A siRNA molecule for inhibiting GRWD1 gene and its application

Abstract

The present invention discloses a siRNA molecule for inhibiting the GRWD1 gene and its application, belonging to the field of biomedical technology. The present invention reveals for the first time that the expression of GRWD1 in the cancerous tissue of liver cancer patients is higher than that in the adjacent cancerous tissue, and the siRNA molecule of the GRWD1 gene provided by the present invention can effectively inhibit the expression of GRWD1, and then can significantly inhibit the proliferation and migration ability of liver cancer cells HepG2 and Huh‑7 cells, and at the same time can also reduce the half inhibition concentration of liver cancer cells to chemotherapeutic drugs. The experiment of this application shows that GRWD1 is a key gene that promotes the occurrence and development of liver cancer, which can be used as a drug design target for the treatment of liver cancer, and the siRNA molecule of the GRWD1 gene provided by the present invention can be used to prepare anti-liver cancer therapeutic drugs, which has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a siRNA molecule for inhibiting GRWD1 gene and application thereof. Background Art

[0002] Primary liver cancer is a common malignant tumor worldwide. The 2020 statistics of the World Health Organization show that the incidence of liver cancer ranks sixth and the mortality rate ranks third in the world. In China, the incidence of liver cancer ranks fifth, and the mortality rate is still second. For early liver cancer, radical surgery is the preferred treatment for liver cancer, but the recurrence rate is as high as 70% 5 years after surgery. Due to the insidious onset of liver cancer and the rapid progression of the tumor, 75% of HCC patients have already developed portal vein cancer thrombosis or distant metastasis and other mid- and late-stage manifestations at the time of diagnosis. The loss of the opportunity for radical surgical resection is the primary reason that affects the overall efficacy of liver cancer in my country. It is difficult to further improve. At present, the vast majority of domestic and foreign guidelines recommend transarterial chemoembolization (TACE) as the standard treatment for patients with mid-stage liver cancer. However, clinically, more than 40% of patients have no objective response to TACE.

[0003] The proliferation, migration and resistance of tumor cells to chemotherapy drugs are important causes of recurrence and metastasis of liver cancer after treatment. The inactivation of the tumor suppressor gene P53 is very common in human tumors. In addition to the inactivation caused by P53 mutation, the reduced stability of wild-type P53 protein is also a major cause of tumor development. About 50% of liver cancer patients do not have P53 mutations, but the reasons why wild-type P53 does not work have not been fully revealed.

[0004] The GRWD1 gene encodes a glutamate-rich ribosomal protein containing five WD-repeat motifs, which may be involved in ribosome biogenesis. Existing reports have shown that GRWD1 is an important factor in negatively regulating the stability of P53, but there are no reports of GRWD1 in liver cancer. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a siRNA molecule that inhibits the GRWD1 gene. Another technical problem to be solved by the present invention is to provide the use of the above siRNA molecule that inhibits the GRWD1 gene in the preparation of a drug for treating liver cancer.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] An siRNA molecule for inhibiting the GRWD1 gene, comprising a sense strand and an antisense strand of the following sequence:

[0008] Justice Chain:

[0009] Antisense strand:

[0010] or

[0011] Justice Chain:

[0012] Antisense strand:

[0013] or

[0014] Justice Chain:

[0015] Antisense strand:

[0016] The application of the siRNA molecule in preparing a drug for inhibiting GRWD1 gene expression.

[0017] Furthermore, in the application, the drug is a drug for treating liver cancer.

[0018] Furthermore, the liver cancer treatment drugs include treatment drugs for liver cancer recurrence and metastasis after surgery or treatment drugs for liver cancer recurrence and metastasis after transarterial chemoembolization.

[0019] Furthermore, the liver cancer therapeutic drug is a drug that reduces the proliferation and migration ability of liver cancer cells and / or reduces the half-inhibitory concentration of liver cancer cells to chemotherapy drugs.

[0020] Furthermore, the chemotherapy drug is cisplatin.

[0021] Furthermore, the medicine is an injection, an intravenous injection or a liquid oral preparation.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention first discovered that the expression of GRWD1 in cancer tissue was higher than that in paracancerous tissue in the database, liver cancer and paracancerous tissue core, and liver cancer and paracancerous tissue slices. The CCK8 experiment was performed using siRNA fragments targeting GRWD1 to transfect cultured liver cancer cells Hep-G2 and Huh-7 cells, and found that GRWD1 promoted the proliferation ability of Hep-G2 and Huh-7 cells. The Transwell migration experiment was used to find that GRWD1 promoted the migration ability of Hep-G2 and Huh-7 cells. Using gradient chemotherapy drug cisplatin treatment, the sensitivity of liver cancer cells to cisplatin was detected, and it was found that GRWD1 inhibited the sensitivity of Hep-G2 and Huh-7 cells to cisplatin. It shows that GRWD1 is a key gene that promotes the occurrence and development of liver cancer and can be used as a potential therapeutic target. The siRNA molecules of the GRWD1 gene provided by the present invention can also be used to prepare anti-liver cancer therapeutic drugs, which have important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The expression of GRWD1 in cancer tissue and adjacent tissue in TCGA database and liver cancer tissue microarray, where: Figure 1 A is the differential expression of GRWD1 in the TCGA database, Figure 1 B is the differential expression of GRWD1 in tissue microarray, Figure 1 C is the expression of GRWD1 in 8 pairs of HCC and adjacent tissues;

[0025] Figure 2 This is the test result of knockdown efficiency of siGRWD1 in liver cancer cells HepG2 and Huh-7;

[0026] Figure 3 The results of the effect of siGRWD1 on the proliferation of liver cancer cells HepG2 and Huh-7 cells are shown in the figure below. The left figure shows the statistics of CCK8 detection data of control and siGRWD1-transfected HepG2 cells, and the right figure shows the statistics of CCK8 detection data of control and siGRWD1-transfected Huh-7 cells.

[0027] Figure 4 This is a cell migration experiment diagram under a high-power microscope (scale bar is 50 μm). Figure 4 A is a representative image of HepG2 and Huh-7 cells transfected with si control and siGRWD1, and the dark color represents the migrated HepG2 or Huh-7 cells; Figure 4 B is the statistical result of migration rate;

[0028] Figure 5 The effect of siGRWD1 transfection on the half-inhibitory concentration of cisplatin in HepG2 and Huh-7 liver cancer cells, where: Figure 5 A and B are the statistics of IC50 test data of control and siGRWD1-transfected HepG2 cells. Figure 5 C and D are the statistics of IC50 detection data of control and siGRWD1-transfected Huh-7 cells. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments.

[0030] Embodiment 1:

[0031] The expression of GRWD1 in hepatocellular carcinoma was queried through the GEPIA tumor database. Figure 1 A is the expression level of GRWD1 in hepatocellular carcinoma tissue (T) and adjacent tissue (N) in the TCGA database. The results showed that GRWD1 was highly expressed in hepatocellular carcinoma.

[0032] Pathological specimens from patients with hepatocellular carcinoma were collected and immunohistochemical staining of GRWD1 was performed to detect the expression level of GRWD1 protein in hepatocellular carcinoma tissues and adjacent tissues. Figure 1 B shows that GRWD1 is highly expressed in tumor tissues. The main steps of immunohistochemistry are as follows:

[0033] 1) After the pathological tissue is dried, dewaxed, hydrated, and washed, high-temperature antigen repair is performed using alkaline repair solution;

[0034] 2) After cooling, wash the bottle with PBST and then block with blocking agent;

[0035] 3) Dilute the GRWD1 primary antibody in antibody diluent and incubate at room temperature for 2 h;

[0036] 4) After washing with PBST, incubate with secondary antibody at room temperature for 1 hour;

[0037] 5) After washing with PBST, the slides were stained with DAB and counterstained with hematoxylin.

[0038] Western blot was used to detect the expression of proteins in cancer tissues and adjacent fresh tissues of patients with hepatocellular carcinoma. Figure 1 As shown in C, GRWD1 is highly expressed in 8 pairs of fresh hepatocellular carcinoma tumor tissues. The main steps of fresh tissue Western blot are as follows:

[0039] 1) Wash fresh tissue with PBS and then lyse the tissue with tissue lysis buffer;

[0040] 2) Quantify the extracted protein using the BCA protein quantification kit;

[0041] 3) Separation of proteins using polyacrylamide gel electrophoresis;

[0042] 4) Block with BSA;

[0043] 5) Incubate with anti-GRWD1 primary antibody at 4°C overnight;

[0044] 6) Incubate with secondary antibody at room temperature for 1 hour;

[0045] 7) ECL chemiluminescence detection.

[0046] Example 2: Design and synthesis of GRWD1 inhibitors

[0047] Double-stranded small interfering RNA was designed based on the antisense sequence of the target gene GRWD1 mRNA target fragment position, and its sense strand sequence is shown in SEQ ID NO. 1, 3 or 5. The inhibitor and inhibitor control of GRWD1 were synthesized by Heyuan Biotechnology Co., Ltd. The sequence is as follows:

[0048] Inhibitors of GRWD1:

[0049] siRNA-1:

[0050]

[0051] siRNA-2:

[0052]

[0053] siRNA-3:

[0054]

[0055] The inhibitor control was a meaningless random sequence.

[0056] Example 3: Cell culture and transfection

[0057] HepG2 and Huh-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI 1640 (Thermo Fisher, USA) containing 5% fetal bovine serum (FBS; Sciencell), 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37°C in a 5% CO2 cell culture incubator and the medium was changed every two days. Chemically synthesized GRWD1 small interfering RNA and controls were transfected using lipofectamine RNAimax reagent (Invitrogen, USA) according to the instructions.

[0058] Example 4: Western Blot Detection of siRNA Knockdown Effectiveness

[0059] HepG2 and Huh-7 cells transfected with control RNA and GRWD1 siRNA for 48 hours were taken, proteins were collected and quantified, and Western Blot was performed using GRWD1 antibody (Abcam, USA) to detect the GRWD1 knockdown efficiency. Figure 2 As shown, the results show that the efficiency of transfection of siGRWD1 sequence 3 in HepG2 and knockdown of GRWD1 is the highest, and the efficiency of siGRWD1 sequence 3 in Huh-7 is also high in knocking down GRWD1, so the following examples all use sequence 3 for transfection.

[0060] Example 5: Cell proliferation assay

[0061] Inoculate HepG2 and Huh-7 cells transfected with siGRWD1 No. 3 and si control respectively in a 96-well plate, adjust the cell density to 5000 / mL with RPMI1640 medium, place the cells in an incubator at 37°C and 5% carbon dioxide when the cells grow to 50%, add 100μL of complete cell culture medium containing 10μL CCK-8 solution to each well after 24h, and return to the incubator to continue culturing for 48h. Take out the 96-well plate and adjust the detection wavelength of the microplate reader to 450nm before detecting the absorbance.

[0062] like Figure 3 As shown, compared with the si control group, the proliferation ability of HepG2 and Huh-7 cells transfected with siGRWD1 was significantly decreased.

[0063] Example 6: Transwell migration assay

[0064] The surface of the lower chamber of the Transwel chamber was coated with Fibronectin, and HepG2 and Huh-7 cells transfected with siGRWD1 and si control were collected, and the cell density was adjusted to 1×10 4 / mL, add 100μL cell suspension to the upper chamber of the transwell. Add 500μL complete culture medium containing 5% fetal bovine serum to the lower chamber. After culturing for 24h at 37℃ and 5% carbon dioxide, stain with crystal violet. After staining, observe and take pictures under an upright microscope, and randomly select 10 fields of view to count the number of cells. Finally, use 33% glacial acetic acid to completely elute the crystal violet. The OD value of the eluate can be measured at 570nm on an enzyme reader, which indirectly reflects the number of migrated cells. Figure 4 This is a cell migration experiment diagram under a high-power microscope (scale bar is 50 μm). Figure 4 A is a representative image of HepG2 and Huh-7 cells transfected with si control and siGRWD1. Figure 4 B is the statistical result of migration rate, showing that the migration rate of HepG2 and Huh-7 transfected with siGRWD1 was lower than that of the si control group. The results showed that knocking down GRWD1 could reduce the migration rate of HepG2 and Huh-7.

[0065] Example 7: Cellular drug sensitivity test

[0066] HepG2 and Huh-7 cells transfected with siGRWD1 and si control, respectively, were seeded in 96-well plates and washed with RPMI. The cell density was adjusted to 5000 cells / mL with 1640 culture medium. When the cells grew to 70%, 0μg / mL, 2μg / mL, 4μg / mL, 6μg / mL, 8μg / mL, 10μg / mL, 12μg / mL, and 14μg / mL of cisplatin were added to each group of 3 wells at 37°C and 5% carbon dioxide after 12 hours. Cisplatin was added to Huh7 cells at 0μg / mL, 1.5μg / mL, 3μg / mL, 4.5μg / mL, 6μg / mL, 7.5μg / mL, 9μg / mL, and 10.5μg / mL. After continued culture for 24 hours, 100μL of complete cell culture medium containing 10μL CCK-8 solution was added to each well. When testing, the 96-well plate was taken out and the detection wavelength of the microplate reader was adjusted to 450nm to detect the absorbance. The absorbance of each group was counted and the half inhibitory concentration of the cells was calculated. Figure 5 As shown, compared with the si control group, the sensitivity of HepG2 and Huh-7 cells transfected with siGRWD1 to cisplatin was significantly increased.

Claims

1. Application of siRNA molecules that inhibit the GRWD1 gene in the preparation of drugs for the treatment of liver cancer; The siRNA molecule for inhibiting the GRWD1 gene is composed of a sense strand and an antisense strand of the following sequence: Sense strand: 5′-CGGACAGAGCUUCCUCUUATT-3′, Antisense strand: 5′-UAAGAGGAAGCUCUGUCCGTT-3′.

2. The use according to claim 1, characterized in that: The drugs for treating liver cancer include drugs for treating the recurrence and metastasis of liver cancer after surgery or drugs for treating the recurrence and metastasis of liver cancer after transarterial chemoembolization.

3. The use according to claim 1, characterized in that: The drug for treating liver cancer is a drug that reduces the proliferation and migration ability of liver cancer cells and / or reduces the half-inhibitory concentration of liver cancer cells to chemotherapy drugs.

4. The use according to claim 3, characterized in that: The chemotherapy drug is cisplatin.

5. The use according to any one of claims 1 to 4, characterized in that: The medicine is an injection, an intravenous injection or a liquid oral preparation.

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