Application of LncRNA XIST in preparation of medicine for preventing myocardial injury caused by angiotensin II

Abstract

The invention discloses application of LncRNA XIST in preparation of a medicine for preventing myocardial injury caused by angiotensin II, and relates to the technical field of biological medicine. According to the application disclosed by the invention, a single molecular mechanism or pathway research is broken through, a complete mechanism pathway of an LncRNA XIST / miR-20a-5p / Nrf2 axis is established, the adsorption capacity of the LncRNA XIST / miR-20a-5p / Nrf2 is up-regulated by overexpressing XIST, and finally, the expression of Nrf2 is up-regulated, so that myocardial oxidative damage caused by angiotensin II is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to the use of LncRNA XIST in preparing a drug for preventing myocardial damage caused by angiotensin II. Background Art

[0002] Angiotensin II (AngII), the primary effector peptide of the renin-angiotensin system (RAS), plays a key role in regulating blood pressure and cardiovascular homeostasis. In addition to circulating AngII produced by the classical pathway, local RAS also exists in tissues such as the heart, blood vessels, kidneys, and testes. Localized cardiac AngII acts directly on cardiomyocytes through autocrine or paracrine pathways, independently of hemodynamic factors, to promote myocardial apoptosis, hypertrophy, and fibrosis. This locally produced AngII contributes to the development and progression of various cardiac diseases, such as diabetic cardiomyopathy, ischemia-reperfusion injury, and ventricular remodeling after myocardial infarction. Therefore, AngII has attracted considerable attention as an independent risk factor for the development of cardiomyopathy.

[0003] Existing studies have confirmed that oxidative stress is the primary pathogenic factor leading to Ang II-induced myocardial injury. Ang II binds to specific type 1 receptors on cardiomyocytes and activates nicotinamide adenine dinucleotide (NADPH) oxidase, generating large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which exceed the body's clearance capacity and lead to oxidative stress. The continued overproduction of ROS / RNS leads to the oxidation and nitration of various proteins and enzymes, resulting in their inactivation, triggering cardiomyocyte apoptosis or necrosis, and promoting inflammatory responses and fibroblast proliferation, ultimately leading to ventricular remodeling and heart failure.

[0004] Nuclear factor erythroid 2-related factor 2 (Nrf2) is the most important regulator of oxidative stress in the body. It mobilizes hundreds of antioxidant genes, phase II detoxification enzymes, and cytoprotective proteins, constituting the body's most crucial antioxidant defense mechanism. Studies have demonstrated that Ang II-induced myocardial oxidative damage is associated with low Nrf2 expression and activity. Activating Nrf2 with Nrf2 agonists or transgenic approaches can effectively prevent Ang II-induced myocardial damage, confirming the crucial role of Nrf2 in myocardial defense against oxidative stress. However, how Ang II inhibits myocardial Nrf2 and exacerbates myocardial oxidative damage remains unclear. Uncovering this mechanism will provide a deeper understanding of the common features of myocardial redox regulation and provide effective guidance for the diagnosis and prevention of oxidative stress-related cardiomyopathies.

[0005] Long non-coding RNA (lncRNA) is a class of functional RNA molecules with transcripts exceeding 200 nucleotides that do not encode proteins. They are present in the nucleus or cytoplasm. They regulate gene expression at epigenetic, transcriptional, post-transcriptional, and translational levels, and participate in numerous pathophysiological processes. LncRNAs, acting as ceRNAs, participate in the development and progression of various cardiac diseases and can serve as biomarkers for cardiac disease, possessing dual value in clinical diagnosis and treatment. In cardiovascular research, lncRNAs are implicated in the development and progression of myocardial infarction, atherosclerotic heart disease, diabetic cardiomyopathy, and heart failure. LncRNAs also play a crucial role in Ang II-induced myocardial injury. It has been reported that the lncRNAs MIAT, CHRF, and SNHG14 act on different miRNAs to promote Ang II-induced cardiac hypertrophy, while the lncRNAs MAGI1-IT1 and Plscr4 compete with miR-302e and miR-214 for binding, inhibiting Ang II-induced cardiomyocyte hypertrophy. However, these studies on Ang II-induced myocardial injury have focused on pressure-induced hypertrophy, but have not examined the pressure-independent stimulation of Ang II alone or its effects on oxidative stress. Therefore, we will investigate whether XIST knockdown exacerbates Ang II-induced myocardial injury and the efficacy of XIST upregulation in preventing Ang II-induced myocardial injury. We will also further elucidate the molecular mechanism by which XIST, as a ceRNA, targets miR-20a-5p to regulate Nrf2. We hope to provide new molecular targets for the diagnosis and prevention of myocardial oxidative damage and cardiomyopathy, laying a theoretical and experimental foundation for the clinical prevention and treatment of cardiomyopathy. Summary of the Invention

[0006] To address the above-mentioned issues, the present invention provides the use of lncRNA XIST (and its associated miR-20a-5p targeting Nrf2) in the preparation of a drug for preventing angiotensin II-induced myocardial injury. This addresses the problems that existing research has mostly focused on a single molecule or pathway (such as miR-20a-5p or Nrf2), lacks systematic research on the synergistic effect of the lncRNA XIST / miR-20a-5p / Nrf2 axis in Ang II myocardial injury, and has not verified its role in in vitro and in vivo models. In addition, current research progress has mostly focused on the study of simple pathological mechanisms or clinical treatment, and cannot achieve the combination of early diagnosis and precise treatment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides an application of LncRNA XIST in preparing a drug for preventing myocardial damage caused by angiotensin II.

[0008] The present invention provides an application of LncRNA XIST and its combined miR-20a-5p targeting Nrf2 in the preparation of a drug for preventing myocardial damage caused by angiotensin II. The mechanism is as follows: Figure 18 shown.

[0009] Furthermore, the nucleotide sequence of the miR-20a-5p is shown in SEQ ID NO: 1, and the nucleotide sequence of the Nrf2 is shown in SEQ ID NO: 2.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This study breaks away from single-molecule mechanism or pathway research and establishes a complete mechanistic pathway for the lncRNA XIST / miR-20a-5p / Nrf2 axis. Overexpressing XIST leads to increased adsorption of miR-20a-5p, ultimately upregulating Nrf2 expression, thereby alleviating angiotensin II-induced myocardial oxidative damage. This study deciphers the molecular mechanism by which myocardial Nrf2 is regulated by XIST / miR-20a-5p, confirming the effectiveness of upregulating XIST in activating the endogenous antioxidant stress factor Nrf2 and preventing Ang II-induced myocardial damage. The lncRNA XIST / miR-20a-5p / Nrf2 axis of this invention can be used in the preparation of drugs to prevent angiotensin II-induced myocardial damage. This technological breakthrough will provide a new perspective for the diagnosis, prevention, and treatment of Ang II-related myocardial oxidative damage and cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The changes in the average blood pressure of mice at different periods, where a is 2 months (2M), b is 4 months (4M), and c is 6 months (6M); Figure 2 The cardiac structures of mice in the control and AngII groups were detected by ultrasound at different time periods, where ac were 2M, 4M, and 6M for the control group, and df were 2M, 4M, and 6M for the AngII group; Figure 3 For ultrasound detection of mouse cardiac function, a is LVID (left ventricular end-diastolic diameter), b is LVPW (left ventricular posterior wall thickness at diastole), c is EF% (ejection fraction), d is FS% (fractional shortening), and e is the ratio of mouse heart weight to body weight at 2M, 4M, and 6M, respectively; Figure 4 qRT-PCR and Western blot were used to detect the expression level and quantitative analysis of Nrf2 in myocardial tissue of 2M, 4M, and 6M mice, where ac are the Nrf2 protein expression levels at 2M, 4M, and 6M, df are the quantitative analysis at 2M, 4M, and 6M, and g is the Nrf2 mRNA expression level; Figure 5 qRT-PCR and Western blot were used to detect the expression level and quantitative analysis of HO-1, a downstream antioxidant gene of Nrf2, in the myocardial tissue of 2M, 4M, and 6M mice. ac are the HO-1 protein expression levels at 2M, 4M, and 6M, respectively; df are the quantitative analysis at 2M, 4M, and 6M, respectively; g is the HO-1 mRNA expression level; Figure 6 Western blot analysis of the protein expression level and quantification of the Nrf2 downstream antioxidant gene Catalase in the myocardial tissue of 2M, 4M, and 6M mice. ac represents the protein expression levels at 2M, 4M, and 6M, and df represents the quantification analysis at 2M, 4M, and 6M. Figure 7 Figure 3 shows high-throughput transcriptome sequencing analysis of differentially expressed lncRNAs in Ang II myocardial injury tissues; a is a heat map of lncRNA expression; b is a map of differentially expressed lncRNAs; c is a volcano map of lncRNA transcriptome sequencing analysis; d is a map of known differentially expressed lncRNAs in myocardial tissues of mice in the Ang II group; Figure 8 AC16 human cardiomyocytes were stably transfected with the recombinant plasmid pPLK / GFP-XIST-shRNA. (a) and (c) show the infection of the control plasmid observed by fluorescence microscopy, and (b) and (d) show the infection of the sh-XIST plasmid observed by fluorescence microscopy. Figure 9 The expression of Nrf2 was detected by qRT-PCR and western blot, where a represents the expression level of Nrf2 mRNA, bc represents the expression level of Nrf2 protein and quantitative analysis respectively; Figure 10 Western Blot analysis of the expression of oxidative damage indicator 4-HNE, where a is 4-HNE expression and b is quantitative analysis of expression levels; Figure 11 Western Blot was used to detect the expression of hypertrophy-related factor ANP and perform quantitative analysis, where a is ANP expression and b is quantitative analysis of expression level; Figure 12 Western Blot detection of the expression of the inflammatory factor Cleaved-caspase3 and quantitative analysis, where a is the expression of Cleaved-caspase3 and b is the quantitative analysis of the expression level; Figure 13 Western Blot was used to detect the expression of TGF-β, a fibrosis indicator, and quantitative analysis was performed, where a represents TGF-β expression and b represents quantitative analysis of expression levels; Figure 14 AC16 human cardiomyocytes were transiently transfected with pcDNA3.1-XIST, and the expression of XIST and Nrf2 mRNA was detected by qRT-PCR. (a) is the expression level of XIST mRNA; (b) is the expression level of Nrf2 mRNA; Figure 15 Western Blot was used to detect the expression of Nrf2 protein and perform quantitative analysis, where a is the expression of Nrf2 protein and b is the quantitative analysis of the expression level; Figure 16 Western blot was used to detect the expression of oxidative damage indicator (4-HNE) and perform quantitative analysis, where a is the expression of 4-HNE protein and b is the quantitative analysis of expression level; Figure 17 The three binding sites prediction of XIST, miR-20a-5p and Nrf2, among which a is the three binding sites prediction in human model, and b is the three binding sites prediction in mouse model; Figure 18 This is the mechanism by which lncRNA XIST regulates angiotensin II-induced myocardial injury by targeting Nrf2 through miR-20a-5p.

[0012] Note: In the figure, all quantitative data are expressed as X±SD (where Figure 1 、 Figure 3 medium, n=5-7; Figure 4 In af, n=6; Figure 4 In g, n=4-5; Figure 5 In af, n=6, Figure 5 In g, n=4-5; Figure 6ns, no statistical difference; * P <0.05 vs. Control; ** P <0.01 vs. Control; *** P <0.001 vs. Control; **** P <0.0001 vs. Control; # P <0.05 vs. AngII; ## P <0.01 vs. AngII; ### P <0.001 vs. AngII. DETAILED DESCRIPTION

[0013] In order to make the objects and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0014] The instruments, reagents, materials and the like involved in the following examples are all conventional instruments, reagents, materials and the like existing in the prior art, and can be obtained through regular commercial channels, unless otherwise specified. The experimental methods, detection methods and the like involved in the following examples are all conventional experimental methods, detection methods and the like existing in the prior art, unless otherwise specified.

[0015] In the following examples, the nucleotide sequence of miR-20a-5p is shown in SEQ ID NO: 1, and the nucleotide sequence of Nrf2 is shown in SEQ ID NO: 2.

[0016] Example 1 1. Research method (1) AngII myocardial injury model Eight-week-old male C57 / BL mice were subcutaneously injected with a small dose of AngII (0.5 mg / kg, which did not cause significant changes in blood pressure) every other day, once every other day, for 2 months, and the observation was stopped after 6 months of drug withdrawal. The control group was subcutaneously injected with the same dose of solvent (0.9% normal saline).

[0017] (2) AngII in vitro model In AC16 human myocardial cells cultured in a constant temperature incubator at 37°C and 5% carbon dioxide, 100 nmol / L of AngII was added, and cultured for 48 hours.

[0018] (3) Lentiviral packaging experiment The shRNA-XIST plasmid was purchased from PPL (the nucleotide sequence of the sh-XIST plasmid is shown in SEQ ID NO: 3). 293T cells were plated in a 6 cm dish and virus packaging was performed when the cell density reached 30%-40%.

[0019] Prepare the virus system in a 6cm dish. Prepare Solution A: Opti-MEM 1100μl, Lipofectamine 2000 8μl, gently flick to mix, and incubate at room temperature for 5 minutes. Prepare Solution B: Opti-MEM 1500μl, pMD2.G 1.2μg, pRSV-Rev 4μg, pMDLg-pRRE 2.4μg, and target plasmid 4μg, gently flick to mix, and incubate at room temperature for 5 minutes. Add Solution B to Solution A, gently flick to mix, and incubate at room temperature for 20 minutes.

[0020] Add the settled solution evenly to the culture dish, shake gently, and place in a 37°C incubator. After 4-6 hours, replace with 4 ml of culture medium containing 10% fetal bovine serum. Collect the virus after 24, 48, and 72 hours.

[0021] (4) Lentivirus infection cell experiment One day before infection, cells were seeded in a 10 cm dish with complete medium and cultured at 37°C for 16–24 hours to a confluency of 60–70%. The following day, the appropriate volume of virus, calculated based on the appropriate MOI and viral titer, was added, along with infection enhancement solution. Culture was continued at 37°C for 12 hours before switching to regular medium. Puromycin selection was initiated 48 hours later.

[0022] (5) OE-XIST transfection into HL-1 mouse cardiomyocytes The OE-XIST plasmid was purchased from Shandong Weizhen Biotechnology Co., Ltd. (the nucleotide sequence of the OE-XIST plasmid is shown in SEQ ID NO: 4). Cells were seeded in 6-well plates one day before transfection and transfection was performed when the cell confluence reached 50%.

[0023] 6-well plate transfection system. Prepare Solution A: 250 μl Opti-MEM, 8 μl Lipofectamine 2000, gently flick to mix, and incubate at room temperature for 5 min. Prepare Solution B: 250 μl Opti-MEM, 2 μg plasmid, gently flick to mix, and incubate at room temperature for 5 min. Add Solution B to Solution A, gently flick to mix, and incubate at room temperature for 20 min.

[0024] Remove the 6-well plate, discard the old medium, and wash twice with PBS. Add 1.5 ml of serum-free medium to each well, followed by 500 μl of the mixture of Solution A and Solution B. Shake the plate crosswise to ensure even coverage. Continue incubating at 37°C for 6 hours, then replace with fresh medium and continue incubation for 48 hours.

[0025] (6) miR-20a-5p inhibition experiment Before the experiment, 1~5×10 5 Transfer cells to a 24-well plate containing 1 ml of culture medium and continue culturing until the cell density reaches 30-50%. Dilute 1.25 μl of 20 μM miRNA inhibitor with 30 μl of Opti-MEM medium, mix gently, and then add 3 μl of Lipofectamine 2000 transfection reagent. Gently pipette to mix and incubate at room temperature for 10 minutes. Then, add culture medium without dual antibody and continue culturing for 24-96 hours before testing.

[0026] (7) Real-time quantitative PCR (qRT-PCR) qRT-PCR was used to analyze the expression of XIST, miR-20a-5p, myocardial fibrosis, inflammatory response, and Nrf2 downstream genes (see Table 1 for primer sequences). Total RNA was extracted using Trizol reagent, and cDNA was synthesized using random primers. qRT-PCR analysis was then performed using a QuantStudio1 quantitative PCR instrument.

[0027] Table 1 Primer sequences

[0028] (8) Western blot Heart tissue was lysed using a high-speed tissue homogenizer in tissue lysis buffer (2% SDS, 10% glycerol, and 62.5 mM Tris, pH 7.0). After centrifugation at 12,000 rpm for 10 minutes at 4°C, the supernatant was collected for total protein extraction and protein concentration determination. Nuclei were extracted from a portion of myocardial tissue using a nuclear extraction kit (purchased from Nanjing KeyGen Biotechnology Co., Ltd.). Nuclear proteins were extracted using SDS lysis buffer containing protease inhibitors, and protein concentration was determined. Samples were then separated by 10% SDS-PAGE electrophoresis, transferred to a membrane, and washed with TBS for 1–2 minutes. The membrane was then blocked in blocking buffer (5% skim milk and 0.5% bovine serum albumin) for 1 hour and washed three times with TBST (containing 0.05% Tween 20). After transfer and blocking, the proteins were incubated with different primary antibodies at room temperature for 2 h, then washed three times as above, and then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature for 1 h. Finally, ECL was used to observe the expression of the proteins.

[0029] 2. Experimental results (1) Establishment of Ang II myocardial injury mouse model Eight-week-old male C57 / BL mice were subcutaneously injected with a small dose of AngII (0.5 mg / kg) every other day for 2 months. The control group was subcutaneously injected with the same dose of normal saline. Blood pressure changes were monitored monthly using a fully automatic non-invasive blood pressure monitoring system. No significant changes in blood pressure were considered a successful modeling condition. After stopping AngII administration, the mice were observed for 6 months. Figure 1 The cardiac structure was examined by cardiac ultrasound at 2 months (2M), 4 months (4M) and 6 months (6M). Figure 2 Mice were killed after cardiac ultrasound at three time points, and myocardial tissues were collected. The experiment was divided into a control group (CON) and an AngII-treated group (AngII); the three time points included 2 months (2M), 4 months (4M), and 6 months (6M).

[0030] (2) AngII causes cardiac function damage and myocardial hypertrophy in mice Cardiac function tests showed that in the AngII group, there was no significant change in 2M, while the LVID (reflecting cardiac dilatation) and LVPW (reflecting myocardial hypertrophy) in the 4M and 6M groups were higher than those in the control group ( Figure 3 ab); EF and FS (reflecting myocardial contractile function) in the AngII group were lower than those in the control group at 4M and 6M ( Figure 3 cd), the differences between the groups were statistically significant. The ratio of mouse heart weight to body weight can reflect the degree of myocardial hypertrophy, so we calculated the ratio of mouse heart weight to body weight ( Figure 3e), compared with the control group of mice, 4M and 6M, AngII group of mice heart weight / body weight ratio increased. This result shows that low-dose AngII can cause myocardial hypertrophy, left ventricular dilation and heart function in mice without causing blood pressure to increase.

[0031] (3) AngII causes changes in myocardial tissue Nrf2 expression and Nrf2 and downstream antioxidant gene HO-1, Catalase expression Using qRT-PCR and Western blot method to detect the expression of Nrf2 mRNA and protein levels in myocardial tissue of mice, compared with the control group, 2M AngII group of mice heart Nrf2 mRNA and protein levels were significantly increased; and in 4M and 6M, Nrf2 mRNA and protein levels decreased, and in 6M expression down-regulation is more obvious ( Figure 4 ). The above results show that the early myocardial injury caused by AngII, without heart failure, Nrf2 expression appears compensatory increase, and then gradually decreased, to the abnormal heart function is significantly lower than the normal level.

[0032] Using qRT-PCR and Western blot method to detect Nrf2 downstream antioxidant gene HO-1, Catalase expression, results as shown in Figure 5 、 Figure 6 compared with the control group, 2M AngII group of mice myocardial cells HO-1, Catalase expression was not significantly changed, and 4M and 6M significantly reduced, which shows that in the long-term low-dose AngII stimulation caused by myocardial injury in mice in the late myocardial cell oxidative damage.

[0033] (4) Screening of differentially expressed LncRNA in AngII myocardial injury tissue According to the previous establishment of AngII (0.5 mg / kg) subcutaneous injection every other day for 2 months, 6 months of continuous observation to establish a mouse model of chronic myocardial injury caused by AngII. The heart tissue of this myocardial injury model and the normal age of the heart tissue were analyzed by high-throughput transcriptome sequencing to screen differentially expressed LncRNA ( Figure 7 a). According to the difference of fold (Fold Change≥2 or ≤0.5) and statistical significance of difference (p≤0.5), 40 differentially expressed LncRNA, of which 22 up-regulated LncRNA, 18 down-regulated LncRNA, of which 13 were known LncRNA ( Figure 7 b-d). We anchored the down-regulated XIST in the differentially expressed LncRNA.

[0034] (5) XIST deficiency can aggravate myocardial damage caused by AngII To verify whether XIST deficiency aggravates myocardial injury induced by AngII stimulation, the recombinant plasmid pPLK / GFP-XIST-shRNA was stably transfected into human cardiomyocytes AC16 ( Figure 8 ). Nrf2 mRNA and protein levels were significantly downregulated ( Figure 9 Compared with the control group, knockdown of XIST aggravated AngII-induced cardiomyocyte hypertrophy ( Figure 11 ), oxidative damage ( Figure 10 ), myocardial cell apoptosis ( Figure 12 ) and structural reconstruction ( Figure 13 These results indicate that XIST deficiency aggravates AngII-induced cardiomyocyte hypertrophy, oxidative damage, cardiomyocyte apoptosis, and structural remodeling.

[0035] (6) Upregulation of XIST inhibits AngII-induced oxidative damage in HL-1 mouse cardiomyocytes In mouse cardiomyocytes HL-1 transiently transfected with pcDNA-XIST, the expression of Nrf2 mRNA and protein increased significantly ( Figure 14-15 ), and inhibited AngII-induced oxidative damage in AC16 human cardiomyocytes ( Figure 16 ).

[0036] (7) Prediction of XIST, miR-20a-5p, and Nrf2 binding sites At the same time, the online bioinformatics database starBase (http: / / starbase.sysu.edu.cn / ) was used to predict the binding sites of miR-20a-5p with the Nrf2 3'UTR region and XIST. Figure 17 ).

[0037] In summary, Example 1, on the one hand, elucidates the molecular mechanism by which myocardial Nrf2 is regulated by XIST / miR-20a-5p: LncRNAs are involved in the development and progression of various heart diseases and can serve as biomarkers for heart disease, possessing dual value in clinical diagnosis and treatment. Building on previous findings identifying differentially expressed lncRNA XIST in Ang II-injured myocardial tissues and miR-20a-5p binding sites identified using bioinformatics websites, we confirmed the existence of the XIST / miR-20a-5p / Nrf2 regulatory axis in myocardial tissue through a series of techniques, including stable transfection of recombinant plasmid shXIST and transient transfection of pcDNA-XIST into human cardiomyocytes AC16. This discovery provides a novel perspective for the diagnosis, prevention, and treatment of Ang II-related myocardial oxidative damage and cardiomyopathy. Furthermore, we demonstrated the effectiveness of upregulating XIST in preventing Ang II-induced myocardial injury. Based on our findings that XIST expression is downregulated in Ang II-induced myocardial injury and our confirmation that it targets the antioxidant Nrf2, we transiently transfected human AC16 cardiomyocytes with pcDNA-XIST to investigate the effectiveness of upregulating XIST in preventing Ang II-induced myocardial injury by activating the endogenous antioxidant Nrf2. Solving this technical problem will provide a new molecular target for the prevention and treatment of oxidative stress-related cardiomyopathy.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of LncRNA XIST in the preparation of drugs to prevent myocardial injury caused by angiotensin II.

2. Application of LncRNA XIST and its bound miR-20a-5p targeting Nrf2 in the preparation of drugs to prevent myocardial damage caused by angiotensin II.

3. The use according to claim 2, characterized in that: The nucleotide sequence of miR-20a-5p is shown in SEQ ID NO: 1, and the nucleotide sequence of Nrf2 is shown in SEQ ID NO: 2.

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