Application of PLCB1 gene in regulation and control of sensitivity of liver cancer cells to lenvatinib drugs

Abstract

The invention discloses an application of a PLCB1 gene in regulating and controlling the sensitivity of liver cancer cells to a lenvatinib drug. The small interfering RNA provided by the invention effectively knocks down PLCB1 expression in drug-resistant HCC cells, significantly improves the sensitivity of the cells to the lenvatinib, and is helpful for reversing the drug resistance of the lenvatinib; after the PLCB1 is knocked down, the proliferation of Hep3B-LR cells is obviously inhibited, so that the treatment effect of the lenvatinib is improved, and the growth and progress of tumors are favorably controlled; compared with a traditional chemotherapeutic drug, the small interfering RNA has the advantage of high targeting property, only influences the expression of the PLCB1 gene, has small side effects on other non-drug-resistant cells, and is higher in safety.

Description

Technical Field

[0001] The present invention relates to the application of PLCB1 gene in regulating the sensitivity of liver cancer cells to lenvatinib drug, and belongs to the field of cancer molecular targeted therapy. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, usually accompanied by poor prognosis and high recurrence rate. As a multi-target kinase inhibitor, lenvatinib is widely used in the targeted treatment of HCC, but in the process of clinical application, many patients will develop acquired resistance to lenvatinib, resulting in a significant decrease in efficacy. At present, the molecular mechanism of drug resistance in HCC patients has not been fully clarified. Studies have shown that the PLCB1 gene is highly expressed in a variety of tumors and may participate in the anti-apoptotic mechanism of cells by activating signaling pathways such as PI3K / AKT and ERK. However, there is no research and application that clearly links PLCB1 gene knockdown with reversing HCC cell resistance to lenvatinib. Therefore, intervention methods targeting the PLCB1 gene are expected to become an important way to overcome lenvatinib resistance. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of the PLCB1 gene in regulating the sensitivity of liver cancer cells to the drug lenvatinib.

[0004] Technical solution: To solve the above technical problems, the present invention provides the application of a primer pair having nucleotide sequences such as SEQ ID NO.3 and SEQ ID NO.4 in regulating the sensitivity of liver cancer cells to the drug lenvatinib.

[0005] Among them, the application includes enhancing or restoring the sensitivity of liver cancer cells to the drug lenvatinib.

[0006] The present invention also provides application of the PLCB1 gene in regulating the sensitivity of liver cancer cells to the drug lenvatinib.

[0007] Among them, the application includes enhancing or restoring the sensitivity of liver cancer cells to the drug lenvatinib.

[0008] Wherein, the application includes knocking down the PLCB1 gene or inhibiting the expression level of the PLCB1 gene.

[0009] The application includes transfecting PLCB1-specific small interfering RNA into liver cancer cells; the nucleotide sequence of the PLCB1-specific small interfering RNA is shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] Acquired resistance to the targeted drug lenvatinib in patients with hepatocellular carcinoma (HCC). HCC patients are prone to develop resistance after long-term use of lenvatinib, which leads to decreased therapeutic efficacy and accelerated disease progression. Studies have shown that the expression of the PLCB1 gene is significantly upregulated in lenvatinib-resistant HCC cells, which may be a key factor leading to resistance. The present invention aims to restore the sensitivity of HCC cells to lenvatinib by knocking down the PLCB1 gene, thereby improving the therapeutic effect of lenvatinib and providing a new treatment strategy for patients with resistant HCC.

[0011] The present invention provides a small interfering RNA (siRNA) specifically targeting the PLCB1 gene and its application, which specifically comprises the following steps:

[0012] 1. Design and preparation of small interfering RNA: Design a small interfering RNA sequence (siPLCB1) that specifically targets the PLCB1 gene to effectively reduce the expression level of PLCB1 in HCC cells.

[0013] 2. Cell experiment: siPLCB1 was transfected into the lenvatinib-resistant HCC cell line Hep3B-LR, and a control group (NC group) without siRNA transfection was set up. 36 hours after transfection, the knockdown effect of the PLCB1 gene was verified by qPCR, and the results showed that siPLCB1 could significantly reduce the mRNA expression level of PLCB1.

[0014] 3. Drug sensitivity test: Hep3B-LR cells transfected with siPLCB1 were inoculated in 96-well plates, and 10 mM concentration of lenvatinib was added and cultured for four consecutive days. The cell proliferation was detected by CCK-8 method every day to observe the inhibitory effect of PLCB1 knockdown on the proliferation of drug-resistant HCC cells.

[0015] 4. Data analysis: The results showed that after knocking down the PLCB1 gene, the sensitivity of Hep3B-LR cells to lenvatinib was significantly improved, as shown by the significantly lower cell proliferation rate in the siPLCB1 group than in the control group, indicating that the therapeutic effect of lenvatinib was partially restored after knocking down PLCB1.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0017] 1. Restoring the drug sensitivity of drug-resistant cells: The small interfering RNA of the present invention effectively knocked down the expression of PLCB1 in drug-resistant HCC cells, significantly increased the sensitivity of these cells to lenvatinib, and helped to reverse lenvatinib resistance;

[0018] 2. Inhibit the proliferation of drug-resistant cells: After PLCB1 knockdown, the proliferation of Hep3B-LR cells was significantly inhibited, thereby improving the therapeutic effect of lenvatinib and helping to control tumor growth and progression;

[0019] 3. Provide a new treatment strategy for drug-resistant HCC patients: The small interfering RNA of the present invention provides a new molecular targeted treatment method for HCC patients with lenvatinib resistance in the clinic, which is expected to improve the prognosis of patients and enhance the treatment effect;

[0020] 4. High specificity and low side effects: Compared with traditional chemotherapy drugs, the small interfering RNA of the present invention has the advantage of strong targeting, only affects the expression of the PLCB1 gene, has less side effects on other non-drug-resistant cells, and is safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is the result of qPCR detection of knocking down PLCB1 in Hep3B-LR cells: The figure shows the mRNA expression level of PLCB1 gene in the siPLCB1 group compared with the control group (NC group);

[0022] Figure 2 Proliferation curves of the two cells after culture with 10 mM lenvatinib: The figure shows the changing trend of OD values ​​of Hep3B-LR.NC group (drug-resistant cells without PLCB1 knockdown) and Hep3B-LR.siPLCB1 group (PLCB1 knockdown group) with the number of cell culture days. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] The reagents and raw materials used in the present invention can be obtained from commercial sources or prepared according to literature methods. Experimental methods without specific conditions specified in the examples of the present invention are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0025] In this example, small interfering RNA (siRNA) was used to knock down the expression of the PLCB1 gene (GeneID: 23236) in the liver cancer cell line Hep3B-LR to verify the expression effect of PLCB1 after knockdown.

[0026] Example 1 Cell culture and transfection

[0027] 1. Cell recovery and culture

[0028] It was determined that the dose response IC50 (48h) value of Hep3B cells to lenvatinib was about 5μM. The Hep3B lenvatinib-resistant cells were constructed by increasing the drug concentration. Hep3B cells in the logarithmic growth phase were taken. When the cell confluence was 60%, 1.25μM lenvatinib was added for 48h, and the drug-containing medium was discarded. After washing with PBS twice, fresh drug-free medium was replaced. After the cells resumed growth, trypsin digestion and re-plating of cells were repeated with 1.25μM lenvatinib for 48h. Repeat the above operation 6 to 8 times. After the cells can grow stably at this concentration, the drug concentration is gradually increased to continue screening and culturing. Hep3B cells were screened at four concentrations of 1.25μM, 2.5μM, 3.75μM and 5μM for 5m, and finally they could grow stably in a medium containing 5μM lenvatinib. The resistant cell line was named Hep3B-LR (lenvatinib resistant, LR). For detailed steps, please refer to the paper: Targeting LEF1-mediated epithelial-mesenchymal transition reverses lenvatinib resistance in he patocellular carcinoma (https: / / doi.org / 10.1007 / s10637-024-01426-2).

[0029] The constructed Hep3B lenvatinib-resistant cells (named Hep3B-LR) and Hep3B were taken out from the -80°C refrigerator, quickly thawed in a 37°C water bath, gently shaken, and then the cell suspension was transferred into DMEM complete medium containing 10% fetal bovine serum and antibiotics. The cells were cultured in a 37°C, 5% CO2 incubator until the cell density was suitable for passage.

[0030] The revived cells were subcultured to the third generation, and when the cells were stable, Hep3B-LR cells were inoculated into two wells of a 6-well plate, and Hep3B cells were inoculated into one well. The inoculation density was about 2×105 cells per well, and transfection experiments were performed when the cell density reached 60-70%.

[0031] 2. Dissolution of small interfering RNA

[0032] (1) Dissolve the siRNA at a concentration of 20 μM / L (i.e., 20 pmol / μL) according to the nmol number marked on the wall of the tube. For example, if each tube contains 2.5 nmol siRNA, add 125 μL of DEPC water; if it is 5 nmol, add 250 μL of DEPC water. After dissolution, shake gently, centrifuge briefly, and store at -80°C for later use after aliquoting. Among them, siRNA is: SEQ ID NO.3: Sense: 5'-GGAUCUCAGCCUUGUCAA-3', SEQ ID NO.4: Anti-Sense: 5'-UUUGACAGGCUCUGAGAUC-3' (synthesized by Shanghai Sangon Biotechnology Co., Ltd.).

[0033] (2) Transfection and detection

[0034] Transfection reagent dilution: Take 7.5 μL Lipofectamine 3000 transfection reagent per well, add 250 μL DMEM medium without serum and antibiotics, and mix gently.

[0035] siRNA dilution: Add 200 pmol siRNA (such as siPLCB1 or control siRNA, i.e., NC) to each well. Take 10 μL of siRNA solution, add 250 μL of DMEM medium without serum and antibiotics, and mix gently.

[0036] Gently mix the diluted Lipofectamine 3000 and the diluted siRNA and let stand at room temperature for 10 minutes to form a transfection mixture.

[0037] Discard the culture medium in the 6-well plate, add 500 μL of transfection mixture to each well, and supplement with 1.5 mL of "double-free" culture medium (without fetal bovine serum and antibiotics). After gently shaking, place in a 37°C, 5% CO2 incubator for 8-10 hours. After 8-10 hours of transfection, discard the mixture and replace with serum-containing complete culture medium DMEM.

[0038] 3. RNA extraction steps

[0039] 36 hours after transfection, the density of cultured cells reached 80% to 90%. Discard the culture medium and gently wash the cells 3 times with PBS to remove the residual culture medium. Add an appropriate amount of Trizol reagent according to the number of cells to lyse the cells. Add 600μL Trizol to each well of the 6-well plate. Use a pipette to gently blow and completely remove the cells, and then transfer the lysate to an EP tube without RNAse contamination. Let it stand for 10 minutes to ensure that the cells are completely lysed. Add chloroform at a volume ratio of 5:1 between Trizol and chloroform. Cover the EP tube tightly, shake vigorously for 15 seconds, mix the aqueous phase and the organic phase thoroughly, and let it stand for 10 minutes. Place the sample at 4°C and centrifuge at 12000rpm for 15 minutes. After centrifugation, the aqueous phase (the layer where the RNA is located) is separated from the organic phase. Note: The aqueous phase is located in the upper layer, colorless and transparent. Carefully pipette the upper aqueous phase into a new EP tube without RNAse contamination. The volume of the upper aqueous phase is about 60% of the volume of the initial Trizol added (i.e., 1 mL Trizol can obtain 600 μL of aqueous phase). Add an equal volume of isopropanol to the collected aqueous phase to precipitate RNA. For example, add 600 μL of isopropanol to 600 μL of aqueous phase. Gently invert and mix 7 to 8 times, and let it stand for 10 minutes after mixing to promote the formation of RNA precipitation. Place it at 4°C and centrifuge it at 12000 rpm for 10 minutes. After centrifugation, there will be a white RNA precipitate at the bottom of the tube. Discard the supernatant and retain the precipitate. Add 1 mL of pre-cooled 70% ethanol (prepared with DEPC water) to the EP tube, gently invert and mix, and suspend the precipitate in ethanol. After standing at -20°C for 10 minutes, centrifuge it at 7500g for 5 minutes at 4°C. Discard the supernatant after washing and use an RNase-free pipette tip to absorb all the residual ethanol. Add an appropriate amount of DEPC water to the RNA precipitate (20-50 μL depending on the sample size), gently pipette to mix, and accelerate the dissolution of RNA. Place the EP tube in a 37°C water bath for 10 minutes to ensure that the RNA is completely dissolved.

[0040] 4. Reverse transcription of cDNA

[0041] The following components were added to each reaction system:

[0042] RNA: 1 μg

[0043] 5xTransScript All-in-One-SperMix:4μL

[0044] gDNARemoverr: 1 μL

[0045] RNase-free Water: Make up to 20 μL

[0046] Incubate at 42°C for 15 min, then heat at 85°C for 5 s to terminate the reaction, and store the generated cDNA at -20°C for later use.

[0047] 5. Fluorescence quantitative PCR

[0048] qPCR reaction system: In the qPCR experiment, PLCB1 specific primers and Green qPCRSuperMix (2×) was used for fluorescence quantitative PCR. The specific reaction system was as follows:

[0049]

[0050] The primer pair is SEQ ID NO.1: 5'-GGACTGACCCTCAGGGATTTT-3', SEQ ID NO.2: 5'-AAGCCAGCAGGATTCAATGGG-3' (synthesized by Shanghai Sangon Biotechnology Co., Ltd.).

[0051] qPCR reaction conditions: according to the sample and target gene, the reaction system is divided into eight tubes, and centrifuged briefly to ensure that the reaction solution is evenly distributed at the bottom of the tube. Place it in the qPCR instrument for amplification, using the following two-step procedure:

[0052] Stage 1: pre-denaturation at 95°C for 30 seconds;

[0053] The second stage (40 cycles): denaturation at 95°C for 5 seconds; annealing at 60°C for 30 seconds (fluorescence signals were collected during this stage).

[0054] The statistical analysis results show that ( Figure 1 ), the expression of PLCB1 gene in the siPLCB1 group was significantly lower than that in the control group NC (P<0.01), indicating that the transfection was successful and the PLCB1 gene was effectively knocked down.

[0055] Example 2 Cell proliferation inhibition experiment

[0056] In this example, Hep3B-LR cells with PLCB1 knocked down were used to study its effect on the drug sensitivity of lenvatinib.

[0057] 1. Experimental Design

[0058] After successful transfection, the cells in the six-well plate were digested and counted. The three groups of cells, Hep3B (NC group), Hep3B-LR (NC group), and Hep3B-LR (siPLCB1 group), were inoculated in 96-well plates, 3000 cells per well, and five rows of four wells were repeated for a total of 20 wells. After culturing for 24 hours, the cells were allowed to adhere to the wall and grow. 100ul of complete medium DMEM containing 10mM concentration of lenvatinib was added to each well of the four rows of cells except the first row. The cells were cultured for four days, and the proliferation of cells in different treatment groups was observed.

[0059] 2. Proliferation Assay

[0060] The cell proliferation was detected using the CCK-8 kit, and the OD values ​​of cells on day 0, day 1, day 2, day 3, and day 4 were recorded. On day 0, the absorbance was directly measured without adding the culture medium containing lenvatinib. The results were as follows: Figure 2 As shown. The experimental results showed that the cell proliferation of the Hep3B-LR.siPLCB1 group was significantly lower than that of the Hep3B-LR.NC group (****P<0.0001), and the cell proliferation of the Hep3B-LR.siPLCB1 group with PLCB1 knockdown was significantly inhibited, while the proliferation of the Hep3B-LR.NC cells in the control group was not significantly affected. This indicates that the knockdown of PLCB1 can significantly restore the sensitivity of liver cancer resistant cells to lenvatinib, and the proliferation of liver cancer resistant cells is significantly inhibited.

[0061] Summary: The specific embodiments of the present invention show that targeted knockdown of the PLCB1 gene by small interfering RNA (siRNA) can restore the resistance of liver cancer cells to lenvatinib, and has potential application value in anti-liver cancer.

[0062] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limitations of the present invention. The protection scope of the present invention should be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims, that is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. Application of the primer pair with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 in regulating the sensitivity of liver cancer cells to lenvatinib.

2. The application according to claim 1, characterized in that: The application includes enhancing or restoring the sensitivity of liver cancer cells to the drug lenvatinib.

3. Application of PLCB1 gene in regulating the sensitivity of liver cancer cells to lenvatinib.

4. The use according to claim 3, characterized in that: The application includes enhancing or restoring the sensitivity of liver cancer cells to the drug lenvatinib.

5. The use according to claim 4, characterized in that: The application includes knocking down the PLCB1 gene or inhibiting the expression of the PLCB1 gene.

6. The use according to claim 4 or 5, characterized in that: The application includes transfecting PLCB1-specific small interfering RNA into liver cancer cells; the nucleotide sequence of the PLCB1-specific small interfering RNA is shown in SEQ ID NO.3 and SEQ ID NO.4.

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