CircSESN1 gene as well as overexpression vector, specific primer pair, detection kit and application thereof

Abstract

The invention discloses a CircSESN1 gene as well as an overexpression vector, a specific primer pair, a detection kit and application of the CircSESN1 gene. The invention also discloses application of the CircSESN1 gene, an overexpression vector, the specific primer pair or the fluorescent quantitative detection kit in regulation and control of growth and development of chicken skeletal muscle. The invention also discloses a method for inhibiting chicken myoblast proliferation and / or promoting differentiation. The detection kit provided by the invention can be used for detecting the expression quantity of the CircSESN1 gene in chicken muscle tissues and myoblasts, and the detection method is simple and quick. The gene can be used as an important non-coding RNA for researching a regulation theory of growth and development of chicken skeletal muscles.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bioengineering, and particularly relates to a CircSESN1 gene, an overexpression vector thereof, a specific primer pair, a detection kit thereof and application. BACKGROUND

[0002] Skeletal muscle is an important component of animal body, accounting for about 40% of body weight. For broiler chickens, the development status of skeletal muscle directly affects the yield of chicken meat and determines the economic value of broiler chickens. Skeletal muscle content is mainly determined by the number, diameter and type of muscle fibers. Myoblasts, as precursor cells of muscle fibers, determine the number of muscle fibers through proliferation and differentiation, and after a series of proliferation and differentiation, they eventually become mature muscle fibers. Therefore, studying the genetic regulation mechanism of myoblast proliferation and differentiation has important scientific significance and guiding role for elucidating the theoretical basis of chicken skeletal muscle growth and development and genetic improvement of growth traits of local chickens in China.

[0003] CircRNA (circular RNA) is a kind of non-coding RNA molecule with closed loop structure without 5' cap and 3' poly(A) tail, which is stable in expression, mainly located in cytoplasm or stored in exosomes, and has been proved to exist widely in various eukaryotes. Studies have shown that CircRNAs have important functions in the regulation of chicken skeletal muscle growth and development, mainly as miRNA molecular sponges to regulate the expression of chicken myoblast proliferation and differentiation related genes.

[0004] A small number of studies have shown that the source gene SESN1 of CircSESN1 can promote the proliferation and differentiation of chicken myoblasts. Another transcription product of SESN1 gene, CircSESN1, is derived from the third, fourth and fifth exons of SESN1 gene (XM_004940327.5), and studies have shown that it can promote the proliferation and differentiation of chicken myoblasts through the CircSESN1 / miR-16-5p / SESN1 pathway.

[0005] However, the current research on the structure and function of CircSESN1 gene is relatively few and not comprehensive, especially the structure of CircSESN1 gene in different breeds of chickens has not been comprehensively and deeply studied. Therefore, it is necessary to explore the structure and function of CircSESN1 gene in different breeds of chickens, so as to deeply study the influence of new CircSESN1 gene on chicken skeletal muscle growth and development, reveal new muscle development regulation mechanism, and possibly provide new valuable molecular markers for broiler chicken breed selection and breeding. SUMMARY

[0006] The technical problem solved by the present application is to provide a new CircSESN1 gene, which has a ring structure and is distributed in the nucleus and cytoplasm of cells.

[0007] The present application also provides an application of the CircSESN1 gene in inhibiting proliferation and / or promoting differentiation of chicken myoblast cells.

[0008] The present application also provides an application of the overexpression vector in inhibiting proliferation and / or promoting differentiation of chicken myoblast cells.

[0009] The present application also provides a specific primer pair for detecting the expression amount of the CircSESN1 gene.

[0010] The present application also provides a fluorescent quantitative detection kit.

[0011] The present application also provides an application of the CircSESN1 gene, the overexpression vector, the specific primer pair or the fluorescent quantitative detection kit in regulating growth and development of chicken skeletal muscle.

[0012] The present application also provides a method for inhibiting proliferation and / or promoting differentiation of chicken myoblast cells.

[0013] In order to achieve the above-mentioned application purposes, the present application provides a CircSESN1 gene, wherein the nucleotide sequence of the CircSESN1 gene is shown as SEQ ID NO. 11.

[0014] The present application provides an application of the CircSESN1 gene in inhibiting proliferation and / or promoting differentiation of chicken myoblast cells.

[0015] The present application provides an overexpression vector, wherein the overexpression vector comprises the CircSESN1 gene, and preferably, the overexpression vector comprises pcDNA3.1-CircSESN1.

[0016] The present application provides an application of the overexpression vector in inhibiting proliferation and / or promoting differentiation of chicken myoblast cells.

[0017] The present application provides a specific primer pair for detecting the expression amount of the CircSESN1 gene, wherein the specific primer pair sequence of the CircSESN1 gene is shown as SEQ ID NO. 1 and SEQ ID NO. 2.

[0018] Further, the application also includes a specific primer pair for detecting the expression of CircSESN1 gene and Linear SESN1 gene in chicken muscle tissue and / or myoblasts.

[0019] Further, the specific primer pair of the CircSESN1 gene has the sequence shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the specific primer pair of the Linear SESN1 gene has the sequence shown as SEQ ID NO. 3 and SEQ ID NO. 4.

[0020] The application provides application of the specific primer pair of the CircSESN1 gene in preparation of a reagent or kit for detecting the expression of CircSESN1 in chicken muscle tissue and / or myoblasts.

[0021] The application provides a fluorescent quantitative detection kit, which comprises the specific primer pair.

[0022] Further, the fluorescent quantitative detection kit further comprises 2×ChamQ SYBR qPCR Master Mix reagent and enzyme-free double distilled water.

[0023] Further, the fluorescent quantitative detection kit further comprises primer pairs of reference genes β-actin, GAPDH and U6, the primer pair of the reference gene β-actin has the sequence shown as SEQ ID NO. 5 and SEQ ID NO. 6, the primer pair of the reference gene GAPDH has the sequence shown as SEQ ID NO. 7 and SEQ ID NO. 8, and the primer pair of the reference gene U6 has the sequence shown as SEQ ID NO. 9 and SEQ ID NO. 10.

[0024] The application provides application of the CircSESN1 gene, the overexpression vector, the specific primer pair or the fluorescent quantitative detection kit in regulation of growth and development of chicken skeletal muscle.

[0025] The application is to detect the expression of CircSESN1 gene in chicken muscle tissue and / or myoblasts by fluorescent quantitative PCR.

[0026] Further, the fluorescent quantitative PCR reaction system comprises 2×ChamQ SYBR qPCR Master Mix, a primer with the sequence shown as SEQ ID NO. 1, a primer with the sequence shown as SEQ ID NO. 2, a template cDNA and enzyme-free double distilled water.

[0027] Further, the fluorescent quantitative PCR reaction system comprises 2*ChamQ SYBR qPCR Master Mix reagent 10 muL, primer with sequence as shown in SEQ ID NO. 1 0.4 muL, primer with sequence as shown in SEQ ID NO. 2 0.4 muL, template cDNA 2 muL and enzyme-free double distilled water 7.2 muL.

[0028] Further, the fluorescent quantitative PCR reaction system further comprises primer with sequence as shown in SEQ ID NO. 3 0.4 muL, primer with sequence as shown in SEQ ID NO. 4 0.4 muL, primer with sequence as shown in SEQ ID NO. 5 0.4 muL, primer with sequence as shown in SEQ ID NO. 6 0.4 muL, primer with sequence as shown in SEQ ID NO. 7 0.4 muL, primer with sequence as shown in SEQ ID NO. 8 0.4 muL, primer with sequence as shown in SEQ ID NO. 9 0.4 muL and primer with sequence as shown in SEQ ID NO. 10 0.4 muL.

[0029] The application provides a method for inhibiting proliferation and / or promoting differentiation of chicken myoblasts, which comprises increasing the expression amount of the CircSESN1 gene.

[0030] The application collects chicken leg muscle tissues, performs fluorescent quantification on gDNA and cDNA by using specific primers for CircSESN1 and Linear SESN1, performs agarose gel electrophoresis and Sanger sequencing, in-vitro cultures chicken embryo leg muscle primary myoblasts and DF-1 cells, and performs RNase R enzyme digestion test, actinomycin D test and nucleolus separation test. -△△CT The application performs fluorescent quantification PCR by using specific primers for CircSESN1 and internal reference beta-actin and GAPDH, and the PCR reaction system and amplification procedure are the same as those of conventional fluorescent quantification PCR. -△△CT The application performs fluorescent quantification PCR by using specific primers for CircSESN1 and internal reference beta-actin and GAPDH, and the PCR reaction system and amplification procedure are the same as those of conventional fluorescent quantification PCR.

[0031] Beneficial effects: Compared with the prior art, the present application has the following advantages: the present application verifies a new CircSESN1 gene with a circular structure distributed in the nucleus and cytoplasm through Sanger sequencing, RNase R enzyme digestion and other tests; the present application also designs and synthesizes specific primers for detecting the expression amount of the new CircSESN1 gene, and a detection kit and a detection method thereof. The detection kit provided by the present application can be used to detect the expression amount of the new CircSESN1 gene in chicken muscle tissue and myoblast cells, and the detection method is simple and fast. The new CircSESN1 gene provided by the present application can be used as an important non-coding RNA for studying the regulation theory of chicken skeletal muscle growth and development. The new CircSESN1 gene provided by the present application can be used for the molecular mechanism research of chicken skeletal muscle growth and development. In subsequent research, the specific regulation mechanism of the new CircSESN1 gene in the process of chicken skeletal muscle growth and development is determined by verifying the target miRNA and the regulated gene after overexpression treatment of the new CircSESN1 gene. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is a CircSESN1 gene ring verification result graph; (A) Sanger sequencing of CircSESN1; (B) agarose gel electrophoresis graph of PCR product of CircSESN1 and Linear SESN1 primers respectively on gDNA and cDNA template; (C) RNase R enzyme digestion experiment of CircSESN1 and Linear SESN1 on chicken embryo leg muscle primary myoblast cells (CPMS cells) and DF-1 cells; (D) actinomycin D experiment of CircSESN1 and Linear SESN1 on chicken embryo leg muscle primary myoblast cells (CPMS cells) and DF-1 cells.

[0033] Figure 2 Figure 2 is a CircSESN1 gene expression distribution graph in the nucleus and cytoplasm;

[0034] Figure 3 Figure 3 is a CircSESN1 gene expression situation result graph in different tissues and different time periods; (A) expression profile of CircSESN1 gene in 13 tissues of 1-day-old chicks; (B) expression rule of CircSESN1 gene in different periods of chicken embryo leg muscle primary myoblast cells (GM-50%, GM-100% respectively represent 50% and 100% density when proliferating; DM1, DM2, DM3, DM4 and DM5 respectively represent induced differentiation for 1-5 days);

[0035] Figure 4 Figure 4 is a CircSESN1 overexpression vector efficiency detection result graph;

[0036] Figure 5 Figure for the results of qRT-PCR and Western blot detection of the effect of CircSESN1 gene on cell proliferation; (A) qRT-PCR detection of the expression changes of proliferation marker genes; (B) the effect of CircSESN1 gene on PCNA protein expression; (C) PCNA protein gray value comparison column chart;

[0037] Figure 6 Figure for the results of EDU detection of the effect of CircSESN1 gene on cell proliferation; (A) fluorescence map after overexpression of CircSESN1 gene; (B) cell proliferation ratio after overexpression of CircSESN1 gene;

[0038] Figure 7 Figure for the effect of CircSESN1 gene on the expression of differentiation marker gene MYHC;

[0039] Figure 8 Figure for the effect of CircSESN1 gene on MYHC protein expression; (A) the effect of CircSESN1 gene on MYHC protein expression; (B) MYHC protein gray value comparison column chart.

[0040] Figure 9 Figure for the results of indirect immunofluorescence detection of the effect of CircSESN1 gene on myotube differentiation; (A) fluorescence microscope results after overexpression of CircSESN1 gene; (B) myotube area statistics. DETAILED DESCRIPTION

[0041] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained by purchase.

[0042] Example 1 Primer design of new CircSESN1 gene and verification of CircSESN1 gene ring formation and subcellular localization

[0043] The present invention screens out differentially expressed CircSESN1 genes by performing RNA-Seq on leg muscle tissues at different time points during the chicken embryonic period. The present invention found through research that the gene is a circRNA ring structure, so the design of specific primers for the CircSESN1 gene needs to span the splicing site, and the specific primer pair sequences of the CircSESN1 gene are shown in SEQ ID NO.1 and SEQID NO.2. The specific primer pair for the Linear SESN1 gene has sequences shown in SEQ ID NO.3 and SEQ ID NO.4, and the primer pair sequence for the internal reference gene β-actin is shown in SEQ ID NO:5 and SEQ ID NO:6. Total RNA and gDNA from the leg muscle tissues of chickens from the Bian Chicken Seed Farm of Shanxi Agricultural University were extracted respectively, and the total RNA was reverse transcribed to obtain cDNA. Using cDNA and gDNA as templates, specific primers were used to perform fluorescence quantification of the CircSESN1 gene and the Linear SESN1 gene, respectively, and the products were subjected to agarose gel electrophoresis ( Figure 1 B), CircSESN1 gene-specific primer pair can only amplify bands on cDNA, but not on gDNA, while Linear SESN1 gene-specific primer pair can amplify bands on both gDNA and cDNA. Sanger sequencing of the specific product of CircSESN1 gene ( Figure 1 A), Figure 1 A and Figure 1 B confirmed the existence of the circular binding site of the CircSESN1 gene. Total RNA extracted from CPMs cells and DF-1 cells was divided into two parts. One part was treated with RNase R, and the other part was not. The digestion reaction was carried out on a PCR instrument. The digestion product was reverse transcribed to obtain cDNA. The cDNA was fluorescently quantified using specific primers for the CircSESN1 gene, Linear SESN1 gene, and β-actin gene ( Figure 1 C), the results showed that the linear SESN1 gene is in the RNase R + The expression levels of the treated groups were significantly lower than those of RNase R - The expression of CircSESN1 did not change significantly in the treatment group (P < 0.01) (P > 0.05). CPMs and DF-1 cells were plated into 6-well plates. After the cell density reached 80%, medium containing actinomycin D was added. Five time periods were set, with 6 replicates each time period. Total RNA was extracted and reverse transcribed into cDNA. The cDNA was fluorescently quantified using CircSESN1 and β-actin specific primers ( Figure 1D), the results showed that the decreasing trend of CircSESN1 expression was significantly smaller than that of Linear SESN1 expression with the extension of the culture time of the actinomycin D medium (P<0.05). The above research results confirmed the true existence of the circular structure of the CircSESN1 gene Figure 1

[0044] The chicken primary myoblasts were extracted from the leg muscle of 12-embryonic-age Beggars chicken embryos and plated into 6-well plates. After the cells grew, the nuclei and cytoplasm were separated by using a nuclei and cytoplasm extraction kit (Novizen Company, item number E101), and the RNA was extracted and reversely transcribed into cDNA. The cDNA was subjected to fluorescent quantification by using specific primer pairs of CircSESN1, GAPDH and U6 Figure 2 The sequence of the specific primer pair of the CircSESN1 gene is shown in SEQ ID NO. 1 and SEQ ID NO. 2, the sequence of the specific primer pair of the Linear SESN1 gene is shown in SEQ ID NO. 3 and SEQ ID NO. 4, the sequence of the primer pair of the reference gene GAPDH is shown in SEQ ID NO: 7 and SEQ ID NO: 8, and the sequence of the primer pair of the reference gene U6 is shown in SEQ ID NO: 9 and SEQ ID NO: 10. The primer sequences were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0045] The above amplification reaction system was 20 μL, containing 10 μL of 2×ChamQ SYBR qPCR Master Mix reagent; specific primer pair or reference gene primer pair (0.4 μL of specific primer each or 0.4 μL of reference gene primer each); template cDNA 2 μL; and 7.2 μL of enzyme-free double distilled water.

[0046] The fluorescent quantification reaction program was as follows: 95℃ pre-denaturation for 30S; 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles; 95℃ for 15 sec, 60℃ for 60 sec, 95℃ for 30 sec, 60℃ for 15 sec to obtain the melting curve.

[0047] The RNase R enzyme reaction system was 20 μL, and the RNase R enzyme group contained 4 μg of RNA sample, 0.6 μL of RNase R enzyme, 2 μL of 10×RNase R Reaction Buffer, and DEPC-treated water up to 20 μL. The group without RNase R enzyme contained 4 μg of RNA sample, 0 μL of RNase R enzyme, 2 μL of 10×RNase R Reaction Buffer, and DEPC-treated water up to 20 μL. ​

[0048] The fluorescence quantitative reaction condition is 37℃ reaction for 15 min and 70℃ inactivation for 10 min.

[0049] The primer sequence is as follows:

[0050]

[0051]

[0052] It can be known from Figure 1 A, Figure 1 B, Figure 1 C, Figure 1 D that the circular structure of the CircSESN1 gene is verified through experiments, and Figure 1 B that the specific primer pair of the CircSESN1 gene amplifies a single band, indicating that the primer design is good.

[0053] It can be known from Figure 2 that the CircSESN1 gene is expressed in the nucleus and cytoplasm.

[0054] 2, The CircSESN1 gene inhibits the proliferation of chicken myoblasts

[0055] The proliferation and differentiation of myoblasts are a basic process of skeletal muscle development, and myoblasts need to proliferate and differentiate to become mature muscle cells. The present application verifies the influence of the CircSESN1 gene on the proliferation and differentiation of chicken myoblasts. The present application entrusts Shanghai Jimma Company to construct a CircSESN1 overexpression vector named pcDNA3.1-CircSESN1, provides the CircSESN1 gene sequence to Shanghai Jimma Company, Shanghai Jimma Company clones the CircSESN1 gene sequence, and subclones it into the pcDNA3.1(+) vector through the BamH1 and EcoRI restriction enzyme cutting sites. In the experiment, the empty pcDNA3.1(+) vector is used as a control.

[0056] The new CircSESN1 gene sequence is as follows (443bp): SEQ ID NO. 11

[0057]

[0058] Thirteen kinds of tissue samples such as heart, liver and spleen are taken from one-day-old female edge chickens, 4 repetitions are set, RNA is extracted, reverse transcribed into cDNA, and the template cDNA is detected by fluorescence quantitative PCR with the CircSESN1 and beta-actin specific primer pairs, and the expression amount of the CircSESN1 gene in different tissues is detected Figure 3A), the results show that the relative expression of CircSESN1 gene is the highest in pectoral muscle and leg muscle. The chicken primary myoblasts are extracted from the leg muscle of 12-embryonic chicken embryos and inoculated in 6-well plates, and the RNA of proliferation period (GM50%, GM100%) and differentiation period (induced differentiation 1 day DM1, induced differentiation 2 days DM2, induced differentiation 3 days DM3, induced differentiation 4 days DM4, induced differentiation 5 days DM5) is extracted, each period has 3 repeats, the specific primers of CircSESN1 and β-actin are used for the fluorescence quantification of the template cDNA, and the expression of CircSESN1 in different periods is detected Figure 3 B), the results show that the relative expression of CircSESN1 gene shows an overall upward trend, and the expression in each time period of the differentiation period is significantly higher than that in the proliferation period (P<0.05).

[0059] The chicken primary myoblasts are extracted from the leg muscle of 12-embryonic chicken embryos and inoculated in 12-well plates, and the cells are transfected when the cell density reaches 70%, each group has four biological repeats, and the transfection steps are as follows: the overexpression vector is adjusted to 250 μg / μl with DEPC water. The overexpression vector pcDNA3.1-CircSESN1 is mixed with transfection buffer and vortexed for 10 s, and then the transfection reagent is added after a short centrifugation, vortexed for 1 s again, and incubated at room temperature for 20 min. Finally, it is added to the cell culture plate to complete the transfection process. The dose is as follows: overexpression vector 3.2 μL, transfection buffer 75 μL, and transfection reagent 2.4 μL. After 48 h of transfection, the cells are collected to detect the overexpression efficiency of CircSESN1, and the results show that the overexpression efficiency is close to 120000 times, reaching a very significant level (P<0.001), which can be used for subsequent experiments Figure 4 )。

[0060] Finally, the pcDNA3.1-CircSESN1 overexpression vector is selected for verifying the influence of CircSESN1 on the proliferation and differentiation of chicken myoblasts.

[0061] The chicken myoblasts are inoculated in 6-well plates, and the pcDNA3.1-CircSESN1 is transfected when the cell density reaches 70%, each group has three biological repeats, and the fluorescence quantitative PCR results show that the mRNA expression of proliferation marker genes PAX7 and PCNA is extremely significantly or significantly reduced (P<0.001 or P<0.01) Figure 5 A); it is found by Western blotting experiment that the transfection of pcDNA3.1-CircSESN1 can inhibit the expression of PCNA protein Figure 5B, C); finally, it was found by EDU detection that the positive cell ratio of pcDNA3.1-CircSESN1 transfection was significantly lower than that of the control group (P<0.001) Figure 6 A, Figure 6 B).

[0062] In addition, the chicken myoblast cells were plated in a 6-well plate, and when the cell density reached 90%, the differentiation medium was replaced and transfected with pcDNA3.1-CircSESN1, and the results of fluorescence quantitative PCR showed that after transfection of pcDNA3.1-CircSESN1, the mRNA expression of cell differentiation marker gene MYHC was significantly increased (P<0.01) Figure 7 ); Western blotting experiment found that transfection of pcDNA3.1-CircSESN1 significantly increased the expression of MYHC protein (P<0.01) Figure 8 A, Figure 8 B); finally, the results of indirect immunofluorescence experiment showed that transfection of pcDNA3.1-CircSESN1 significantly increased the myotube area (P<0.001) Figure 9 A, Figure 9 B).

[0063] In summary, the CircSESN1 gene newly discovered by the application has the functions of inhibiting chicken myoblast cell proliferation and promoting differentiation, while the previously reported CircSESN1 gene with another transcript has the functions of promoting chicken myoblast cell proliferation and differentiation at the same time. In the process of muscle growth and development, inhibiting excessive cell proliferation is a key link to ensure normal muscle differentiation and functional maturation, and excessive cell proliferation may lead to abnormal muscle hyperplasia (such as fibrosis) and also has the risk of tumor. After the cell stops proliferating, energy and raw materials (such as amino acids) can be diverted to the differentiation process such as myotube fusion and muscle fiber hypertrophy. Therefore, CircSESN1 can regulate cell cycle, signal pathway and differentiation program, etc. to ensure that muscle tissue stops dividing at the right time and turns to functional maturation, so as to breed new lines of high-yield broilers. The new CircSESN1 provided by the application can be used as an important non-coding RNA for studying the regulation theory of chicken skeletal muscle growth and development.

Claims

1. CircSESN1 A gene characterized by described CircSESN1 The nucleotide sequence of the gene is shown in SEQ ID NO.

11.

2. CircSESN1 Application of a gene in inhibiting the proliferation and / or promoting the differentiation of chicken myoblasts.

3. Overexpression vector, characterized in that, The overexpression vector comprises the CircSESN1 Gene, preferably, the overexpression vector includes pcDNA3.1-CircSESN1.

4. Use of the overexpression vector according to claim 3 in inhibiting cell proliferation and / or promoting chicken myoblast differentiation.

5. For detecting the CircSESN1 A gene-specific primer pair, characterized in that described CircSESN1 The gene-specific primer pair sequences are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

6. The method according to claim 5 CircSESN1 Gene-specific primer pairs for detecting the gene in chicken muscle tissue and / or myoblasts CircSESN1 The application of the reagent or kit for expressing the expression amount.

7. A fluorescence quantitative detection kit, characterized in that: The kit comprises the specific primer pair according to claim 5.

8. The method according to claim 1 CircSESN1 Use of the gene, the overexpression vector according to claim 3, the specific primer pair according to claim 5 or the fluorescent quantitative detection kit according to claim 7 in regulating the growth and development of chicken skeletal muscle.

9. The use according to claim 8, characterized in that The application is to detect the presence of fibroblasts in chicken muscle tissue and / or myoblasts by fluorescence quantitative PCR. CircSESN1 Gene expression level.

10. A method for inhibiting the proliferation and / or promoting the differentiation of chicken myoblasts, characterized in that: The method comprises improving the CircSESN1 Gene expression level.

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