Application of transcription and expression products of TMEM63C gene in breast cancer diagnosis products

Abstract

The invention relates to the technical field of biological medicines, in particular to application of transcription and expression products of a TMEM63C gene in breast cancer diagnosis products. The DNA (Deoxyribose Nucleic Acid) sequence of the TMEM63C gene is as shown in SEQ ID NO: 1; the amino acid sequence of the protein translated from the TMEM63C gene is as shown in SEQ ID NO: 2; the invention further provides a product for diagnosing the breast cancer. The product comprises a chip or a kit for detecting the mRNA or protein expression level of the TMEM63C gene. The invention also provides a pharmaceutical composition for treating tumors, and the pharmaceutical composition is characterized by comprising an inhibitor of TMEM63C. Compared with a traditional means, the molecular marker is used for providing diagnosis, prevention and treatment schemes, and higher sensitivity is achieved. And a new target is provided for targeted drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the application of the transcription and expression products of the TMEM63C gene in breast cancer diagnosis products. Background Art

[0002] Female breast cancer is the most common type of cancer. Clinically, breast cancer is generally divided into four molecular subtypes: TNBC, HER2, LumA, and LumB based on the gene expression profiles of estrogen receptor α (Erα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). Drug therapy, radiotherapy, and surgery are the main treatments for breast cancer. Although LumA has a better prognosis compared with other breast cancer subtypes, it remains the most important subtype because LumA patients account for the highest proportion of the total number of breast cancer patients.

[0003] In the process of detecting tumor development and tumor treatment, it is very important to determine effective treatment strategies. Developing targeted drugs for dysregulated genes is one of the effective means of treating tumors. However, as the treatment progresses, tumor cells may develop drug resistance, resulting in the failure of targeted drugs; secondly, some targeted drugs have some toxic side effects; thirdly, as the research deepens, there are not many targets that are easy to find, and the discovery and verification of new targets has become increasingly difficult. Therefore, it is urgent to find new targets or markers for drug development and improving prognosis. Summary of the invention

[0004] The purpose of the present invention is to provide the application of the transcription and expression products of the TMEM63C gene in breast cancer diagnosis products to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Application of the transcription and expression products of the TMEM63C gene in breast cancer diagnostic products.

[0007] Furthermore, the DNA sequence of the TMEM63C gene is shown in SEQ ID NO:1; the amino acid sequence of the protein translated from the TMEM63C gene is shown in SEQ ID NO:2.

[0008] The transcription and expression products of the TMEM63C gene were constructed by the following steps: RNA from breast cancer cells was extracted and reverse transcribed into cDNA, and the TMEM63C gene was cloned using the following primers;

[0009] Forward primer: 5′-cttgcggccgcagaattcatgtctgcctcaccagacgac-3′ (SEQ ID NO: 3);

[0010] Reverse primer: 5′-ggtaccgatatcagatcttcagtggtactggttctggcc-3′ (SEQ ID NO: 4);

[0011] The TMEM63C gene was cloned into the p3×FLAG-CMV-10 cell expression vector to construct the recombinant plasmid p3FLAG-TMEM63C.

[0012] A product for diagnosing breast cancer, comprising a chip or a kit for detecting the mRNA and / or protein expression level of the TMEM63C gene.

[0013] Furthermore, the chip or kit diagnoses breast cancer by detecting the expression level of TMEM63C mRNA and / or protein.

[0014] The method for detecting the expression level of TMEM63C mRNA and / or protein by the chip or kit comprises:

[0015] Extract RNA from cells, reverse transcribe into cDNA, and perform RT-qPCR detection using the following primers:

[0016] 5'-gctagaggagcagctaacgg-3' (SEQ ID NO: 5);

[0017] 5'-tggagtcctggaaggtgaca-3' (SEQ ID NO: 6); and / or

[0018] The proteins of the cells were extracted and detected by western blot using anti-TMEM63C antibody.

[0019] A pharmaceutical composition for treating tumors, comprising an inhibitor of TMEM63C.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discovered for the first time a molecular marker TMEM63C gene related to the occurrence and development of breast cancer. By detecting the TMEM63C gene in the test tissue, it is possible to determine whether breast cancer exists and the effect of breast cancer treatment, thereby providing clinicians with prevention and treatment plans. Compared with traditional methods, the use of molecular markers to provide diagnosis, prevention and treatment plans has higher sensitivity, providing new targets for the development of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a WB detection image of the protein Flag antibody in Example 1 of the present invention;

[0023] Figure 2 This is a cell viability detection diagram of tumor cells overexpressing the TMEM63C gene in Example 2 of the present invention;

[0024] Figure 3 This is a graph showing the proliferation ability of tumor cells overexpressing the TMEM63C gene in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] See also Figures 1 to 3 , the present invention provides a technical solution:

[0027] Specifically, the transcription and expression products of the TMEM63C gene are used in breast cancer diagnosis products, wherein the DNA sequence of the TMEM63C gene SEQ ID NO: 1 is described as follows:

[0028]

[0029] The amino acid sequence of the protein translated from the TMEM63C gene is described in SEQ ID NO:2 as follows:

[0030] MSASPDDLSTGGRLQNMTVDECFQSRNTVLQGQPFGGVPTVLCLNIALWVLVLVVYSFLRKAAWDYGRLALLIHNDSLTSLIYGEQSEKTSPSETSLEMERRDKGFCSWFFNSITMKDEDLINKCGDDARIYIVFQYHLIIFVLIICIPSLGIILPINYTGSVLDWSSHFARTTIVNVSTESKLLWLHSLLSFFYFITNFMFMAHHCLGFAPRNSQKVTRTLMITYVPKDIEDPELIIKHFHEAYPGSVVTRVHFCYDVRNLIDLDDQRRHAMRGRLFYTAKAKKTGKVMIRIHPCARLCFCKCWTCFKEVDAEQYYSELEEQLTDEFNAELNRVPLKRLDLIFVTFQDSRMAKRVRKDYKYVQCGVQPQQSSVTTIVKSYYWRVTMAPHPKDIIWKHLSVRRFFWWARFIAINTFLFFLFFFLTTPAIIMNTIDMYNVTRPIEKLQNPIVTQFFPSVMLWGFTVILPLIVYFSAFLEAHWTRSSQNLVMVHKCYIFLVFMVVILPSMGLTSLDVFLRWLFDIYYLEQASIRFQCVFLPDNGAFFVNYVITAALLGTGMELLRLGSLFCYSTRLFFSRSEPERVNIRKNQAIDFQFGREYAWMMNVFSVVMAYSITCPIIVPFGLLYLCMKHLTDRYNMYYSFAPTKLNEQIHMAAVSQAIFAPLLGLFWMLFFSILRLGSLHAITIFSLSTLLIAMVIAFVGIFLGKLRMVADYEPEEEEIQTVFDMEPSSTSSTPTSLLYVATVLQEPELNLTPASSPARHTYGTMNNQPEEGEEESGLRGFARELDSAQFQEGLELEGQNQYH。

[0031] Example 1: Construction of TMEM63C overexpressing cells

[0032] RNA was extracted from breast cancer cells and reverse transcribed into cDNA. The TMEM63C gene was cloned using the following P1 / P2 primers:

[0033] The sequence of the P1 forward primer was 5′-cttgcggccgcagaattcatgtctgcctcaccagacgac-3′;

[0034] The sequence of the P2 reverse primer was 5′-ggtaccgatatcagatcttcagtggtactggttctggcc-3′;

[0035] The TMEM63C gene was cloned into the p3×FLAG-CMV-10 cell expression vector to construct the recombinant plasmid p3FLAG-TMEM63C. After the cells were digested into a single cell suspension, they were seeded into a 6-well plate at a density of 1×106 cells / well. The cells were transfected with the recombinant plasmid p3FLAG-TMEM63C using Lipofectamine 2000 and cultured for 24 hours. The RNA of the transfected cells was extracted and reverse transcribed into cDNA. The following P3 / P4 primers were used for RT-qPCR detection, where:

[0036] The sequence of the P3 primer was 5′-gctagaggagcagctaacgg-3′;

[0037] The sequence of the P4 primer was 5′-tggagtcctggaaggtgaca-3′;

[0038] Proteins from transfected cells were extracted and detected by WB using anti-TMEM63C antibody. Figure 1 As shown, the transcription and expression levels of TMEM63C in transfected T-47D and MCF-7 cells were significantly increased.

[0039] Example 2: Cell viability detection of tumor cells overexpressing the TMEM63C gene

[0040] The tumor cells overexpressing the TMEM63C gene and the control cells were digested into a single cell suspension and inoculated into a 96-well plate at a density of 5000 cells / well, 100 μL per well, and the well plate was placed in a cell culture incubator (37°C and 5% CO2) for culture. Every 24 hours, a 96-well plate was taken out of the incubator, 10 μL of CCK-8 was added to the well to be tested, and the plate was returned to the cell culture incubator for further culture for 4 hours. The 96-well plate with CCK-8 added was taken out, and the OD value of the well to be tested was detected at OD450 using a microplate reader, and the results were recorded. Figure 2 As shown, the cell viability of the cell line overexpressing the TMEM63C gene was significantly increased.

[0041] Example 3: Detection of proliferation ability of tumor cells overexpressing the TMEM63C gene

[0042] Tumor cells overexpressing the TMEM63C gene and control cells were digested into single cell suspensions and inoculated into 12-well plates at a density of 1000 cells / well, 1 mL per well. The well plates were placed in a cell culture incubator (37°C and 5% CO2) for culture, and fresh culture medium was replaced every 2 to 3 days. After continuous culture for 14 days, the 12-well plates were removed, washed twice with PBS, fixed with 4% paraformaldehyde for 10 minutes, and then washed twice with PBS. Crystal violet was added and stained for 30 minutes. The well plates were washed with PBS until the well plate background was colorless, photographed and counted, and the results were as follows: Figure 3 As shown, the clone-forming ability of the cell line overexpressing the TMEM63C gene was significantly improved.

[0043] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of TMEM63C gene transcription and expression products in breast cancer diagnostic products.

2. The use according to claim 1, characterized in that: The DNA sequence of the TMEM63C gene is shown in SEQ ID NO: 1; the amino acid sequence of the protein translated from the TMEM63C gene is shown in SEQ ID NO:

2.

3. A product for diagnosing breast cancer, characterized in that: The product includes a chip or a kit for detecting the mRNA and / or protein expression level of the TMEM63C gene.

4. The product for diagnosing breast cancer according to claim 3, characterized in that: The chip or kit diagnoses breast cancer by detecting the expression level of TMEM63C mRNA and / or protein.

5. The product for diagnosing breast cancer according to claim 3, characterized in that: The method for detecting the expression level of TMEM63C mRNA and / or protein by the chip or kit comprises: Extract RNA from cells, reverse transcribe into cDNA, and perform RT-qPCR detection using the following primers: 5′-gctagaggagcagctaacgg-3′; 5'-tggagtcctggaaggtgaca-3'; and / or The proteins of the cells were extracted and detected by western blot using anti-TMEM63C antibody.

6. A pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition includes an inhibitor of TMEM63C.

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