CN112553330A - Novel tumor-associated transcription factor ZSCAN16 and application thereof in tumor inhibition - Google Patents

Abstract

The invention relates to a novel tumor-related transcription factor ZSCAN16 and an effect thereof in tumor inhibition, belonging to the technical field of biological medicines. The ZSCAN16 gene is used as a bladder cancer treatment target. The invention has the following advantages: the invention discloses that ZSCAN16 may play an important biological role in the process of bladder cancer development and is closely related to the development of tumors. The results of the invention show that ZSCAN16 is a key novel oncogene for the occurrence and development of bladder cancer. In vitro experimental results show that silencing of ZSCAN16 inhibited proliferation, colony formation, apoptosis, migration and invasion of T24 cells. The ZSCAN16 can be used as a research target for bladder cancer regulation, can be used as a marker for tumor diagnosis and prognosis evaluation, and can be used as a target for developing a tumor-inhibiting medicament.

Description

Novel tumor-associated transcription factor ZSCAN16 and application thereof in tumor inhibition

Technical Field

The invention relates to a novel tumor-related target ZSCAN16 and application thereof in tumor inhibition, belonging to the technical field of biological medicines.

Background

Bladder cancer is one of the most common genitourinary malignancies in the world, with a total of 340 million people worldwide suffering from bladder cancer since 2015, with an additional 43 million cases annually. The morbidity and mortality of bladder cancer in china has risen rapidly in the last decade. Smoking, family history, radiotherapy history, repeated bladder infections, and chemical exposure have been shown to be high risk factors for the development and progression of bladder cancer. Standard treatments for patients with bladder cancer include radiation therapy, chemotherapy, and surgical resection. The treatment of non-muscle invasive bladder cancer comprises endoscopic bladder tumor resection, postoperative auxiliary intravesical perfusion chemotherapy, and common medicines comprise epirubicin, adriamycin, BCG and mitomycin. Whereas for muscle-invasive bladder cancer, MAVC regimens, i.e., cisplatin, doxorubicin, vinblastine in combination with methotrexate, are recommended as first-line chemotherapy regimens. However, despite advances in the diagnosis and treatment of bladder cancer, the overall survival of patients has not improved over the past few decades. Therefore, the research of new molecular therapeutic targets is imperative, which is beneficial to the deeper understanding of the pathogenesis of bladder cancer so as to establish a more effective molecular therapeutic strategy.

In recent years, immunotherapy has become a focus of cancer research. Evasion of the immune system is a hallmark of cancer. Tumor cells can express a series of immunosuppressive related proteins to induce immune cell dysfunction and apoptosis. The NF-. kappa.B (nuclear factor kappa light chain enhancer of activated B cells) family of transcription factors plays a key role in tumor progression and metastasis, as well as in immune regulation. All proteins of the NF-. kappa.B family have a relatively homologous domain at their N-terminus. p50 and p65 are closely related to tumors as heterotrimers of the most common NF-. kappa.B signaling pathway. Inhibition of p50 and p65 is mediated primarily through binding to the arrestin ikba to inhibit nuclear signaling. Phosphorylation, degradation and ubiquitination, as the major modification following I κ B α transcription, results in the release of either the p50 or p65 subunits from the I κ B complex, causing nuclear translocation and ultimately activation of a range of oncogenes. A number of studies have demonstrated that PRMT5 induces symmetric demethylation of NF-. kappa.B to enhance its activity. The apoptosis of the bladder cancer cells is mainly controlled by NF-kB related signal paths and is closely related to the cell proliferation process. In view of the large number of potentially unknown oncogenes, the search for new transcription factors, or new genes that can regulate key transcription factors, contributes to providing a powerful molecular basis for future targeted therapies.

ZSCAN16 (containing zinc finger and 16 structural domains) is a protein coding gene and belongs to a DNA binding transcription factor family. As a "new" transcription factor, the function and mechanism of ZSCAN16 is rarely reported. The SCAN domain family is a subset of the highly conserved zinc finger transcription factor family. In bladder cancer, the only reported member of the SCAN domain family is ZSCAN12, a potential biomarker for bladder cancer, and can be used for advanced bladder cancer diagnosis.

However, no research report about the association of the ZSCAN16 gene with bladder cancer exists at present.

Disclosure of Invention

The invention aims to provide a novel tumor-related transcription factor ZSCAN 16.

The invention also aims to solve the technical problem of providing the function of the tumor-associated transcription factor ZSCAN16 in inhibiting tumors.

In order to achieve the purpose, the invention adopts the following technical scheme:

the ZSCAN16 gene is used as a bladder cancer treatment target.

Use of an inhibitor of ZSCAN16 protein in the preparation of a medicament for the prevention and/or treatment of bladder cancer.

The medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of ZSCAN16 protein.

The inhibitor of the ZSCAN16 protein is selected from an antibody of ZSCAN16 protein and/or a binding protein of ZSCAN16 protein.

Application of an inhibitor of ZSCAN16 gene expression in preparation of medicines for preventing and/or treating bladder cancer.

The medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of ZSCAN16 gene expression.

The inhibitor of the ZSCAN16 gene is selected from one or more of RNAi specific to the ZSCAN16 gene, microRNA specific to the ZSCAN16 gene or an inhibitor for inhibiting a ZSCAN16 gene promoter.

A medicament for preventing and/or treating bladder cancer, which comprises a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an inhibitor of the ZSCAN16 protein and/or an inhibitor of the ZSCAN16 gene.

The inhibitor of the ZSCAN16 protein is selected from an antibody of ZSCAN16 protein and/or a binding protein of ZSCAN16 protein; the ZSCAN16 gene expression inhibitor is selected from one or more of RNAi specific to ZSCAN16 gene, microRNA specific to ZSCAN16 gene or an inhibitor inhibiting ZSCAN16 gene promoter.

An in vitro non-therapeutic method for inhibiting tumor cells comprises culturing tumor cells in the presence of a ZSCAN16 protein inhibitor or a ZSCAN16 gene inhibitor, thereby inhibiting the tumor cells. Wherein said tumor cell is a bladder cancer cell.

Based on our previous studies, we found that ZSCAN16 is highly expressed in bladder cancer, suggesting that this may be a potential oncogene. In the invention, the role of ZSCAN16 in bladder cancer is researched, the research result shows that ZSCAN16 is highly expressed in bladder cancer cells, and the proliferation of the bladder cancer cells is inhibited by silencing ZSCAN16, and further Western Blot research shows that ZSCAN16 plays a role in bladder cancer by regulating NF-kB signaling pathways. These results reveal great promise of ZSCAN16 as a new therapeutic target in bladder cancer.

It is well known that the development and progression of bladder cancer involves a variety of pathological processes involving large-scale genetic changes, where activation of oncogenes and inactivation of tumor suppressor factors are key events in bladder tumorigenesis. The intracellular mechanism of bladder cancer, one of the most fatal cancers in the world, is still unclear. There is a need to develop new oncogenes as therapeutic targets for bladder cancer. In our previous studies (data not published), we found that ZSCAN16 is highly expressed in bladder cancer. Therefore, to further investigate the role of ZSCAN16 in bladder cancer, we used subsequent studies on the effect of ZSCAN16 on T24 cell function by designing and screening shRNA to silence ZSCAN16 in T24 bladder cancer cell line and finally determining the two most effective shRNA targets KD1 and KD 2.

The results of our study show that ZSCAN16 is a novel oncogene in bladder cancer. We found that the mRNA expression of ZSCAN16 was positively correlated with the progression of bladder cancer disease, i.e. the expression level of ZSCAN16 in bladder cancer-like was significantly higher in TCGA database than in paracancerous normal tissue levels. To functionally explore the effect of ZSCAN16 on bladder cancer cell proliferation, we used dual shRNA targets to silence ZSCAN16 in T24 cells, respectively. In vivo and in vitro experimental results show that ZSCAN16 is a transcription factor with strong function, and can influence various cell functions such as cell proliferation, clone formation and apoptosis.

Bladder cancer is generally considered to be susceptible to metastasis, an important factor that is closely related to patient prognosis. The carcinogenic effect of ZSCAN16 in bladder cancer metastasis and invasion was confirmed by wound-healing test, scratch-migration test and invasion test. In conclusion, the ZSCAN16 may be a potential oncogene of bladder cancer, and in order to further study the intracellular mechanism of ZSCAN16 oncogene, 22 genes closely related to cell proliferation and metastasis were selected for western blot analysis and functional recovery confirmation experiments were performed. Research development, NF-kB down-regulation is the most significant in ZSCAN16 silencing regulation. NF- κ B is a pleiotropic transcription factor, expressed in almost all cell types, and is the end point of a series of signaling events initiated by a number of stimuli associated with many biological processes such as inflammation, immunity, differentiation, inflammation, cell proliferation, carcinogenesis and apoptosis. NF-. kappa.B has been shown to be a homodimer or heterodimer complex formed by proteins containing REL-like domains such as RELA/p65, RELB, NFKB1/p105, NFKB1/p50, RE and NFKB2/p 52. Dimers bind at the Kappa-B site of their target DNA and exhibit different binding capacities to the Kappa-B site due to the differences in molecular specificity and affinity of the individual dimers. Thus, in addition to direct binding to transcriptional agonists, NF-. kappa.B family members are also capable of regulating promoter accessibility to transcriptional factors, and thus indirectly gene expression. In this experiment, we successfully recovered the function of ZSCAN16 gene silencing by over-expressing NF-. kappa.B-p 65, which demonstrates that ZSCAN16 exerts its function through or partially through NF-. kappa.B-related signaling pathway.

In addition, we examined other marker genes associated with cancer progression and metastasis in addition to NF- κ B. The result shows that ZSCAN16 has certain inhibition effect on P-P38, P-ERK and P-mTOR. As the two most common regulators in the mTOR-MAPK pathway, p38 and ERK play important regulatory roles in the tumor's multicellular process. In addition, the correlation between the activation of AKT-mTOR-MAPK signaling pathway and tumorigenesis progression in a variety of cancers, such as prostate cancer, lung cancer and ovarian cancer, has been well studied. Accordingly, we found that in our study, AKT and mTOR signals were also activated by ZSCAN 16. In summary, we conclude that: the AKT-mTOR-MAPK signaling pathway is activated by ZSCAN16, indicating that the NF- κ B mediated AKT-mTOR-MAPK network is consistent with previous reports in intracellular mechanisms.

In an in-vivo experiment of a nude mouse bladder cancer model, nude mice in an experimental group and a control group are respectively injected with ZSCAN16 shRNA (T24 ZSCAN16 KD2 group) and T24 cells transduced by non-silencing siRNA (T24 ZSCAN16 ND group) to establish a tumor forming model. And calculating the tumor volumes of the two groups, drawing a tumor curve, and measuring the fluorescence expression quantity in the tumor bodies of the two groups of nude mice. The results show that compared with the ND group, the tumor body weight and the fluorescence expression amount in the tumor body area of the KD group are obviously reduced (P < 0.05).

The invention has the following advantages: the invention discloses that ZSCAN16 may play an important biological role in the process of bladder cancer development and is closely related to the development of tumors. The results of the invention show that ZSCAN16 is a novel key oncogene for the occurrence and development of bladder cancer. In vitro experimental results show that silencing of ZSCAN16 inhibited proliferation, colony formation, apoptosis, migration and invasion of T24 cells. The ZSCAN16 suggests that ZSCAN16 can be used as a key target for bladder cancer treatment and even immune regulation treatment through the regulation and control effect on NF-kB mediated AKT-mTOR-MAPK signal pathways, can be used as a marker for tumor diagnosis and prognosis evaluation, and can be used as a target for developing a tumor inhibition drug.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Fig. 1 shows the expression profile of ZSCAN16 in TCGA tumor and bladder cancer cells. FIG. 1A is a comparison of the expression levels of mRNA from 19 pairs of bladder cancer tissues and their adjacent normal bladder tissues ZSCAN16 in TCGA sample pools. FIG. 1B shows the mRNA expression levels of ZSCAN16 in T24 and 5637 cell lines detected by qRT-PCR. Fig. 1C and 1D demonstrate the silencing efficacy of shZSCAN16 in T24 cells by qPCR and Western Blot, respectively. KD1 is an abbreviation for silent shZSCAN16 target 1(GAGTAAGTTCAGCTTAT), and KD2 is an abbreviation for silent shZSCAN16 target 2 (TAGAGTACACCGGAGTA). GAPDH was used as a reference for qPCR and Western blot experiments. P < 0.01.

FIG. 2 shows the effect of ZSCAN16 silencing on T24 cell proliferation, apoptosis, and clonal function. Experimental and control groups were shZSCAN16 with KD1 and KD2, respectively, and negative shRNA lentivirus transduced T24 cells. As shown in fig. 2A, cell count and doubling changes were calculated by an automated GFP-labeled fluorescence image detector for comparative analysis of cell growth rate. FIG. 2B comparison of cell viability by MTT assay (OD490) was performed 3 days after shRNA transfection for 5 consecutive days. Figure 2C shows the evaluation of the effect on apoptotic function by CASPASE3/7 activity comparison. FIG. 2D shows comparative T24 cell colony forming ability to assess the impact of clonal function. The result shows that the shZSCAN16 of KD1 and KD2 can obviously inhibit the proliferation and cloning of T24 bladder cancer cell strains and promote apoptosis compared with a negative shRNA control group. Error bars represent standard deviation. P-is less than 0.01.

Fig. 3 shows the effect of ZSCAN16 silencing on the metastatic and invasive function of T24 cells. Experimental and control groups were shZSCAN16 for KD1 and KD2, respectively, and negative control shRNA transduced T24 cells. Fig. 3A is a healing test, and the scratch width was measured after 24 hours using Celigo, and the result showed that shZSCAN16 significantly inhibited the migration ability of T24 cells. As shown in fig. 3B and 3C, shZSCAN16 significantly inhibited T24 cell migration and invasion ability as shown by the membrane-bottom cell staining counts in Trans-well and invasion experiments. Error bars represent standard deviation. P-is less than 0.01.

The shZSCAN16 shown in fig. 4 inhibits NF- κ B signaling pathway conduction. Fig. 4A and 4B show Western blot analysis of 22 tumor-associated key genes, shZSCAN16 down-regulated gene: NF- κ B-P65, FN1, CDH2, Twist, VIM, MMP2, Slug, mTOR, P38, MyC, P-AKT; up-regulation of genes: P-NF-kB-P65, MMP9, CDH1, P-P38, ERK1/2, P-ERK1/2 and P-mTOR; invariant genes: beta-Catenin, P-beta-Catenin, Snail, AKT. Of the above, the expression level of NF-kB-p 65 is changed most remarkably, which suggests that NF-kB-p 65 may be a key downstream signal path of ZSCAN 16. To verify the key role of NF-. kappa.B signaling in ZSCAN16 signaling, we performed functional recovery experiments by overexpressing NF-. kappa.B-p 65 while silencing ZSCAN 16. FIGS. 4C and 4D are functional recovery experiments with Trans-well and MTT, respectively, and show that the group of KD + OE can significantly restore the T24 cell proliferation and transfer ability of shZSCAN16 by over-expressing NF- κ B-p 65. Error bars represent Standard Deviation (SD). P-is less than 0.01.

FIG. 5 is an in vivo experiment: the relevant figures of the nude mouse bladder cancer model. The experimental group (KD) is a shZSCAN 16T 24 tumor formation model, and the control group (NC) is a negative shRNA transduction tumor formation model. FIG. 5A is a whole image of a nude mouse bladder cancer model, and FIG. 5B is a tumor tissue of the nude mouse bladder cancer model. FIG. 5C is a comparison of the two sets of tumor formation curves, and FIG. 5D is a comparison of the two sets of tumor volumes. FIG. 5E is a graph showing the in vivo fluorescence expression level of nude mouse tumor in experimental group, and FIG. 5F is a graph showing the in vivo fluorescence expression level of nude mouse tumor in control group. Fig. 5G shows the difference in mean radiant efficiency between the two groups, T test, with a P value of 0.00003.

Detailed Description

Example 1:

materials and methods

TC6A database: from the TCGA database, ZSCAN16 expression profiles of 19 paired cancer and adjacent normal TCGA samples and clinical information of bladder cancer samples were obtained. The clinical data for 19 patients are shown in table 1.

Table 1: clinical data for 19 patients

2. Cell culture and assay

2.1 cell culture and reagents

Two human bladder cancer cell lines (T24 and 5637) were first selected, purchased from ATCC, and cultured and passaged as per the instructions. Briefly described as follows: t24 and 5637 cells at 5x10⁴The cells were inoculated at a concentration of/ml into a T-25 culture dish containing 10ml of RPMI-1640 (purchased from Gibco Co., Ltd.) + 10% (v/v) FBS (purchased from Sigma Aldrich Co., Ltd.) and incubated at 37 ℃ in a 5% carbon dioxide incubator. After verifying the silencing efficiency of ZSCAN16 with qPCR (real-time fluorescent quantitative nucleic acid amplification detection), we selected T24 cells for the following experiments. Lentiviral packaging HEK293T cells were selected and cultured in DMEM complete medium containing 10% FBS.

2.2 Lentiviral RNAi

Two candidate siRNA sequences are pre-screened, and the gene silencing efficiency is verified. Lentiviral vectors were purchased from Shanghai Gene chemistry, Inc., China. Non-silencing siRNA (5'-gccactgtcagaagaa-3', SEQ ID NO: 1) was used as Negative Control (NC). Finally, two sequences were selected for this study, 5'-gagtaaagttcagcttat-3', (KD1), SEQ ID NO: 2 and 5'-tagagtacacacggagta-3' (KD2), SEQ ID NO: 3 in a pharmaceutically acceptable carrier.

2.3 Celigo imaging

T24 cells were seeded in 96-well plates in triplicate (2000 cells/well). And counting the cells for 1-5 days by adopting a Celigo imaging cytometer. Two candidate siRNA sequences are pre-screened, and the gene silencing efficiency is verified. Lentiviral vectors were purchased from Shanghai Gene chemistry, Inc., China. Non-silencing siRNA (5'-gccactgtcagaagaa-3', SEQ ID NO: 1) was used as Negative Control (NC). Finally, two sequences 5'-gagtaaagttcagcttat-3' (KD1) SEQ ID N0: 2 and 5'-tagagtacacacggagta-3' (KD2) SEQ ID N0: 3 in a pharmaceutically acceptable carrier.

2.4MTT cell viability assay

For the MTT assay, 20. mu.l of 5mg/ml MTT was added to the cells over 4 hours. The medium was then removed and 150. mu.l DMSO was added to each well. The 96-well plate was gently shaken in parallel for 3 to 5 minutes and then read at 490nm/570nm using a VICTORNivo multimode plate reader (available from PERKIN Elmer Co., Ltd.).

2.5 Caspase3/7 assay

T24 bladder cancer cells were seeded onto 96-well plates (5X 10)⁴100u1) and culturing in an incubator at 37 ℃ for 3-5 days. Caspase3/7 assays were performed according to the Caspase-Glo 3/7 kit (purchased from Promega) instructions. Caspase-Glo 3/7 reagent was added at a ratio of 1: 1 to the sample volume, and after incubation at 37 ℃ for 1 hour, the light flux measurement was carried out at 490nm/520nm using a Victor Nivo multimode plate reader (PERKIN Elmer Co., Ltd.).

2.6 colony formation assay

T24 cells transduced with shRNA resistant ZSCAN16 were seeded into 6-well plates (600 cells/well) and cultured in an incubator containing 5% carbon dioxide at 37 degrees celsius for 14 days to form colonies. The medium was changed every 3 days. Virus without anti-ZSCAN 16 transduced T24 cells as a negative control. Cells were fixed with 4% paraformaldehyde for 45 minutes and stained with Giemsa. Clone images were collected with IX71(Olympus, Inc.) and the number of clones was calculated.

2.7 scratch healing test

We used T24 cell line 1X10⁵At a concentration of 100ul in 96-well plates, incubate overnight until the cells grow to 90% saturation. The cell monolayer was scratched with a 96-well plate scratch replicator (VP Scientific), and after washing off the suspension cells, the culture was again replaced with complete medium and continued for 24 hours before image acquisition and analysis using Celigo software (Nexcelom).

2.8 cell migration and invasion assay

The cell migration ability was examined by the Trans-well method. T24 cells were trypsinized and resuspended in serum-free medium. Will 100ul 5x10⁴T24 cells were gently added to the upper chamber of a Trans-well (Corning Corp.). 600 μ l DMEM with 30% FBS was added to the lower compartment of the Trans-well. Then, after further culturing at 37 ℃ in an incubator containing 5% carbon dioxide for 24 hours, the cells remaining in the upper layer were removed with a cotton ball. Staining with 0.5% crystal violet for 5 minutes and randomly selecting 4 different views at 100 times magnification and 9 different views at 200 times magnification. Images of the underlying stained cells were obtained under a microscope,the number of cells averaged from the 200XMagnition microscopic view was used as the number of migrated cells.

3. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and immunoblot experiment (Western blot)

3.1 real-time qRT-PCR analysis

The relative expression of ZSCAN16 was detected by qRT-PCR. cDNA was synthesized from 1ug of total RNA sample using the Invitrogen M-MLV cDNA kit. The qRT-PCR assay was performed using Roche real-time PCR 384 (Roche Ltd.). GAPDH is used as an internal reference, and the expression of related genes is quantitatively analyzed by a delta CT method. Each experiment was repeated three times. The primer sequences used were: GAPDH F-TGACTTCAACGGACACCA (SEQ ID NO: 4) should be changed to capitalized, GAPDH R-CACCTGTTGCTGTAGCCAAA (SEQ ID N0: 5); ZSCAN16F-TTCTCTAAGACCTGCAAGC (SEQ ID NO: 6), ZSCAN16R-TGTCCATGAGTCCCGTC (SEQ ID N0: 7).

3.2 protein isolation and immunoblotting experiments

T24 cells were dissolved in RIPA buffer (Beyotime Co., Ltd.). In case of degradation, a protease cocktail (Roche) was added. Protein concentration determination was performed using a BCA kit (Beyotime Co., Ltd.). Cell lysates were separated on 10% SDS-PAGE gels and subjected to protein transfer using a BiO-RAD apparatus (Bio RAD Co. Ltd.) at 80V for 1.5-2 hours. Immunoblots were then performed in PBS containing 0.1% Tween and 5% (w/v) milk according to standard protocols. The main antibodies were ZSCAN16, ERK, etc. from Abcam, Inc., and goat anti-rabbit and goat anti-mouse IgG antibodies from Santa Cru, Inc., and imaged using ECL Plus Kit Regents (Thermo, Inc.).

3.3 statistical analysis

Statistical analysis was performed using Graphpad prism (version 8.01; Graphpad software) and SPSS (version 20.0, SPSS Co., Ltd.) statistical software. The significance of the independent group and the matched material was analyzed by student's t-test and matched t-test, respectively. Data are presented as mean ± SD. Treatment groups were compared using independent sample t-tests. P < 0.05(, P < 0.01(, P) is considered statistically significant. Three replicates were set up for all experiments and at least three independent experiments were performed.

Second, experimental results

ZSCAN16 is widely expressed in TCGA tumor and bladder cancer cells.

To verify the expression profile of ZSCAN16 in bladder cancer, we tested the expression level of ZSCAN16 in 19 pairs of TCGA samples and 2 classical bladder cancer cell lines (T24 and 5637). As shown in fig. 1A and 1B, ZSCAN16 was highly expressed in tumor samples as well as in two selected bladder cancer cell lines. It is evident that ZSCAN16 was highly expressed in both T24 and 5637 cells. Finally, we selected T24 cells expressing moderate abundance for subsequent experiments.

TCGA analysis of ZSCAN16 showed that ZSCAN16 may also play a key role in the progression of bladder cancer. First, ZSCAN16 was silenced with shRNA lentivirus and tested for silencing efficiency. As shown in fig. 1C and D, both qPCR and Western blot demonstrated high silencing efficiency of shRNA targets in T24 cells (close to 60% in KD1 and 80% in KD 2). The relative expression of the ZSCAN16 protein was consistent with the qPCR results. These results indicate that ZSCAN16 was successfully silenced in T24 cells.

ZSCAN16 for promoting T24 cell proliferation

Next, we investigated the effect of ZSCAN16 silencing on T24 cell proliferation-related cell function. For proliferation-related assays, we first applied the Celigo cell proliferation assay to T24 cells transduced with two ZSCAN16 shrnas (KD1 and KD2) or negative control shrnas. T24 cells were labeled with GFP fluorescence tags and were automatically imaged 3 days after shRNA transfection for 5 consecutive days. Fig. 2A shows typical images, cell counts and doubling changes of T24 cells. MTT assay results as shown in figure 2B, ZSCAN16 silencing significantly inhibited cell proliferation.

The inhibition effect of the ZSCAN16 gene silencing on T24 cell proliferation suggests the potential influence on cell apoptosis, and we next research the activity of Caspase 3/7. As shown in FIG. 2C, the Caspase3/7 activity of the shZSCAN16 lentivirus-transduced T24 cells was significantly increased compared to the control group. The shRNA targets (KD1 and KD2) have significant difference (P < 0.01) compared with the control group.

To further investigate the mechanism of action of ZSCAN16 in regulating T24 cell proliferation, we examined the effect of silencing ZSCAN16 on T24 cell clonality. As shown in fig. 2D, the number of T24 cell colonies formed after silencing of the ZSCAN16 gene was significantly reduced.

Taken together, the above results clearly show that silencing ZSCAN16 will inhibit the proliferation of bladder cancer cells.

ZSCAN16 promoting migration and invasion of T24 cells

Previous studies confirmed that metastases account for more than 90% of solid tumor-associated deaths, and we then verified whether ZSCAN16 affected the metastatic potential of bladder cancer cells by in vitro scratch testing and Trans-well metastatic invasion testing. shZSCAN16(KD1 and KD2) significantly reduced the migratory capacity of T24 cells in the scratch experiment (fig. 3A). Through migration and invasion experiments, we observed fewer cells at the bottom of the cell membrane, indicating that the migration and invasion capacity of T24 cells was significantly reduced at shZSCAN16(KD1 and KD2) (fig. 3B and 3C).

Taken together, the above results clearly show that silencing ZSCAN16 inhibits migration and invasion of T24 cells.

Silencing of ZSCAN16 inactivated NF- κ B pathway signals

To further validate the mechanism of regulation of proliferation, migration and invasion within ZSCAN16 cells. We performed 22 key gene panel studies using Western blotting. As shown in FIG. 4A, among the 22 selected key genes, 11 genes (NF-. kappa.B-P65, Fn1, CDH2, Twist, VIM, MMP2, slug, mTOR, P38, myc, P-AKT) were down-regulated, and 7 genes (P-NF-. kappa.B-P65, MMP9, Cdh1, P-P38, ERK1/2, P-ERK1/2, P-mTOR) were up-regulated. 4 genes (beta-Catenin, P-beta-Catenin, Snail, AKT) were unchanged. The multiplicative changes in protein expression associated with the control group (see FIG. 4B) showed that NF-. kappa.B-p 65 was the most significant, with expression levels downregulated by more than 50%. This suggests that ZSCAN16 may be involved in NF-. kappa.B-p 65 mediated signaling pathway activation. The details of the antibodies used in this study are shown in table 2.

Table 2: antibodies used in this study

To further investigate how NF-. kappa.B signaling affects the mechanism of ZSCAN16 function, we performed functional recovery experiments including the Tran-swell assay (FIG. 4C) and the MTT assay (FIG. 4D) by over-expressing NF-. kappa.B-p 65. The results are consistent with the results of the Westernblot in FIG. 4A, and we can clearly observe that overexpression of NF- κ B-p65 can significantly restore the inhibition effect of shZSCAN16 on the proliferation and migration of T24 cells.

The above results indicate that ZSCAN16 mediates its function by activating NF- κ B and its downstream signaling pathway.

Example 2: animal experiments

Materials and methods

Nude mice of 4 weeks were purchased from Shanghai Ling Biotech, Inc., approximately the same weight and size before the experiment, and randomly divided into 2 groups of 10 cells each, 1 × 107 non-silencing siRNA T24 cell lines (T24 ZSCAN16 NC group of example 1) and T24 cell lines transfected with lentivirus and anti-ZSCAN 16 shRNA (T24 ZSCAN16 KD2 group of example 1), and injected subcutaneously into the right axilla of nude mice respectively, and the average weight difference of each group was not statistically significant (P < 0.05). Tumor volume and mouse volume were measured twice weekly after nodulation (30 days after cell seeding). Tumor volume ═ pi/6 × L × W, i.e., ═ 3.14/6 × L × W. Wherein L represents a long diameter and W represents a short diameter. After 6 weeks, all nude mice were subjected to fluorescence expression in tumor region (Total radial Efficiency, uW/cm) by Lumina LT Living body imager (purchased from PERKIN Elmer Co., Ltd.)²) Measurements were taken and subsequently sacrificed for measurement.

Secondly, the results

See fig. 5A-5G for illustration: in vivo experiments in nude mouse bladder cancer models, nude mice in experimental and control groups were subcutaneously injected with shZSCAN16(KD2 group) and non-silencing siRNA transduced T24 cells (T24 ZSCAN16 ND group), respectively, to establish a tumor formation model (fig. 5A and 5B). The tumor formation curve was plotted according to the tumor formation volume (see fig. 5C), and the tumor weights of the two groups were compared (fig. 5D), and the amount of fluorescence expression in the tumors of the two groups of nude mice was measured and analyzed (see fig. 5E, fig. 5F). Statistical analysis showed that there was a significant difference in mean radiant efficiency between the two groups (P-value 0.00003) (fig. 5G).

The above listing of a series of detailed descriptions is merely a detailed description of possible embodiments of the present invention and is not intended to limit the scope of the invention, and one skilled in the art may devise many other modifications and embodiments that will fall within the spirit and scope of the principles disclosed herein. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure, the drawings and the appended claims. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.

Sequence listing

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Claims (10)

1. The application of the ZSCAN16 gene as a bladder cancer treatment target.
2. Use of an inhibitor of ZSCAN16 protein for the preparation of a medicament for the prevention and/or treatment of bladder cancer.
3. 3. Use according to claim 2, characterized in that: the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of ZSCAN16 protein.
4. 4. Use according to claim 3, characterized in that: the inhibitor of the ZSCAN16 protein is selected from an antibody of ZSCAN16 protein and/or a binding protein of ZSCAN16 protein.
5. Use of an inhibitor of ZSCAN16 gene expression in the preparation of a medicament for the prevention and/or treatment of bladder cancer.
6. 6. Use according to claim 5, characterized in that: the medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of ZSCAN16 gene expression.
7. 7. Use according to claim 6, characterized in that: the ZSCAN16 gene expression inhibitor is selected from one or more of ZSCAN16 gene 5 'UTR, promoter, CDS and 3' UTR region-specific RNAi, ZSCAN16 gene-specific microRNA, or an inhibitor for inhibiting ZSCAN16 gene promoter.
8. 8. A medicament for preventing and/or treating bladder cancer, characterized in that: comprises a pharmaceutically acceptable carrier and effective amount of the following active ingredients: an inhibitor of the ZSCAN16 protein and/or an inhibitor of the ZSCAN16 gene.
9. 9. The medicament of claim 7, wherein: the inhibitor of the ZSCAN16 protein is selected from an antibody of ZSCAN16 protein and/or a binding protein of ZSCAN16 protein; the inhibitor of the ZSCAN16 gene is selected from one or more of 5 'UTR, promoter, CDS and 3' UTR region-specific RNAi of the ZSCAN16 gene, microRNA specific to the ZSCAN16 gene, or an inhibitor for inhibiting the ZSCAN16 gene promoter.
10. 10. A method of non-therapeutically inhibiting tumor cells in vitro comprising: culturing a tumor cell in the presence of a ZSCAN16 protein inhibitor or a ZSCAN16 gene expression inhibitor, thereby inhibiting the tumor cell, wherein the tumor cell is a bladder cancer cell.

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