SEQ ID NO: 43-65 on the 5' untranslated region of the virus. (See Figure 1(A)).

[Second embodiment]

To further confirm, this project constructed a luciferase reporter plastid that binds directly to Let-7g (hepatitis C virus 5'UTR-WT) and lacks the Let-7g binding sequence (hepatitis C virus 5'UTR-DEL). . In Figure 1(B), Let-7g mimic (■) or negative control (NC) mimic and hepatitis C virus 5'UTR-WT luciferase report plastid transfer into the C-hepatitis virus gene pool In AVA5 cells. In Figure 1(C), Let-7g mimic co-transforms with the luciferase reporter plastid of hepatitis C virus 5'UTR-WT (□) or 5'UTR-DEL (■) into C-type hepatitis Viral gene pool in AVA5 cells. The luciferase activity value of AVA cells was measured after 48 hours and was controlled internally by green fluorescent protein (GFP). The data of this example are the mean ± standard deviation obtained from three experiments, * P < 0.05.

The results of the experiment showed that when the transfection of the hepatitis C virus 5'UTR-WT plastid containing the Let-7g mimic was compared with the negative control (NC) mimic, the luciferase activity was decreased (see Figure 1(B)). ).

However, the plastids that simultaneously transfected Let-7g mimic and contained the lack of the Let-7g binding sequence (hepatitis C virus 5'UTR-DEL) contained Let-7g mimic and contained hepatitis C virus 5'UTR-WT At the time of the body, it was found that the activity of the luciferase was increased (see Fig. 1(C)). This indicates that Let-7g does bind directly to the position of the 5' untranslated region of the hepatitis C virus. Therefore, this case demonstrates that the hepatitis C virus 5'UTR contains a Let-7g binding sequence.

[Third embodiment]

To investigate the relationship between Let-7g and hepatitis C virus infection, this study examined the expression levels of Let-7g in hepatitis C virus replicons and infected cells. In Fig. 2(A), Huh7 and AVA5 cells were cultured for 72 hours and then their RNA was isolated. In Fig. 2(B), Huh7.5.1 cells were cultured for 24 hours and then added to cell culture medium (HCV cell-culture derived HCV). Hepatitis C virus infects cells JFH1 HCVcc and isolates its RNA 72 hours after infection. The expression of Let-7g was detected using timely quantitative PCR and the snU6B level was used as internal control. In addition, Let-7g mimic or negative control mimic (NC) was transfected into AVA5 cells (Fig. 2 (C)) or JFH infectious cells (Fig. 2 (D)) and RNA was isolated 72 hours after infection. The expression of the hepatitis C virus NS5B protein was detected using timely quantitative PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels as internal controls and the horizontal axis is unit concentration (nM). The data of this example is the mean ± standard deviation obtained from three experiments, * P < 0.05.

In this case, it was found that Let-7g significantly decreased the expression level of Let-7g in cells carrying the hepatitis C virus gene pool (AVA5) and hepatitis C virus infection (JFH) (see Figure 2(A) and Figure 2 ( B)). After transfection with Let-7g mimic, the level of hepatitis C virus gene in AVA5 and JFH-infected cells was reduced, that is, Let-7g mimic down-regulated the NS5B gene. It can be further inferred that the expression level of Let-7g is decreased in cells carrying the hepatitis C virus gene pool and infected with hepatitis C virus (see Fig. 2(C) and Fig. 2(D)).

Fourth Embodiment

In order to clarify the role of Let-7g in hepatitis C virus infection, the cell model of Let-7g infection in vitro with hepatitis C virus JFH1 virus was carried out, and cells transfected with miR-122 inhibitor were used as a positive control group.

In this example, cells of Huh7.5.1 were cultured for 24 hours, followed by transfection with Let-7g mimic, miR-122 inhibitor or negative control (NC), infected with JFH1 HCVcc 24 hours after transfection and 72 hours after infection. Cells were treated with interferon (100 IU/ml) or ribavirin (40 mM) for 24 hours and supernatants were collected and proteins were isolated 96 hours after infection. In Fig. 3 (A) and Fig. 3 (C), the hepatitis C virus core protein was analyzed by Western blotting. In Fig. 3 (B) and Fig. 3 (D), hepatitis C virus RNA in the cell culture supernatant was analyzed by Abbott Timed Quantitative Polymerase Chain Reaction (RT-PCR). The data of this example is the average value ± standard obtained from three experiments. Deviation, **P<0.005 and #P<0.05 and ##P<0.005.

The results showed that both Let-7g mimic and miR-122 inhibitor reduced the expression of the core protein of hepatitis C virus, ie, down-regulated the core protein (see Figure 3(A)).

Let-7g mimic reduced the viral load of supernatants from JFH1 virus-infected cells, accounting for approximately 73% (p < 0.0005) (see Figure 3(B)).

Both Let-7g mimic and IFN/RAB reduce C-type hepatitis C core protein expression (see Figure 3(C)).

Let-7g mimic combined with IFN/RBV reduced the viral load of JFH1 virus-infected cell supernatant by approximately 99% (p<0.0004). It can be seen that Let-7g can reduce the infectivity of HCVcc, that is, reduce the viral load. (See Figure 3(D)).

[Fifth Embodiment]

Because of the current clinical treatment, long-acting interferon (IFN-α) and ribavirin (Ribavirin) mixed treatment. To further understand whether there is a synergistic effect between Let-7g and interferon/rebavirin. In Fig. 4(A), after 24 hours of AVA5 cell culture, RNA was isolated after treatment of cells with interferon (100 IU/ml) or ribavirin (40 mM) for 24 hours. Hepatitis C virus gene levels were detected using timely quantitative PCR and snU6B levels were used as internal controls. In Fig. 4(C) to Fig. 4(F), Let-7g mimic or negative control mimic (NC) was transfected into AVA5 cells and RNA was isolated 72 hours after infection. The expression of the hepatitis C virus NS5B protein was detected using timely quantitative PCR and GAPDH levels were used as internal controls. Detection of hepatitis C virus gene levels using timely quantitative PCR (Fig. 4(C)), mucinous viral protein MxA (Fig. 4(D)), 2'-5' oligoadenylate synthetase OAS (Fig. 4(E) And expression in the protein kinase R PKR (Fig. 4 (F)) and GAPDH levels were used as internal controls. Figure 4 (B) uses Western blotting to determine the expression of the hepatitis C virus NS5B protein. The relative level of NS5B protein was quantified using a number of 1-D analysis software. The data of this example are the mean ± standard deviation obtained from three experiments, * P < 0.05, ** P < 0.05, # P < 0.05.

The results of the experiment show that IFN-α 2A combined with RBV can increase the level of hepatitis C virus gene (see Figure 4 (A)).

Treatment of IFN/RBV with AVA5 cells transfected with Let-7g reduced the expression levels of the hepatitis C virus gene and NS5B protein (see Figure 4(B)).

This case indicates that type I and type II interferons up-regulate interferon-stimulated genes (ISGs) such as protein kinase R (PKR), 2'-5' oligoadenylate synthetase (OAS) or myxovirus (MxA). It is an important anti-viral factor. The results of the experiment showed that the expression of MxA gene was approximately 2-fold (see Figure 4(D)) and the expression of OAS gene was approximately 17-fold when transfected with Let-7g mimic alone (see Figure 4(E)), but did not affect the PKR gene. Expression (see Figure 4(F)). This case further examined whether Let-7g mimic combined with IFN/RBV affects the synergistic effect between these interferon-stimulated genes. The data showed that Let-7g mimic combined with IFN/RBV caused MxA gene expression about 110-fold and OAS gene expression about 226-fold (see Figure 4(D)~(E)). This indicates that there is a synergistic induction effect between Let-7g mimic and IFN/RBV, and the antiviral effect of Let-7g is dependent on the interferon pathway.

[Sixth embodiment]

This case detects the expression level of Let-7g before drug treatment in patients with hepatitis C virus infection. In Fig. 5(A), pre-drug sera of patients with hepatitis C virus infection were divided into viral persistent response group (SVR) (n=15) and non-viral persistent response group (non-SVR) (n=16). The expression of Let-7g was detected using timely quantitative PCR and the snU6B level was used as an internal control. On the Y-axis, the expression level of Let-7g is expressed as LOG (2-ΔCT) and ΔCT = CT (LET-7G)-CT (snU6B). *P<0.05, **P<0.005.

The results of the experiment showed that: in the serum of patients with hepatitis C virus infection before drug treatment, it was found that Let-7g had a more significant decrease in non-SVR patients than in SVR patients (p=0.001).

[Seventh embodiment]

In the present example, the expression level of liver tissue Let-7g was measured before drug treatment in patients infected with hepatitis C virus. In Figure 5(B), liver tissue was divided into nonalcoholic fatty liver group (NAFLD) (n=17) and viral persistent response group (SVR) (n=11) before drug treatment in patients with hepatitis C virus infection. Non-SVR (n=6), expression of Let-7g was detected using timely quantitative PCR and RNU44 levels were used as internal controls. On the Y-axis, the expression level of Let-7g is represented by LOG (2-ΔCT) and ΔCT=CT(LET-7G)-CT (RNU44). *P<0.05, **P<0.005.

The results of the experiment showed that in the living liver tissue, the Let-7g showed a more significant decrease in the non-SVR patients than in the SVR patients (P=0.04). In addition, the Let-7g of patients infected with hepatitis C virus also showed a significant decrease when compared with Let-7g of patients with nonalcoholic fatty liver disease (NAFLD). The decrease of NAFLD for SVR is P=0.021; the decrease of NAFLD for non-SVR is P=0.004 (see Figure 5(B)).

The above data indicate that low expression levels of Let-7g may be associated with a therapeutic response. Therefore, the expression level of Let-7g can be used as a predictive measure of the efficacy of drugs for patients with chronic hepatitis C virus infection. For example, when the expression level of Let-7g is relatively high, it means the efficacy of interferon/rebavirin in the treatment of hepatitis C. May be better.

[Eighth Embodiment]

This case further explores how to regulate the activity of Let-7g. Figure 6 is mainly concerned with whether the nucleic acid binding protein lin28, lin28 small interfering nucleic acid (siRNA) can regulate the expression of Let-7g.

In Fig. 6(A), Huh7 and AVA5 cell lines were isolated after 72 hours of culture.

In Fig. 6(B), Huh7.5.1 cell line (1x105 cells) was infected with JFH1 HCVcc after being cultured for 24 hours, and its RNA was isolated 72 hours after infection. Expression of the nucleic acid binding protein lin28A or lin28B can be detected by RT-PCR.

In Figure 6 (C), lin28A siRNA, lin28B siRNA or negative control (NC) siRNA was transfected into control group Con1 cells. In Fig. 6(D), lin28A siRNA, lin28B siRNA or negative control (NC) siRNA was transfected into the J6/JFH1 cell line. The luciferase activity test was performed 48 hours after the transfection, and the cell proliferation assay reagent (WST1) was used as the internal control, and the horizontal axis was a unit concentration (nM). The data of this example are the mean ± standard deviation obtained from three experiments, * P < 0.05, ** P < 0.005.

In Figure 6 (E), lin28B siRNA or negative control (NC) siRNA was transfected into the AVA5 cell line. Relative Let-7g levels were determined 48 hours after transfection. The mean ± standard deviation of the data from the three experiments, **P < 0.005.

In Figure 6 (F), lin28B siRNA or negative control (NC) siRNA was transfected into JFH cell lines. Relative Let-7g levels were determined 48 hours after transfection. The mean ± standard deviation of the data from the three experiments, **P < 0.005.

The results showed that the expression levels of the nucleic acid binding proteins lin28A and lin28B were lower in the AVA5 cell line than in the Huh7 cell line (see Figure 6(A)).

The expression levels of the nucleic acid binding proteins lin28A and lin28B in the Huh7.5.1 cell line were lower than those of the infected JFH1 virus cell line (see Figure 6(B)).

Con1 cell luciferase activity transfected with lin28 siRNA was substantially reduced with siRNA concentration compared to the control group (see lin28A siRNA and lin28B siRNA in Figure 6 (C)).

The luciferase activity in the J6/JFH1 cell line transfected with lin28 siRNA was significantly reduced with the concentration of siRNA compared to the negative control (NC) siRNA (see Figure 6(D)).

The relative Let-7g levels were higher in the AVA5 cell line transfected with lin28 siRNA compared to the negative control (NC) siRNA (see Figure 6(E)).

Compared to the negative control (NC) siRNA, it was transfected with Let-7g relatively high levels in the infected cell line JFH lin28 siRNA (see FIG. 6 (F)).

Therefore, the use of the nucleic acid binding protein lin28 can be first inferred, and the expression level of a small ribonucleic acid, Let-7g, can be downregulated in a replicon or a sample infected with hepatitis C virus. Furthermore, lin28 small interfering nucleic acid (siRNA) or up-regulated the expression level of the small ribonucleic acid Let-7g down-regulated the activity of hepatitis C virus.

Ninth Embodiment

Please refer to FIG. 7. FIG. 7 shows an embodiment of a method for pre-evaluating the efficacy of interferon/rebavirin in the treatment of hepatitis C. In step S701, a sample is provided, and the sample may be derived from a living or in vitro cultured cell; in step S702, the sample is paired with a microRNA (miRNA) Let-7g. And a polymerase chain reaction (PCR) reagent mixing; and in step S703, the expression level of the microRNA lactic-7t-7g of the sample detected by the polymerase chain reaction reagent can be pre-evaluated for the test The efficacy of interferon/rebavirin in the treatment of hepatitis C.

[Tenth embodiment]

Please refer to FIG. 8. FIG. 8 shows an embodiment of a method for evaluating the efficacy of interferon/rebavirin in the treatment of hepatitis C. In step S801, a sample sampled from a hepatitis C patient who has used interferon/rebavirin is provided; in step S802, the sample is associated with a microRNA (miRNA) Let-7g a primer pair and a polymerase chain reaction (PCR) reagent are mixed; and in step S803, the expression level of the microribonucleic acid Let-7g of the sample is detected according to the polymerase chain reaction reagent, and can be evaluated The hepatitis C patient is treated with interferon/rebavirin for the treatment of hepatitis C.

[Eleventh Embodiment] A method for pre-evaluating the therapeutic effect of Interferon/Ribavirin for treating hepatitis C, comprising the steps of: providing a sample; and detecting the sample with a picoruclear acid ( miRNA) a primer pair of Let-7g and a polymerase chain reaction (PCR) The reagent is mixed; and according to the expression level of the microribonucleic acid Let-7g of the sample detected by the PCR reagent, the therapeutic effect of the interferon/rebavirin on the hepatitis C for the specimen can be evaluated in advance.

[Twelfth Embodiment] A kit for pre-evaluating the efficacy of Interferon/Ribavirin in the treatment of hepatitis C, comprising: a primer pair of a small ribonucleic acid Let-7g, used for Mixing with a sample; a polymerase chain reaction (PCR) reagent for mixing with the primer pair of the microribonucleic acid Let-7g and the sample, wherein the tiny amount of the sample is detected according to the PCR reagent The expression level of ribonucleic acid Let-7g can be pre-evaluated for the efficacy of interferon/rebavirin in the treatment of hepatitis C for this sample.

[Thirteenth Embodiment] The use of a small ribonucleic acid, Let-7g, is mainly used for down-regulating an NS5B gene expression level, a core protein expression level, and a viral load in a hepatitis C virus (HCV) ( Load) at least one of the three.

[Fourteenth Embodiment] The use of the piconucleotide Let-7g according to the thirteenth embodiment, wherein the piconucleotide Let-7g and the 5' untranslated region of the hepatitis C virus gene pool ( 5' UTR) The nucleotides of SEQ ID NO: 43-65 are combined.

[Fifteenth Embodiment] The use of the piconucleotide Let-7g according to the fourteenth embodiment further comprises up-regulating 2'-5' oligoadenylate synthetase (OAS) or myxovirus (MxA) Interferon-stimulated genes (ISGs).

[16th embodiment] The use of the piconucleotide Let-7g according to the thirteenth embodiment, wherein the hepatitis C virus is derived from an in vivo replicon or an infected cell.

[17th embodiment] The use of the piconucleotide Let-7g according to the thirteenth embodiment, wherein the hepatitis C virus is derived from an in vitro replicon or an infected cell.

[Eighteenth Embodiment] The use of the piconucleotide Let-7g according to the sixteenth and seventeenth embodiments further includes synergistic use of Interferon/Ribavirin, In order to down-regulate the activity of hepatitis C virus.

[Nineteenth embodiment] The use of a nucleic acid binding protein lin28 is mainly for downregulating the expression level of a small ribonucleic acid Let-7g in a replicon or a sample infected with hepatitis C virus.

[Twentieth embodiment] The use of the nucleic acid binding protein lin28 according to the nineteenth embodiment, wherein a lin28 small interfering nucleic acid (siRNA) is upregulated in the replicon or a cell infected with hepatitis C virus The expression level of the small ribonucleic acid Let-7g down-regulates the activity of the hepatitis C virus.

"Materials and Methods"

All cell culture related reagents were purchased from GIBCO-BRL. Interferon alpha-2a interferon (ROFERON®-A) was purchased from Roche. Rebavirin and all chemical reagents were purchased from Sigma-Aldrich. A solution that enhances chemiluminescence (ECL) was purchased from Millipore Corporation. The TRIZOL® kit was purchased from Invitrogen. The luciferase assay system was purchased from Promega. The primer pair was purchased from Genomic Biotechnology Corporation (Taipei, Taiwan).

『Cell culture』

Hepatoma cell lines Huh7, Huh7.5.1 and cell line AVA5 (type 1b) carrying the hepatitis C virus gene pool were obtained from Apath (St. Louis, Mo.). The cell line was cultured in DMEM medium containing 10% fetal bovine serum, 5% antibiotics and 5% non-essential amino acids. The AVA5 cell line was additionally supplemented with 1 mg/mL aminoglycoside antibiotic (G418).

"In vitro hepatitis C virus infection"

The JFH1 cDNA was obtained from Dr. Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan). Hepatitis C virus infection in vitro method of JFH1 culture system containing liquid (equivalent to a concentration of ¹⁰⁵ parts by number of hepatitis C viral RNA in a titration contained), added directly to Huh7.5.1 cell lines. After 6 hours, the cells were washed 3 times with phosphate buffered saline (PBS), and then the cells were maintained in growth medium.

"microRNA transfection"

mirVana ^TM Let-7g mimic the negative control group and were purchased from Ambion, Inc. ®. miRNA transfected into cells using Oligofectamine ^TM transfection reagent (commercially available from Life Technologies, Inc.).

"Construction of C-type hepatitis 5' UTR plastid"

Hepatitis C virus 5' UTR-WT and hepatitis C virus 5' UTR-DEL plastids were synthesized by GENEWIZ (South Plainfield, NJ, USA).

"Use timely quantitative PCR to detect small RNAs"

The Let-7g assay was the synthesis of cDNA from the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The expression of Let-7g was performed using the Gene Amplifier 7900® Sequence Detection System (Applied Biosystems). The relative expression level of Let-7g in each sample required correction of its own snU6B or RNU44.

"Western Blot"

Western blotting was used to assess the protein expression described. Hepatitis C virus Core antibody was purchased from a company of Thermo Scientific, a hepatitis C virus NS5B protein antibody was purchased from ViroStat, and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein antibody was purchased from Millipore Corporation.

"Measure the amount of hepatitis C virus load in the supernatant"

The Hepatitis C virus load in the supernatant was measured using an Abbott mSample Preparation System reagent M2000 instrument (Abbott, North Chicago, IL) using the method according to the manufacturer's instructions.

"RNA separation and timely quantitative polymerase chain reaction (RT-PCR)"

Total RNA was extracted using TRIzol according to the manufacturer's instructions. The quality of the RNA was confirmed using the A260/A280 reading. Reverse Transcription Kit Using High Capacity cDNA (Applied) Biosystems, CA, USA). cDNA was synthesized from 100 ng of total RNA. The PCR reaction (ABI PRISM 7900 system) was carried out using SYBR® Green PCR Master Mix (Applied Biosystems). All primers used are listed in Table 1 below.

"Experimental examination of clinical samples"

17 cases of chronic hepatitis C (CHC) clinical specimens and 17 non-alcoholic fatty liver disease (NAFLD) specimens were used as control group. A biopsy of patients with chronic hepatitis C demonstrated that PEG-IFN/Ribavirin was treated at 24 or 48 weeks depending on the viral genotype and maintained at least 80% of the assigned duration of treatment and treatment regimen: 11 patients achieved a sustained viral response (SVR) (meaning that hepatitis C virus RNA was not detected 24 weeks after treatment) and 6 non-viral sustained responses (non-SVR). All patients with nonalcoholic fatty liver disease were anti-C hepatitis virus antibodies and hepatitis C virus RNA negative status. Relevant human subjects' clinical specimens have been approved by the Kaohsiung Medical University Hospital Ethics Committee and have been guided by an international coordination meeting for good clinical practice. All subjects signed informed consent.

"Statistical Analysis"

Data for one continuous variable is expressed as mean ± standard deviation (SD). A paired Student's t-test was used to compare the differences between the two groups. The Studden's t-test and χ 2 were used to analyze the characteristics of the patient. A multivariate logistic regression model was used to determine the outcome of the relevant factors. All P values were bilaterally less than 0.05 and were considered statistically significant. All statistical calculations were performed on the 9th edition of the JMP software.

To predict complementarity between Let-7g and the hepatitis C virus genome, Miranda was used in this case to calculate and predict whether Let-7g binds to the hepatitis C virus genome sequence. In most of the hepatitis C virus genotypes, Let-7g is predicted to bind to the 5' untranslated region (5'UTR) in the hepatitis C virus gene pool. Table 2 shows that Let-7g may bind. To the 5' untranslated region of different genotypes of hepatitis C virus.

In summary, experimental data indicate that non-coding microRNAs, Let-7g, are associated with infection with hepatitis C virus and can provide another new targeted therapy for hepatitis C virus. However, how to further regulate the activity of Let-7g, such as detecting the presence of a transcriptional core region among the promoters of Let-7g, is one of the future efforts.

In addition, the above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, but the scope of the present invention is not limited to the specific embodiments described above; and the present invention is to be considered by those skilled in the art. All kinds of modifications, but do not deviate from the scope of the application of the case.

<110> Kaohsiung Medical University

<120> Down-regulation of NS5B gene, core protein and viral load of hepatitis C virus by Let-7g

<211> 20

<212> DNA

<213> HCV

<223> HCV NS5B primer

<400> 1

<110> Kaohsiung Medical University

<120> Down-regulation of NS5B gene, core protein and viral load of hepatitis C virus by Let-7g

<211> 21

<212> DNA

<213> HCV

<223> HCV 5’UTR primer

<400> 1

<110> Kaohsiung Medical University

<120> Down-regulation of NS5B gene, core protein and viral load of hepatitis C virus by Let-7g

<211> 20

<212> DNA

<223> MxA primer

<400> 1

<110> Kaohsiung Medical University

<120> Down-regulation of NS5B gene, core protein and viral load of hepatitis C virus by Let-7g

<211> 29

<212> DNA

<223> PKR primer

<400> 1

<110> Kaohsiung Medical University

<120> Down-regulation of NS5B gene, core protein and viral load of hepatitis C virus by Let-7g

<211> 21

<212> DNA

<223> 2'5'-OAS primer

<400> 1

Claims (3)

Use of a small ribonucleic acid, Let-7g, for the preparation of a synergistic up-regulation of 2'-5' oligoadenylate synthetase (OAS) or adhesion in combination with interferon and ribavirin A viral protein (MxA) acts to inhibit a pharmaceutical composition of hepatitis C virus (HCV). The use of the piconucleotide Let-7g according to claim 1, wherein the hepatitis C virus is derived from an in vivo replicon or an infected cell. The use of the piconucleotide Let-7g according to claim 1, wherein the hepatitis C virus is derived from an in vitro replicon or an infected cell.