CN114561389A

CN114561389A - VRK1 expression inhibitor and application thereof

Info

Publication number: CN114561389A
Application number: CN202210255062.1A
Authority: CN
Inventors: 吴佳成; 胡宝英; 王小林
Original assignee: Nantong Tumor Hospital
Current assignee: Nantong Tumor Hospital
Priority date: 2022-03-15
Filing date: 2022-03-15
Publication date: 2022-05-31

Abstract

The invention belongs to the technical field of biology, and particularly discloses a VRK1 expression inhibitor and application thereof. shRNA molecules, wherein the sequence of a sense strand is 5'-GCAAGGAACCTGGTGTTGA-3' (SEQ ID NO: 1), and the sequence of an antisense strand is 5'-TCAACACCAGGTTCCTTGC-3' (SEQ ID NO: 2). A recombinant plasmid or construct comprising SEQ ID NO: 3, or consists of the shRNA molecule and the plasmid. The application of the plasmid in preparing the medicine for preventing and treating bladder cancer. The application of the plasmid in preparing the medicine for preventing and treating the diseases induced by the over-expression of VRK 1. The medicine for preventing and treating bladder cancer contains the VRK1 gene expression inhibitor. The invention provides a novel shRNA molecule, and researches show that the shRNA molecule has a good inhibition effect on VRK1 gene expression; and researches find that the compound has a better treatment effect on tumors (such as bladder cancer) and can play a better regulation effect on tumor cells. Meanwhile, the inhibition of the expression of the VRK1 gene is also found to have a better therapeutic effect on the prevention and treatment of bladder cancer, and can control the transformation of bladder cancer cells.

Description

VRK1 expression inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a VRK1 expression inhibitor and application thereof.

Background

VRK1 is a multifunctional protein that is a member of the vaccinia-associated kinase (VRK) family of serine/threonine protein kinases. Most of the serine-Thr kinase, nucleosome kinase or chromatin kinase which is positioned in the nucleus and has atypical activity can directly and stably interact with different chromatin proteins, and has close relation with cell cycle entry, promotion of DNA damage repair and drug resistance; a small fraction is located at the plasma membrane and is involved in cell migration. It regulates a variety of transcription factors including SOX2, ATF2, p53 and c-Jun. Research shows that VRK1 and SOX2 form a complex to activate the expression of CCND1 and promote cell cycle process. DNA damage by ionizing radiation triggers phosphorylation of NBS1 by VRK1, thereby facilitating the DNA repair process. Doxorubicin induces the formation of a complex between VRK1 and 53BP1, thereby initiating DNA damage repair and allowing the cells to develop drug resistance. VRK1 is overexpressed on the plasma membrane and part of the PAK1-paxillin signaling complex, promoting metastasis of breast cancer cells by modulating the transcriptional repressors snail, slug and twist 1. The prior research shows that the over-expression of VRK1 can induce some diseases, and the corresponding diseases can be treated by controlling VRK1, but the research of VRK1 as a bladder cancer treatment target is still blank.

The structures of three members of the VRK family have been established with several structural differences in their kinase folding, predicting that they should be insensitive to most inhibitors currently available. The shRNA aiming at the VRK1 mRNA can inhibit the translation of the VRK1, has low toxicity and is a potential targeting intervention strategy.

Over 43 million patients are diagnosed with bladder cancer each year, of which about 16.5 will die as a result. Although the use of radical cystectomy has made great progress in survival, its 5-year survival rate is still relatively low. Recurrence and progression of postoperative tumors are major clinical events affecting the prognosis of these patients. Recent molecular profiling studies prove that in fact, a disease with high somatic mutation rate is caused, and high mutation load of expressed protein on tumor cell membranes forms multiple tumor antigens, so that an effective clinical treatment scheme is still lacked at present to thoroughly kill and eliminate tumor cells, and the finding of targets which can prompt bladder cancer prognosis indexes and can intervene multiple tumor cell behavioral characteristics at one time is very important.

Disclosure of Invention

Aiming at the problems, the invention provides a VRK1 expression inhibitor and application thereof, mainly relating to a novel shRNA molecule, regulating action in VRK1 and solving the problem of prevention and control regulation of tumors (including bladder cancer).

In order to solve the problems, the invention adopts the following technical scheme:

shRNA molecule in the sequence thereof

The sense strand sequence is 5'-GCAAGGAACCTGGTGTTGA-3' (SEQ ID NO: 1),

the antisense strand sequence is 5'-TCAACACCAGGTTCCTTGC-3' (SEQ ID NO: 2).

In some embodiments, it has the following sequence:

5'-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3'(SEQ ID NO：3)。

a recombinant plasmid or construct comprising SEQ ID NO: 3, or consists of the shRNA molecule and the plasmid.

An inhibitor of expression of VRK1 gene, comprising at least one of

a. The aforementioned shRNA molecule;

b. the above recombinant plasmid.

A VRK1 gene silencing kit comprising the foregoing inhibitor.

Application of VRK1 gene expression inhibitor in preparing medicine for preventing and treating bladder cancer.

In one mode, the VRK1 gene expression inhibitor is at least shRNA or siRNA or miRNA or polypeptide.

In some embodiments, the VRK1 gene expression inhibitor comprises at least one of

a. The aforementioned shRNA molecule;

b. the above recombinant plasmid.

In some embodiments the inhibitor modulates bladder cancer by at least any one of

i) Inhibiting the proliferation and cloning formation of bladder cancer cells;

ii) inhibiting the metastasis and invasion of bladder cancer cells.

Application of plasmid in preparation of drugs for preventing and treating bladder cancer, wherein the plasmid is at least one of the following plasmids

a. Any of the foregoing shRNA molecules;

b. the recombinant plasmid as described above.

Application of plasmid in preparing medicine for preventing and treating diseases induced by over-expression of VRK1, wherein the plasmid is at least one of the following plasmids

a. Any of the foregoing shRNA molecules;

b. the above recombinant plasmid.

Use of a plasmid for the manufacture of a medicament for inhibiting a disease resulting from a change of tumor cells to a mesenchymal type, wherein said plasmid is at least one of

a. Any of the foregoing shRNA molecules;

b. the above recombinant plasmid.

The medicine for preventing and treating bladder cancer contains the VRK1 gene expression inhibitor.

An inhibitor of expression of VRK1 protein, comprising at least two of

I).siVRK1:5′-GCAAGGAACCTGGTGTTGA-3′，

5′-TCAACACCAGGTTCCTTGC-3′；

II).shVRK1-1:5′-tGAAAGAGAGTCCAGAAGTAttcaagagaTACTTCTGGACTCTCTTTCtttttggaa ac-3’(SEQ ID NO：4)；

III).shVRK1-2:5′-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3′(SEQ ID NO：3)。

The mixed VRK1 protein expression inhibitor is applied to the preparation of medicaments for preventing and treating bladder cancer.

The term "construct"Refers to intermediates containing the corresponding specific molecular sequence and used for constructing plasmids, such as recombinant plasmids or protein expression vectors.

The term "shVRK 1"Refers to VRK1 specific shRNA molecule.

Term "Gene (protein) over-expression induced disease "It means that overexpression is pathogenic to the disease and inhibition of the corresponding disorder can be achieved by inhibiting this overexpression mechanism.

The term "control"Refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the amelioration of a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to the removal of the cause of the associated disease, pathological condition, or disorder. In addition, the term also includes palliative treatments, i.e., treatments designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e. treatment aimed at minimising or partially or completely inhibiting the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e. treatment for supplementing another specific therapy directed to an improvement of the relevant disease, pathological condition or disorder.

The invention has the beneficial effects that:

provides a new shRNA molecule, and researches show that the shRNA molecule has better inhibition effect on VRK1 gene expression; and researches find that the compound has a better treatment effect on tumors (such as bladder cancer) and can play a better regulation effect on tumor cells. Meanwhile, the inhibition of the expression of the VRK1 gene is also found to have a better therapeutic effect on the prevention and treatment of bladder cancer, and can control the transformation of bladder cancer cells.

Drawings

FIG. 1 shows the real-time fluorescent quantitative polymerase chain reaction and Western immunoblotting to detect the expression level of VRK 1;

FIG. 2 is a graph showing specific inhibition of expression of VRK1 gene in bladder cancer cell lines by means of shRNA interference;

FIG. 3 shows that the CCK-8 experiment verifies that shVRK1 inhibits the proliferation of bladder cancer;

FIG. 4 is a clone formation experiment demonstrating that shVRK1 inhibits bladder cancer proliferation;

fig. 5 is a graph demonstrating the ability of shVRK1 to inhibit bladder cancer metastasis;

FIG. 6 is a Transwell invasion assay demonstrating that shVRK1 inhibits bladder cancer invasion;

FIG. 7 shows that the expression of mesenchymal marker molecules in epithelial-mesenchymal transition (EMT) signaling pathway of tumor cells can be inhibited by shVRK1 verified by Western Blot experiment.

Detailed Description

In a first aspect of this section, shRNA molecules and corresponding plasmids are described：

First, it relates to shRNA molecules, in the sequence thereof

The sense strand sequence is 5'-GCAAGGAACCTGGTGTTGA-3' (SEQ ID NO: 1),

the antisense strand sequence is 5'-TCAACACCAGGTTCCTTGC-3' (SEQ ID NO: 2).

The sense and antisense strands are linked by a loop sequence, controlled by a promoter, and finally linked to a transcription terminator.

In some embodiments, a specific complete sequence is as follows:

second, it relates to a recombinant plasmid or construct comprising the nucleotide sequence of SEQ ID NO: 3, or consists of the shRNA molecule and plasmid;

wherein, the recombinant plasmid is a plasmid formed by combining an expression vector formed by recombination with a target sequence. The construct is an intermediate which is loaded with the aforementioned sequences, but has not yet been able to form a plasmid for direct use, and still requires further processing, one of which may be to load the sequence of interest during the preparation of a recombinant plasmid for use in linking the intermediate to the plasmid.

In the second aspect of this section, shRNA molecules and corresponding plasmids and related products are described：

An inhibitor of expression of VRK1 gene, comprising at least one of

a. The aforementioned shRNA molecule;

b. the above recombinant plasmid.

One of the two components of a \ b can be selected or coexist, and plays a role in inhibiting the expression of the VRK1 gene.

A VRK1 gene silencing kit comprising the aforementioned inhibitor of VRK1 gene expression. The gene silencing kit is mainly used for silencing target genes, and one application scenario of the gene silencing kit is control variables in commercial experiments.

The medicine for preventing and treating bladder cancer comprises the VRK1 gene expression inhibitor.

In the third aspect of this section, some applications of shRNA molecules and corresponding plasmids and related product applications are introduced:

first, the application of VRK1 gene expression inhibitor in preparing medicine for preventing and treating bladder cancer. It effects modulation of mechanisms in bladder cancer by inhibiting VRK1 gene expression.

More specifically, the VRK1 gene expression inhibitor comprises at least one of the following

a. The aforementioned shRNA molecule;

b. the aforementioned recombinant plasmid;

c. other existing inhibitors, such as polypeptides and other inhibitors that other existing studies have demonstrated to be feasible; on the premise that the invention proves that the inhibition of the expression of the VRK1 gene can prevent and treat bladder cancer, other inhibitors are not described in detail.

The VRK1 polypeptide 1-polypeptide 1 can be used as an inactive polypeptide sequence to antagonize the kinase function of endogenous protein, so that the kinase function can not carry out phosphorylation modification on downstream protein, and the amino acid sequence is as follows:

WKVGLPIGQGGFGCIYLADMNSSESVGSDAPCVVKVEPSDNGPLFTELKFYQRAAKPEQIQKWIRTRKLKYLGVPKYWGSGLHDKNGKSYRFMIMDRFGS DLQKIYEANA KRFSRKTVLQLSLR；

VRK1 polypeptide 2-polypeptide 2 can compete with endogenous VRK1 for binding to downstream effector molecules, thereby blocking its binding to downstream molecules and inhibiting its activity, and its amino acid sequence is:

KPGEIAKYMETVKLLDYTEKPLYENLRDILLQGLKAIGSKDDGKLDLSVVENGGLKAKTITKKRKKEIEESKEPGVEDTEWSNTQTEEAIQTRSRTRKRV QK。

in some embodiments, the modulating effect of the inhibitor on bladder cancer is at least one of

i) Inhibiting the proliferation and cloning formation of bladder cancer cells;

ii) inhibiting the metastasis and invasion of bladder cancer cells.

Secondly, the application of the plasmid in preparing the medicine for preventing and treating the bladder cancer is not strictly limited and must be realized by inhibiting the expression of VRK1 gene, wherein the plasmid is at least one of the following plasmids

a. The shRNA molecule is recombined;

b. the above recombinant plasmid.

Thirdly, the application of the plasmid in preparing the medicine for preventing and treating the diseases induced by the over-expression of the VRK1, wherein the plasmid is at least one of the following plasmids

a. Any of the foregoing shRNA molecules;

b. the above recombinant plasmid.

Fourthly, the application of the plasmid in preparing the medicine for inhibiting the disease of transforming the tumor cells to the interstitial type, wherein the plasmid is at least one of the following plasmids

a. Any of the foregoing shRNA molecules;

b. the above recombinant plasmid.

The fourth aspect of this section introduces a mixed VRK1 protein expression inhibitor：

An inhibitor of expression of VRK1 protein, comprising at least two of

I).siVRK1:5′-GCAAGGAACCTGGTGTTGA-3′，

5'-TCAACACCAGGTTCCTTGC-3', respectively; siRNA is chemically synthesized directly, and can be used directly or mixed with transfection reagents.

III), shVRK1-2: 5'-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3' (SEQ ID NO: 3) (ii) a Two shrnas are typically constructed as plasmids mixed with other materials. Particularly, the combination effect of the two shRNAs is better, and the effect is better than the sum of the effects generated by the two shRNAs which are independently used.

After the combination, the effect of the inhibitor is better than that of any one, and the inhibition effect is obviously improved and even better than the sum of the original single use effects.

The fifth aspect of this section is presented in conjunction with a detailed study：

Experiment one: this example uses real-time fluorescent quantitative polymerase chain reaction and western blotting to detect the expression level of VRK1 in bladder cancer and paracarcinoma.

The patients with bladder cancer had 10 pairs of fresh tissues beside and after the cancer operation. Tissue total RNA was extracted at a rate of 1ml Trizol to lyse 100mg of tissue. 1ug of total RNA was reverse transcribed into cDNA and real-time fluorescent quantitative polymerase chain reaction was performed to analyze the expression of mRNA from VRK1 in paracancerous and cancerous tissues. Primer: VRK1, 5'-CTTCAGAGTCAGTTGGCAGTG-3'; 5'-CTTCAGCTTACGGGTACGAAT-3', respectively; GAPDH, 5 'CAT GGG TGT GAA CCA TGA GAA GTA-3'.

Total tissue protein was extracted at a rate of 1ml of protein lysate to lyse 100mg of tissue. Protein immunoblotting experiments were performed at 100ug protein loading to analyze the expression of VRK1 protein in the paracarcinoma and cancer tissues of the bladder.

And (4) analyzing results: as shown in fig. 1, both mRNA and protein levels of VRK1 were significantly higher in bladder cancer tissue than in paracancerous tissue.

Experiment two: this example specifically inhibits expression of VRK1 gene in bladder cancer cell lines using both siRNA and shRNA interference.

The specific sequence is constructed as follows:

siVRK1:5′-GCAAGGAACCTGGTGTTGA-3′，

5′-TCAACACCAGGTTCCTTGC-3′；

shVRK1-1:5′-tGAAAGAGAGTCCAGAAGTAttcaagagaTACTTCTGGACTCTCTTTCtttttggaa ac-3’；

shVRK1-2:5′-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3′；

shRNA-NC:5′-tCAGTCGCGTTTGCGACTGGttcaagagaCCAGTCGCAAACGCGACTGtttttggaaac-3′。

in T24 and 5637 cell lines with a fusion degree of 50% -70% in 6-well plates, each well was transfected separately (shRNA-NC, siVRK1, shVRK1-1 and shVRK 1-2). After 8h of transfection, the medium containing the complex was discarded and replaced with new medium. On the third day after transfection, cell samples were collected and analyzed for expression of VRK1 using western blot assay.

And (4) analyzing results: as shown in FIG. 2, the expression level of VRK1 protein in cells of shVRK1-1 and shVRK1-2 groups (T24, 5637) was significantly lower than that of control shRNA-NC, siVRK1 and shVRK1-1 groups. The shVRK1-1 and shVRK1-2 sequences have better inhibiting effect on the VRK1 gene than siVRK1 small interfering fragments. Indicating that of the two interference approaches, the shRNA approach is more effective than the siRNA approach.

Experiment three: shVRK1-2 inhibits bladder cancer proliferation and clonogenic.

The experiment of CCK-8 proves that shVRK1-2 inhibits the proliferation of bladder cancer.

Bladder cancer cells T24 and 5637 are respectively inoculated on a 96-well culture plate at the density of 2000 cells/well, shRNA-NC, shVRK1-1 and shVRK1-2 are respectively transfected, 10 mu L of CCK-8 solution is added into each well after 0h, 20h, 40h, 60h and 80h, the wavelength is selected to be 450nm after incubation for 2 hours at 37 ℃, the light absorption value of each well at different time points is measured on an enzyme-linked immunosorbent assay instrument, and the result is recorded. The magnitude of the absorbance reflects the cellular activity.

The results show that: as shown in fig. 3, the experimental group (shVRK1-2) significantly inhibited proliferation of T24 and 5637 cells, compared to the control group (shRNA-NC) and the experimental group (shVRK 1-1).

② clone formation experiments prove that shVRK1-2 can inhibit the proliferation of bladder cancer.

The log-phase bladder cancer cells T24 and 5637 transfected with shRNA-NC, shVRK1-1 and shVRK1-2 were digested into single cell suspensions, 100 cells of each group were inoculated into 6-well plates containing 2.5ml of 37 ℃ pre-warmed culture medium, and gently rotated to disperse the cells uniformly. Placing in a 5% CO2 incubator at 37 deg.C, and standing for 2-3 weeks. When macroscopic colonies appeared in the culture dish, the culture was terminated. Discarding the supernatant, carefully washing with PBS for 2 times, adding methanol for fixation for 15min, discarding methanol, adding Giemsa for dyeing for 10-30min, slowly washing off the dyeing solution with running water, standing and drying. The number of clones larger than 10 cells was counted by taking pictures under a microscope.

The results show that: as shown in fig. 4, the experimental group (shVRK1-2) significantly inhibited clonogenic capacity of T24 and 5637 cells, compared to the control group (shRNA-NC) and the experimental group (shVRK 1-1). And the inhibition capacity of the compound against T24 cells is obviously better than that of other compounds.

Experiment four: shVRK1-2 can inhibit the metastasis and invasion of bladder cancer.

Firstly, subjecting logarithmic bladder cancer cells T24 and 5637 transfected with shRNA-NC, shVRK1-1 and shVRK1-2 to serum starvation for 12 hours, digesting the cells into single cell suspension, suspending the single cell suspension into 2 x 105 cells/ml by using serum-free culture solution containing 0.1% BSA, taking 200ul of the cell suspension, and inoculating the cell suspension into the upper chamber of a Transwell, wherein the lower layer of the culture solution contains 10% FBS complete culture medium. The plates were incubated for 24h in CO2 medium at 37 ℃. The chamber was removed, washed 3 times with PBS, the cells in the upper microporous membrane layer of the chamber were carefully wiped off with a cotton swab, fixed with 4% paraformaldehyde for 20min, stained with 0.1% crystal violet for 15min, and then air-dried. And (4) taking a picture under an inverted microscope, counting 5 fields of view at random for each sample, taking an average value, and carrying out statistical analysis.

The results show that: as shown in fig. 5, the experimental group (shVRK1-2) significantly inhibited the metastatic ability of T24 and 56373 cells, compared to the control group (shRNA-NC and experimental group (shVRK 1-1)).

② Transwell invasion experiments prove that shVRK1 can inhibit the invasion of bladder cancer.

The day before the experiment, Matrigel was placed in a refrigerator at 4 ℃ overnight from-20 ℃ in advance, and was melted from a solid state to a liquid state. Mixing matrigel with a mixture of 1: 8, coating the upper surface of a basement membrane at the bottom of the Transwell chamber after dilution, and air-drying for 3 h. The residual liquid in the culture plate was aspirated, 50ul of serum-free culture medium was added to each well, and the basement membrane was hydrated at 37 ℃ for 30 min. The log-phase T24 and 5637 bladder cancer cells transfected with shRNA-NC, shVRK1-1 and shVRK1-2 were serum-starved for 12h and then digested into single cell suspensions, adjusted to a density of 5X 105 cells/ml. 200ul of cell suspension was added to the upper chamber of the Transwell chamber. 600ul of medium containing 10% FBS was added to the lower chamber. The culture plate was placed in CO2 medium at 37 ℃ and cultured for 48 hours. The chamber was removed, washed 3 times with PBS, the cells in the upper microporous membrane layer of the chamber were carefully wiped off with a cotton swab, fixed with 4% paraformaldehyde for 20min, stained with 0.1% crystal violet for 15min, and then air-dried. And (4) taking a picture under an inverted microscope, counting 5 fields of view at random for each sample, taking an average value, and carrying out statistical analysis.

The results show that: as shown in fig. 6, the experimental group (shVRK1-2) significantly inhibited the invasive ability of T24 and 56373 cells, compared to the control group (shRNA-NC) and the experimental group (shVRK 1-1).

Experiment five: shVRK1-2 can inhibit bladder cancer invasion and metastasis by inhibiting epithelial-mesenchymal transition (EMT) signaling pathway in bladder cancer cells.

In T24 and 5637 cell lines with a fusion degree of 50% -70% in 6-well plates, each well was transfected separately (shRNA-NC, siVRK1, shVRK1-1 and shVRK 1-2). After 8h of transfection, the medium containing the complex was discarded and replaced with new medium. On the third day after transfection, cell samples were collected and analyzed for the expression of representative molecules in the EMT signaling pathway, such as epithelial markers (E-cadherin), mesenchymal markers (N-cadherin, Vimentin, and Fibronectin).

The results show that: as shown in FIG. 7, the epithelial marker E-cadherin was up-regulated in the experimental group (shVRK1-1 and shVRK1-2) and highest in the shVRK1-2 group, compared to the control group (shRNA-NC); the expression of the mesenchymal markers (N-cadherin, Vimentin and Fibronectin) was all down-regulated, with shVRK1-2 group expressing the lowest. It was shown that shVRK1-2 was able to shift bladder cancer cells to an epithelial type that was less prone to metastasis and invasion. In addition, the part has verified the transformation regulation effect on the tumor cells, the regulation effect on other types of tumor cells is not repeated, and all diseases which can be prevented and treated by regulating corresponding markers are within the scope of the invention.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims

shRNA molecule characterized in that it is in its sequence

The sense strand sequence is SEQ ID NO: 1: 5'-GCAAGGAACCTGGTGTTGA-3' the flow of the air in the air conditioner,

the antisense strand sequence is SEQ ID NO: 2: 5'-TCAACACCAGGTTCCTTGC-3' are provided.
2. An shRNA molecule according to claim 1, characterized in that it has the following sequence SEQ ID NO: 3:

5'-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3'。
3. a recombinant plasmid or construct comprising the nucleic acid sequence of SEQ ID NO: 3, or a shRNA molecule and plasmid according to any of claims 1-2.
An inhibitor of expression of VRK1 protein, comprising at least one of the following

a. The shRNA molecule of claim 1 or 2;

b. the recombinant plasmid of claim 3.
A VRK1 gene silencing kit comprising the inhibitor of claim 4.
Application of VRK1 gene expression inhibitor in preparing medicine for preventing and treating bladder cancer.
7. The use of claim 6, wherein the inhibitor of VRK1 gene expression is at least an shRNA or siRNA or miRNA or polypeptide.
8. The use according to claim 7, wherein the inhibitor of VRK1 gene expression comprises at least one of

a. The shRNA molecule of claim 1 or 2;

b. the recombinant plasmid of claim 3.
9. The use of claim 6, wherein the inhibitor modulates bladder cancer by at least any one of

i) Inhibiting the proliferation and cloning formation of bladder cancer cells;

ii) inhibiting the metastasis and invasion of bladder cancer cells.
10. Application of plasmid in preparation of drugs for preventing and treating bladder cancer, wherein the plasmid is at least one of the following plasmids

a. The shRNA molecule of claim 1 or 2;

b. the recombinant plasmid of claim 3.

Application of plasmid in preparing medicine for preventing and treating diseases induced by over-expression of VRK1, wherein the plasmid is at least one of the following plasmids

a. The shRNA molecule of claim 1 or 2;

b. the recombinant plasmid of claim 3.

Use of a plasmid for the manufacture of a medicament for inhibiting a disease resulting from a change of tumor cells to a mesenchymal type, wherein said plasmid is at least one of

a. The shRNA molecule of claim 1 or 2;

b. the recombinant plasmid of claim 3.

A drug for preventing and treating bladder cancer, which comprises the VRK1 gene expression inhibitor according to claim 4.

The VRK1 protein expression inhibitor is characterized by comprising at least two of the following

I).siVRK1:5′-GCAAGGAACCTGGTGTTGA-3′，

5′-TCAACACCAGGTTCCTTGC-3′；

II).shVRK1-1:5′-tGAAAGAGAGTCCAGAAGTAttcaagagaTACTTCTGGACTCTCTTTCtttttggaa ac-3’(SEQ ID NO：4)；

III).shVRK1-2:5′-tGCAAGGAACCTGGTGTTGAttcaagagaTCAACACCAGGTTCCTTGCtttttggaaac-3′(SEQ ID NO：3)。

Use of the VRK1 protein expression inhibitor of claim 14 in the preparation of a medicament for the prevention and treatment of bladder cancer.