Application of HAUS7 protein in preparation of lymphoma treatment medicine

Abstract

The invention discloses an application of HAUS7 protein in preparation of a medicine for treating lymphoma. The invention provides the application of the HAUS7 protein as a drug target to preparation of the drug for resisting malignant proliferation of lymphoma for the first time. Furthermore, after the expression of the HAUS7 gene is down-regulated, the proliferation and clone formation capabilities of lymphoma cells are remarkably inhibited. Therefore, the invention not only discloses the application of the HAUS7 gene, but also provides a new therapeutic target for lymphoma. The invention provides possibilities for preparing a novel lymphoma medicine, improving the curative effect of a patient and improving prognosis and survival.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and particularly relates to an application of HAUS7 protein in preparing a drug for treating lymphoma. Background Art

[0002] Lymphoma is one of the ten most common malignant tumors in my country. According to the World Health Organization, the annual incidence of lymphoma is increasing at a rate of 7.5%, making it one of the fastest-growing malignancies, with approximately 350,000 new cases worldwide each year. China, which accounts for approximately one-fifth of the world's population, faces a rapidly increasing burden of lymphoma. Chemotherapy, radiotherapy, targeted therapy, and immunotherapy are all used to treat lymphoma, but approximately one-third of lymphoma patients still experience relapse or refractory disease. CAR-T cell therapy has been approved for the treatment of certain types of relapsed lymphoma, but these newer therapies remain incurable in many patients with relapsed lymphoma. Relatively few oncogenic driver genes have been identified in lymphoma, with only a few clear therapeutic targets, such as MYC and BCL2 translocations. Furthermore, the selection of drugs targeting these driver events is very limited. Therefore, current clinical treatment options for lymphoma remain limited. Identifying new potential targets and interventions for lymphoma treatment is crucial for advancing clinical treatment.

[0003] The HAUS7 gene encodes HAUS Augmin-like complex subunit 7 (UIP1). This complex plays a key role in the cell cycle, primarily regulating centrosome stability and mitotic spindle integrity, and is essential for the completion of cytokinesis. Current research on HAUS7 primarily focuses on male reproductive diseases, with mutations in the HAUS7 gene associated with congenital bilateral aplasia of the vas deferens (CBAVD). However, in the field of cancer research, exploration of HAUS7 is very limited. Current findings indicate that HAUS7 expression levels are significantly higher in low-grade gliomas (LGGs) than in normal brain tissue, suggesting that HAUS7 may play a role in tumor growth. However, there are no literature reports on the relationship between HAUS7 and lymphoma, and therefore, the role of HAUS7 in lymphoma requires further investigation. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of HAUS7 protein in the preparation of lymphoma therapeutic drugs, which is an application of a strategy of downregulating HAUS7 expression level in the preparation of anti-lymphoma malignant progression drugs to improve patient efficacy, prognosis and survival.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides the use of HAUS Augmin-like complex subunit 7 (HAUS7) protein as a drug target in the preparation of a drug for treating lymphoma malignant proliferation. Specifically, the drug is a drug for downregulating the level of HAUS7 protein.

[0006] Furthermore, the nucleotide sequence of the gene encoding the HAUS7 protein is shown in SEQ ID NO: 1, and the amino acid sequence of the HAUS7 protein is shown in SEQ ID NO: 2.

[0007] The present invention shows through cell proliferation experiments and clone formation ability experiments that upregulation of HAUS7 expression can significantly promote the proliferation and clone ability of lymphoma cells, which is specifically manifested in a significant increase in the proliferation and clone ability of lymphoma OCI-LY8 cells.

[0008] The present invention uses interfering RNA targeting the HAUS7 gene to downregulate HAUS7, thereby studying the role of targeted HAUS7 intervention in lymphoma treatment. Two interfering RNAs targeting different HAUS7 sequences (SEQ ID NO:3 and SEQ ID NO:4) both reduced HAUS7 expression, significantly inhibiting lymphoma growth. Therefore, the present invention not only discloses the use of interfering RNA targeting the HAUS7 gene but also provides a new therapeutic target for treating lymphoma malignant progression.

[0009] Specifically, targeting HAUS7 protein, a corresponding pharmaceutical preparation is developed to downregulate the expression of the protein, thereby achieving the purpose of anti-lymphoma by downregulating the expression of HAUS7 protein.

[0010] Furthermore, the drug is an interfering RNA targeting HAUS7.

[0011] Furthermore, the targeting nucleotide sequence of the interfering RNA is shown in SEQ ID NO: 3 or SEQ ID NO: 4. The above two interfering RNAs targeting the HAUS7 gene can inhibit the expression level of the HAUS7 protein, thereby inhibiting the proliferation and clone formation ability of lymphoma cells.

[0012] The results of this study show that downregulating HAUS7 expression can significantly inhibit the proliferation and clonogenicity of lymphoma OCI-LY10 and U2932 cells. Therefore, we propose for the first time that interfering RNA targeting the HAUS7 gene can be used as a therapeutic agent for the malignant progression of lymphoma and play an important role in the treatment of lymphoma.

[0013] Furthermore, the anti-lymphoma drug includes: double-stranded RNA that inhibits HAUS7 gene expression through RNA interference, or a polypeptide for inhibiting HAUS7 protein activity, or a small molecule compound for inhibiting HAUS7 protein function.

[0014] Furthermore, the drug may include a pharmaceutically acceptable excipient. The excipient is any formulation or carrier medium that can deliver an effective dose of the active substance without interfering with the biological activity of the active substance and without toxic side effects on the host or subject. The excipient includes a filler, wetting agent, binder, disintegrant, or lubricant.

[0015] The drug can be prepared into a preparation according to the preparation method described in pharmaceutics. The preparation form of the above-mentioned drug is a liquid preparation or a solid preparation.

[0016] The present invention has the following beneficial effects: This invention, for the first time, uses the HAUS7 protein as a drug target for the development of drugs to combat lymphoma progression. Further downregulating HAUS7 expression can effectively inhibit lymphoma cell proliferation, providing a new direction for drug development to combat lymphoma progression. This invention opens the door to the development of new lymphoma treatments, enhancing efficacy and improving prognosis and survival in lymphoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The RT-qPCR method was used to detect the effect of overexpression of HAUS7 on the expression level of HAUS7 protein in lymphoma cells, among which A was targeted at OCI-LY8 cells and had the effect of significantly upregulating the level of HAUS7 protein.

[0018] Figure 2 To investigate the effect of upregulating HAUS7 protein expression on the malignant progression of lymphoma, A and B target the proliferation and clone-forming ability of OCI-LY8 cells, which significantly improve the proliferation and clone-forming ability of lymphoma cells.

[0019] Figure 3 Two shRNAs targeting different HAUS7 sequences (SEQ ID NO: 3; SEQ ID NO: 4) were applied to lymphoma cells, wherein A was directed against OCI-LY10 cells and B was directed against U2932 cells, both of which had the effect of significantly downregulating the protein level of HAUS7.

[0020] Figure 4Two shRNAs targeting different HAUS7 sequences (SEQ ID NO: 3; SEQ ID NO: 4) were applied to lymphoma cells, where A and C were targeted at OCI-LY10 cells, and B and D were targeted at U2932 cells. The proliferation and clone-forming ability of lymphoma cells were significantly inhibited. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0022] Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0023] The application of the drug for downregulating HAUS7 protein expression of the present invention can refer to conventional drug formulation methods and actual development. The drug dosage form and biological preparation can be any medically approved dosage form, such as powder, injection, capsule, tablet or oral solution.

[0024] Example 1: Using molecular cloning techniques, the HAUS7 cDNA was cloned into the lentiviral expression vector PCDH to construct the lentiviral recombinant plasmid PCDH-HAUS7. After identification, the recombinant plasmid was packaged into the lentivirus. PCDH-HAUS7 and the corresponding vector PCDH were introduced into OCI-LY8 cells (purchased from the Chinese Academy of Sciences Cell Bank) via lentiviral infection. Three days after infection, cells were selected with puromycin to identify cells expressing HAUS7. RNA was extracted from the cells using Trizol lysis buffer, and transcript levels were detected using RT-qPCR.

[0025] Results see Figure 1 The results showed that HAUS7 was significantly overexpressed in OCI-LY8.

[0026] Example 2: PCDH-HAUS7 and the corresponding vector PCDH were introduced into OCI-LY8 cells (purchased from the Chinese Academy of Sciences Cell Bank) by lentiviral infection. After three days of infection, puromycin was used to select cells that successfully expressed HAUS7. The cells were counted using a hemocytometer and 5×10 4 The cells were plated in 6-well plates. The day of inoculation was designated as day 0, and the cell proliferation capacity was subsequently evaluated on days 1, 3, 5, and 7.

[0027] PCDH-HAUS7 and the corresponding vector PCDH were introduced into OCI-LY8 cells (purchased from the Chinese Academy of Sciences Cell Bank) by lentiviral infection. After three days of infection, puromycin was used to select cells that successfully expressed HAUS7. 2×10 3 The cells were mixed with semi-solid agar gel and inoculated into 6-well plates. After colonies were formed, NBT staining was used to examine the colony-forming ability of the cells.

[0028] Results see Figure 2 The results showed that upregulation of HAUS7 significantly promoted the proliferation and clone-forming ability of OCI-LY8 cells.

[0029] Example 3: Using synthesized oligonucleotide sequences, the sequences encoding two shHAUS7s were cloned into the lentiviral expression vector pLKO.1 to construct the lentiviral recombinant plasmid pLKO.1-shHAUS7. Simultaneously, a control plasmid pLKO.1-shCtrl was constructed. The correctly identified plasmids were packaged into lentivirus. Two shRNAs targeting different HAUS7 sequences (SEQ ID NO: 3; SEQ ID NO: 4) and the negative control shCtrl were lentivirally transfected into OCI-LY10 and U2932 cells (purchased from the Chinese Academy of Sciences Cell Bank). After 96 hours, the cells were harvested and lysed with Trizol buffer to extract RNA. Transcription levels were then detected by RT-qPCR.

[0030] The nucleotide sequences targeted by the two shRNAs are: 5'-CGGGAAGCTGAAGGACCTAAA-3' (SEQ ID NO: 3); 5'-CGCTTAGAACGGAGTACTTTG-3' (SEQ ID NO: 4).

[0031] Results see Figure 3 The results showed that all of the above HAUS7 shRNAs could effectively inhibit the expression of HAUS7 protein.

[0032] Example 4: Two shRNAs targeting different HAUS7 sequences (SEQ ID NO: 3; SEQ ID NO: 4) and a negative control shCtrl were introduced into OCI-LY10 and U2932 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) via lentiviral infection. On the second day of treatment, the cells were counted using a hemocytometer and 6.6×10 4The cells were plated in 6-well plates. The day of inoculation was the first day, and the cell proliferation ability was subsequently examined on the 3rd, 5th, and 7th days.

[0033] Two shRNAs targeting different HAUS7 sequences (SEQ ID NO: 3; SEQ ID NO: 4) and a negative control shCtrl were introduced into OCI-LY10 and U2932 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) via lentiviral infection. On the second day of treatment, 2×10 3 The cells were mixed with semi-solid agar gel and inoculated into 6-well plates. After colonies were formed, NBT staining was used to examine the colony-forming ability of the cells.

[0034] Results see Figure 4 The results showed that all of the above HAUS7 shRNAs could significantly inhibit the proliferation and colony formation ability of lymphoma OCI-LY10 and U2932 cells.

[0035] The above examples are only partial examples of the present invention and should not be construed as limiting the present invention. The above examples utilize overexpression and RNA interference techniques to verify that upregulating and downregulating HAUS7 gene expression can regulate the malignant proliferation of lymphoma cells. Furthermore, other techniques for regulating gene expression also fall within the scope of the present invention.

Claims

1. A use of HAUS7 protein in preparing a drug for treating lymphoma, characterized in that: The nucleotide sequence of the gene encoding the HAUS7 protein is shown in SEQ ID NO: 1, and the amino acid sequence of the HAUS7 protein is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The drug is an interfering RNA targeting HAUS7.

3. The use according to claim 2, characterized in that The targeting nucleotide sequence of the interfering RNA is shown in SEQ ID NO: 3, and the targeting nucleotide sequence of the interfering RNA is shown in SEQ ID NO:

4.

4. The use according to claim 1, characterized in that Interfering RNA targeting the HAUS7 gene inhibits the expression level of HAUS7 protein, thereby suppressing the proliferation and clone formation ability of lymphoma cells.

5. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that The drug is in the form of a liquid preparation or a solid preparation.

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