KR20210108326A - Composition for preventing, improving or treating chronic kidney disease or renal fibrosis comprising Lin28a gene or protein - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating chronic kidney diseases or renal fibrosis, including Lin28a gene or protein. Particularly, the present invention relates to: a pharmaceutical composition for preventing or treating chronic kidney diseases or renal fibrosis, including Lin28a gene or protein, or a material enhancing mRNA of Lin28a gene or protein expression thereof; a health-aid food composition for preventing or treating chronic kidney diseases or renal fibrosis, including the same active ingredient; a composition for diagnosis of chronic kidney diseases or renal fibrosis, including a formulation for measuring the level of mRNA of Lin28a gene or protein expressed therefrom, and a kit using the same; a method for providing information for diagnosis of chronic kidney diseases or renal fibrosis, including a step of measuring the level of mRNA of Lin28a gene or protein expressed therefrom; and a method for screening a material for preventing or treating chronic kidney diseases or renal fibrosis.

Description

Composition for preventing, improving or treating chronic kidney disease or renal fibrosis disease comprising Lin28a gene or protein {Composition for preventing, improving or treating chronic kidney disease or renal fibrosis comprising Lin28a gene or protein}

The present invention relates to a composition for preventing, improving or treating chronic kidney disease or renal fibrosis disease, comprising the Lin28a gene or protein, and more particularly, the Lin28a gene or Lin28a protein; Or a pharmaceutical composition for the prevention or treatment of chronic kidney disease or renal fibrosis disease, comprising a substance that increases mRNA or protein expression of Lin28a gene, or a health function for preventing or improving chronic kidney disease or renal fibrosis disease comprising the same composition A food composition, a composition for diagnosing chronic kidney disease or renal fibrosis disease comprising an agent for measuring the level of mRNA of the Lin28a gene or a protein expressed therefrom, a kit, measuring the level of mRNA of the Lin28a gene or a protein expressed therefrom It provides a method for providing information for diagnosis of chronic kidney disease or renal fibrosis disease, and a method for screening a substance for preventing or treating chronic kidney disease or renal fibrosis disease, including.

Chronic kidney disease (CKD) is recognized as a serious disease worldwide, and in recent decades, the main causes of disease development and death have changed to overnutrition/deficiency and progressive chronic inflammatory disease. The increasing incidence of chronic kidney disease is one aspect of this transition.

Endstage renal disease (ESRD) is a condition in patients suffering from chronic kidney disease requiring renal replacement therapy such as dialysis or transplantation. There is no therapeutic agent showing an effect other than an angiotensin converting enzyme (ACE) inhibitor alone or in combination with a renin-angiotensin-aldosterone system (RAS) inhibitor.

However, even these treatments are effective in delaying the onset of the end-stage renal disease (ESRD) in some CKD patients or suppressing the decrease in glomerular filtration rate (GFR), but insignificant in most CKD patients. .

If various chronic kidney diseases persist, eventually end-stage renal failure is reached. Chronic renal failure is caused by chronic glomerulonephritis, diabetes, high blood pressure, urinary tract obstruction, renal tuberculosis, and hereditary renal disease. It shows the limit of not being able to.

Accordingly, renal replacement therapy is being used as a treatment for chronic renal failure. Renal replacement therapy is a treatment that can replace some of the functions of the kidneys, and there are three types of hemodialysis, peritoneal dialysis, and kidney transplantation. However, in the case of kidney replacement therapy, the treatment procedure is cumbersome, it has to be carried out for a lifetime, and it is difficult to seek help from a nephrologist due to the high risk of side effects. There are problems such as

Therefore, while overcoming the limitations of renin-angiotensin-aldosterone system (RAS) inhibitors, a genetic approach that can more fundamentally prevent or treat chronic kidney disease is needed.

On the other hand, Korean Patent Application Laid-Open No. 10-2014-0124930 discloses a composition for diagnosing renal fibrosis comprising an agent capable of detecting Runx2, and Korean Patent Publication No. 10-2017-0139747 discloses periostin pressure Although a composition for diagnosing or treating renal fibrosis including a tamer is disclosed, the role of Lin28a in relation to chronic kidney disease and renal fibrosis disease has not been revealed.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of chronic kidney disease or renal fibrosis disease using the Lin28a gene or protein.

Another object of the present invention is to provide a health functional food composition for preventing or improving chronic kidney disease or renal fibrosis disease using the Lin28a gene or protein.

Another object of the present invention is to provide a composition and/or kit for diagnosing chronic kidney disease or renal fibrosis disease, comprising an agent for measuring the level of mRNA of Lin28a gene or protein expressed therefrom.

Another object of the present invention is to provide an information providing method for diagnosing chronic kidney disease or renal fibrosis disease, comprising measuring the level of mRNA of the Lin28a gene or a protein expressed therefrom.

Another object of the present invention is to provide a method for screening a substance for preventing or treating chronic kidney disease or renal fibrosis disease, comprising measuring the level of the mRNA of the Lin28a gene or a protein expressed therefrom.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a Lin28a gene or Lin28a protein; Or it provides a pharmaceutical composition for the prevention or treatment of chronic kidney disease or renal fibrosis disease, comprising a substance that increases the mRNA or protein expression of the Lin28a gene.

According to a preferred embodiment of the present invention, the renal fibrosis disease may be tubular interstitial fibrosis.

The present invention also provides Lin28a gene or Lin28a protein; Or it provides a health functional food composition for the prevention or improvement of chronic kidney disease or renal fibrosis disease, comprising a substance that increases the mRNA or protein expression thereof of the Lin28a gene.

In addition, the present invention provides a composition for diagnosing chronic kidney disease or renal fibrosis disease, comprising an agent for measuring the level of mRNA of Lin28a gene or protein expressed therefrom.

According to a preferred embodiment of the present invention, the agent for measuring the mRNA level of the Lin28a gene may include a primer or probe specifically binding to the gene, and the agent for measuring the level of the Lin28a protein is the protein may contain an antibody specific for

The present invention also provides a kit for diagnosing chronic kidney disease or renal fibrosis disease, comprising the composition for diagnosing the aforementioned chronic kidney disease or renal fibrosis disease.

According to a preferred embodiment of the present invention, the kit may be an RT-PCR kit, a DNA chip kit or a protein chip kit.

Further, the present invention provides an information providing method for diagnosing chronic kidney disease or renal fibrosis disease, comprising the steps of:

(a) measuring the mRNA level of the Lin28a gene or protein expressed therefrom from a biological sample isolated from the subject; and

(b) comparing the measured level of mRNA or the level of a protein expressed therefrom with a level measured from a sample of a normal control.

According to a preferred embodiment of the present invention, the method (c) is diagnosed as chronic kidney disease or renal fibrosis disease when the mRNA level measured in the biological sample isolated from the subject or the level of the protein expressed therefrom is lower than that of the normal control. It may further include the step of

According to another preferred embodiment of the present invention, the mRNA level of the gene is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time reverse transcriptase polymerase reaction (real time) It can be measured using any one selected from the group consisting of quantitative RT-PCR, RNase protection method, Northern blotting, and DNA chip technology assay.

According to another preferred embodiment of the present invention, the level of the protein is western blotting (western blotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmuno-diffusion (radial immunodiffusion), oh Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence , immunochromatography, FACS analysis (fluorescenceactivated cell sorter analysis), and protein chip technology assay.

The present invention also provides a method for screening a substance for preventing or treating chronic kidney disease or renal fibrosis disease, comprising the following steps:

(a) treating a candidate material in a biological sample isolated from a patient with chronic kidney disease or renal fibrosis disease;

(b) comparing the mRNA level of Lin28a in the sample of the patient treated with the candidate or the level of the protein expressed therefrom with the level measured from the sample of a normal control; and

(c) when the level of mRNA or protein expressed therefrom in step (b) is increased than before treatment with the candidate substance, determining the candidate substance as a substance for preventing or treating chronic kidney disease or renal fibrosis disease.

The composition for preventing, improving, or treating chronic kidney disease or renal fibrosis disease using the Lin28a gene or Lin28a protein according to the present invention reduces the expression of p-smad3 in the fibrosis induction process by TGF-β, thereby reducing the cell epithelial-mesenchymal By suppressing the expression of proteins (vimentin, α-SMA) involved in the transition to cells, and also suppressing the expression of fibronectin and collagen type 1, which are representative genes for inducing renal fibrosis, the limitations of existing renal replacement therapy are overcome. It can be used as a more fundamental genetic treatment that can be treated.

1A is a photograph of observing cells after inducing fibrosis by TGF-β in renal proximal tubular epithelial cells overexpressing Lin28a protein.
FIG. 1b shows the epithelial-mesenchymal cell migration and fibrosis-induced fibrosis in renal proximal tubular epithelial cells overexpressing Lin28a protein after induction of fibrosis by TGF-β. Vimentin, α-SMA, and collagen It is the result of confirming the protein expression change of type 1 (Type 1 collagen) and fibronectin (Fibronectin) by Western blot.
Figure 2a shows the epithelial cells in the renal proximal tubular epithelial cells induced by fibrosis by TGF-β collagen type 1 (Type 1 collagen), vimentin, fibronectin and This is the result of confirming the protein expression changes of α-SMA and the target protein, Lin28a, by Western blot.
2B is a quantitative graph showing the results of FIG. 2A.
Figure 3a confirms the mechanism of inhibition of fibrosis induction according to Lin28a protein overexpression. In the process of inducing fibrosis by TGF-β, p-smad3 expression and p-ERK, p-JNK, and p-p38 expression of the MAPK pathway were confirmed by Western blot. show the results.
3B is a quantitative graph showing the result of FIG. 3A.
Figure 4a shows the Lin28a protein and epithelial-mesenchymal cells after fibrosis induction by unilateral ureteral ligation (UUO), and the representative genes vimentin, α-SMA, collagen type 1 (Type 1 collagen), fibronectin (Fibronectin) and the target protein Lin28a expression changes were confirmed by Western blot.
4B is a quantitative graph showing the results of FIG. 4A.
5 is hematoxylin & eosin staining, Mason's trichrome staining, Sirius red staining for histological examination of kidney tissue extracted from a unilateral ureteral ligation (UUO) mouse model. staining) and Lin28a immunohistochemical staining are shown.

As described above, a more fundamental genetic approach for the prevention or treatment of chronic kidney disease is needed due to the absence of an effective therapeutic agent for chronic kidney disease, including chronic renal failure, and the limitations of kidney replacement therapy and kidney transplantation.

Accordingly, the present inventors have made diligent efforts to develop gene therapy for chronic kidney disease. As a result, the use of Lin28a protein in the renal tubule inhibits the transition of renal tubule cells to epithelial-mesenchymal cells (EMT: epithelial-to-mesenchymal transition). And, by suppressing the expression of proteins involved in fibrosis of the interstitial tubules to confirm that it exhibits a fundamental therapeutic effect on chronic kidney diseases including renal fibrosis disease, the present invention has been completed.

A first aspect of the present invention is a Lin28a gene or Lin28a protein; Or a pharmaceutical composition for preventing or treating chronic kidney disease or renal fibrosis disease comprising a substance that increases mRNA or protein expression of the Lin28a gene and/or Lin28a gene or Lin28a protein; Or it relates to a health functional food composition for preventing or improving chronic kidney disease or renal fibrosis disease, comprising a substance that increases mRNA or protein expression of Lin28a gene.

Lin-28 homolog A (Lin-28 homolog A, LIN28A) is a protein encoded by the LIN28 gene in humans. LIN28 encodes an RNA-binding protein that binds to insulin-like growth factor 2 mRNA and enhances its translation. Lin28 blocks the production of mature let-7 microRNA by binding to let-7 pre-microRNA in mouse embryonic stem cells. In pluripotent embryonal carcinoma cells, LIN28 is localized to ribosomes, P-bodies and stress granules. LIN28 is highly expressed in human embryonic stem cells, and it is known that it can improve the formation efficiency of induced pluripotent stem cells (iPS cells) from human fibroblasts.

In the present invention, the Lin28a gene may be composed of the nucleotide sequence represented by SEQ ID NO: 1. In addition, the protein encoded by the Lin28a gene includes a Lin28a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the proteins. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution or deletion of amino acids. is 95, 96, 97, 98, or 99% or more of sequence homology, and refers to a protein that exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2.

In the present invention, the term "chronic kidney disease (CKD)" refers to a state in which proteinuria is continuously coming out, there is evidence of kidney damage such as hematuria, or kidney function is continuously decreased for 3 months or more. Chronic renal failure (CRF) is called chronic renal failure when renal function has fallen below 50% for 3 months or more, and has a narrower meaning than chronic kidney disease. Subjective symptoms include polyuria, swelling around the eyes and lower extremities, drowsiness, easily fatigued, no appetite, itchy skin, bad breath (a symptom of ammonia odor), etc., accompanied by anemia and high blood pressure. In children, development is delayed and the complexion may be accompanied by symptoms.

In the composition of the present invention, the chronic kidney disease may be selected from the group consisting of diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, interstitial nephritis, acute renal failure and chronic renal failure.

In the present invention, the term “renal fibrosis disease” refers to a symptom in which kidney tissue and/or blood vessels are hardened. The renal fibrosis disease may be tubular interstitial fibrosis. In the present invention, the term “tubular interstitial fibrosis” refers to tubular interstitial fibrosis between tubules and tubules, ie tubulointerstitial fibrosis. Most chronic kidney disease progresses to tubular interstitial fibrosis.

In the present invention, the term "prevention" refers to any action that suppresses or delays the onset of symptoms caused by chronic kidney disease and/or renal fibrosis disease by administration of the composition, and "treatment" means chronic kidney disease by administration of the composition. It refers to any action in which symptoms caused by kidney disease and/or renal fibrosis disease are improved or changed to a beneficial effect.

In the pharmaceutical composition of the present invention, a substance that increases Lin28a gene or Lin28a protein and mRNA of Lin28a gene or protein expression thereof is included in a "therapeutically effective amount" required to alleviate one or more symptoms of a disease or disorder to be treated, and , which refers to a sufficient amount of the pharmaceutical composition to provide the desired effect. The term "therapeutically effective amount" refers to an amount of a substance that increases the expression of the Lin28a gene or Lin28a protein or mRNA of the Lin28a gene or protein thereof, sufficient to provide a particular effect when administered to a typical subject. As used herein, an effective amount is sufficient to delay the development of symptoms of a disease, alter the development of symptoms of a disease (eg, but not limited to, slow the progression of symptoms of a disease), or reverse the symptoms of a disease. amount will be included. Thus, while it is impossible to specify an exact "effective amount", for any particular case, an appropriate "effective amount" can be readily determined by one of ordinary skill in the art through routine experimentation.

The pharmaceutical compositions of the present invention may be administered to a patient in need thereof by any suitable route that results in effective treatment in the subject. The term “administration” refers to introduction into a subject by a method or route that results in at least partial localization at the desired site such that the desired effect(s) occur.

In some embodiments, the pharmaceutical composition of the present invention is administered to a subject having chronic kidney disease by any mode of administration, which may include injection, infusion, instillation, and inhalation administration. If the Lin28a gene, the Lin28a protein or the mRNA of the Lin28a gene or a substance that increases the protein expression thereof can be protected from inactivation in the digestive tract, an oral dosage form may be considered. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, spinal canal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, capsular subarachnoid, intrathecal, intracerebro spinal, and intrasternal injections and infusions. In some embodiments, the pharmaceutical composition of the present invention is administered by intravenous injection or infusion.

In the present invention, the term "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection. As used herein, the phrases "systemic administration" and "peripheral intravenous administration" refer to administration that is not directed to a target site, tissue, or organ, such as a tumor site, such that it enters the circulatory system of a subject and is provided for metabolism and other processes. do.

For clinical use, administration of the Lin28a gene, Lin28a protein or mRNA of the Lin28a gene or a substance that increases protein expression thereof is administered by parenteral administration, for example intravenously; mucous membranes, eg, intranasal; formulation into pharmaceutical compositions or into pharmaceutical formulations for ocular, or other modes of administration. In some embodiments, any pharmaceutically acceptable carrier compound, substance, or composition that results in effective treatment in a subject may be administered. Accordingly, the pharmaceutical formulation may contain a substance that increases the Lin28a gene, Lin28a protein or mRNA of Lin28a gene or protein expression thereof in combination with one or more pharmaceutically acceptable ingredients.

In the present invention, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, in humans and animals without excessive toxicity, irritation, allergic reaction, or other problems or complications, conforming to an appropriate benefit/risk ratio. refers to such compounds, substances, compositions, and/or formulations suitable for use in contact with tissue. In the present invention, the term "pharmaceutically acceptable carrier" refers to Lin28a gene, Lin28a protein or mRNA of Lin28a gene, or a pharmaceutically acceptable carrier related to maintaining stability, solubility, or activity of a substance that increases protein expression thereof. Possible substances, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids (eg, lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulation means material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can serve as pharmaceutically-acceptable carriers include:

(1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients such as cocoa butter and suppository waxes; (8) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols such as propylene glycol; (10) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters such as ethyl oleate and ethyl laurate; (12) agar; (13) buffers such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19) pH buffered solutions; (20) polyesters, polycarbonates and/or polyanhydrides; (21) bulking agents such as polypeptides and amino acids; (22) serum components such as serum albumin, HDL and LDL; (23) C2-C12 alcohols such as ethanol; and (24) other non-toxic commercial substances used in pharmaceutical formulations. Release agents, coating agents, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

The pharmaceutical compositions of the present invention may be specially formulated for administration to a subject in solid, liquid or gel form, including those applied for: (1), for example, a sterile solution or suspension, or sustained release parenteral administration, eg by subcutaneous, intramuscular, intravenous or epidural injection, such as in dosage forms; (2) topical applications such as, for example, creams, ointments, or controlled release patches or sprays applied to the skin; (3) vaginal or rectal, for example as a pessary, cream or foam; (4) eyeball; (5) transdermal; (6) mucosal ducts; or (7) nasal.

Additional embodiments and modes of administration of the pharmaceutical compositions of the present invention are described below.

parenteral formulation . Parenteral formulations of the pharmaceutical compositions of the present invention may be administered to subjects with chronic renal impairment by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. can

Because administration of parenteral formulations must bypass the patient's natural defenses against contaminants, parenteral formulations are preferably sterile or can be sterilized prior to administration to a patient. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry products that can be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions for injection, controlled-release parenteral formulations, and emulsions. do.

Suitable vehicles that can be used to provide parenteral formulations are well known to those skilled in the art. Examples include, but are not limited to: sterile water; water for injection USP; saline; glucose solution; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

In some embodiments, a composition comprising an effective amount of a Lin28a gene, Lin28a protein, or an agent that increases mRNA of Lin28a gene or protein expression thereof is administered in separate formulations, such as, but not limited to, purification (without limitation). divided or coated tablets), pills, dragees, capsules, chewable tablets, powder packets, vials, troches, wafers, aerosol sprays, or liquids such as, but not limited to, syrups, elixirs, solutions in aqueous liquids or as a suspension, non-aqueous liquid, oil-in-water emulsion, or water-in-oil emulsion, formulated for oral administration. Such compositions contain pre-determined amounts of pharmaceutically acceptable salts of the disclosed compositions and may be prepared by pharmaceutical methods well known to those skilled in the art. In general, see Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990)].

Due to their ease of administration, tablets and capsules represent the most advantageous solid oral dosage unit forms, in which case solid pharmaceutical excipients are employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such formulations may be prepared by any pharmaceutical method. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers, finely divided solid carriers, or both, and then, if necessary, forming the product in the desired shape. In some embodiments, the oral formulation is not used for antibiotics.

A typical oral dosage form of a composition comprising an effective amount of an effective amount of a Lin28a gene, Lin28a protein or an mRNA of a Lin28a gene or a protein expression thereof is a pharmaceutical formulation of a Lin28a gene, Lin28a protein or Lin28a gene mRNA or a substance that increases the protein expression thereof. It is prepared by intimately mixing an acceptable salt with one or more excipients according to conventional pharmaceutical formulation techniques. Excipients can take a wide variety of forms depending on the form of composition desired for administration. For example, excipients suitable for use in oral liquid or aerosol formulations include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Excipients suitable for use in solid oral dosage forms (eg, powders, tablets, capsules, and dragees) include, but are not limited to, starch, sugar, microcrystalline cellulose, kaolin, diluents, granulating agents, lubricants, binders, and Contains disintegrants.

Binders suitable for use in the pharmaceutical formulations described herein include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (eg ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose , (eg, Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical formulations described herein include, but are not limited to, talc, calcium carbonate (eg, granules or powder), microcrystalline cellulose, powdered cellulose, dextrate, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical compositions described herein typically represents from about 50 to about 99 weight percent of the pharmaceutical composition.

Disintegrants are used in the oral pharmaceutical formulations described herein to provide tablets that disintegrate when exposed to an aqueous environment. A sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) is a solid oral dosage of the Lin28a gene, Lin28a protein, or mRNA of Lin28a gene described herein or a substance that increases protein expression thereof. It should be used to form the formulation. The amount of disintegrant used will vary based on the type of formulation and can be readily distinguished by one skilled in the art. Disintegrants that may be used to form the oral pharmaceutical formulation include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrylline potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used to form oral pharmaceutical formulations of substances that increase the expression of the Lin28a gene, Lin28a protein or Lin28a gene or mRNA of the Lin28a gene described herein include, but are not limited to, calcium stearate, magnesium stearate, minerals Oils, light mineral oils, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g. peanut oil, cottonseed oil, sunflower oil) weaning, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. Additional lubricants include, for example, syloid silica gel (AEROSIL 200, manufactured by WR Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (Degussa Co. of Piano, Tex). ), CAB-O-SIL (pyrogenic silicon dioxide sold by Cabot Co. of Boston, Mass.), and mixtures thereof.

In another embodiment, lactose-free pharmaceutical formulations and compositions are provided, which compositions preferably contain, if any, small amounts of lactose or other mono- or di-saccharides. As used herein, the term “lactose-free” means that the amount of lactose present is insufficient to substantially increase the rate of degradation of the active ingredient. The lactose-free compositions of the present disclosure may include excipients well known in the art and are listed in USP (XXI)/NF (XVI), incorporated herein by reference.

Oral formulations of substances that increase the Lin28a gene, Lin28a protein, or mRNA of Lin28a gene or protein expression thereof, are, in some embodiments, the Lin28a described herein as an active ingredient, because water can promote the degradation of this compound. It further includes anhydrous pharmaceutical compositions and formulations comprising a gene, Lin28a protein or mRNA of Lin28a gene or a substance that increases the protein expression thereof. For example, the addition of water (eg, 5%) is widely accepted in the pharmaceutical art as a means of simulating long-term storage to determine characteristics such as shelf life or stability of a formulation over time. See, eg, Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and formulations described herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and formulations comprising one or more active ingredients comprising lactose and a primary or secondary amine are preferably free of moisture and/or moisture when substantial contact with moisture and/or moisture is expected during manufacture, packaging, and/or storage. Mercury. Anhydrous compositions are preferably packaged using materials known to prevent exposure to water so that they can be included in a suitable formulary kit. Examples of suitable packaging include, but are not limited to, hermetically sealed foil, plastic, unit dose containers (eg, glass bottles) with or without desiccant, blister packs, and strip packs. do.

aerosol formulation . Lin28a gene, Lin28a protein or a substance increasing mRNA of Lin28a gene or protein expression thereof is prepared in a pressurized aerosol container with a suitable propellant, for example a hydrocarbon propellant such as propane, butane, or isobutane with a conventional adjuvant. can be packed in A substance that increases the Lin28a gene, Lin28a protein or mRNA of Lin28a gene or protein expression thereof may also be administered in a non-pressurized form such as a nebulizer or nebulizer. Lin28a gene, Lin28a protein or a substance that increases mRNA of Lin28a gene or protein expression thereof can also be administered directly into the respiratory tract in the form of a dry powder, for example, by use of an inhaler.

Suitable powder compositions include, for example, a powdered agent of a substance that increases the expression of the Lin28a gene, Lin28a protein or Lin28a gene or its protein, which is thoroughly mixed with lactose, or other inert powders acceptable for intrabronchial administration. include The powder composition may be administered via an aerosol container or may be wrapped in a crushable capsule that may be inserted by a subject with a device that punctures the capsule to inflate the powder in a continuous flow suitable for inhalation. The composition may include a propellant, a surfactant, and a co-solvent, and may be filled into a conventional aerosol container closed by a suitable metering valve.

Aerosols for delivery to the respiratory tract are known in the art. See, eg, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemic delivery of peptides and proteins as well (Patton and Platz, Advanced Drug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J. Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, D. A., Drug Delivery, 2: 101-20 1995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), the entire contents of which are incorporated herein by reference in their entirety.

Controlled and delayed release formulations . In some embodiments of aspects described herein, the Lin28a gene, Lin28a protein, or a substance that increases mRNA of Lin28a gene or protein expression thereof can be administered to the patient by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled-release formulation in medical treatment is characterized by a minimum of drug substance used to cure or control a disease in a minimum amount of time. Advantages of controlled-release formulations include: 1) prolonged activity of the drug; 2) reduced dosing frequency; 3) increased patient compliance; 4) less total drug usage; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) potentiation or reduction of loss of drug activity; and 10) improvement in the rate of control of the disease or disorder. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to modulate the onset of action, duration of action, plasma levels at a therapeutic period, and peak blood levels of a substance that increases the Lin28a gene, Lin28a protein or mRNA of the Lin28a gene or protein expression thereof. .

In particular, controlled- or extended-release formulations or formulations are designed to achieve the maximal effect of a substance that increases the Lin28a gene, Lin28a protein or mRNA of Lin28a gene or its protein expression, while underdosing the drug ( That is, it can be used to ensure the minimization of potential side effects and safety concerns that can arise from both (below minimum therapeutic levels) as well as exceeding toxic levels for the drug.

Such formulations may be formulated with, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or combinations thereof, to provide slow or controlled-release of one or more active ingredients. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus affect controlled delivery of drugs. Examples of specific anion exchangers include, but are not limited to, DUOLITE A568 and DUOLITE AP143 (Rohm & Haas, Spring House, Pa. USA).

In some embodiments of the methods described herein, the agent that increases the Lin28a gene, Lin28a protein or mRNA of Lin28a gene or protein expression thereof for use in the methods described herein is administered by sustained release or in pulses. administered to the subject. Pulse therapy does not take the form of discontinuous administration of the same amount of the composition over time, but includes the administration of the same dose of the composition at a reduced frequency or in reduced dose administration. Sustained release or pulse administration is particularly preferred, for example, when the disorder continues to develop in a subject with chronic kidney disease. Each pulse dose can be reduced and the total amount of a substance that increases the expression of the Lin28a gene, Lin28a protein or Lin28a gene or mRNA of the Lin28a gene described herein administered over the course of treatment to the subject or patient is is minimized

If necessary, the interval between pulses can be determined by a person skilled in the art. Often, the interpulse interval can be calculated by administering different doses of the composition when the composition or active ingredient of the composition can no longer be detected in the subject prior to delivery of the next pulse. The interval can also be calculated from the in vivo half-life of the composition. The interval can be calculated to be greater than the half-life in vivo, or 2, 3, 4, 5 and even 10 times greater than the half-life of the composition.

In some embodiments, a sustained release agent can be prepared comprising a substance that increases the expression of the Lin28a gene, the Lin28a protein, or the mRNA of the Lin28a gene or protein thereof. Suitable examples of sustained release agents include semipermeable matrices of solid hydrophobic polymers containing the inhibitor, which matrices are in the form of molded articles, such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactide, L-glutamic acid and y ethyl-L- copolymers of glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-) -3-hydroxybutyric acid.

The formulation comprising the Lin28a gene, the Lin28a protein or the mRNA of the Lin28a gene or a substance that increases the protein expression thereof used for in vivo administration is preferably sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes, and other methods known to those skilled in the art.

In the present invention, the term "health functional food" includes both the meanings of "functional food" and "health food".

In the present invention, the term "functional food (functional food)" is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it has medical and medical effects processed to efficiently exhibit bioregulatory functions. It means high food.

As used herein, the term "health food" refers to food having an active health maintenance or promotion effect compared to general food, and health supplement food refers to food for the purpose of health supplementation. In some cases, the terms functional food, health food, and dietary supplement are preferred. The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, and the like.

As a specific example of such a functional food, by using the composition, it is possible to manufacture a processed food with good storage properties while at the same time being transformed by taking advantage of the characteristics of agricultural products, livestock products or aquatic products.

The health functional food composition of the present invention may also be prepared in the form of nutritional supplements, food additives and feed, etc., and is intended to be eaten by animals, including humans or livestock.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. Common foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, canned fruit, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, sausages) corn beef, etc.), breads and noodles (eg udon noodles, soba noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine , vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) can be prepared by adding a substance that increases Lin28a gene, Lin28a protein, or Lin28a gene mRNA or protein expression thereof.

In addition, the nutritional supplement is not limited thereto, but it can be prepared by adding a substance that increases Lin28a gene, Lin28a protein or Lin28a gene mRNA or protein expression thereof to capsules, tablets, pills, etc.

In addition, the health functional food is not limited thereto, but for example, the Lin28a gene, the Lin28a protein or the Lin28a gene mRNA or a substance that increases the protein expression thereof is prepared in the form of tea, juice, and drink for drinking (health drink) It can be ingested by liquefying, granulating, encapsulating and powdering. In addition, in order to use the Lin28a gene, Lin28a protein, or the mRNA of the Lin28a gene, or a substance that increases the protein expression thereof, in the form of a food additive, it may be prepared in the form of a powder or a concentrate. In addition, the Lin28a gene, Lin28a protein or Lin28a gene mRNA or a substance that increases the protein expression thereof and a known active ingredient known to be effective in preventing or improving chronic kidney disease or renal fibrosis disease in the form of a composition can be manufactured.

When the food composition of the present invention is used as a health drink composition, the health drink composition may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatin, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

Lin28a gene, Lin28a protein or Lin28a gene mRNA or a substance that increases the protein expression thereof may be contained as an active ingredient in a food composition for the prevention or improvement of chronic kidney disease or renal fibrosis disease, the amount of which is the prevention or improvement effect. An effective amount to obtain, for example, preferably 0.01 to 100% by weight based on the total weight of the composition, but is not particularly limited thereto. The food composition of the present invention is mixed with other active ingredients known to be effective in preventing or improving chronic kidney disease or renal fibrosis disease together with a substance that increases the Lin28a gene, Lin28a protein or Lin28a gene mRNA or protein expression thereof. can be manufactured.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives , glycerin, alcohol, or a carbonation agent. In addition, the health food of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The second aspect of the present invention, based on the expression profile or sequence information of the Lin28a gene or a protein expressed therefrom as a biomarker for the diagnosis of chronic kidney disease or renal fibrosis disease, the subject is chronic kidney disease or renal fibrosis disease Compositions, kits, assays, methods, and systems for determining whether a person has a predisposition.

Lin28a is useful as a biomarker to identify subjects at risk of developing chronic kidney disease or renal fibrosis disease or to monitor the effectiveness of treatment on the progression of chronic kidney disease or renal fibrosis disease.

As used herein, a “biomarker” is an organic substance that is differentially present in a sample taken from a subject of one phenotypic state (eg, having a disease) compared to another phenotypic state (eg, not having the disease). refers to biomolecules. A biomarker exists differently between different phenotypic states if the mean or median expression level of the biomarker in different groups is calculated to be statistically significant. General tests for statistical significance include, inter alia, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds. ratio) is included.

Accordingly, the present invention provides a composition for diagnosing chronic kidney disease or renal fibrosis disease, a kit, and/or a method for providing information for diagnosis, comprising an agent for measuring the level of mRNA of the Lin28a gene or a protein expressed therefrom.

Specifically, the present invention relates to a composition for diagnosing chronic kidney disease or renal fibrosis disease, comprising an agent for measuring the level of mRNA of Lin28a gene or a protein expressed therefrom, and/or a kit for diagnosing chronic kidney disease or renal fibrosis disease comprising the same provides

In the composition and kit for diagnosing chronic kidney disease or renal fibrosis disease of the present invention, the agent for measuring the mRNA expression level of the gene includes a primer pair, a probe or an antisense nucleotide that specifically binds to the gene, and the protein The agent for measuring the expression level may include an antibody specific for the protein of the biomarker gene.

In the present invention, the term "agent for measuring the mRNA level of a gene" refers to an agent used in a method for measuring the level of mRNA transcribed from the target gene in order to check whether the target gene contained in the sample is expressed. However, it is not particularly limited thereto, but preferably RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), It may be a probe or primer capable of specifically binding to a target gene used in methods such as Northern blotting and DNA chip analysis.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template and is a starting point for template strand copying. It refers to a short nucleic acid sequence that functions as Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

For the purpose of the present invention, the primer is used as a means for detecting Lin28a mRNA, preferably after synthesizing cDNA from mRNA obtained from a patient's sample, amplifying and detecting the Lin28a gene included in the cDNA. A primer may be used. In this case, the polynucleotide sequence of the primer is not particularly limited as long as it can amplify the Lin28a gene included in the cDNA.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to a gene or mRNA, an oligonucleotide probe, It may be manufactured in the form of a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

As used herein, the term "agent for measuring the level of a protein" refers to an agent used in a method for measuring the level of a target protein included in a sample, but is not particularly limited thereto, but preferably by western blotting. , ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), may be an antibody used in methods such as FACS and protein chip assay (protein chip assay).

As used herein, the term "antibody" refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody is obtained by cloning each gene into an expression vector according to a conventional method. A protein encoded by a marker gene can be obtained, and it can be prepared from the obtained protein by a conventional method. For the purpose of the present invention, the antibody is used as a means for detecting Lin28a protein contained in a patient's sample, and preferably a polyclonal antibody, monoclonal antibody or antigen binding agent capable of specifically binding to Lin28a protein. Part of the antibody of the present invention may include not only all immunoglobulin antibodies, but also special antibodies such as humanized antibodies. In addition, the antibody includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2 and Fv.

The kit of the present invention may be an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit or a protein chip kit.

The kit of the present invention measures the mRNA level of the gene encoding Lin28a or the level of the protein expressed therefrom from a sample of a patient suspected of chronic kidney disease or renal fibrosis disease to diagnose the onset of chronic kidney disease or renal fibrosis disease. It may be used, but is not particularly limited thereto, but one or more other component compositions, solutions or Devices may be included.

According to an embodiment of the present invention, the kit for measuring the mRNA expression level of the Lin28a gene of the present invention may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit contains, in addition to each pair of primers specific for the gene, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. enzymes such as DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. In addition, a pair of primers specific to a gene used as a quantitative control may be included.

In another embodiment of the present invention, the kit of the present invention may include essential elements necessary for performing DNA chip analysis. The DNA chip analysis kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof.

In another embodiment of the present invention, the kit of the present invention may be a protein chip analysis kit for measuring the level of a protein expressed from the Lin28a gene, the kit is not particularly limited thereto, For detection, a substrate, an appropriate buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like may be included. The substrate is not particularly limited thereto, but a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a glass slide glass, etc. may be used, and the chromogenic enzyme is not particularly limited thereto. However, peroxidase or alkaline phosphatase may be used, and the fluorescent material may be FITC, RITC, etc., but is not particularly limited thereto, and the color substrate solution is not particularly limited thereto, but ABTS (2 ,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine).

The present invention also provides an information providing method for diagnosing chronic kidney disease or renal fibrosis disease, comprising the steps of:

(a) measuring the mRNA level of the Lin28a gene or protein expressed therefrom from a biological sample isolated from the subject; and

(b) comparing the measured level of mRNA or the level of a protein expressed therefrom with a level measured from a sample of a normal control.

In the present invention, the terms "subject" or "individual" are used interchangeably and refer to an animal, such as a human. The terms “non-human animal” and “non-human mammal” are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. do. The term “subject” also includes any vertebrate including, but not limited to, mammals, reptiles, amphibians and fish. Advantageously, however, the subject is a mammal such as a human, or other mammal such as a domesticated mammal, eg, a dog, cat, horse, or the like.

In the present invention, the term "sample" refers to any substance containing nucleic acids, DNA or RNA, or amino acids. In general, such material may be in the form of a blood sample, fecal sample, tissue sample, cell, bacterial, microtissue section, or buccal swab. The sample may be prepared, for example, the sample may be fresh, fixed, frozen, or embedded in paraffin. As used herein, the term “biological assay” refers to a cell or population of cells or an amount of tissue or body fluid from a subject. Most often, a sample is isolated from a subject, but the term “biological sample” may also refer to a cell or tissue that is analyzed in vivo, ie, has not been isolated from the subject. Often a "biological sample" may contain cells isolated from an animal, but the term is also a non-cellular fraction of blood, saliva, or urine, which may be used to determine the level of expression of or non-cellular biological material, such as a gene. can refer to Biological samples include, but are not limited to, tissue biopsies, scrapes, whole blood, plasma, serum, urine, saliva, cell culture, or cerebrospinal fluid. Biological samples also include tissue biopsies, cell cultures. Biological samples or tissue samples include, but are not limited to, for example, urine, blood, plasma, serum, kidney biopsy, feces, sputum, spinal fluid, pleural fluid, ductal fluid, lymph, external sections of skin, respiratory, intestinal and genital tract; may refer to a sample of tissue or body fluid isolated from a subject, including samples of tears, saliva, milk, cells (including but not limited to blood cells), tumors, organs, and/or ex vivo cell culture components. . In some embodiments, when a urine sample is obtained, the urine sample is centrifuged to pellet any kidney cells, upon which the assays and methods disclosed herein can be performed. In some embodiments, the sample is from a kidney biopsy, such as a needle biopsy of a kidney or a portion thereof, such as a podocyte sample. Additionally, a fine needle aspirate sample is used. In some embodiments, a biological sample may be prepared, eg, the biological sample may be fresh, fixed, frozen, or embedded in paraffin. The sample may be obtained by isolating a sample of cells from a subject, but also by using previously isolated cells (eg, isolated by another person), or by performing a method disclosed herein in vivo. can be achieved.

In the present invention, the term "expression" may interchangeably refer to expression of a polypeptide or protein or expression of a polynucleotide or expression of a gene. Expression also refers to expression of pre- and post-translationally modified proteins, and expression of precursor-mRNA (pre-mRNA) molecules, optionally spliced mature mRNA molecules. Expression of polynucleotides can be determined, for example, by measuring the production of RNA transcription molecules, eg, messenger RNA (mRNA) transcription levels. Expression of a protein or polypeptide can be determined, for example, by an immunoassay using antibody(s) that bind to the polypeptide. The term "encode" as applied to polynucleotides means that when manipulated in its native state or by methods well known to those skilled in the art, it will produce RNA that can be transcribed into an amino acid sequence that can be translated into a polypeptide and/or fragment thereof. Refers to a polynucleotide that is said to "encode (encode)" a polypeptide or protein when possible. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced therefrom. The term “endogenously expressed” or “endogenous expression” refers to the expression of a gene product at normal levels and under normal control for this cell type.

In one embodiment of the present invention, after fibrosis induction in vitro, Lin28a protein and epithelial-mesenchymal cell migration and expression change of representative fibrosis-inducing factors were confirmed. As a result, as shown in FIGS. 2a and 2b Likewise, it was confirmed that the expression of the corresponding markers was changed to the EMT expression pattern by TGF-β treatment, and the expression of the target protein, Lin28a, was decreased.

In another embodiment of the present invention, after induction of fibrosis by unilateral ureteral ligation (UUO), it was confirmed that the Lin28a protein and the transition to epithelial-mesenchymal cells and the expression change of fibrosis-inducing representative factors were changed, and as a result, as shown in FIGS. 4a and 4b , Similarly, in the unilateral ureteral ligation (UUO) group, the expression of Vimentin and α-SMA was significantly increased compared to the normal control group (CON), and the EMT expression pattern was changed, and the expression of the target protein, Lin28a, was also significantly increased. was found to decrease. In addition, as a result of histological examination of the kidneys, it was confirmed that collagen accumulation was increased by the UUO procedure compared to the normal control (CON) as shown in FIG. 5, and as a result of immunostaining, the expression of Lin28a This renal fibrosis was reduced in the advanced tissue.

Therefore, the information providing method of the present invention (c) adding the step of diagnosing chronic kidney disease or renal fibrosis disease when the mRNA level measured in the biological sample isolated from the subject or the level of the protein expressed therefrom is lower than that of the normal control. can be included as

In the information providing method of the present invention, the mRNA level of the gene is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT -PCR), RNase protection method (RNase protection method), Northern blotting (Northern blotting) and DNA chip analysis method (DNA chip technology assay) can be measured using any one selected from the group consisting of.

In the information providing method of the present invention, the level of the protein is western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoassay, radial immunodiffusion, oukteroni Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromato The measurement may be performed using any one selected from the group consisting of immunochromatography, fluorescenceactivated cell sorter analysis, and protein chip technology assay.

A third aspect of the present invention relates to a method for screening a substance for preventing or treating chronic kidney disease or renal fibrosis disease, comprising the following steps:

(a) treating a candidate material in a biological sample isolated from a patient with chronic kidney disease or renal fibrosis disease;

(b) comparing the mRNA level of Lin28a in the sample of the patient treated with the candidate or the level of the protein expressed therefrom with the level measured from the sample of a normal control; and

(c) when the level of mRNA or protein expressed therefrom in step (b) is increased than before treatment with the candidate substance, determining the candidate substance as a substance for preventing or treating chronic kidney disease or renal fibrosis disease.

In the method of screening a substance for preventing or treating chronic kidney disease or renal fibrosis disease of the present invention, the description of the "biological sample" is the same as that described above, and thus the description thereof will be omitted.

In addition, since the method for measuring the mRNA level of Lin28a or the level of the protein expressed therefrom is also the same as described above, the description thereof is also omitted.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

cell preparation

1-1. cell culture

Human Renal Proximal Tubular Epithelial cells (HK-2) were purchased from Korea Cell Line Bank (KCLB). Renal proximal tubule epithelial cells were cultured in RPMI 1640 medium containing 10% Fetal bovine serum (FBS) and 1% antibiotic (Gibco) at 37° C. and 5% CO ₂ conditions. Renal proximal tubular epithelial cells ^{were cultured to a density of about 70-80% in culture medium in 1x10 6} cells/100 mm dish, subcultured once every 2 days, and cell lines subcultured 3-6 times were used for the experiment. .

1-2. Lin28a protein overexpression and fibrosis induction

In a 6-well plate, the cells of Example 1-1 were cultured at ^{an amount of 1x10 5} cells/well for 24 hours, and the cultured cells were cultured in a serum-free medium for 24 hours after removing the existing culture medium, Ad_Lin28a adenovirus was transfected for 2 hours, and it was replaced with fresh serum-free medium, treated with TGF-β 10 ng/mL, a renal fibrosis inducer, and cultured for 24 hours. The cultured cell line was washed with PBS and used for the experiment after recovery.

Confirmation of expression change in epithelial-mesenchymal cell migration and fibrosis-induced representative gene according to Lin28a protein overexpression in vitro

2-1. Observation of cell morphology

As described in Example 1-2, the morphology of proximal renal tubular epithelial cells induced by fibrosis by treatment with TGF-β after overexpressing Lin28a protein was observed. Differentiation of tubular epithelial cells into fibroblasts was induced by TGF-β treatment, and the cell shape changed to a flat and elongated appearance. It was confirmed that the number of cells was higher.

2-2. western blot

For protein samples, antibodies from Lin28, collagen type 1 (Type 1 collagen), vimentin, α-SMA (cell signaling Technology Inc., Danvers, MA) and fibronectin (Abcam, Cambridge, MA, USA) were used. Thus, protein electrophoresis was performed. After binding a secondary antibody (cell signaling Technology Inc., Danvers, MA) to which horseradish peroxidase was bound to the primary antibody used above, a chemiluminescent material (Bio-Rad Laboratories, Hercules, CA) was After treatment and reaction, immunoblot was confirmed using Chemi Doc (Bio-Rad Laboratories, Hercules, CA). The concentration of the immunoblot was measured using ImageJ (NIH), an image analysis software.

As shown in Fig. 1b, in the group overexpressing Lin28a protein, the expression of Vimentin and α-SMA, proteins involved in the transition of cells to epithelial-mesenchymal cells, which is the mechanism of renal fibrosis, in a concentration-dependent manner of Lin28a protein. was inhibited, and it was confirmed that the expression of collagen type 1 (Type 1 collagen) and fibronectin, which are representative factors inducing renal fibrosis, was also reduced.

Confirmation of expression change of Lin28a protein and epithelial-mesenchymal cell migration and fibrosis-induced representative gene after induction of fibrosis in vitro

After the cells of Example 1-1 were treated with 10 ng/mL of TGF-β and cultured for 24 hours, changes in the expression of Lin28a protein and representative genes related to the transition of cells to epithelial-mesenchymal cells were confirmed.

More specifically, as a result of performing Western blotting in the same manner as in Example 2-2, the expression of the corresponding markers was changed to the EMT expression pattern by TGF-β treatment as shown in FIGS. 2a and 2b. It was confirmed that the expression of the protein Lin28a was decreased.

Confirmation of the mechanism of inhibition of fibrosis induction by Lin28a protein overexpression in vitro

In this example, in order to confirm the mechanism of inhibition of fibrosis induction by Lin28a protein overexpression, p-smad3 expression and p-ERK, p-JNK, and p-p38 expression in the MAPK pathway during fibrosis induction by TGF-β were analyzed by Western blot. was confirmed as

Specifically, the protein was extracted from the cells of Example 1-2 and Western blot was performed in the same manner as in Example 2-2, wherein the antibody was phosphorylated or total ERK, JNK, smad3 and p38 antibodies (Cell signaling, Beverly , MA, USA) were used.

As a result, as shown in FIGS. 3a and 3b , in the group overexpressing intracellular Lin28a protein, MAPK-related p-ERK, p-JNK, and p-p38 were not affected and TGF-b compared to the control group expressing GFP. By decreasing the expression of p-smad3 increased by , a pathway for inhibiting renal fibrosis was identified.

Animal model preparation

As one of the chronic kidney disease models, a unilateral ureteral obstruction (UUO) model that induces renal fibrosis was manufactured as follows.

C57BL/6 male mice 10 weeks old (20-30 g) were used, and the mice were maintained in a place controlled by temperature (21±2° C.) and light (12 hours light/dark cycle). Anesthesia was induced by intraperitoneal administration of zoletyl and rumpun mixed anesthetic (100 g/mL), and after laparotomy, the left ureter of the mouse was tied using a silk thread and sutured again. The experiment was divided into a total of 2 groups, that is, the 14-day control group and the 14-day UUO group, and the experiment was conducted using 5 mice in each group. At 14 days after UUO, the mouse kidneys were removed and the experimental samples were obtained by freezing storage (-80°C) or tissue fixation for use in the experiment.

After induction of fibrosis by unilateral ureteric ligation (UUO), confirmation of changes in the expression of Lin28a protein, transition to epithelial-mesenchymal cells and fibrosis-induced representative genes

6-1. western blot

With the kidney tissue extracted from each group in Example 5, Western blotting was performed in the same manner as in Example 2-2.

As a result, as shown in Figures 4a and 4b, the unilateral ureteral ligation (UUO) group significantly increased the expression of vimentin and α-SMA compared to the normal control group (CON), and the EMT expression pattern was changed. , It was confirmed that the expression of collagen type 1 (Type 1 collagen) and fibronectin, which are representative factors inducing renal fibrosis, also significantly increased.

6-2. histological examination

After measuring the weight of the kidney tissue removed from the mouse, some were fixed in 10% formalin for immunostaining and then embedded in paraffin. Tissue sections were cut to a thickness of 4 μm, stained with hematoxylin & eosin, and then observed with an optical microscope, and some were stained with Mason's trichrome and Sirius red. staining) was performed, and it was confirmed whether collagen was accumulated due to unilateral ureter ligation.

As a result, as shown in FIG. 5 , it was confirmed that collagen accumulation was increased by the UUO procedure compared to the normal control group (CON).

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Composition for preventing, improving or treating chronic kidney disease or renal fibrosis comprising Lin28a gene or protein <130> PD20-D027-P1 <150> KR 10-2020-0023253 <151> 2020-02-25 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 630 <212> DNA <213> Lin28a <400> 1 atgggctcgg tgtccaacca gcagtttgca ggtggctgcg ccaaggcagc ggagaaggcg 60 ccagaggagg cgccgcctga cgcggcccga gcggcagacg agccgcagct gctgcagggg 120 gccggcatct gtaagtggtt caacgtgcgc atggggttcg gcttcctgtc tatgaccgcc 180 cgcgctgggg tcgcgctcga ccccccggtg gacgtctttg tgcaccagag caagctgcac 240 atggaagggt tccgaagcct caaggagggt gaggcggtgg agttcacctt taagaagtct 300 gccaagggtc tggaatccat ccgtgtcact ggccctggtg gtgtgttctg tattgggagt 360 gagcggcggc caaaagggaa gaacatgcag aagcgaagat ccaaaggaga caggtgctac 420 aactgcggtg ggctagacca tcatgccaag gaatgcaagc tgccacccca gcccaagaag 480 tgccactttt gccaaagcat caaccatatg gtggcctcgt gtccactgaa ggcccagcag 540 ggccccagtt ctcagggaaa gcctgcctac ttccgggagg aagaggaaga gatccacagc 600 cctgccctgc tcccagaagc ccagaattga 630 <210> 2 <211> 209 <212> PRT <213> Lin28a <400> 2 Met Gly Ser Val Ser Asn Gln Gln Phe Ala Gly Gly Cys Ala Lys Ala 1 5 10 15 Ala Glu Lys Ala Pro Glu Glu Ala Pro Pro Asp Ala Ala Arg Ala Ala 20 25 30 Asp Glu Pro Gln Leu Leu His Gly Ala Gly Ile Cys Lys Trp Phe Asn 35 40 45 Val Arg Met Gly Phe Gly Phe Leu Ser Met Thr Ala Arg Ala Gly Val 50 55 60 Ala Leu Asp Pro Pro Val Asp Val Phe Val His Gln Ser Lys Leu His 65 70 75 80 Met Glu Gly Phe Arg Ser Leu Lys Glu Gly Glu Ala Val Glu Phe Thr 85 90 95 Phe Lys Lys Ser Ala Lys Gly Leu Glu Ser Ile Arg Val Thr Gly Pro 100 105 110 Gly Gly Val Phe Cys Ile Gly Ser Glu Arg Arg Pro Lys Gly Lys Asn 115 120 125 Met Gln Lys Arg Arg Ser Lys Gly Asp Arg Cys Tyr Asn Cys Gly Gly 130 135 140 Leu Asp His His Ala Lys Glu Cys Lys Leu Pro Pro Gln Pro Lys Lys 145 150 155 160 Cys His Phe Cys Gln Ser Ile Asn His Met Val Ala Ser Cys Pro Leu 165 170 175 Lys Ala Gln Gln Gly Pro Ser Ser Gln Gly Lys Pro Ala Tyr Phe Arg 180 185 190 Glu Glu Glu Glu Glu Ile His Ser Pro Ala Leu Leu Pro Glu Ala Gln 195 200 205 Asn

Claims (12)

Lin28a gene or Lin28a protein; or
A pharmaceutical composition for the prevention or treatment of chronic kidney disease or renal fibrosis disease, comprising a substance that increases the expression of Lin28a gene mRNA or protein thereof. According to claim 1, wherein the renal fibrosis disease is tubular interstitial fibrosis, chronic kidney disease or a pharmaceutical composition for the prevention or treatment of renal fibrosis disease. Lin28a gene or Lin28a protein; or
A health functional food composition for preventing or improving chronic kidney disease or renal fibrosis disease, comprising a substance that increases mRNA or protein expression of Lin28a gene. A composition for diagnosing chronic kidney disease or renal fibrosis disease, comprising an agent for measuring the level of Lin28a gene mRNA or protein expressed therefrom. According to claim 4, wherein the agent for measuring the mRNA level of the Lin28a gene comprises a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene, and the agent for measuring the level of the Lin28a protein is specific to the protein A composition for diagnosing chronic kidney disease or renal fibrosis disease, comprising an antibody. A kit for diagnosing chronic kidney disease or renal fibrosis disease, comprising the composition of claim 4 or 5. The diagnostic kit of claim 6, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit. (a) measuring the mRNA level of the Lin28a gene or protein expressed therefrom from a biological sample isolated from the subject; and
(B) comprising the step of comparing the measured mRNA level or the level of the protein expressed therefrom with the level measured from a sample of a normal control, information providing method for the diagnosis of chronic kidney disease or renal fibrosis disease. The method of claim 8, (c) further comprising the step of diagnosing chronic kidney disease or renal fibrosis disease when the mRNA level measured in the biological sample isolated from the subject or the level of the protein expressed therefrom is lower than that of a normal control. , a method of providing information for the diagnosis of chronic kidney disease or renal fibrosis disease. The method of claim 8, wherein the mRNA level of the gene is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR reaction (real time quantitative RT-PCR) , RNase protection assay (RNase protection method), Northern blotting (Northern blotting) and DNA chip analysis method (DNA chip technology assay) will be measured using any one selected from the group consisting of, chronic kidney disease or renal fibrosis disease A method of providing information for diagnosis of The method according to claim 8, wherein the level of the protein is determined by western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion method (radial immunodiffusion), Oukteroni immune diffusion method ( Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography ( immunochromatography), FACS analysis (fluorescenceactivated cell sorter analysis), and protein chip technology assay, which is measured using any one selected from the group consisting of, providing information for the diagnosis of chronic kidney disease or renal fibrosis disease Way. (a) treating a candidate material in a biological sample isolated from a patient with chronic kidney disease or renal fibrosis disease;
(b) comparing the mRNA level of Lin28a or the protein expressed therefrom in the sample of the patient treated with the candidate substance with the level measured from the sample of the normal control group; and
(c) when the level of mRNA or protein expressed therefrom in step (b) is increased than before treatment with the candidate substance, determining the candidate substance as a substance for preventing or treating chronic kidney disease or renal fibrosis disease; including; A method of screening a substance for the prevention or treatment of chronic kidney disease or renal fibrosis disease.

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