CN115998846B - Application of SMIM26 protein and SLC25A11 protein in renal cancer - Google Patents

Abstract

The present invention discloses the application of SMIM26 protein and SLC25A11 protein in renal cancer. In the present invention, experiments have shown that the mitochondrial SMIM26 protein encoded by lncRNA is related to renal cancer. The SMIM26 protein can effectively inhibit the growth, proliferation, migration and invasion of renal cancer cells, and can be used as a tumor suppressor protein to develop drugs for treating renal cancer or renal cancer metastasis. The present invention also found that overexpression of SMIM26 protein enhances the mitochondrial function of renal cancer cells, while interfering with SLC25A11 protein inhibits the function of mitochondria. Therefore, mitochondrial homeostasis can be regulated by the interaction between SMIM26 and SLC25A11, thereby regulating renal cancer metastasis.

Description

Application of SMIM26 protein and SLC25A11 protein in renal cancer

Technical Field

The present invention belongs to the field of biomedicine technology, and particularly relates to the application of SMIM26 protein and SLC25A11 protein in renal cancer.

Background Art

Renal cancer is a malignant tumor that seriously endangers human life safety. In recent years, the incidence of renal cancer has shown an increasing trend year by year. Since the early symptoms of renal cancer are not very obvious, 30% of patients have reached the late stage when they develop the disease, thus missing the best treatment time for renal cancer. Local renal cancer can be treated by resection or radical nephrectomy, cryoablation. Although nephrectomy has a significant effect on the treatment of local renal cancer, about 30% of renal cancer patients will develop metastatic lesions, which requires systemic treatment and is accompanied by a high mortality rate, and the 5-year survival rate of patients with metastatic renal cancer is less than 10%. At present, the drug treatment for renal cancer is mainly targeted therapy for vascular endothelial growth factor (VEGF) and mTOR pathways, but the treatment effect is still not very ideal. The occurrence of chemotherapy resistance and toxic side effects affects the prognosis of cancer patients. Therefore, it is imperative to further develop new oncogene targeted drugs.

Renal cancer is a disease with severe metabolic disorders, and mitochondrial proteins actively participate in the malignant transformation of renal cancer. In renal cancer, the mitochondrial genome often mutates, but these mutations do not lead to the inactivation of mitochondrial energy metabolism, but cause changes in mitochondrial bioenergetics. These changes will be transmitted to the nucleus through reverse signal transduction, and through a series of signal transduction mechanisms, the metabolism of tumor cells will be reprogrammed to balance the rapid proliferation and metastasis of tumor cells. The demand for energy and metabolic intermediates. Therefore, given the close connection between mitochondrial dysfunction and tumor deterioration, more and more studies have begun to focus on targeting and restoring the stable state of mitochondria as a strategy for cancer control and treatment. At present, there are many drugs targeting mitochondrial metabolism-related pathways. By specifically blocking the aerobic glycolysis pathway and restoring mitochondrial oxidative phosphorylation (OXPHOS), it has been proven that they have a relatively good therapeutic effect on the treatment of various cancers in vitro and in vivo. However, the relevant drugs targeting mitochondrial pathways are still very limited. One of the main reasons is that the regulatory factors related to mitochondrial metabolic regulation have not yet been fully discovered.

With the rapid development of sequencing technology and proteomics technology, more and more research reports show that some of the non-coding genes that account for about 98% of the human genome can encode new proteins. These new proteins are usually translated from small open reading frames (sORFs) on lncRNAs, have small molecular weights, and play a very important regulatory role in tumor biology. Proteomics mass spectrometry technology is considered an important technical means to identify proteins, and shotgun mass spectrometry (shotgun MS) is the most direct method for discovering new proteins. Translationomics (RNC-seq and Ribo-seq) can discover RNA with translation function in cells on a large scale, making up for the lack of new protein databases in mass spectrometry technology. Therefore, the combined use of proteomics and translationomics technologies provides a basis for discovering new coding proteins.

SMIM26 (Small Integral Membrane Protein 26) is a protein encoded by LINC00493 and localized in mitochondria, but there are still no reports on the function of SMIM26 protein in the human body and its relationship with tumors.

Summary of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide the use of SMIM26 protein in the preparation of a drug for treating renal cancer and/or a drug for preventing renal cancer metastasis.

Another object of the present invention is to provide a use of SMIM26 protein and SLC25A11 protein in combination for preparing a drug for treating renal cancer and/or a drug for preventing renal cancer metastasis.

The purpose of the present invention is achieved through the following technical solutions:

Use of SMIM26 protein (as a drug target) in preparing drugs for treating renal cancer and/or drugs for preventing renal cancer metastasis.

The coding gene sequence of the SMIM26 protein is shown in SEQ ID NO.1.

The treatment of renal cancer or the prevention of renal cancer metastasis is achieved by overexpressing SMIM26 protein and/or its mRNA; overexpressing SMIM26 protein and/or its mRNA can inhibit the proliferation and growth of renal cancer cells (inhibit the cloning of renal cancer cells), and can inhibit the migration and invasion ability of renal cancer cells.

The overexpression of SMIM26 protein is preferably achieved by the following method:

(1) The coding gene sequence of SMIM26 protein as shown in SEQ ID NO.1 and the pLVX-Puro Vector plasmid were digested with restriction endonucleases EcoR I and BamH I, and then ligated with T4 ligase to obtain the recombinant plasmid pLVX-Puro-SMIM26-flag;

(2) The recombinant plasmid pLVX-Puro-SMIM26-flag and the packaging plasmid were transfected into 293T cells, and then the supernatant of the transfected cells was taken out, centrifuged and filtered to remove cell debris, and then infected with renal cancer cells. After resistance screening, a SMIM26 overexpressing cell line was obtained.

The resistance screening described in step (2) is performed by screening with 2 μg/mL of puromycin.

The renal cancer cells described in step (2) are human renal cancer cells; preferably human renal cancer cells ACHN and/or human renal clear cell adenocarcinoma cells 769-P.

The administration method of the anti-renal cancer metastasis drug is preferably subcutaneous injection or intravenous injection.

Use of a reagent for detecting the expression level of SMIM26 protein in preparing a product for diagnosing renal cancer metastasis.

Application of SMIM26 protein and SLC25A11 protein in combination in the preparation of a drug for treating renal cancer and/or a drug for preventing renal cancer metastasis.

The coding gene sequence of the SMIM26 protein is shown in SEQ ID NO.1, and the accession number of the coding gene sequence of the SLC25A11 protein in uniprot (protein database) is: Q02978 (https://www.uniprot.org/uniprotkb/Q02978/entry).

The treatment of renal cancer or the prevention of renal cancer metastasis is achieved by overexpressing SMIM26 protein and/or its mRNA, and interfering with SLC25A11 protein; overexpressing SMIM26 protein and/or its mRNA can enhance the mitochondrial function of renal cancer cells, while interfering with SLC25A11 can inhibit the function of mitochondria, that is, SMIM26 regulates mitochondrial homeostasis by interacting with SLC25A11, thereby regulating renal cancer metastasis.

The interference with the SLC25A11 protein is achieved by introducing a SLC25A11 interference fragment; wherein the sequence of the SLC25A11 interference fragment is preferably as shown in SEQ ID NO.2-5.

The anti-renal cancer metastasis drug includes a drug for regulating the mitochondrial homeostasis (stable state) of renal cancer cells.

Compared with the prior art, the present invention has the following advantages and effects:

1. The present invention discovered for the first time that the mitochondrial SMIM26 protein encoded by lncRNA is related to the metastasis of renal cancer. SMIM26 interacts with SLC25A11 to maintain mitochondrial homeostasis and inhibit the AKT pathway. Therefore, SMIM26 can be used as an effective drug target for the clinical diagnosis of renal cancer.

2. The present invention found that SMIM26 protein can effectively inhibit the proliferation and growth of renal cancer and can be used as a tumor suppressor protein to provide new support for the development of drugs for the clinical treatment of renal cancer.

3. The present invention found that the migration and invasion abilities of the cell lines overexpressing SMIM26 were significantly weaker than those of the control group, indicating that SMIM26 inhibited the metastatic ability of renal cancer cells in the form of protein. Therefore, SMIM26 protein can be used to prevent renal cancer metastasis.

4. The present invention discovered that SMIM26 protein can regulate tumor metastasis: by regulating the expression of SMIM26 mRNA or protein, tumor metastasis can be regulated; or SMIM26 can regulate tumor metastasis by interacting with SLC25A11 and regulating mitochondrial homeostasis; SMIM26 regulates mitochondrial homeostasis through SLC25A11 and can serve as a target for regulating cancer cell metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing the effect of SMIM26 protein on the growth, migration and invasion of renal cancer cells; wherein A is a lncRNA in RNA form that constructs SMIM26 protein and mutates the start codon and stop codon; B is the successful overexpression of SMIM26 protein in renal cancer cells; C is the inhibition of renal cancer cell cloning by overexpression of SMIM26 protein; D is the inhibition of renal cancer cell migration and invasion by overexpression of SMIM26 protein; E is the inhibition of renal cancer cell growth in mice by overexpression of SMIM26 protein; F is the inhibition of renal cancer growth in mice by overexpression of SMIM26; and G is the inhibition of renal cancer cell metastasis in mice by overexpression of SMIM26.

FIG2 is a diagram showing the results of a protein immunoblotting experiment of the interaction between SMIM26 and SLC25A11; wherein A is SMIM26 immunoprecipitated with SLC25A11; and B is SLC25A11 immunoprecipitated with SMIM26.

Figure 3 is a diagram showing the effects of SMIM26 and SLC25A11 on mitochondrial oxygen consumption rate; wherein A shows that overexpression of SMIM26 in ACHN cells enhances the oxygen consumption rate (interference with SLC25A11 inhibits the increase in oxygen consumption rate); B shows that overexpression of SMIM26 in 769-P cells enhances the oxygen consumption rate (interference with SLC25A11 inhibits the increase in oxygen consumption rate).

Figure 4 is a diagram showing the effects of SMIM26 and SLC25A11 on the migration and invasion of renal cancer cells; (A) and (C) show the effects of SMIM26 and SLC25A11 on the migration and invasion of ACHN cells; (B) and (D) show the effects of SMIM26 and SLC25A11 on the migration and invasion of 769-P cells.

DETAILED DESCRIPTION

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

The plasmid construction-related reagents in the present invention are as follows: DNA polymerase (PrimeSTAR Max DNA Polymerase), QuickCut ^™ EcoR I and QuickCut ^™ BamH I restriction endonucleases, and T4 DNA ligase were all purchased from TAKARA.

Example 1 Plasmid Construction

The recombinant plasmids pLVX-Puro-SMIM26-flag and pLVX-Puro-Mut were constructed on the backbone of pLVX-Puro Vector (Addgene). The specific steps are as follows:

1. Design primers: Design corresponding primers based on the sequences of SMIM26 (NM_001348957.2) and LINC00493 (as a control, i.e., based on SMIM26, the start codon ATG was mutated to GGA, and the stop codon TAA was mutated to GCT), where:

The SMIM26 gene sequence (SEQ ID NO.1) is as follows:

ATGTATCGAAATGAGTTCACGGCCTGGTACCGGCGGATGTCGGTGGTCTACGGGATCGGCACCTGGTCTGTGTTGGGCTCACTGCTTTACTATAGCCGGACAATGGCGAAGTCGTCAGTAGACCAAAAGGATGGCTCAGCAAGTGAAGTACCCAGTGAACTCTCTGAACGCCCAAAAGGATTTTATGTGGAAACAGTTGTCACATATAAAGAAGATTTTGTTCCAAATACAGAAAAGATCCTCAACTATTGGAAATCATGGACT GGTGGCCCTGGTACAGAACCATAA.

The primer sequences of pLVX-Puro-SMIM26-flag (experimental group) are as follows:

Upstream primer F1: 5′-GCGAATTCATGTATCGAAATGAGTTCACGG-3′ (SEQ ID NO. 6);

Downstream primer R1: 5′-GCGGATCCTTACTTATCGTCGTCATCCTTGTAATCTGGTTCTGTACCAGGGCCAC-3′ (SEQ ID NO. 7).

The primer sequences of pLVX-Puro-Mut (control group) are as follows:

Upstream primer F2: 5′-GCGAATTCGGATATCGAAATGAGTTCACGG-3′ (SEQ ID NO. 8);

Downstream primer R2: 5′-GCGGATCCAGCCTTATCGTCGTCATCCTTGTAATCTGGTTCTGTACCAGGGCCAC-3′ (SEQ ID NO. 9).

2. Preparation of cDNA template: Total RNA was extracted from human renal cancer ACHN cells (purchased from the cell bank of the Chinese Academy of Sciences), and its reverse transcription product was used as a cDNA template.

3. PCR amplification: using the above upstream and downstream primers (F1 and R1, F2 and R2), respectively, and cDNA as a template, PCR amplification is performed to obtain PCR products; wherein:

The PCR system is:

PrimeSTAR Max DNA Polymerase 10μL cDNA template 2μL Upstream primer 1μL Downstream primer 1μL Sterile pure water 6μL

The PCR program is:

Step 1: 95°C, 5 minutes; Step 2: 56°C, 30 seconds; Step 3: 72°C, 30 seconds; Step 4: 72°C, 10 minutes; Step 5: 4°C, 30 minutes; set 30 cycles from step 2 to step 4.

4. Gel running and gel cutting: The PCR product is subjected to agarose gel electrophoresis, and the gel is cut and purified according to the theoretical position of the target fragment and the actual gel running position;

5. Enzyme digestion: The PCR product purified and recovered in step 4 and the plasmid pLVX-Puro Vector were subjected to restriction endonuclease digestion reaction respectively, placed in a 37°C metal bath for 15 minutes, and purified and recovered; wherein the enzyme digestion system is shown in Table 1;

Table 1 Enzyme digestion system

PCR product or vector 1 μg Upstream endonuclease EcoR I 2μL Downstream endonuclease BamH I 2μL Enzyme digestion buffer (10×) 2μL Sterile water to 20μL

6. Ligation: After purification and recovery, calculate the actual mass of plasmid and PCR product to be added according to the molar ratio, i.e., M _empty : M _product ≈ 1:6 (the amount of plasmid is controlled at 100 μg ± 50) (corresponding to X and Y in Table 2), prepare the ligation system according to the system in Table 2, and ligate at 16°C for 16 h;

Table 2 Connection system

Carrier XμL Target gene YμL T4 DNA Ligase 2μL T4 DNA Ligation Buffer (10×) 2μL Sterile water to 20μL

9. Transformation: Gently mix the ligation product with 50 μL of E. coli DH5α competent cells, and place on ice for 10 min; incubate in a 42°C water bath for 90 s, and place on ice for 10 min; add 400 μL of LB medium, shake at 37°C for 45 min; take 200 μL of bacterial solution and plate;

10. Pick single clones: Select 5 single clones and inoculate them into a 12-well plate in LB medium supplemented with 100 mg/L ampicillin antibiotics, and shake the culture at 37°C for 4-5 hours;

11. Colony PCR: Use bacterial solution as template and upstream and downstream primers (F1 and R1, F2 and R2) of target gene as colony PCR primers to perform PCR amplification, and check whether it meets expectations by gel electrophoresis;

12. Extract plasmids and send them for sequencing: The positive clones were expanded and cultured, and a portion of them were extracted with the instructions in the plasmid extraction kit, and sent to the company for bidirectional sequencing using the universal primers on the vector; the other portion was preserved in a -80°C liquid nitrogen tank to obtain the recombinant plasmid pLVX-Puro-SMIM26-flag and the control plasmid pLVX-Puro-Mut. A simple schematic diagram of the recombinant plasmid pLVX-Puro-SMIM26-flag and the control plasmid pLVX-Puro-Mut is shown in Figure 1A.

Example 2 Construction of stable cell lines using lentivirus

(1) Plating 293T cells: Plating 293T cells (purchased from ATCC) in a six-well plate so that each well contains about 80% of the cells;

(2) 1 μg of each of the pLVX-Puro-SMIM26-flag, pLVX-Puro-Mut plasmid and pLVX-PuroVector plasmid constructed in Example 1 and the packaging plasmid pMD2.G and psPAX2 (purchased from Quanyang Biotechnology Co., Ltd.) were transfected into the cell dish using Lip3000 (thermo) at 4 μL/tube;

(3) 48 h after transfection, 3 mL of cell supernatant was taken out and centrifuged at 300 g to remove cell debris;

(4) Infecting target cells ACHN and 769-P (purchased from the Chinese Academy of Sciences Cell Bank): Filter the virus obtained in step (3) with a 0.45 μM filter to remove cell debris, etc., and then add it to the target cells ACHN and 769-P respectively;

(5) Resistance screening: After two days of infection, the medium was changed, and then 2 μg/mL of puromycin was used to screen the renal cancer ACHN and 769-P cell lines until there was no significant change in the cell number, and then the drug was withdrawn;

(6) Western blotting to detect overexpression or interference effects: 3 days after drug withdrawal, cells were collected and Western blotting was performed to verify overexpression or interference effects;

(7) The successfully constructed SMIM26 overexpression cell line, control cell line (Mut), and blank vector control cell line (NC) were frozen and stored in a -80°C liquid nitrogen tank.

(8) Western blot was used to detect the overexpression effect of SMIM26 protein. The results are shown in Figure 1B: SMIM26 protein was successfully overexpressed in the SMIM26 overexpressing cell line, and there was no overexpression effect in the control cell line (Mut) and the blank vector control cell line (NC).

Example 3 Experiment on the inhibition of SMIM26 protein on renal cancer cell clone formation, migration and invasion

(1) Clone formation experiment: The SMIM26 overexpressing renal cancer cell line, control cell line (Mut), and empty vector control cell line (NC) constructed in Example 2 were digested into 1.5 mL centrifuge tubes, and the cell number was counted using a hemocytometer. 1000 cells were taken and plated in a 6-well plate, and 2 ml of DMEM medium (Thermo) was added. The number of tumor cell clones was observed after 10 days. This was repeated three times.

(2) Migration experiment: SMIM26 overexpressing renal cancer cell lines, control cell lines (Mut), and empty vector control cell lines (NC) were digested into 1.5 mL centrifuge tubes, and the supernatant was removed after centrifugation. The cells were resuspended in DMEM medium without serum (Thermo Company), mixed, and then the cells were aspirated and added to a cell counting slide for counting. After counting, 150,000 cells per well were taken into a new Ep tube, centrifuged, and resuspended in 200 μL of DMEM medium without serum per well. After the cells were mixed, they were added to the upper chamber of the chamber respectively. After adding 600 μL of DMEM complete medium to the lower chamber, the 24-well plate was placed in a cell culture incubator and incubated to wait for cell migration. The chamber was collected according to the migration ability of the cell line. In this experiment, the most suitable migration time for the ACHN cell line was about 9 to 11 hours, and that for the 769-P cell line was about 3 to 4 hours. When the most suitable migration time was reached, the chamber was taken out from the clean bench and placed in a 24-well plate containing anhydrous methanol for fixation for 10 minutes. After fixation, stain with crystal violet for 10 min, then rinse the crystal violet in pure water and gently wipe off the cells in the upper chamber with a cotton swab. After the chamber is inverted to dry, take pictures under a microscope and count using Image J software. Repeat three times.

(3) Invasion assay: The invasion assay uses Martrigal matrix gel to simulate the extracellular matrix environment in vivo. Cells can only pass through the matrix gel after secreting proteases to dissolve the matrix gel. Based on this principle, the invasion ability of cells is evaluated. Martrigal matrix gel and DMEM medium without serum are diluted on ice at a volume ratio of 1:19. 100 μL is drawn with a pre-cooled pipette tip and added to the upper chamber of the chamber without generating bubbles. The 24-well plate is placed in a cell culture incubator and allowed to stand for 1 hour to solidify. After solidification, the operation is the same as the above migration assay. In this experiment, the most suitable invasion time for the ACHN cell line is about 32 to 34 hours, and for the 769-P cell line is about 16 to 17 hours.

The results are shown in Figures 1C and 1D: The clone formation ability (Figure 1C), cell migration and invasion ability (Figure 1D) of ACHN and 769-P cell lines overexpressing SMIM26 instead of LINC00493 (Mut) were significantly weaker than those of the control group (NC), indicating that SMIM26 inhibits the metastasis ability of renal cancer cells in the form of protein and is a potential protein drug with hope for clinical treatment.

Example 4 In vivo animal experiment

Twenty 6-week-old male NOD-SCID mice (the male nude mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) were selected, and the SMIM26 overexpression ACHN cell line constructed in Example 2 and the empty vector control ACHN cell line were used to construct a tumor subcutaneous growth model and a tail vein metastasis model. The specific steps are as follows:

(1) Subcutaneous growth model:

① 5 million empty vector control ACHN renal cancer cells (NC) were inoculated subcutaneously in 10 mice, and 5 million corresponding SMIM26-overexpressing ACHN renal cancer cells were inoculated subcutaneously in another 10 mice;

②Observe the growth of mice and remove subcutaneous tumors after 4 weeks for follow-up research.

(2) Tail vein metastasis model:

① Before the experiment, the NOD-SCID mice were anesthetized, and the degree of anesthesia was assessed by painless and painful stimulation to ensure that the mice were in an anesthetized state;

②10 NOD-SCID mice were injected with 10 ⁶ empty vector control ACHN renal cancer cells (NC), and the other 10 mice were injected with 10 ⁶ corresponding SMIM26 overexpressing ACHN cells. The resuspended cells were injected into the tail vein of nude mice using a microsyringe with a 25G needle.

③Observe the growth of mice and remove the tumors on day 35 for follow-up research.

The results are shown in Figures 1E, 1F and 1G: Figures 1E and 1F show that the growth of renal cancer cells overexpressing SMIM26 in nude mice was significantly inhibited. Figure 1G shows that the metastatic ability of renal cancer cells overexpressing SMIM26 in NOD-SCID immunodeficient mice was inhibited. This indicates that overexpression of SMIM26 can inhibit the subcutaneous growth ability of renal cancer cells in nude mice and the metastatic ability of mice, indicating that SMIM26 is a biomarker related to renal cancer metastasis and has clinical application value for renal cancer.

Example 5 Interaction between SMIM26 and SLC25A11

1. Co-immunoprecipitation experiment:

The pLVX-Puro-SMIM26-flag plasmid constructed in Example 1 was transfected into the cells, and the cell lysate was subjected to immunoprecipitation experiment (three replicates were set) 48 hours after transfection. The specific steps are as follows:

(1) pLVX-Puro-SMIM26-flag (1.5 μg) was overexpressed in 293T cells (purchased from ATCC) and transfected for 48 h;

(2) Cell lysis: Wash three times with pre-cooled PBS, add an appropriate amount of IP lysis buffer (Biyuntian, P0013), lyse on ice for 30 minutes, gently invert and mix every 10 minutes. Centrifuge at 4°C, 13200 rpm, 30 minutes, and collect the supernatant;

(3) Pre-washing lysate: Measure the concentration using the BCA method, take 1 mg of protein, add 2.5 μg of IgG antibody and 30 μL of ProteinA/G beads (Santa Cruz, Cat#sc-2003), and then incubate in a rotating mixer at 4°C for 30 min; centrifuge at 2500 rpm at 4°C for 5 min, and take the supernatant;

(4) Pre-wash the lysate again: add 30 μL of Protein A/G beads, centrifuge at 2500 rpm for 5 min at 4°C, and take the supernatant;

(5) Antibody incubation: Add 2.5 μg of flag antibody (Proteintech, Cat#20543-1-AP) and incubate at 4°C in a rotating mixer for 16 h;

(6) Incubate beads: add 40 μL of Protein A/G beads and incubate at 4°C in a rotating mixer for 4 h;

(7) Removal of impurities: Centrifuge at 4°C, 2500 rpm for 5 min and discard the supernatant; Gently resuspend the beads in 1 mL of IP lysis buffer, centrifuge at 4°C, 2500 rpm for 5 min and discard the supernatant;

(7) Remove impurities again: Gently resuspend the beads in 1 mL IP lysis buffer, centrifuge at 2500 rpm for 5 min at 4°C, remove the supernatant, and repeat twice;

(8) Sample preparation: Add 20 μL of 1× protein loading buffer to resuspend the beads, mix well, and boil in a boiling water bath for 10 min; the samples were used for protein immunoblotting experiments.

2. Western Blotting

(1) Selecting a separation gel of appropriate solubility according to the molecular weight of the protein to be detected and preparing the separation gel;

(2) Take 20ul of cell lysate, add 5ul of 5× loading buffer (Fuder Biotech), boil in boiling water for 5min, place on ice for 2min, centrifuge for 10s to allow the liquid on the side wall to return to the bottom of the EP tube, and prepare for sample loading;

(3) Assemble the electrophoresis apparatus, add Running Buffer, load the sample and pre-stained protein marker;

(4) After loading, use 70-80V voltage for the concentration gel stage and 80-120V voltage for the separation gel stage until the bromophenol blue band approaches the end, and then end the electrophoresis; cut the PVDF membrane to the corresponding size according to the size of the gel strip;

(5) Activate the PVDF membrane by soaking it in methanol until it changes color, and arrange it in the order of filter paper-gel-PVDF membrane-filter paper. Connect the transfer power supply and transfer the membrane on ice at a constant current of 235 mA for 60 min.

(6) After transfer, remove the PVDF membrane, rinse with TBST buffer to remove the residual transfer buffer, remove the transfer buffer, and then block with 5% (v/v) skim milk at room temperature for 2 h;

(7) After blocking, rinse with TBST buffer to remove residual milk, cut the membrane according to the protein marker, add the corresponding primary antibody (Proteintech, Cat#12253-1-AP), and incubate at 4°C overnight (12-16 h);

(8) Recover the primary antibody, wash the membrane with TBST buffer for 30 min, change the buffer every ten minutes, add rabbit secondary antibody (Proteintech, Cat#SA00001-2), and incubate at room temperature for 2 h;

(9) Recover the secondary antibody and wash the membrane with TBST buffer for 30 min, changing the buffer every ten minutes;

(10) Reasonably control the exposure time and development, crop the images using Photoshop, and calculate the grayscale value using ImageJ.

The experimental results are shown in Figure 2: The results showed that SMIM26 protein and SLC25A11 protein had a significant interaction, suggesting that SMIM26 regulates renal cancer cell metastasis by interacting with SLC25A11.

Example 6 Effects of SMIM26 and SLC25A11 on Mitochondrial Oxygen Consumption Rate

First, pLVX-Puro Vector, pLVX-Puro-SMIM26-flag, pLVX-Puro Vector and SLC25A11 interference fragment, pLVX-Puro-SMIM26-flag and SLC25A11 interference fragment were transfected into renal cancer cells (ACHN and 769-P), respectively (the sequences of the interference fragments were: #1 upstream F: 5'-CCCGCCUUGGCAUCUAUACTT-3' (SEQ ID NO.2); #1 downstream R: 5'-GUAUAGAUGCCAAGGCGGGUA-3' (SEQ ID NO.3); #2 upstream F: 5'-CAAUCCAAGCAGUUCUUACTT-3' (SEQ ID NO.4); #2 downstream R: 5'-GUAAGAACUGCUUGGAUUGGG-3' (SEQ ID NO.5)), then a cell energy metabolism analyzer (Agilent, USA) was used to detect the oxygen consumption rate of the mitochondria of the renal cancer cells. The experiment was repeated three times. The specific steps are as follows:

(1) Hydrate the probe plate. Add XF calibration solution (1 ml/well) to the wells of the XF24 Utility plate (Agilent, USA). Cover with Hydro Booster. Then carefully place the Sensor Cartridge with probes back on the Utility plate, ensuring that all probes are completely immersed in the liquid. Finally, cover with the Cartridge lid and place in a 37°C incubator overnight.

(2) Turn on the Seahorse instrument and computer, wait for the instrument to gradually heat up to 37℃ and keep preheating. Prepare an appropriate amount of Cell-Tak working solution, immediately add it evenly to the XF24 cell culture plate (50μl/well), place it at room temperature for 20 minutes, then aspirate it, wash it twice with sterile water, and place it at room temperature for use. Collect renal cancer cells in the logarithmic growth phase by trypsin digestion, plate them on the Cell-Tak-coated XF 24 cell culture plate, and place them in an incubator at 37℃ for 25-30 minutes. Add 400μl XF detection solution and place them in an incubator at 37℃ for 25-30 minutes. Dilute the stock solutions of oligomycin, FCCP, and rotenone/antimycin A (stock solution concentration is 100 micromolar) 10 times respectively; remove the probe card, remove the Hydro Booster, and carefully add the above-prepared working solution to the drug addition well of the Sensor Cartridg. After the calibration is completed, the software automatically ejects the Utility Plate and replaces it with the Cell Plate, after which the software automatically runs the Cell OCR real-time measurement program.

The experimental results are shown in Figure 3: The results show that overexpression of SMIM26 enhances the mitochondrial function of renal cancer cells, while interference with SLC25A11 inhibits mitochondrial function, suggesting that SMIM26 regulates mitochondrial homeostasis in renal cancer cells through interaction with SLC25A11.

Example 7 Effects of SMIM26 and SLC25A11 on migration and invasion of renal cancer cells

First, pLVX-Puro Vector, pLVX-Puro-SMIM26-flag, pLVX-Puro Vector and SLC25A11 interference fragment (same as Example 6) were transfected into renal cancer cells (ACHN and 769-P) respectively, and empty vector control cell line (NC), SMIM26 overexpressed renal cancer cell line, and SMIM26 overexpressed and SLC25A11 knocked down cell line were obtained in turn, and then the migration and invasion ability of renal cancer cells were detected. The experiment was repeated three times, and the specific steps were as follows:

(1) Migration experiment: The empty vector control cell line (NC), the renal cancer cell line overexpressing SMIM26, and the cell line overexpressing SMIM26 and knocking down SLC25A11 were digested into 1.5 mL centrifuge tubes, the supernatant was removed after centrifugation, the cells were resuspended with DMEM medium without serum (Thermo), mixed and then the cells were aspirated and added to the cell counting slide for counting. After counting, 150,000 cells per well were taken into a new Ep tube, centrifuged, and resuspended with 200 μL of DMEM medium without serum per well. After the cells were mixed, they were added to the upper chamber of the chamber. After adding 600 μL of complete culture medium to the lower chamber, the 24-well plate was placed in a cell culture incubator and incubated to wait for cell migration. The chamber was collected according to the migration ability of the cell line. In this experiment, the most suitable migration time for the ACHN cell line was about 9 to 11 hours, and that for the 769-P cell line was about 3 to 4 hours. When the most suitable migration time comes, take out the chamber from the clean bench and place it in a 24-well plate containing anhydrous methanol for 10 minutes. After fixation, place it in crystal violet for staining for 10 minutes, then rinse the crystal violet in pure water and gently wipe off the cells in the upper chamber with a cotton swab. After the chamber is inverted to dry the water, take a picture under a microscope and count it using Image J software.

(2) Invasion assay: The invasion assay uses Martrigal matrix gel to simulate the extracellular matrix environment in vivo. Cells can only pass through the matrix gel after secreting proteases to dissolve the matrix gel. Based on this principle, the invasion ability of cells is evaluated. Martrigal matrix gel and DMEM medium without serum are diluted on ice at a volume ratio of 1:19. 100 μL is drawn with a pre-cooled pipette tip and added to the upper chamber of the chamber without generating bubbles. The 24-well plate is placed in a cell culture incubator and allowed to stand for 1 hour to solidify. After solidification, the operation is the same as the above migration assay. In this experiment, the most suitable invasion time for the ACHN cell line is about 32 to 34 hours, and that for the 769-P cell line is about 16 to 17 hours.

The experimental results are shown in Figure 4: The results showed that interfering with the expression of SLC25A11 could reverse the inhibitory effect of overexpression of SMIM26 on the migration and invasion ability of renal cancer cells.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (6)

1. Use of smim26 protein for the preparation of a medicament for the treatment of renal cancer and/or a medicament for the prevention of metastasis of renal cancer.
2. 2. The use according to claim 1, characterized in that: the coding gene sequence of the SMIM26 protein is shown as SEQ ID NO. 1.
3. 3. The use according to claim 1, characterized in that: the treatment of renal cancer or the anti-renal cancer metastasis is realized by the mode of over-expressing SMIM26 protein and/or mRNA thereof; the over-expression SMIM26 protein and/or mRNA thereof can inhibit proliferation and growth of kidney cancer cells and inhibit migration and invasion capacity of the kidney cancer cells.
4. 4. Use of an agent that detects the expression level of SMIM26 protein in the manufacture of a product for diagnosing metastasis of renal cancer.
5. Use of a combination of smim26 protein and SLC25a11 protein for the preparation of a medicament for the treatment of renal cancer and/or a medicament for the prevention of metastasis of renal cancer.
6. 6. The use according to claim 5, characterized in that: the coding gene sequence of the SMIM26 protein is shown as SEQ ID NO.1, and the accession number of the SLC25A11 protein sequence in uniprot database is: q02978.