CN115948410A

CN115948410A - Application of TMEM11 gene and TMEM11 gene inhibitor and product

Info

Publication number: CN115948410A
Application number: CN202211566704.6A
Authority: CN
Inventors: 王昆; 陈鑫哲; 刘翠云; 李新敏
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-04-11

Abstract

The invention provides an application and a product of a TMEM11 gene and a TMEM11 gene inhibitor, and relates to the technical field of biology. The inventor researches show that the expression activity of the protein level of TMEM11 is obviously improved from the newborn to the adult, but the regeneration of the myocardium of the adult is limited; the inhibition of TMEM11 can obviously improve the expression level of the cardiac muscle cell proliferation marker, so that TMEM11 plays an important role in the regulation of cardiac muscle cell proliferation. The TMEM11 gene inhibitor provided by the invention can be used as a medicine for proliferation and regeneration repair of myocardial cells, and can remarkably increase the number of myocardial cells by inhibiting the protein expression of TMEM11, activate the repair of damaged myocardial tissues, and enable the myocardial cells to reenter the cell cycle and the proliferation window period. The discovery of the inhibition of the TMEM11 gene expression effectively relieves the difficult problems of poor prognosis of patients with myocardial infarction and treatment of heart diseases.

Description

Application of TMEM11 gene and TMEM11 gene inhibitor and product

Technical Field

The invention relates to the technical field of biology, in particular to application and a product of a TMEM11 gene and a TMEM11 gene inhibitor.

Background

Cardiovascular disease is the leading cause of death worldwide, the occurrence of which is highly age-related and heart failure is the leading cause of death. Ischemic heart disease is a global disease challenge, and is the result of death or injury to the heart muscle cells, most likely caused by myocardial infarction. After myocardial infarction, cardiac function is impaired, resulting in chronic heart failure. It is a severe myocardial ischemia syndrome, an obstruction of the blood supply to the heart, resulting in massive death and loss of cardiomyocyte function. Myocardial infarction, which results in a large loss of functional cells of the heart, is the basic pathological process for inducing ischemic heart disease. In the past, complex signaling processes that regulate cardiomyocyte death have been identified, but new therapeutic approaches remain lacking.

The proliferation of the existing adult myocardial cells (ACMs) is a feasible source for generating new myocardial cells, and provides a new direction for the treatment of myocardial infarction. The ability of the heart of a newborn mouse to regenerate after removal of the apex has been demonstrated, while the ability of the heart of an adult mouse to self-renew is very limited. Mature cardiomyocytes can re-enter the cell cycle by dedifferentiation, proliferation and redifferentiation, forming new cardiomyocytes. Studies have shown that Hippo and Wnt signaling pathways play important roles in cardiomyocyte proliferation. The Hippo signaling pathway is a common evolutionary pathway that limits development and reproduction primarily by inhibiting the activity of the transcriptional coactivator Yap. There is a great deal of evidence that many proteins, micrornas and non-coding RNAs control proliferation of adult mammalian cardiomyocytes. Gp130, which is a co-receptor of Oncostatin M (OSM), is completely activated during cardiac regeneration, and can activate Yap through Src, thereby promoting cardiomyocyte proliferation. The extracellular matrix protein Agrin promotes the regeneration of the heart of the mouse. Mir-128 and Mir-17-92 are key regulators of endogenous CM proliferation. Similarly, mir302-367 plays a crucial role in the proliferation of cardiac muscle cells, and is sufficient to induce adult cell proliferation and promote cardiac regeneration. In addition, lncRNA is involved in regulating cardiomyocyte proliferation and cardiac repair, and CPR is an inhibitor of cardiomyocyte proliferation.

Mitochondria are highly dynamic organelles involved in various physiological activities such as cell cycle, development and morphological changes. The mitochondria of cardiomyocytes produce most of the chemical energy in the form of adenosine triphosphate, which is required for cardiac contractility and strong cardiac function. Mitochondria have become a central factor in the onset and progression of Heart Failure (HF) and other cardiovascular diseases (CVD), but there is currently no method of treating mitochondrial dysfunction. The family of mitochondrial transmembrane proteins (TMEM) is involved in certain important physiological functions, mainly including autophagy, apoptosis, signal transduction pathways, and plasma membrane ion channels. Among the TMEM protein family members, TMEM 45A6, TMEM 977 and TMEM 1408 are found in human glioblastoma multiforme. The TMEM family acts as a channel, allowing transport of specific substances through biological membranes. However, the biological capabilities of many proteins remain unclear, primarily because of the difficulty in extracting and purifying these proteins. Proteins of this family may be part of various membranes, such as the mitochondrial, endoplasmic reticulum, lysosomal and golgi membranes. The TMEM family is an important group in cancer. In addition, some proteins also act on tumor suppressor factors. Some of which are used as precursor biomarkers. For example, in kidney cancers, some TMEMs have been demonstrated with predicted endoplasmic reticulum localization. The down-regulation of TMEM 88 gene can inhibit the differentiation of myocardial cells and promote the differentiation of endothelial cells. TMEM 215 is an important factor involved in angiogenesis by regulating endothelial cell survival.

Delivery of therapeutic drugs (e.g., protein, drug or gene and cell loaded particles) by direct injection into the myocardium is a promising clinical intervention. Among them, hydrogel has achieved remarkable effect as a better biological carrier, and the three-dimensional structure of hydrogel is similar to the extracellular matrix of many tissues, so that hydrogel is an ideal scaffold material. In a drug delivery system, the hydrogel can be used for drug slow release and site-specific release, and the porous structure of the hydrogel can carry various drugs, migrate to a specific part and generate different swelling responses after being stimulated by environment such as pH, enzyme and the like, so that the drugs are released at different rates. However, most hydrogel systems have a low retention rate in the infarcted area of the heart, losing as much as 60-90% in 24 hours, and the beating of the heart may be the major cause of this condition.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide the application of TMEM11 gene in preparing products for regulating and controlling the proliferation of myocardial cells.

The second object of the present invention is to provide a marker for cardiomyocyte proliferation ability.

The third object of the present invention is to provide a kit for detecting the proliferative capacity of cardiomyocytes.

The fourth purpose of the invention is to provide the application of the TMEM11 gene inhibitor in preparing the medicine for preventing and/or treating heart diseases.

The fifth purpose of the invention is to provide a medicament for preventing and/or treating heart disease, so as to improve the prognosis of the clinical long-term cardiac function of patients with heart disease such as myocardial infarction.

In a first aspect, the invention provides an application of TMEM11 gene in preparing a product for regulating and controlling myocardial cell proliferation.

As a further technical scheme, the TMEM11 gene is highly expressed, and the proliferation of myocardial cells is inhibited;

inhibiting and expressing TMEM11 gene and inducing the proliferation of myocardial cells;

preferably, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO. 1.

In a second aspect, the present invention provides a marker for cardiomyocyte proliferative capacity, said marker being selected from the group consisting of RNA transcribed from the TMEM11 gene or a protein expressed from the TMEM11 gene;

In a third aspect, the present invention provides a kit for detecting the proliferative capacity of cardiomyocytes, wherein the kit is used to detect the marker.

As a further technical scheme, the kit comprises a primer pair for amplifying RNA transcribed by the TMEM11 gene, wherein the primer pair has a sequence shown as SEQ ID NO.5 and a sequence shown as SEQ ID NO. 6.

In a fourth aspect, the present invention provides the use of an inhibitor of the TMEM11 gene in the manufacture of a medicament for the prevention and/or treatment of cardiac disease.

As a further technical scheme, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO.1, and the TMEM11 gene inhibitor has the nucleic acid sequences shown as SEQ ID NO.3 and SEQ ID NO. 4;

preferably, the cardiac disease comprises coronary artery disease or myocardial ischemia-reperfusion.

In a fifth aspect, the present invention provides a medicament for preventing and/or treating heart disease, the medicament comprising a TMEM11 gene inhibitor;

As a further technical scheme, the TMEM11 gene inhibitor has a nucleic acid sequence shown in SEQ ID NO.3 and SEQ ID NO. 4.

As a further technical scheme, the medicament further comprises: a hydrogel consisting essentially of hyaluronic acid and elastin-like proteins.

Compared with the prior art, the invention has the following beneficial effects:

the inventor researches show that the expression activity of the protein level of TMEM11 is obviously improved from the newborn to the adult, but the regeneration of the myocardium of the adult is limited; the inhibition of TMEM11 can obviously improve the expression level of the myocardial cell proliferation markers (Ki 67, PH3 and auroraB), so that TMEM11 plays an important role in the regulation of myocardial cell proliferation.

The TMEM11 gene inhibitor provided by the invention can be used as a medicine for proliferation and regeneration repair of myocardial cells, and can remarkably increase the number of the myocardial cells by inhibiting the protein expression of the TMEM11, activate the repair of damaged myocardial tissues, and enable the myocardial cells to re-enter a cell cycle and a proliferation window period. The discovery of the inhibition of the TMEM11 gene expression effectively relieves the difficult problems of poor prognosis of patients with myocardial infarction and treatment of heart diseases. The Hyaluronic Acid (HA) and elastin-like protein (HELP) gel are used as a carrier, an inhibitor for inhibiting the TMEM11 gene is delivered to the position of a focus of a heart, and the Hyaluronic Acid (HA) and elastin-like protein (HELP) gel can be used as a medicine for treating coronary artery diseases or myocardial ischemia-reperfusion, so that the focus can be effectively treated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the expression levels of mRNA in TMEM11 at different developmental stages in mice;

FIG. 2 shows the distribution of TMEM11 protein in cardiomyocytes;

FIG. 3 shows the knockdown effect of TMEM11 adenovirus;

FIG. 4 shows the proliferation rate of myocardial cells Ki67 detected by immunofluorescence after TMEM11 inhibition;

FIG. 5 shows the proliferation rate of myocardial cells auroraB detected by immunofluorescence after TMEM11 inhibition;

FIG. 6 shows the proliferation rate of myocardial cell PH3 detected by immunofluorescence after TMEM11 inhibition;

FIG. 7 shows the expression level of TMEM11 in a TMEM11 knockout mouse;

FIG. 8 is the ultrasonic cardiac function FS index of TMEM11Ko mice with myocardial infarction for 60 days;

FIG. 9 is the ultrasonic cardiac function EF index of TMEM11Ko mice with myocardial infarction for 60 days;

FIG. 10 is a graph of the fibrotic scar and infarct size after myocardial infarction measured after TMEM11 inhibition;

FIG. 11 shows the proliferation rate of PH3 of the myocardial cells of TMEM11 knockout mice with myocardial infarction for 60 days;

FIG. 12 shows the proliferation rate of Ki67 cells in TMEM11 knockout mice with myocardial infarction for 60 days;

FIG. 13 shows the proliferation rate of auroraB in the TMEM11 knockout mouse cardiomyocytes for 60 days of myocardial infarction.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

Wherein, the TMEM11 gene is highly expressed, and the proliferation of myocardial cells is inhibited;

The inventor researches to find that the expression activity of TMEM11 on the protein level is obviously increased from the newborn to the adult; the inhibition of TMEM11 obviously improves the expression quantity of the cardiac muscle cell proliferation marker, and influences the positive expression rate of Ki67, PH3 and auroraB in the cardiac muscle cells. It is noted that since adult myocardial regeneration is limited, TMEM11 plays an important role in the regulation of cardiomyocyte proliferation, and can regulate cardiomyocyte proliferation and cardiac regeneration repair by participating in the regulation of cardiomyocyte cycle activity, including key events such as DNA synthesis, nuclear division and cytokinesis, thereby improving the clinical prognosis of myocardial infarction.

preferably, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO. 1;

preferably, the amino acid sequence of the protein expressed by the TMEM11 gene is shown in SEQ ID NO. 2.

As described above, since the TMEM11 gene is involved in the proliferation ability of cardiomyocytes, the proliferation ability of cardiomyocytes can be characterized using, as a marker, RNA transcribed from the TMEM11 gene or a protein expressed from the TMEM11 gene.

In some preferred embodiments, the kit comprises a primer pair for amplifying the RNA transcribed from the TMEM11 gene, said primer pair having a sequence as shown in SEQ ID No.5 and having a sequence as shown in SEQ ID No. 6.

In some preferred embodiments, the TMEM11 gene has the nucleic acid sequence shown in SEQ ID No.1, and the TMEM11 gene inhibitor has the nucleic acid sequence shown in SEQ ID No.3 and SEQ ID No. 4:

GUACAAGUACAUUGUGAUAGA(SEQ ID NO.3)；

UAUCACAAUGUACUUGUACUG(SEQ ID NO.4)。

in some preferred embodiments, the cardiac disease comprises coronary artery disease or myocardial ischemia reperfusion, preferably myocardial infarction.

In a fifth aspect, the present invention provides a medicament for preventing and/or treating heart diseases, which comprises a TMEM11 gene inhibitor;

In some preferred embodiments, the TMEM11 gene inhibitor has a nucleic acid sequence as shown in SEQ ID No.3 and SEQ ID No. 4.

The TMEM11 gene inhibitor provided by the invention can be used as a medicine for proliferation and regeneration repair of myocardial cells, and can remarkably increase the number of myocardial cells by inhibiting the protein expression of TMEM11, activate the repair of damaged myocardial tissues, and enable the myocardial cells to reenter the cell cycle and the proliferation window period. The discovery of the inhibition of the TMEM11 gene expression effectively relieves the difficult problems of poor prognosis of patients with myocardial infarction and treatment of heart diseases.

In some preferred embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient;

the carrier is preferably: a hydrogel consisting essentially of hyaluronic acid and elastin-like proteins.

The Hyaluronic Acid (HA) and elastin-like protein (HELP) gel are used as a carrier, an inhibitor for inhibiting the TMEM11 gene is delivered to the position of a focus of a heart, and the Hyaluronic Acid (HA) and elastin-like protein (HELP) gel can be used as a medicine for treating coronary artery diseases or myocardial ischemia-reperfusion, so that the focus can be effectively treated.

In addition, the administration of the drug is preferably by direct injection of the therapeutic drug into the myocardial ischemic tissue by non-invasive catheter surgery.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Materials and methods

1 culture of mouse Primary cardiomyocytes

Centrifuging myocytes from 1-2 day old newborn mice (C57 BL/6), washing the newborn mice with 75% alcohol, taking out the heart with surgical scissors in a petri dish with precooled PBS, washing the heart three times with PBS, cutting the heart tissue with surgical scissors, transferring the heart tissue to 10mL of digestive juice (1.2 mg/mL pancreatin and 0.14mg/mL collagenase II), shaking gently in a water bath at 37 ℃ for 6min, transferring the supernatant to a centrifuge tube with serum after each digestion, storing the centrifuge tube on ice, adding new mouse again, washing the heart with 75% alcohol with water, and removing the serum from the centrifuge tubeUntil the heart tissue disappears. Centrifuging at 1000rpm/min for 10min, collecting supernatant, resuspending with 10% serum-containing F12/DMEM, centrifuging again at 1000rpm/min for 10min, collecting supernatant, filtering with 70 mesh cell filter sieve, placing the filtered cardiomyocytes in 10cm culture dish, and placing at 37 deg.C 5% CO ₂ Culturing for 1.5h in a humidified incubator, adhering fibroblasts, collecting the culture medium containing the cardiomyocytes into a new centrifuge tube, centrifuging at 1000rpm/min for 10min, discarding the supernatant, resuspending with a new culture medium containing serum, and adding 0.1mM 5-bromodeoxyuridine (BrdU). Plates were split according to the experimental design and placed at 37 ℃ 5% CO ₂ Culturing in a humidified incubator. The next day, the culture medium in the petri dish was changed and the culture was continued for one day.

2

3000 mediated cell transfection

(1) Separating primary myocardial cells of the mice suckling mice, and treating after liquid change.

(2) One 1.5mL EP tube was taken and labeled, and 250. Mu.l of Opti-MEM was pipetted into the 1.5mL EP tube and 5. Mu.L

3000, gently sucking, mixing, standing at room temperature for 5min.

(3) A new 1.5mL EP tube is taken and marked, 250 μ L of Opti-MEM is sucked and added into the 1.5mL EP tube, 5 μ L of siRNA (the nucleic acid sequences are shown as SEQ ID NO.3 and SEQ ID NO.4, the inventor finds that the siRNA with the nucleic acid sequences shown as the above can realize the inhibition of a target gene through research, and the siRNA with the nucleic acid sequences shown as SEQ ID NO.3 and SEQ ID NO.4 is used for testing) or plasmid DNA is lightly sucked, stirred, placed at room temperature and kept for 5min.

(4) Two 1.5mL EP tubes of (2) and (3) were mixed and gently pipetted well and allowed to stand at room temperature for 20min.

(5) And (3) taking out the six-hole plate in the cell incubator until the standing is completed, discarding the original culture medium, and washing with PBS.

(6) After the completion of the standing of the transfection reagent, (4) is added to the corresponding six-well plate, and then the 5% FBS-containing solution is filled,

A DMEM high-sugar culture medium containing 1% double-antibiotic sterilizing liquid.

(7) The six-well plate was placed in an incubator for 6h.

(8) After culturing for six hours, removing the original culture medium, adding a new culture medium, and placing the six-hole plate in an incubator to continue culturing for 24-48 hours.

3 immunoblot assay

Total protein was extracted from cardiomyocytes or tissues using RIPA and PMSF, the total protein concentration was measured using BCA kit, and the OD value was read by a microplate reader. Protein loading buffer was added to the protein samples according to concentration at 95 ℃ for 10min. Protein samples were subjected to SDS-PAGE and transferred to PVDF membrane, and protein expression was detected using specific antibodies. The antibodies used were as follows: TMEM11 (Proteitech, cat:16564-1-AP, 1; GAPDH (ABClonal, cat: A19056, 1.

4 immunofluorescence

Fixing the treated cardiomyocytes or cardiac tissue sections with 4% paraformaldehyde for 15min, PBS washing three times, 0.1% Triton X-100 for 15min, PBS washing three times, 5% incubation with BSA solution for 30min, binding the protein signal with specific antibodies using the following antibodies: anti-Ki67 (abcam, cat: ab16667, 1; anti-Aurora B (abcam, cat: ab2254, 1; anti-Histone H3 (abcam, cat: ab5176, 1; anti-Cardiac Troponin T (abcam, cat: ab8295, 1; TMEM11 rabbitpab (Abclonal, a17578, 1. Fluorescence signals were detected using a Leica inverted two-photon laser confocal scanning microscope.

5 mouse echocardiography assessment

The Vevo2100 imaging system is adopted to carry out echocardiography, and by combining Electrocardiogram (ECG) acquisition analysis and an anatomical M-type ultrasonic technology, the dynamic change of the morphological structure of each chamber of the heart in a cardiac cycle, the relation between the change of blood pressure and the change of the volume of a heart cavity can be effectively recorded in real time, and quantitative study on cardiac muscle can be carried out.

6Masson trichrome staining

Dewaxing paraffin sections, staining with Weigart hematoxylin for 5min, differentiating with acidic ethanol for 10s, washing with acidic ethanol with distilled water, staining with Masson's blue solution for 2min, washing with Masson's blue solution with distilled water, staining with ponceau fuchsin for 5min, washing with weak acid solution for 1min, washing with phosphomolybdic acid for 2min, washing with weak acid solution for 2min, staining with aniline blue for 1min, washing with weak acid solution for 2min, dehydrating, transparentizing, and mounting.

7 mouse myocardial infarction model

We selected 6 to 8 week old mice (C57 BL/6) for the experiments. Anesthesia was performed by intraperitoneal injection of 4% chloral hydrate, and chest and axillary hair were removed with surgical scissors (fully exposing the surgical field) and the surgical field was sterilized with 75% ethanol. The ventilator was turned on, the parameters (respiratory rate 110 bpm) were set, and the mice were intubated with trachea. The chest was opened between the three and four ribs with ophthalmic scissors to fully expose the heart and the left anterior descending coronary artery (LAD) or area. The needle holder takes 6-0 suture with needle, and the needle is inserted at 2mm position of the lower edge of the left auricle, and the suture passes through the LAD to completely block the blood flow of the LAD. After ligation, the opening of the thoracic cavity is completely sutured by 4-0 suture (no gap and no dislocation are ensured), the thoracic cavity is closed, and each layer of muscle and skin is sutured layer by layer from inside to outside.

8 mitochondrial isolation

The myocytes in the culture dish were washed with PBS, centrifuged at 800g for 5min to collect the cells, 1.0mL of precooled lysine Buffer was added to resuspend the cells, the cell suspension was transferred to a small volume glass homogenizer and ground 30 times in an ice bath. The cell homogenate was transferred to a centrifuge tube and centrifuged at 1000g for 5min at 4 ℃. The supernatant was removed and transferred to a new centrifuge tube and centrifuged at 12,000g for 10min at 4 ℃. The supernatant after centrifugation contains cytoplasmic components from which cytoplasmic proteins can be extracted. The supernatant was transferred to a fresh centrifuge tube and the mitochondria settled at the bottom of the tube. Adding 0.5ml of Wash Buffer into the mitochondrial precipitate, and centrifuging at 4 ℃ for 5min at 1000 g. The supernatant was removed and transferred to a new centrifuge tube and centrifuged at 12,000g for 10min at 4 ℃. The supernatant was discarded and high purity mitochondria were precipitated at the bottom of the tube.

9 nuclear matter separation

Nucleoplasmic isolation was performed using the BioVision kit. First, the cells were washed with PBS, cardiomyocytes were collected, centrifuged at 4 ℃ and 600g/min for 5min. The supernatant was discarded and 200. Mu.L of CEB-A (containing DDT and protease inhibitor) was added, shaken vigorously for 5s and left on ice for 10min. Add 11. Mu.L of LCEB-B, shake vigorously for 5s, and incubate on ice for 1min. Vigorous shaking was continued for 5s. Centrifugation is carried out for 5min at 16000g/min at 4 ℃. The supernatant was collected in a new Eppendorf tube and placed on ice. Add 100. Mu.L NEB to the precipitate; the precipitate was vigorously shaken for 15s and then placed on ice for 10min. Centrifugation is carried out for 10min at 16000g/min and 4 ℃. The supernatant was transferred to a new Eppendorf tube and stored in a refrigerator at-80 ℃.

Construction of 10TMEM11 knockout mice

The Nanjing Collection drug Corjus utilizes CRISPR/Cas9 technology to carry out gene modification on TMEM11 gene, and a TMEM11 gene knockout mouse is obtained. There are 3 transcripts of the TMEM11 gene. Based on the structure of the TMEM11 gene, exon 2 of the TMEM11-201 transcript was used as the knockout region. This region contains most of the coding sequence. Knocking out this region will result in disruption of protein function. A simple procedure is the in vitro transcription of sgRNAs. Cas9 and sgRNA were microinjected into C57BL/6 mouse zygotes. F0 positive mice are obtained by transplanting fertilized eggs and are verified by PCR and sequencing. F0 positive mice were mated with C57BL/6 mice to obtain stable F1 mouse models.

11 real-time fluorescent quantitative PCR (RT-qPCR)

TRIZOL reagent extracts total RNA from mouse cardiomyocytes or tissues and determines the concentration and purity of total RNA using nanodrop One. The AG Evo M-MLV RT Kit reverse transcription Kit converts total RNA into cDNA. Fluorescent quantitative PCR was performed using SYBR Green Kit. The RT-qPCR adopts a CFX96 real-time fluorescent quantitative PCR detection system. Reaction conditions are as follows: 10min at 95 ℃;95 ℃ for 10s and 60 ℃ for 30s, for a total of 40 cycles. The experimental results were qualitatively analyzed for relative expression using the 2- Δ Ct method. The qPCR primer sequences were:

name (R)	Numbering	Nucleotide sequence
			TMEM11-F	SEQ ID NO.5	AACGCCCAGGACCAGTTTG
TMEM11-R	SEQ ID NO.6	GTGCTGTCTCGTCTCCAATTC
			GAPDH-F	SEQ ID NO.7	AGGTCGGTGTGAACGGATTTG
GAPDH-R	SEQ ID NO.8	TGTAGACCATGTAGTTGAGGTCA

Note: TMEM11-F and TMEM11-R are used for amplification of RNA transcribed from the TMEM11 gene; GAPDH-F and GAPDH-R are used for amplification of RNA transcribed from the GAPDH gene.

12 hyaluronic acid and elastin-like protein (HELP) hydrogels

12.1 expression of elastin-like proteins

Elastin-like protein (ELP) is expressed in BL21 (DE 3) pLysS E.coli under the control of the T7 promoter, as described previously. Briefly, the stock containing the ELP plasmid was streaked onto agar plates and incubated overnight at 37 ℃. A single colony was selected and incubated overnight at 37 ℃ with constant stirring. Expression was induced by addition of 1mM IPTG. After 7 hours of expression at 32 ℃ the ELP-containing E.coli cells were harvested by centrifugation, resuspended in buffer (0.1M Tris-Cl,0.01M EDTA,1M sodium chloride, pH 8.0) and then frozen at-80 ℃.

12.2 functionalization of elastin-like proteins

ELP contains a central lysine (K) in the elastin-like domain. The hydrazine group is functionalized according to the following scheme. First, ELP was dissolved in anhydrous 6% (w/v) Dimethylsulfoxide (DMSO). After the ELP was completely dissolved, an equal volume of N, N-Dimethylformamide (DMF) was added to the reaction mixture. In a separate round bottom flask: (1) Triborohydrazineacetic acid (Tri-Boc), (2) Azobenzotriazoltetramethyluranium Hexafluorophosphate (HATU), (3) 4-methylmorpholine, dissolved in DMF at Room Temperature (RT) to allow activation of the free acid groups on the TriBoc molecule by HATU. The reagent solution was added dropwise to the ELP to a final ELP concentration of 2% (w/v). The reaction was carried out overnight at room temperature with constant stirring. The reaction solution was then added dropwise to pre-cooled diethyl ether in a final volume ratio of 1:5. the modified ELP was collected by centrifugation at 4 ≧ 18000g for 30min and dried overnight to afford the Boc protected intermediate. To remove the Boc protecting group, boc protected ELP was dissolved at 2% (w/v) in a 1:1 mixture of dichloromethane and trifluoroacetic acid with 2.5% (v/v) triisopropylsilane and then stirred at room temperature for 4 hours. The deprotected product was then precipitated in chilled ether and centrifuged as above. The final product was then dissolved in 2% (w/v) deionized water and dialyzed against 4L of deionized water at 4 ℃.4L of dialysis water was refreshed routinely every 12 hours over 3 days. The final dialysis product was sterilized, frozen and finally lyophilized for 3 days.

12.3 functionalization of hyaluronic acid with aldehydes, benzaldehyde

Hyaluronic Acid (HA) functionalization achievesbase:Sub>A two-part reaction with aldehyde (HA-A) or benzaldehyde (HA-B) groups via copper click chemistry. First, HA (Lifecore) was functionalized by EDC chemistry containing an alkynyl group. Second, commercial small molecules with pendant aldehyde or benzaldehyde groups were clicked onto the HA molecule by standard copper click chemistry. Briefly, 100kDa HA (sodium salt) was dissolved in MES buffer (0.2M MES,0.15M sodium chloride; pH 4.5) at a concentration of 1% (w/v). Once dissolved, enough propynylamine was added directly to functionalize 12% of the available carboxylic acid groups, and the pH was immediately adjusted to 6.0 with 1M sodium hydroxide. N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added in this order and the reaction was continued with stirring at room temperature for 4 hours. The reaction was then dialyzed against 4L of deionized water at 4 ℃.4L of dialysis water was refreshed routinely every 12 hours for 3 days. The final dialysis product was sterile filtered, frozen, and lyophilized for 3 days to yield HA-Alkyne.

And attaching the pendant aldehyde or benzaldehyde group on the HA-Alkyne by standard copper click chemistry to obtain HA-A and HA-B respectively. Briefly, HA-alkyne was dissolved at 2.5% (w/v) in 10 XPBS and 0.85mg/mL of cyclodextran supplement was added. After the reaction mixture was completely dissolved, it was degassed with nitrogen. A2.4 mM copper sulfate and 45.2mM sodium ascorbate solution was additionally prepared and degassed. According to the expected modification scheme, ald-CH2-PEG 3-azide or 4-azidobenzaldehyde was dissolved in DMSO (100 mg/mL). Sodium ascorbate and copper stock solutions were then added to final concentrations of 4.52mM and 0.24mM, respectively. Finally, small molecules containing aldehyde or benzaldehyde are added, the solution is degassed, and the reaction is carried out for 24 hours in the dark at room temperature. After 24 hours, an equal amount of 50mM EDTA, pH 7.0, was added and stirred at room temperature for 1 hour. The solution was then diluted with deionized water and sterile filtered to remove any copper precipitate. The resulting solution was dialyzed against 4L of deionized water at 4 ℃.4L of dialysis water was refreshed routinely every 12 hours for 3 days. The final dialysis product was then sterilized with a 0.22 μm syringe filter, frozen, and lyophilized for 3 days.

12.4 Oxidation of hyaluronic acid

1.5MDa hyaluronic acid (sodium salt) was first dissolved in 4mg/mL overnight with constant stirring at 4 ℃. The next day, sodium periodate (2:1; sodium periodate: HA) was dissolved in deionized water to a final concentration of 0.1M, and then added dropwise to the HA solution in a final volume ratio of 1:5. after the addition of sodium periodate, the reaction flask was left at room temperature in the dark for the time required for the reaction. After the desired oxidation time, ethylene glycol was added to inactivate any unreacted periodate. After 1 hour of inactivation, the reaction volume was transferred to a dialysis tube and dialyzed against 4L of deionized water at 4 ℃.4L of dialysis water was refreshed routinely every 12 hours over 3 days. The final dialysis product was sterile filtered, frozen and then lyophilized for 3 days.

12.5 hydrogel formation

For the HELP system, ELP was dissolved at a concentration of 4% (w/v) in sterile 1 × PBS, overnight at 4 ℃. HA was dissolved overnight in 4% (w/v) 10 XPBS. The next day, equal amounts of HA and pre-cooled 1 × PBS were mixed on ice. The 2-vol HA solution was then mixed with an equal volume of 4-vol ELP on ice. The hydrogel was thawed overnight for the matrigel. Then, 1 XPBS (final concentration: 12.5% (v/v)) was added to the thawed matrix gel, and mixing on ice ensured that no bubbles were present.

Example 1 Regulation of cardiomyocyte proliferation and cardiac repair by mitochondrial transmembrane protein 11 (TMEM 11)

As shown in the figure, in order to detect the expression change of TMEM11 in the development process of the myocardial cells, heart tissues of the mice at embryonic stage of 15 days, birth of 1 day, birth of 7 days and birth of 30 days are collected, and the expression level of TMEM11 in each development stage is detected by RT-qPCR (figure 1). The results showed that the expression level of TMEM11 increased gradually with the development of mouse cardiomyocytes. The adult mouse heart has little capacity for regeneration, and may be associated with upregulation of TMEM11 expression. Nuclear and cytoplasmic isolation, mitochondrial isolation and immunofluorescence results showed that TMEM11 was expressed in the nucleus, cytoplasm and mitochondria (figure 2). The expression level of TMEM11 in each tissue of the mouse was measured by Western Blot, and the results showed that the expression level of TMEM11 in the heart was relatively high. These results indicate that TMEM11 is highly expressed in mouse cardiomyocytes and may be involved in regulating cardiomyocyte proliferation.

Adenovirus AdV-shTMEM11 is constructed, and the knocking-down effect is verified through RT-qPCR. The results showed that adenovirus AdV-shTMEM11 was able to knock down the expression of TMEM11 (as shown in FIG. 3, in the figure, shRNA-NC indicates that TMEM11 knockdown adenovirus negative control, shRNA-TMEM11 indicates that TMEM11 knockdown adenovirus, + indicates that corresponding adenovirus was transfected into cardiomyocytes, and-indicates that corresponding adenovirus was not transfected into cardiomyocytes). The effect of TMEM11 on cardiomyocyte proliferation was examined by immunofluorescence. Serine 10 phosphorylated histone H3 (PH 3) is used as a marker for commonly used cardiomyocyte proliferation. Aurora B is a marker of cytokinesis; the Ki67 protein is a cell cycle-associated nucleoprotein expressed during cell division G1, S, G and M. Compared with the myocardial cells transfected with AdV-NC, the transfected AdV-shTMEM11 promotes the proliferation of mouse mammary mouse myocardial cells. After knocking down the expression of TMEM11, the number of myocardial cells was significantly increased (FIG. 4, FIG. 5, FIG. 6, in which NC represents the control group and TMEM11 represents the knocking down TMEM11 group). These data indicate that knock-down TMEM11 is effective in promoting myocardial nuclear formation and cell division. Thus, TMEM11 is able to inhibit proliferation of cardiomyocytes.

TMEM11 knock-out mice (TMEM 11 Ko) were constructed to verify the role of TMEM11 in cardiac regeneration, and the knock-out effect of TMEM11Ko mice was confirmed by Western Blot (fig. 7). To explore the effects of TMEM11Ko on mouse cardiomyocyte proliferation and cardiac injury repair, a model of adult mouse (6-8 weeks old) myocardial infarction was constructed and analyzed 60 days after myocardial infarction surgery. Echocardiographic results in mice showed improved myocardial function in TMEM11Ko mice with myocardial infarction for 60 days compared to wild type mice with myocardial infarction for 60 days (fig. 8, fig. 9). Masson staining showed that the myocardial infarct size of TMEM11Ko mice with myocardial infarct for 60 days was significantly smaller than that of wild-type mice (fig. 10). Immunofluorescence assays of the cardiomyocyte proliferation markers PH3, auroraB and Ki67 showed that TMEM11Ko mice with 60 days of myocardial infarction promoted cardiomyocyte proliferation compared to wild-type mice with 60 days of myocardial infarction (fig. 11, 12, 13). Taken together, these data indicate that TMEM11Ko promotes cardiomyocyte proliferation, improves cardiac function after myocardial infarction, and promotes cardiac repair.

In conclusion, the invention discloses a new mechanism for recovering myocardial infarction injury and promoting myocardial cell proliferation, and TMEM11 plays an important role in the regulation of myocardial cell proliferation. The knockout of TMEM11 significantly improves the cardiac function after myocardial infarction injury, reduces the myocardial infarction area and promotes the proliferation of myocardial cells, and the results show that TMEM11 plays an important role in the regulation and control of the proliferation of myocardial cells and suggest that TMEM11 has great application potential in the aspects of cardiac repair and regeneration.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims

Application of TMEM11 gene in preparing products for regulating and controlling myocardial cell proliferation.
2. The use according to claim 1, wherein the TMEM11 gene is highly expressed to inhibit cardiomyocyte proliferation;

inhibiting and expressing TMEM11 gene and inducing the proliferation of myocardial cells;

preferably, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO. 1.
3. A marker of cardiomyocyte proliferative capacity, wherein said marker is selected from the group consisting of RNA transcribed from the TMEM11 gene or a protein expressed from the TMEM11 gene;

preferably, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO. 1.
4. A kit for detecting the proliferative capacity of cardiomyocytes, wherein the kit is used to detect the marker of claim 3.
5. The kit according to claim 4, wherein the kit comprises a primer pair for amplifying the RNA transcribed from the TMEM11 gene, the primer pair having a sequence as shown in SEQ ID No.5 and having a sequence as shown in SEQ ID No. 6.
Use of a tmem11 gene inhibitor for the preparation of a medicament for the prevention and/or treatment of heart disease.
7. The use according to claim 6, wherein the nucleic acid sequence of the TMEM11 gene is shown in SEQ ID No.1, and the TMEM11 gene inhibitor has the nucleic acid sequences shown in SEQ ID No.3 and SEQ ID No. 4;

preferably, the cardiac disease comprises coronary artery disease or myocardial ischemia reperfusion.
8. A medicament for preventing and/or treating heart disease, which comprises a TMEM11 gene inhibitor;

preferably, the nucleic acid sequence of the TMEM11 gene is shown as SEQ ID NO. 1.
9. The medicament of claim 8, wherein the TMEM11 gene inhibitor has a nucleic acid sequence as shown in SEQ ID No.3 and SEQ ID No. 4.
10. The medicament of claim 8, further comprising: a hydrogel consisting essentially of hyaluronic acid and elastin-like proteins.