Application of SIRT6 protein as a marker for detecting diabetic cataract and preparing drugs for preventing and treating diabetic cataract

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of SIRT6 protein as a marker for detecting diabetic cataracts and in the preparation of drugs for preventing and treating diabetic cataracts. The SIRT6 protein provided by the present invention is significantly downregulated in the target cell lens epithelial cells of diabetic cataracts, and thus can be used as a marker for detecting diabetic cataracts. At the same time, overexpression of SIRT6 protein in diabetic cataract target cells can promote the expression of autophagy factors in diabetic cataract target cells and increase the proliferation activity of lens epithelial cells, protect the normal metabolism of lens epithelial cells, and play a role in alleviating the progression of the disease in diabetic cataracts. Therefore, SIRT6 protein can be used as a therapeutic target for diabetic cataracts.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of SIRT6 protein as a marker for detecting diabetic cataracts and preparing drugs for preventing and treating diabetic cataracts. Background Art

[0002] Diabetic cataract is a common eye disease in clinic, with the main feature of visual impairment caused by lens opacity. The patient's visual function decreases, and in severe cases, the vision is completely lost. The pathogenesis of diabetic cataract is complex. The main reason is that blood sugar is higher than normal, the glucose concentration in the aqueous humor of the eye increases, and a large amount of glucose accumulates in the lens, which affects the physiological environment of the lens epithelial cells and the sugar metabolism pathway, causing the lens transparency to decrease or lose, thus causing cataracts. With the improvement of social living standards and the change in people's dietary structure, the incidence of diabetes has increased repeatedly. Relevant epidemiological studies have shown that the number of people with diabetes in the world currently reaches 425 million, and by 2040, the number of diabetic patients may reach about 642 million. According to statistics, 63% of diabetic patients will have diabetic cataracts. Because there is no effective drug for the treatment of diabetic cataracts in clinic, surgery is the only way to solve the blindness caused by diabetic cataracts. Although the surgical methods, auxiliary equipment and artificial lenses for cataract treatment have developed rapidly in recent years, postoperative complications such as vitreous opacity and fundus hemorrhage are difficult to avoid. Therefore, it is very important to develop an effective drug target for the prevention and treatment of diabetic cataracts, thereby delaying the occurrence and development of diabetic cataracts.

[0003] Silent information regulator protein 6 (SIRT6) is a member of the longevity protein Sirtuins family (SIRT1-SIRT7). SIRT6 is mainly located in the cell nucleus, 39.1kb, located on chromosome 19p13.3, and is involved in regulating multiple cell signaling pathways. Studies have found that SIRT6 protein can regulate the expression of certain genes and DNA repair by deacetylation of histones. It can also improve the nucleic acid repair function after DNA double-strand breaks caused by oxidative stress and maintain genome stability through ribosylation modification. Some studies have knocked out SIRT1-SIRT7 in experimental mice, and only SIRT6-deficient mice showed obvious aging phenotypes. In the study of chronic obstructive pulmonary disease, it was found that SIRT6 can regulate Akt and other pathways to affect the autophagy level and activity of its cells. However, there have been no reports on the application of SIRT6 protein in diabetic cataracts. Therefore, finding safe and effective methods to prevent and delay the occurrence and development of diabetic cataracts has important clinical significance and socioeconomic benefits. Summary of the invention

[0004] The purpose of the present invention is to provide the use of SIRT6 protein as a marker for detecting diabetic cataracts and in the preparation of drugs for preventing and treating diabetic cataracts. SIRT6 protein can be used as a marker for detecting diabetic cataracts and a therapeutic target, thereby increasing its medical use.

[0005] The present invention provides the use of SIRT6 protein as a marker for detecting diabetic cataract.

[0006] Preferably, the expression level of the SIRT6 protein is downregulated in diabetic cataract target cells.

[0007] Preferably, the diabetic cataract target cells include lens epithelial cells.

[0008] The present invention provides application of SIRT6 protein in preparing medicine for preventing and treating diabetic cataract.

[0009] Preferably, the SIRT6 protein is overexpressed in diabetic cataract target cells.

[0010] Preferably, the diabetic cataract target cells include lens epithelial cells.

[0011] Preferably, the lens epithelial cells include SRA01 / 04 cells.

[0012] The present invention provides the use of SIRT6 protein in preparing a drug for promoting the expression of autophagy factors in lens epithelial cells of diabetic cataract and / or improving the proliferation activity of lens epithelial cells.

[0013] Preferably, the amino acid sequence of the SIRT6 protein is as shown in SEQ ID No.1.

[0014] Preferably, the nucleotide sequence encoding the SIRT6 protein is shown as SEQ ID No.2.

[0015] Beneficial effects:

[0016] The present invention provides SIRT6 protein as a marker for detecting diabetic cataracts. The expression level of SIRT6 protein in lens epithelial cells, the target cells of diabetic cataracts, is significantly downregulated. Therefore, SIRT6 protein can be used as a marker for detecting diabetic cataracts.

[0017] At the same time, overexpression of SIRT6 protein in diabetic cataract target cells can promote the expression of autophagy factors in diabetic cataract target cells and increase the proliferation activity of lens epithelial cells, which protects the normal metabolism of lens epithelial cells and can alleviate the progression of diabetic cataract. Therefore, SIRT6 protein can be used as a therapeutic target for diabetic cataract. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The effect of different concentrations of glucose on the proliferation activity of SRA01 / 04 cells in Example 1, 20× scale;

[0020] Figure 2 The effect of different concentrations of glucose on SIRT6 protein expression in SRA01 / 04 cells in Example 1;

[0021] Figure 3 The effect of high glucose intervention at different times on SIRT6 protein expression in SRA01 / 04 cells in Example 1, compared with the 0h group, *P<0.05; **P<0.01;

[0022] Figure 4 The expression level of SIRT6 protein in each group detected by Western blot in Example 2, ****P<0.0001;

[0023] Figure 5 The expression level of SIRT6 mRNA in each group was detected by fluorescence quantitative PCR in Example 2, ****P<0.0001;

[0024] Figure 6 The effect of overexpression of SIRT6 on the proliferation activity of SRA01 / 04 cells treated with high glucose was detected by CCK-8 method in Example 2, *P<0.05, ****P<0.0001;

[0025] Figure 7 The effect of overexpression of SIRT6 on the expression of LC3A / B, p62, and Beclin1 proteins in SRA01 / 04 cells treated with high glucose by Western blot in Example 2, A. Statistical results of LC3A / B protein expression levels, B. Statistical results of p62 protein expression levels, C. Statistical results of Beclin1 protein expression levels, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0026] Figure 8The effect of overexpression of SIRT6 on the expression of Atg5 and Atg12 proteins in SRA01 / 04 cells treated with high glucose by Western blot in Example 2, A. Western blot electrophoresis results, B. Statistical results of Atg5 protein expression level, C. Statistical results of Atg12 protein expression level, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0027] Fig. 9 The effect of overexpression of SIRT6 on the expression of t-mTOR and p-mTOR proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B statistical results of p-mTOR protein expression levels, C statistical results of t-mTOR protein expression levels, **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0028] Fig.10 The effect of overexpression of SIRT6 on the expression of t-Akt and p-Akt proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B statistical results of p-Akt protein expression levels, C statistical results of t-Akt protein expression levels, **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0029] Fig.11 The effect of overexpression of SIRT6 on the expression of p-AMPK and t-AMPK proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B statistical results of p-AMPK protein expression level, C statistical results of t-AMPK protein expression level, **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0030] Fig.12 The effect of overexpression of SIRT6 on the expression of p-MAPK and t-MAPK proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B statistical results of p-MAPK protein expression levels, C statistical results of t-MAPK protein expression levels, **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0031] Fig.13The effect of overexpression of SIRT6 on the expression of p-p53, t-p53 and proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B statistical results of p-P53 protein expression level, C statistical results of t-P53 protein expression level, ***P < 0.001, ****P < 0.0001;

[0032] Fig.14 The effect of overexpression of SIRT6 on the expression of Bax and Bcl-2 proteins in SRA01 / 04 cells treated with high glucose in Example 2, A. Western blot electrophoresis results, B. Statistical results of Bcl-2 protein expression levels, C. Statistical results of Bax protein expression levels, ***P < 0.001, ****P < 0.0001.

[0033] Fig.15 The effect of knocking down SIRT6 on the proliferation activity of SRA01 / 04 cells detected by CCK-8 method in Example 3, *P<0.05, ****P<0.0001;

[0034] Fig.16 The effect of knocking down SIRT6 on the expression of p62, Beclin1, Atg5, Atg12, and LC3A / B proteins in SRA01 / 04 cells in Example 3, A. Western blot electrophoresis results, B. Statistical results of p62 protein expression level, C. Statistical results of Beclin1 protein expression level, D. Statistical results of Atg5 protein expression level, E. Statistical results of Atg12 protein expression level, F. Statistical results of LC3A / B protein expression level, **P<0.01, ***P<0.001, ****P<0.0001;

[0035] Fig.17 The effect of knocking down SIRT6 on the expression of p-mTOR / t-mTOR, p-AMPK / t-AMPK, p-Akt / t-Akt, and p-MAPK / t-MAPK proteins in SRA01 / 04 cells in Example 2, A. Western blot electrophoresis results, B statistical results of p-mTOR protein expression levels, C statistical results of t-mTOR protein expression levels, D statistical results of p-AMPK protein expression levels, E statistical results of Et-AMPK protein expression levels, F statistical results of Fp-Akt protein expression levels, Gt-Akt protein expression levels, H statistical results of Hp-MAPK protein expression levels, It statistical results of MAPK protein expression levels, **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0036] Fig.18 The effect of knocking down SIRT6 on the expression of p-p53, t-p53, Bcl-2 and Bax proteins in SRA01 / 04 cells in Example 3, A. Western blot electrophoresis results, B. Statistical results of p-p53 protein expression level, C. Statistical results of t-P53 protein expression level, D. Statistical results of Bcl-2 protein expression level, E. Statistical results of Bax protein expression level, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0037] The present invention provides the use of SIRT6 protein as a marker for detecting diabetic cataracts. The SIRT6 protein of the present invention is a member of the longevity protein Sirtuins family (SIRT1-SIRT7). The SIRT6 protein is mainly localized in the cell nucleus, 39.1 kb, and located on chromosome 19p13.3. Its amino acid sequence is preferably as shown in SEQ ID No.1, specifically: MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS; the nucleotide sequence encoding the SIRT6 protein is preferably as shown in SEQ ID No.2, and the accession number in NCBI is NM_016539. The SIRT6 protein of the present invention is down-regulated in diabetic cataract target cells, and can be used as a detection target for detecting diabetic cataracts. The diabetic cataract target cells of the present invention preferably include lens epithelial cells, and more preferably include lens epithelial cells damaged by high concentration glucose.

[0038] The present invention also provides the use of SIRT6 protein in the preparation of a drug for preventing and treating diabetic cataracts. The present invention preferably overexpresses the SIRT6 protein in diabetic cataract target cells, increases the expression of SIRT6 protein in diabetic cataract target cells, improves the damage to lens epithelial cells caused by diabetes, and thus achieves the effect of preventing and treating diabetic cataracts. The diabetic cataract target cells of the present invention preferably include lens epithelial cells, and more preferably include SRA01 / 04 cells. The drug of the present invention preferably also includes pharmaceutically acceptable excipients.

[0039] The present invention also provides the use of SIRT6 protein in the preparation of a drug for promoting the expression of autophagy factors in lens epithelial cells of diabetic cataracts and / or increasing the proliferation activity of lens epithelial cells. The present invention can promote the expression of autophagy factors in lens epithelial cells of diabetic cataracts, inhibit the apoptosis of lens epithelial cells, and increase the proliferation activity of lens epithelial cells by overexpressing the SIRT6 protein in lens epithelial cells, thereby protecting the normal metabolism of lens epithelial cells and alleviating the progression of the disease in diabetic cataracts.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Specifications and sources of each antibody in the examples:

[0042] SIRT6 polyclonal antibody (Affinity, DF12739), β-actin polyclonal antibody (Biossion, bs-0061R), LC3 antibody polyclonal antibody (proteintech, 14600), Beclin1 monoclonal antibody (proteintech, 66665), ATG5 polyclonal antibody (proteintech, 60061), ATG12 polyclonal antibody (proteintech, 11122), BCL-2 polyclonal antibody (proteintech , 12789), BAx polyclonal antibody (proteintech, 50599), P62 (Affinity, AF5384), p-AMPK / t-AMPK (Affinity, AF3423 / AF6423), p-p53 / t -p53 (Affinity, AF3075 / AF0879), p-MAPK / t-MAPK (Affinity, AF3455 / AF6456) and p-Akt / t-Akt (Affinity, AF0832 / AF8355).

[0043] Example 1

[0044] SIRT6 protein expression is downregulated in lens epithelial cells damaged by high concentration of glucose

[0045] (I) Experimental cell lines and main reagents: human lens epithelial cell immortalized line SRA01 / 04 (Peking Union Medical College Cell Center), DMEM culture medium (Hyclone, USA), fetal bovine serum (BI, Israel), 0.25% trypsin (Hyclone, USA), protein content detection kit and protein extraction kit (Keygen Biotech Co., Ltd., China), SIRT6 polyclonal antibody (Affinity, DF12739) and β-actin polyclonal antibody (Biossion, bs-0061R).

[0046] (II) Cell culture: After sterilizing the clean bench and culture flasks and centrifuge tubes used in the cell culture room, take out the cryovials of SRA01 / 04 cells from the cryopreservation box in the liquid nitrogen tank, transfer them to an ice box, and quickly place them in a 37°C water bath. After thawing, sterilize the cryopreservation tubes with 70% alcohol, and then blend the cell cryopreservation solution in twice the volume of DMEM complete culture medium. Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, add 4 mL of complete culture medium, and gently pipet to mix. Then transfer the solution to a 25 mL culture flask and gently shake the cell suspension by the cross-shaking method. Culture the suspension in a 5% CO2, 37°C cell culture incubator. Change the medium after the cells adhere for 24 hours.

[0047] (III) Cell passaging: When the cells grow to about 85% and are in good condition (high transparency and strong refractive index) under a microscope, they are passaged at a ratio of 1:4. When passaging, first use a 3mL bus pipette to suck out the culture medium, then add 2mL PBS buffer and rinse to remove the metabolites at the bottom of the culture bottle by cross-shaking method, wash 3 times with PBS, add 700μL 0.25% trypsin without EDTA, shake well, and place in a 37℃ incubator for digestion for 1min until the cells become round and partly fall off. Use the left hand hypothenar to gently tap the bottom of the culture bottle until the cells completely fall off and flow like sand. Add 2mL of complete culture medium containing 10% FBS to terminate digestion, mix well with a pipette, and transfer to a 15mL centrifuge tube. After centrifugation at 1000rpm for 5min, discard the supernatant, add 4mL of complete culture medium and mix well. Take 1mL of cell suspension and transfer to a 25mL culture bottle with 3mL culture medium, shake well, and place in the incubator for continued culture.

[0048] (IV) Effect of different concentrations of glucose on the proliferation activity of lens epithelial cells: After conventional culture of SRA01 / 04 cells adhered to the wall for 24 hours, DMEM culture medium containing 5.5mmol / L, 15.5mmol / L, 25.5mmol / L, 35.5mmol / L, 45.5mmol / L, 55.5mmol / L, and 75.5mmol / L glucose was added to intervene in each group of cells. After 48 hours, the cell morphology was observed under a microscope and photographed. 3 Each group of cells was inoculated in a 96-well plate, and 4 replicate wells were set up in each well for 48 hours. After the cell status was observed under a light microscope, 10 μL of CCK-8 reagent was added to each well in the dark, and the well was returned to the 37°C cell culture medium for incubation for 2 hours. The optical density (OD) value of each well was measured at a wavelength of 450 nm using an enzyme marker. The average value of the 4 replicate wells was taken for each group. The experiment was repeated 3 times. The results are shown in the table. Figure 1 As shown, A shows the cell morphology changes at a glucose concentration of 5.5 mmol / L; B shows the cell morphology changes at a glucose concentration of 55.5 mmol / L; C shows the cell morphology changes at a glucose concentration of 75.5 mmol / L; D shows the results of the proliferation activity of cells in different glucose concentration groups detected by CCK-8 method, *P<0.05, **P<0.01.

[0049] (V) WB detection of changes in SIRT6 protein expression in SRA01 / 04 cells cultured at different glucose concentrations

[0050] 1. Effect of different concentrations of glucose on SIRT6 expression in SRA01 / 04 cells: After intervention with different concentrations of glucose for 48 hours, cells from each group were collected, and whole proteins were extracted using the Keygen protein extraction kit according to the instructions. Protein quantification kits were used to quantify the extracted proteins of each group. After quantification, the corresponding lodding buffer and ddH2O were added to adjust to an equal volume and mass system. Each group of samples was subjected to 10% SDS-PAGE electrophoresis, wet transfer at 280 mA for 45 minutes, and blocked with 10% skim milk powder at room temperature for 4 hours. After washing the membrane three times with TBST, primary antibodies were incubated: rabbit anti-SIRT6 antibody (1:1000 dilution) and rabbit anti-β-actin antibody (1:7000 dilution), respectively, and incubated at 4°C overnight or at room temperature for two hours. After washing the membrane three times with TBST, goat anti-rabbit IgG antibody was used for hybridization at room temperature for 1.5 hours. After washing the membrane three times with TBST, 1:1 ECL working solution was used to develop the color using the Image Lab gel imaging image analysis system and collect the grayscale value for image analysis.

[0051] Western blot experiments confirmed that with the increase of glucose concentration, the expression of SIRT6 protein in SRA01 / 04 cells gradually decreased in a dose-dependent manner. Compared with the expression of SIRT6 in the glucose concentration 5.5mmol / L group, there was no statistically significant difference between the glucose concentration 15.5mmol / L group and the glucose concentration 25.5mmol / L group (all P>0.05, such as Figure 2 ) There were statistically significant differences between the glucose concentration 45.5mmol / L group, the glucose concentration 55.5mmol / L group and the glucose concentration 75.5mmol / L group (compared with the glucose concentration 5.5mmol / L group (NC group), *P<0.05; **P<0.01, such as Figure 2 ).

[0052] 2. Effect of high-glucose intervention at different times on the expression of SIRT6 in lens epithelial cells: After conventional culture SRA01 / 04 cells were fixed to the wall for 24 hours, the SRA01 / 04 cells were intervened with culture medium containing 55.5 mmol / L glucose for 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h, and the cells were collected. The above method (Part V, Step 1) was used to extract protein, quantify protein, and use Western blot to detect the expression of SIRT6 and β-actin in each group of cells.

[0053] Western blot results showed that under the same high concentration of glucose intervention, the protein expression of SIRT6 in SRA01 / 04 cells gradually decreased with time, showing a time-dependent manner. Compared with the SIRT6 protein expression in the 0h group, there was no statistically significant difference between the 6h group and the 12h group (all P>0.05, Figure 3 ) The differences between the 24h group, 48h group and 72h group were statistically significant (all P < 0.05, Figure 3 ). This indicates that the longer the high glucose intervention time, the lower the SIRT6 protein expression in SRA01 / 04 cells.

[0054] Example 2

[0055] Effects of SIRT6 overexpression on SRA01 / 04 cells under high glucose (55.5mmol / L) conditions and its mechanism

[0056] The SIRT6 overexpression lentivirus SRA01 / 04 stable cell line was constructed, and Western blotting and fluorescence quantitative PCR were used to identify whether the SIRT6 overexpression SRA01 / 04 stable cell line was successfully constructed. The upstream sequence of the primer pair used for fluorescence quantitative PCR was: Forward primer: 5'-GGAGGAGCTGGAGCGGAAGG-3' (SEQ ID No. 3), and the downstream sequence was: Reverse primer: 5'-TGAAGTCGGGGATGCCAGAGG-3' (SEQ ID No. 4). Then the cells were divided into a normal control group (CON), a high glucose group (HG+LV-CON), and a high glucose+SIRT6 overexpression group (HG+LV-SIRT6). After 48 hours of intervention in each group, the CCK-8 experiment was used to detect the cell proliferation activity of each group. The high glucose group (HG+LV-CON) was compared with the normal control group (CON) to observe the effects of high glucose intervention on autophagy and related apoptosis factors in lens epithelial cells; the high glucose+SIRT6 overexpression group (HG+LV-SIRT6) was compared with the high glucose group (HG+LV-CON) to observe the effects of overexpression of SIRT6 on autophagy and related apoptosis factors in SRA01 / 04 cells under high glucose conditions.

[0057] Western blotting technology was used to detect the expression of LC3A / B, Beclin1, p62, Atg5, Atg12, p-mTOR / t-mTOR, p-Akt / t-Akt, p-MAPK / t-MAPK, p-AMPK / t-AMPK, p-p53 / t-p53, Bcl-2 and Bax proteins in each group of cells.

[0058] Method for constructing SIRT6 overexpression lentivirus SRA01 / 04 stable cell line: First, construct a human SIRT6 cDNA overexpression lentivirus vector, and use PCR to identify whether it is successfully constructed. At the same time, compare and analyze it with the SIRT6 cDNA standard sequence on GenBank. The identified lentivirus recombinant expression plasmid LV-SIRT6 and the packaging plasmid were co-transfected into 293T cells to prepare lentivirus LV-SIRT6 carrying SIRT6. After discarding the regular cell culture medium, the SRA01 / 04 cells in each group were rinsed 3 times with PBS to remove the culture medium containing double antibodies. 2 mL of normal culture medium was added to the CON group, and 1.2 μL SIRT6 overexpression lentivirus (virus titer was 1.5×10 9 TU / mL), 40 μL virus infection enhancement solution HiTransGA (HA), 958.8 μL culture medium without double antibody and containing 10% FBS; 1.8 μL SIRT6 overexpression empty vector lentivirus (virus titer was 1.0×10 9TU / mL), 40μL virus infection enhancement solution HA, 958.2μL double antibody-free medium containing 10% FBS. 1000μL double antibody-free medium was added 4 hours after virus infection. After 12 hours, the virus-containing medium was aspirated and 2mL of medium containing 20% ​​FBS and 1% double antibody was added. The medium was changed every 48h to maintain cell activity, and the cells were cultured for 72 hours, and the infection efficiency was observed by fluorescence microscopy based on the fluorescence intensity. When the cell fusion reached about 85% and the infection efficiency was determined to be 80% by fluorescence microscopy, it was cultured with 2ug / mL concentration of puromycin medium. The purpose was to screen out normal cells and leave SRA01 / 04 cells successfully infected with SIRT6 overexpression and empty lentivirus.

[0059] Western blotting and fluorescence quantitative PCR showed that compared with the CON group, the expression levels of SIRT6 protein and mRNA in SRA01 / 04 cells in the LV-SIRT6 group were significantly increased (P < 0.05), confirming that the SIRT6 overexpression lentivirus-transfected SRA01 / 04 cell line was successfully constructed ( Figure 4-5 );

[0060] After high-concentration glucose intervention, CCK-8 results showed that compared with the CON group, the cell proliferation activity of the HG+LV-CON group was significantly decreased; compared with the HG+LV-CON group, the cell proliferation activity of the HG+LV-SIRT6 group was significantly increased (P<0.05) ( Figure 6 ); at the same time, the results of Western blotting showed that compared with the CON group, the expression of LC3A / B, Beclin1, Atg5, Atg12, p-AMPK / t-AMPK, and Bcl-2 in the HG+LV-CON group was downregulated, and the expression of p62, p-mTOR / t-mTOR, p-Akt / t-Akt, p-MAPK / t-MAPK, p-p53 / t-p53, and Bax was upregulated (all P<0.0 5); Compared with the HG+LV-CON group, the expressions of LC3A / B, Beclin1, Atg5, Atg12, p-AMPK / t-AMPK, and Bcl-2 in the HG+LV-SIRT6 group were significantly upregulated, and the expressions of p62, p-mTOR / t-mTOR, p-Akt / t-Akt, p-MAPK / t-MAPK, p-p53 / t-p53, and Bax were significantly downregulated (all P<0.05)( Figure 7-14 ).

[0061] Example 3

[0062] Effects of knocking down SIRT6 expression on SRA01 / 04 cells and its mechanism

[0063] The SRA01 / 04 cell line stably transfected with SIRT6 interference lentivirus was constructed, and a normal control group (CON-lens epithelial cells cultured in conventional culture medium), a negative control group (LV-si-CON-lens epithelial cells infected with SIRT6 interference lentivirus empty plasmid) and a SIRT6 interference group (LV-si-SIRT6-SIRT6-cells infected with SIRT6 interference lentivirus and stably knocked down SIRT6) were set up. After 48 hours of cell culture, the proliferation activity of cells in each group was detected by CCK-8 method; immunofluorescence technique and Western blotting was used to detect the expression level of SIRT6 protein in each group of cells to identify whether the SRA01 / 04 cell line with stable knockdown of SIRT6 expression was successfully constructed; at the same time, Western blotting was used to detect the expression of LC3A / B, Beclin1, p62, Atg5, Atg12, p-mTOR / t-mTOR, p-Akt / t-Akt, p-MAPK / t-MAPK, p-AMPK / t-AMPK, p-p53 / t-p53, Bcl-2 and Bax proteins in each group of cells.

[0064] The negative control group and the normal control group showed that the empty plasmid had no effect on the autophagy and apoptosis of the lens epithelial cells; the comparison between the SIRT6 interference group and the normal control group (CON) showed the effect of knocking down SIRT6 expression on the autophagy and apoptosis-related proteins of SRA01 / 04 cells; the comparison between the SIRT6 interference group and the negative control group (LV-si-CON) showed that the effect of knocking down SIRT6 expression on the autophagy and apoptosis of SRA01 / 04 cells was not related to the infection of SIRT6 with the empty plasmid.

[0065] The results showed that the results of western blotting and immunofluorescence techniques showed that SIRT6 protein was localized in the cell nucleus. Compared with the CON group and LV-si-CON group, the expression level of SIRT6 in the LV-si-SIRT6 group was significantly decreased (P < 0.05), confirming that the SIRT6 interference lentiviral stable SRA01 / 04 cell line was successfully constructed; at the same time, the CCK-8 results showed that compared with the LV-si-CON group and the CON group, the cell proliferation activity in the LV-si-SIRT6 group was significantly decreased (P < 0.05) ( Fig.15 ); Western blotting results showed that compared with the LV-si-CON and CON groups, the expressions of LC3A / B, Beclin1, Atg5, Atg12, p-AMPK / t-AMPK, and Bcl-2 proteins in the LV-si-SIRT6 group were significantly downregulated (all P < 0.05), and the expressions of p62, p-mTOR / t-mTOR, p-Akt / t-Akt, p-MAPK / t-MAPK, p-p53 / t-p53, and Bax were significantly upregulated (all P < 0.05). Figure 16-18 ).

[0066] The above examples show that autophagy may play a positive role in diabetic complications, and SIRT6 can protect cells by activating autophagy. High glucose intervention in SRA01 / 04 cells reduces the expression of SIRT6, promotes apoptosis and inhibits its autophagy level. SIRT6 overexpression lentivirus does not affect the proliferation activity of normal lens epithelial cells, but can increase the proliferation activity of lens epithelial cells under high glucose intervention.

[0067] At the same time, this study showed that the LC3Ⅱ / Ⅰ(A / B) of SRA01 / 04 cells in the high glucose group decreased, and overexpression of SIRT6 could reverse the changes of LC3, suggesting that SIRT6 could upregulate the conversion of LC3Ⅰ to LC3Ⅱ in SRA01 / 04 cells treated with high glucose ( Figure 7 A).

[0068] This study showed that p62 expression in the high glucose group was upregulated, and overexpression of SIRT6 could reverse the changes in p62, suggesting that SIRT6 could downregulate the expression of p62 in SRA01 / 04 cells treated with high glucose ( Figure 7 B).

[0069] The experimental results showed that Beclin1 in SRA01 / 04 cells in the high glucose group was reduced, and overexpression of SIRT6 could reverse the changes in Beclin1, suggesting that SIRT6 could upregulate the expression of Beclin1 in SRA01 / 04 cells treated with high glucose ( Figure 7 C).

[0070] This study showed that the expression of Atg5 and Atg12 in the high glucose group was downregulated, and overexpression of SIRT6 could reverse the changes of Atg5 and Atg12, suggesting that SIRT6 could upregulate the expression of Atg5 and Atg12 in SRA01 / 04 cells treated with high glucose ( Figure 8 ).

[0071] This study showed that the expression of activated mTOR in the high glucose group was upregulated, and overexpression of SIRT6 could reverse the changes in activated mTOR, suggesting that SIRT6 could downregulate the expression of activated mTOR in SRA01 / 04 cells treated with high glucose and promote autophagy ( Fig. 9 ).

[0072] The experimental results showed that Akt expression was upregulated in the high glucose group, and overexpression of SIRT6 could reverse the expression of Akt, suggesting that SIRT6 could downregulate the expression of Akt in SRA01 / 04 cells treated with high glucose and promote autophagy ( Fig.10 ).

[0073] This study showed that AMPK expression was downregulated in the high glucose group, and overexpression of SIRT6 could reverse the expression of AMPK, suggesting that SIRT6 could upregulate the expression of AMPK in SRA01 / 04 cells treated with high glucose and activate autophagy ( Fig.11 ).

[0074] The experimental results showed that the expression of MAPK in the high glucose group was upregulated, and overexpression of SIRT6 could reverse the expression of MAPK, suggesting that SIRT6 could downregulate the expression of MAPK in SRA01 / 04 cells treated with high glucose and promote autophagy ( Fig.12 ).

[0075] The experimental results showed that the expression of phosphorylated p53 in the high glucose group was upregulated, and overexpression of SIRT6 could reverse the changes in phosphorylated p53, suggesting that SIRT6 could downregulate the expression of phosphorylated p53 in SRA01 / 04 cells treated with high glucose ( Fig.13 ).

[0076] This study showed that Bcl-2 expression was downregulated and Bax expression was upregulated in the high glucose group, and overexpression of SIRT6 could reverse the expression of Bcl-2 and Bax ( Fig.14 ).

[0077] In conclusion, SIRT6 in SRA01 / 04 cells treated with high concentrations of glucose can promote the expression of autophagy-related factors and affect cell apoptosis, thereby increasing the proliferation activity of SRA01 / 04 cells and protecting their normal metabolism. This suggests that SIRT6 may play a role in alleviating the progression of diabetic cataracts and can be used as a therapeutic target for diabetic cataracts.

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention. Sequence Listing <110> Ningxia Medical University <120> Application of SIRT6 protein as a marker for detecting diabetic cataract and preparing drugs for preventing and treating diabetic cataract <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 355 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Val Asn Tyr Ala Ala Gly Leu Ser Pro Tyr Ala Asp Lys Gly 1 5 10 15 Lys Cys Gly Leu Pro Glu Ile Phe Asp Pro Pro Glu Glu Leu Glu Arg 20 25 30 Lys Val Trp Glu Leu Ala Arg Leu Val Trp Gln Ser Ser Ser Val Val 35 40 45 Phe His Thr Gly Ala Gly Ile Ser Thr Ala Ser Gly Ile Pro Asp Phe 50 55 60 Arg Gly Pro His Gly Val Trp Thr Met Glu Glu Arg Gly Leu Ala Pro 65 70 75 80 Lys Phe Asp Thr Thr Phe Glu Ser Ala Arg Pro Thr Gln Thr His Met 85 90 95 Ala Leu Val Gln Leu Glu Arg Val Gly Leu Leu Arg Phe Leu Val Ser 100 105 110 Gln Asn Val Asp Gly Leu His Val Arg Ser Gly Phe Pro Arg Asp Lys 115 120 125 Leu Ala Glu Leu His Gly Asn Met Phe Val Glu Glu Cys Ala Lys Cys 130 135 140 Lys Thr Gln Tyr Val Arg Asp Thr Val Val Gly Thr Met Gly Leu Lys 145 150 155 160 Ala Thr Gly Arg Leu Cys Thr Val Ala Lys Ala Arg Gly Leu Arg Ala 165 170 175 Cys Arg Gly Glu Leu Arg Asp Thr Ile Leu Asp Trp Glu Asp Ser Leu 180 185 190 Pro Asp Arg Asp Leu Ala Leu Ala Asp Glu Ala Ser Arg Asn Ala Asp 195 200 205 Leu Ser Ile Thr Leu Gly Thr Ser Leu Gln Ile Arg Pro Ser Gly Asn 210 215 220 Leu Pro Leu Ala Thr Lys Arg Arg Gly Gly Arg Leu Val Ile Val Asn 225 230 235 240 Leu Gln Pro Thr Lys His Asp Arg His Ala Asp Leu Arg Ile His Gly 245 250 255 Tyr Val Asp Glu Val Met Thr Arg Leu Met Lys His Leu Gly Leu Glu 260 265 270 Ile Pro Ala Trp Asp Gly Pro Arg Val Leu Glu Arg Ala Leu Pro Pro 275 280 285 Leu Pro Arg Pro Pro Thr Pro Lys Leu Glu Pro Lys Glu Glu Ser Pro 290 295 300 Thr Arg Ile Asn Gly Ser Ile Pro Ala Gly Pro Lys Gln Glu Pro Cys 305 310 315 320 Ala Gln His Asn Gly Ser Glu Pro Ala Ser Pro Lys Arg Glu Arg Pro 325 330 335 Thr Ser Pro Ala Pro His Arg Pro Pro Lys Arg Val Lys Ala Lys Ala 340 345 350 Val Pro Ser 355 <210> 2 <211> 1068 <212> DNA <213> Artificial Sequence <400> 2 atgtcggtga attacgcggc ggggctgtcg ccgtacgcgg acaagggcaa gtgcggcctc 60 ccggagatct tcgacccccc ggaggagctg gagcggaagg tgtgggaact ggcgaggctg 120 gtctggcagt cttccagtgt ggtgttccac acgggtgccg gcatcagcac tgcctctggc 180 atccccgact tcaggggtcc ccacggagtc tggaccatgg aggagcgagg tctggccccc 240 aagttcgaca ccacctttga gagcgcgcgg cccacgcaga cccacatggc gctggtgcag 300 ctggagcgcg tgggcctcct ccgcttcctg gtcagccaga acgtggacgg gctccatgtg 360 cgctcaggct tccccaggga caaactggca gagctccacg ggaacatgtt tgtggaagaa 420 tgtgccaagt gtaagacgca gtacgtccga gacacagtcg tgggcaccat gggcctgaag 480 gccacgggcc ggctctgcac cgtggctaag gcaagggggc tgcgagcctg caggggagag 540 ctgagggaca ccatcctaga ctgggaggac tccctgcccg accgggacct ggcactcgcc 600 gatgaggcca gcaggaacgc cgacctgtcc atcacgctgg gtacatcgct gcagatccgg 660 cccagcggga acctgccgct ggctaccaag cgccggggag gccgcctggt catcgtcaac 720 ctgcagccca ccaagcacga ccgccatgct gacctccgca tccatggcta cgttgacgag 780 gtcatgaccc ggctcatgaa gcacctgggg ctggagatcc ccgcctggga cggcccccgt 840 gtgctggaga gggcgctgcc acccctgccc cgcccgccca cccccaagct ggagcccaag 900 gaggaatctc ccacccggat caacggctct atccccgccg gccccaagca ggagccctgc 960 gcccagcaca acggctcaga gcccgccagc cccaaacggg agcggcccac cagccctgcc 1020 ccccacagac cccccaaaag ggtgaaggcc aaggcggtcc ccagctga 1068 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ggaggagctg ggaggagg <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 tgaagtcggg gatgccagag g

Claims

1. Use of an agent for overexpressing SIRT6 protein in diabetic cataract target cells in the preparation of a drug for preventing and treating diabetic cataract.

2. The use according to claim 1, characterized in that: The diabetic cataract target cells include lens epithelial cells.

3. The use according to claim 2, characterized in that: The lens epithelial cells include SRA01 / 04 cells.

4. Use of an agent that causes SIRT6 protein to be overexpressed in diabetic cataract lenses in the preparation of a drug for promoting the expression of autophagy factors in epithelial cells in diabetic cataract lenses and / or increasing the proliferation activity of lens epithelial cells.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of the SIRT6 protein is shown in SEQ ID No.

1.

6. The use according to claim 5, characterized in that: The nucleotide sequence encoding the SIRT6 protein is shown in SEQ ID No.2.

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