CN117187246B - SiRNA for inhibiting CKAP5 gene expression and application thereof - Google Patents

Abstract

The invention discloses siRNA for inhibiting CKAP5 gene expression and application thereof. The siRNA provided by the invention can inhibit CKAP5 expression in a targeted manner, effectively inhibit gastric cancer cell proliferation and gastric cancer cell clone formation capacity, promote the decrease of gastric cancer cell mitochondrial membrane potential and promote gastric cancer cell copper death, provide a new target and hope for gastric cancer treatment, and have important medical application value.

Description

SiRNA for inhibiting CKAP5 gene expression and application thereof

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to siRNA for inhibiting CKAP5 gene expression and application thereof.

Background

Gastric cancer (Gastric carcinoma, GC) is a clinically common tumor of the digestive tract. Among them, helicobacter pylori (Hp) infection, genetic factors and environmental factors are important factors that lead to the development and progression of gastric cancer. The clinical treatment of gastric cancer adopts the means such as radiotherapy, chemotherapy, etc., but the curative effect is often poor due to the influence of drug resistance and side effects.

CKAP5 (Cytoskeleton-associatedprotein) is a cytoskeletal related protein. It plays an important role in cell division and cell movement. Several studies have shown that CKAP5 expression levels in a variety of tumors are significantly increased and correlated with malignancy and prognosis of the tumor. For example, high expression of CKAP5 is associated with the occurrence and prognosis of multiple cancers such as breast cancer, lung cancer, colorectal cancer, and the like. Overexpression of CKAP5 may promote proliferation, invasion and metastatic capacity of tumor cells. Furthermore, some studies have found that by inhibiting the expression or function of CKAP5, the proliferation, invasion and metastatic capacity of tumor cells can be inhibited. This suggests that CKAP5 may be a potential therapeutic target, helping to develop new antitumor drugs.

There is also limited current research on the relationship between CKAP5 and gastric cancer. However, some studies indicate that CKAP5 expression levels in gastric cancer are significantly increased and correlated with malignancy and prognosis of tumors. However, inhibitors of CKAP5 have not been discovered.

Nucleic acid drugs are a good choice for drugs for which inhibitors have not yet been developed, and are a class of therapeutic methods that utilize nucleic acid molecules (e.g., DNA and RNA) as drugs. Compared with the traditional small molecule compound medicine, the nucleic acid medicine has the following advantages:

1. High specificity: nucleic acid drugs can recognize target molecules, such as specific genes or RNA sequences, through sequence specificity, thereby achieving highly specific therapeutic effects.

2. Targeted therapy: nucleic acid drugs can realize accurate targeted therapy by designing a sequence targeting a specific gene or protein, and reduce the influence on normal cells.

Copper death (copper death) refers to the death of tumor cells in cancer treatment by increasing the concentration of bulk copper ions. Copper plays an important role in cells and is involved in a variety of biological processes including cellular respiration, redox reactions, DNA synthesis, and the like. The mechanism of action of CKAP5 in copper death has not been reported.

Disclosure of Invention

Therefore, the embodiment of the invention provides siRNA for inhibiting CKAP5 gene expression and application thereof.

In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:

according to a first aspect of embodiments of the present invention, there is provided an siRNA that inhibits CKAP5 gene expression, the siRNA being siCKAP-1, siCKAP5-2 or siCKAP5-3; wherein,

The sense strand of siCKAP-1 is shown as SEQ ID NO.1, and the antisense strand is shown as SEQ ID NO. 2;

The sense strand of siCKAP-2 is shown as SEQ ID NO.3, and the antisense strand is shown as SEQ ID NO. 4;

The sense strand of siCKAP-3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6.

Further, the 3' end of the siRNA sequence is 2 dT modifications.

According to a second aspect of embodiments of the present invention, the present invention provides the use of an siRNA that inhibits CKAP5 gene expression as described above in the manufacture of a medicament for preventing or treating gastric cancer.

According to a third aspect of embodiments of the present invention, there is provided an anti-gastric cancer drug comprising an siRNA as described above.

The embodiment of the invention has the following advantages:

The siRNA provided by the invention can inhibit CKAP5 expression in a targeted manner, effectively inhibit gastric cancer cell proliferation and gastric cancer cell clone formation capacity, promote the decrease of gastric cancer cell mitochondrial membrane potential and promote gastric cancer cell copper death, provide a new target and hope for gastric cancer treatment, and have important medical application value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

FIG. 1 shows CKAP5 expression in a gastric cancer patient sample, wherein (a) a non-paired sample and (b) a paired sample;

FIG. 2 shows the expression of CKAP5 in 3 gastric cancer cell lines;

FIG. 3 shows the expression of CKAP5 mRNA in gastric cancer cells after siCKAP action;

FIG. 4 shows the expression of CKAP5 protein in gastric cancer cells after siCKAP action;

FIG. 5 shows the results of activity detection of gastric cancer cells after siCKAP action;

FIG. 6 shows the effect of siCKAP on the proliferation potency of gastric cancer cells;

FIG. 7 shows the decrease in mitochondrial membrane potential of gastric cancer cells after siCKAP action;

FIG. 8 shows the expression of copper death-related proteins after siCKAP action.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

According to a plurality of double-stranded siRNA sequences designed by CKAP5 genes, through experimental verification, the following 3 siRNAs which effectively inhibit CKAP5 expression are screened, and the sequence information is as follows:

siCKAP5-1：

sense strand 5'-3': GCUCCUAGACCUACUCGAU (SEQ ID NO. 1);

Antisense strand 5'-3': AUCGAGUAGGUCUAGGAGC (SEQ ID NO. 2).

siCKAP5-2：

Sense strand 5'-3': GAGUCUGUAGAAGUACUAA (SEQ ID NO. 3);

antisense strand 5'-3': UUAGUACUUCUACAGACUC (SEQ ID NO. 4).

siCKAP5-3：

Sense strand 5'-3': GCCUGUAUAGAGACACUGA (SEQ ID NO. 5);

antisense strand 5'-3': UCAGUGUCUCUAUACAGGC (SEQ ID NO. 6).

The 3' -end of the above sequence was modified with (dT) (dT), and this part was synthesized by the Optimago family.

Example 2

1. Materials and methods

1. Material

1.1 Cell lines

Human gastric cancer cell line (HGC 27) was purchased from Saiborin (Shanghai) Biotechnology Co., ltd.

1.2 Experiment related Agents

DMEM high sugar medium (available from Gibco); opti-MEM reduced serum medium (available from Gibco); 1640 medium (available from Gibco) anti-CKAP5 antibody (available from Proteintech); anti-MTF1, anti-PDHB, anti-beta-tublin antibody (purchased from CST); negative control sequences (provided by the qing family); cell Counting Kit-8 cell counting reagent (CCK-8, available from biomake); lipo2000 transfection reagent (available from thermo); real-time fluorescent quantitative PCR kit (qPCR, biomake).

2. Method of

2.1 Cell culture

HGC 27 was cultured in DMEM high-sugar incomplete medium, counted, cell density was adjusted, inoculated in a petri dish, and finally placed in a cell incubator (37 ℃,5% CO ₂) for stationary culture. Periodically, fresh culture medium was changed and HGC 27 cells in the logarithmic phase were selected for the experiment.

2.2QPCR detection of CKAP5-mRNA expression level after HGC 27 transfection of siRNA

① HGC 27 transfected siRNA: siCKAP5-1, siCKAP-2, siCKAP5-3 freeze-dried mother solution (20 mu M) and negative control freeze-dried mother solution (20 mu M) are dissolved in opti-MEM serum-free medium, and diluted and adjusted to 100nM (solution A); preparing a mixed liquid (liquid B) of Lipo2000:opti-MEM serum-free medium according to a ratio of 1:49 (v/v); finally, mixing and incubating A and B for 20min (solution C) according to the proportion of 1:1 (v/v). The concentration of HGC 27 cells is regulated by using the dilution of a culture medium, the HGC 27 cells are planted in six-hole plates at the density of 25 x 10 ⁴ cells/hole, 500 mu l of C solution is dripped into each hole, the mixture is placed in a cell culture box for culturing for 6 hours, and finally 2ml of 20%1640 complete culture medium is added into each hole for continuous culturing.

② QPCR (quantitative polymerase chain reaction) detection of CKAP5-mRNA expression level: qPCR was used to detect expression of CKAP5-mRNA in each group of cells. Cellular RNA was extracted separately with Trizol. And (3) judging the integrity of the RNA by agarose gel electrophoresis, detecting the purity and the concentration of the total RNA by an ultra-micro spectrophotometer, performing reverse transcription on the RNA to synthesize cDNA, operating according to a kit instruction, designing a primer corresponding to real-time quantification, and performing PCR amplification by using a fluorescent light quantitative PCR instrument. The expression of the target gene and the reference gene in each group is relatively quantified by comparing the Ct value difference between the target gene and the reference gene by using beta-tublin as the internal reference.

2.3Westernblot detection of CKAP5 and copper death-related protein levels after HGC27 transfection of siRNA

Each group of cells was tested for CKAP5 expression using Westernblotting. The total proteins of each group are extracted according to the reagent kit instructions, the protein content in the sample is detected by a BCA method after centrifugation, the protein is separated by 10% SDS-PAGE electrophoresis of the sample, the sample is transferred onto a PVDF membrane, and the sample is blocked by 5% skimmed milk powder for 60 minutes at room temperature. Adding CKAP5 and MTF1, PDHB antibodies after washing the membrane, placing the membrane on a shaking table at 4 ℃ for incubation overnight, adding beta-tublin antibodies after washing the membrane, placing the membrane on a room temperature for incubation for 1h, and fully washing. And (3) preparing ECL luminous solution from the solution A and the solution B according to the proportion of 1:1 (v/v), removing excessive moisture on the film by using absorbent paper, placing the film in a transparent clean dish, adding the luminous solution, developing by using a universal imaging system (BIO-RAD), and further comparing protein expression differences among cell groups.

2.4CCK-8 detection of viability of cells after HGC 27 transfection with siRNA

HGC 27 cells transfected with negative control-siRNA (100 nM), siCKAP-1 (100 nM), siCKAP5-2 (100 nM), siCKAP5-3 (100 nM) were planted in 96-well plates at a density of 5000 cells/well, 4 duplicate wells were placed in each group, 20. Mu.l of CCK-8 detection reagent was added to each well after 72 hours, incubated at 37℃for 1 hour, placed on an oscillator and shaken for 10 minutes, absorbance was detected at 450nM using an enzyme-labeled instrument, and cell viability= (drug well OD-blank well OD)/(blank control well OD-blank well OD).

2.5 Cloning formation experiments to test the clonality of cells after transfection of HGC 27 with siRNA

HGC 27 cells transfected with negative control-siRNA (100 nM), siCKAP-1 (100 nM), siCKAP5-2 (100 nM), siCKAP5-3 (100 nM) were planted in 6-well plates at a cell/well density of 1000 cells/well, 3 duplicate wells were set per group, cultured in a cell incubator for 14 days, liquid changes were performed every 3 days and cell states were observed, medium was removed, cells were washed with PBS, and 4% paraformaldehyde was used to fix the cells for 20min; removing paraformaldehyde, and dyeing with 0.2% crystal violet for 5min; the cells were washed with water, air dried, scanned, photographed, imagej counted for clone number, graphPad treated for analytical data.

2.6 Flow cytometry detection of mitochondrial Membrane potential of cells following transfection of siRNA with HGC 27

HGC 27 cells transfected with negative control-siRNA (100 nM), siCKAP-1 (100 nM), siCKAP5-2 (100 nM), siCKAP5-3 (100 nM) were seeded at a density of 2X 10 ⁵ cells/well in 6-well plates, 3 replicate wells were placed in each group, and cultured in a cell culture incubator for 48 hours. Cells were resuspended in 1mL JC-1 working solution, incubated in an incubator for 30min, centrifuged at 400g for 5min at room temperature, and immediately the fluorescence intensity was measured by flow cytometry and data collected and analyzed.

2. Results

(1) CKAP5 is remarkably and highly expressed in gastric cancer

The expression of CKAP5 protein in normal human and gastric cancer patient samples was analyzed by TCGA on-line database (https:// www.cancer.gov/ccg/research/genome-sequencing/TCGA), and the result indicated that CKAP5 was significantly highly expressed in gastric cancer patient samples (see FIG. 1). In vitro experiments, western blot detection and analysis indicate that CKAP5 protein is highly expressed in 3 gastric cancer cell lines (BGC-803, MGC-803 and HGC-27) (see figure 2).

(2) CKAP5-siRNA can inhibit expression of CKAP5 in HGC 27 gastric cancer cells

Sequences siCKAP-1, siCKAP5-2, and siCKAP5-3 all significantly reduced expression of mRNA and protein levels of CKAP5 in HGC 27 gastric cancer cells (see fig. 3 and 4).

(3) CKAP5-siRNA obviously inhibits activity of HGC 27 gastric cancer cells

After siCKAP-1, siCKAP5-2, or siCKAP5-348h of HGC 27 gastric cancer cell transfection sequences, cell activity was examined using CCK-8, and the results showed that siCKAP-1, siCKAP5-2, and siCKAP5-3 groups were significantly lower than the negative control sequences (see FIG. 5).

(4) CKAP5-siRNA obviously inhibits proliferation formation capacity of HGC 27 gastric cancer cells

Proliferation forming ability of HGC 27 gastric cancer cells transfected with CKAP5-siRNA/NC was examined by using a cell cloning experiment. The results showed that the proliferation formation ability of HGC 27 gastric cancer cells was significantly reduced compared with the control group after transfection siCKAP-1, siCKAP5-2 or siCKAP5-3, wherein the gastric cancer cell mass stained with crystal violet was not substantially seen after transfection siCKAP-3, indicating that the sequence siCKAP5-3 was most potent in inhibiting gastric cancer cell proliferation (see FIG. 6).

(5) CKAP5-siRNA obviously reduces mitochondrial membrane potential of HGC 27 gastric cancer cells

And (3) detecting the mitochondrial membrane potential condition of the HGC 27 gastric cancer cells by using JC-1 reagent staining and a flow cytometer. The results showed that siCKAP-1, siCKAP5-2, and siCKAP5-3 significantly reduced the mitochondrial membrane potential of the HGC 27 gastric cancer cells compared to NC group (see fig. 7).

(6) CKAP5-siRNA for improving expression of HGC 27 gastric cancer cell copper death related protein

And detecting the expression of HGC 27 gastric cancer cell copper death related proteins (MTF 1, PDHB) by using western blot. The results showed that siCKAP-1, siCKAP5-2, and siCKAP5-3 increased the expression of MTF1, PDHB (copper death associated protein) in HGC 27 gastric cancer cells (see FIG. 8).

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (2)

1. An application of siRNA for inhibiting CKAP5 gene expression in preparing a medicament for preventing or treating gastric cancer is characterized in that the siRNA is siCKAP-3, wherein,

The sense strand of siCKAP-3 is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6.

2. The use of claim 1, wherein the 3' end of the siRNA sequence is 2 dT modifications.