CN110960546A - Application of MicroRNAs in preparation of reinforcing agent for treating liver cancer by sorafenib - Google Patents

Abstract

The invention provides application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib, and belongs to the technical field of tumor treatment, wherein the MicroRNAs comprise miR-15a and/or miR-20 b. In the invention, the expression of miR-15a and/or miR-20b enhances the effect of sorafenib on treating liver cancer by inhibiting sorafenib to induce proliferation of liver cancer cells and promoting apoptosis of the liver cancer cells. The miR-15a and miR-20b in the invention participate in the drug resistance of liver cancer cells, and provide new potential drug targets for treating liver cancer and reversing the drug resistance of sorafenib. Therefore, the invention has stronger theoretical and practical significance.

Description

Application of MicroRNAs in preparation of reinforcing agent for treating liver cancer by sorafenib

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib.

Background

Liver cancer (hepatocellular carcinoma) is a common malignant tumor in China, and the incidence rate and the mortality rate of the liver cancer (hepatocellular carcinoma) are fourth and second according to a cancer statistical report issued by the national cancer center in 2019 in 1 month. Liver cancer is also one of the 5 most frequent types of cancer worldwide, and is a serious threat to human health and life. Liver cancer is hidden, early symptoms are not obvious, and diagnosis is often completed in middle and late stages. Surgery, liver transplantation and ablation are the primary treatment for early stage patients, however over 70% of patients with surgery and ablation relapse within 5 years. Similarly, postoperative recurrence is also a major problem in liver transplantation. Therefore, systemic chemotherapy and targeted therapy become the main treatment means for patients with middle and late stage liver cancer. However, the liver cancer cells have strong drug resistance, and the effect of the existing liver cancer treatment drugs is not ideal. Therefore, the research and exploration of the drug resistance mechanism of the liver cancer cells and the discovery of drug resistance molecular targets and markers have important significance for research and development of new liver cancer drugs and medication guidance.

The traditional chemotherapeutic drugs have little effect on the treatment of liver cancer, and the advent of sorafenib makes a great breakthrough on the treatment of liver cancer. Sorafenib (Sorafenib) is a small molecule, multi-target oral anticancer drug approved by the FDA in the united states for the treatment of unresectable hepatocellular carcinoma in 2007. Is officially approved to enter the market of China in 2008, and becomes the first-line medicament for treating the late-stage liver cancer and the auxiliary medicament after the operation and liver transplantation. In the mechanism, sorafenib can not only inhibit the activities of VEGFR, PDGFR, FLT3 and KIT receptor tyrosine kinases, but also can strongly inhibit RAF kinases. Therefore, sorafenib can inhibit angiogenesis and directly inhibit tumor cell proliferation. Clinical studies of shrarp, Oriental, etc. showed that sorafenib treatment of advanced liver cancer significantly prolonged patient survival compared to placebo (shrarp study: 10.7 months vs. 7.9 months, P0.00058; Oriental study: 6.5 months vs. 4.2 months, P0.014). These key studies establish the first-line position of sorafenib in treating advanced liver cancer, make it become the standard first-line therapy of advanced liver cancer, and recommended by many domestic and foreign guidelines as the standard for liver cancer treatment. Our 2011 version of the 'primary liver cancer diagnosis and treatment standard' also recommends sorafenib as a preferred scheme for whole-body treatment.

In the past decade, sorafenib becomes the only first-line drug to be selected when the traditional treatment means of a liver cancer patient is missed or the traditional treatment effect is poor. Although the second-line target drug regorafenib (regorafenib) and the second-line immune drug PD-1 inhibitor have attracted attention in recent two years, earlier clinical data indicate that the second-line drug clinical trial on patients who failed sorafenib treatment ends up failing in many cases. However, the curative effect of sorafenib is always unsatisfactory due to primary and acquired drug resistance.

Disclosure of Invention

In view of the above, the invention aims to provide application of MicroRNAs in preparation of an enhancer for treating liver cancer by sorafenib, and the expression of miR-15a and/or miR-20b in the MicroRNAs enhances the effect of the sorafenib on treating the liver cancer.

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

the invention provides application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib;

the MicroRNAs comprise miR-15a and/or miR-20 b.

The invention also provides application of the MicroRNAs in preparation of a medicament for promoting sorafenib to induce liver cancer cell apoptosis;

the MicroRNAs comprise miR-15a and/or miR-20 b.

The invention also provides application of the MicroRNAs in preparing a medicament for inhibiting sorafenib-induced liver cancer cell proliferation;

the MicroRNAs comprise miR-15a and/or miR-20 b.

Preferably, the dosage form of the medicine comprises one or more of tablets, granules, capsules and emulsion.

The invention provides application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib, wherein the MicroRNAs comprise miR-15a and/or miR-20b, and the expression of the miR-15a and/or miR-20b enhances the effect of the sorafenib on treating the liver cancer by inhibiting sorafenib to induce proliferation of liver cancer cells and promoting apoptosis of the liver cancer cells.

Drawings

FIG. 1-1 is a schematic diagram showing a process for screening sorafenib drug-resistant (sensitization) genes by a Cas9 library;

FIG. 1-2 is a schematic diagram showing stem-loop sequences of miR-15a and miR-20 b;

FIG. 2-1 shows the overexpression of the transcription level of the hepatoma cell line MHCC-LM3 transfected with miR-15a and 20b mimics respectively;

FIG. 2-2 is a graph showing that miR-15a and 20b overexpression increases cell mortality caused by sorafenib treatment;

FIGS. 2-3 show that the sensitivity of miR-15a and miR-20b transfected Focus liver cancer cells to sorafenib action is increased;

FIGS. 2-4 are graphs showing statistics of the percentage of apoptotic cells in the experiments of FIGS. 2-3;

FIGS. 2-5 are graphs showing that Huh7 hepatoma cells transfected with miR-15a and miR-20b have increased sensitivity to sorafenib;

FIGS. 2-6 are graphs showing statistics of the percentage of apoptotic cells in the experiments of FIGS. 2-5;

FIG. 3-1 is a graph showing the effect of CCK8 experiments miR-15 and miR-20b on the survival ability of hepatoma cells MHCC-LM3 under sorafenib treatment;

FIG. 3-2 is a graph showing the effect of CCK8 experiments miR-15 and miR-20b on the survival ability of liver cancer cell Focus under sorafenib treatment;

FIG. 3-3 shows the effect of MHCC-LM3 of liver cancer cells transfected with miR-15a and miR-20b and the ability of control cells to form plate clones under the action of sorafenib;

FIGS. 3-4 are graphs showing statistics of the number of clumps formed by MHCC-LM3 cells in FIGS. 3-3;

FIG. 4 shows the effect of miR-15a and miR-20B on the curative effect of sorafenib treatment on transplanted tumors in animal experiments, wherein A and B respectively show that the overexpression of miR-15a and 20B weakens the capacity of MHCC-LM3 cells to form tumors subcutaneously in nude mice under the treatment of sorafenib.

Detailed Description

The invention provides application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib; the MicroRNAs comprise miR-15a and/or miR-20 b. In the present invention, when miR-15a and miR-20b are included in the enhancer, the two are mixed in an arbitrary mass ratio. The dosage form of the enhancer is not particularly limited in the present invention, and a pharmaceutically acceptable dosage form may be used. The content of miR-15a and/or miR-20b in the enhancer, the auxiliary materials and the auxiliary material content are not particularly limited, the auxiliary material types and the auxiliary material dosage used for conventionally preparing various dosage forms can be adopted, and the content of active substances conventionally contained in various dosage forms can be adopted. The preparation method of the enhancer is not particularly limited, and the enhancer can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention provides application of MicroRNAs in preparation of a medicament for promoting sorafenib to induce liver cancer cell apoptosis; the MicroRNAs comprise miR-15a and/or miR-20 b. In the present invention, when miR-15a and miR-20b are included in the drug, the two genes are mixed in an arbitrary mass ratio. In the invention, the dosage form of the medicine comprises one or more of tablets, granules, capsules and emulsion. The content of miR-15a and/or miR-20b in the medicament and the content of the adopted auxiliary materials and auxiliary materials are not particularly limited, the types and the use amounts of the auxiliary materials used for conventionally preparing various dosage forms are only required, and the content of active substances conventionally contained in various dosage forms is only required. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The invention provides application of MicroRNAs in preparation of a medicament for inhibiting sorafenib-induced liver cancer cell proliferation; the MicroRNAs comprise miR-15a and/or miR-20 b. In the present invention, when miR-15a and miR-20b are included in the drug, the two genes are mixed in an arbitrary mass ratio. In the invention, the dosage form of the medicine comprises one or more of tablets, granules, capsules and emulsion. The content of miR-15a and/or miR-20b in the medicament and the content of the adopted auxiliary materials and auxiliary materials are not particularly limited, the types and the use amounts of the auxiliary materials used for conventionally preparing various dosage forms are only required, and the content of active substances conventionally contained in various dosage forms is only required. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a conventional preparation formulation.

The experimental procedures used in examples 1-4 were as follows:

(1) cell lines and reagents

MHCC-LM3, Huh7 and FOCUS hepatoma cells are from the Shanghai cell Bank of Chinese academy of sciences. DMEM medium containing 10% fetal calf serum is adopted and placed at 37 ℃ with 5% CO₂Culturing the liver cancer cells in an incubator with saturated humidity. 0.25% trypsin (with EDTA) was used to digest the cells.

Sorafenib (Sorafenib; Cell Signaling Technology) was prepared as a 10mM stock solution in which DMSO was dissolved. For experimental purposes, the drug was diluted in fresh medium prior to addition to the cell culture. In the experiment for detecting the effect of miR-15a-5p and miR-20b-5p on the sensitivity of liver cancer cells to sorafenib, a control group is treated by DMSO instead of sorafenib. Mimics (GenePharma; Shanghai, China) synthesized corresponding to human miR-15a-5p and miR-20b-5p and a negative control miR-NC (GenePharma) were prepared as 20. mu.M stock solutions in water without RNase.

(2) Lentivirus package and construction of liver cancer overexpression stable strain

1) 293T cells were cultured in DMEM medium containing 10% FBS, 100IU/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ with 5% CO₂And conventionally culturing in a saturated humidity incubator.

2) When the strain grows to about 80% of density, empty plasmids CMV-PGK-Puro, recombinant plasmids CMV-hsa-miR-15a-PGK-Puro, CMV-hsa-miR-20b-PGK-Puro (Genomeditech; shanghai, China) and the packaging plasmids psPAX2, pMD2.G were added in proportion to a centrifuge tube containing 1.5mL of serum-free medium and mixed well. The transfection reagent

3000 to 1.5mL of serum-free medium, mixing the two tubes of reagents, standing at room temperature for 5min, adding to the cell culture solution, and culturing in a cell culture box for 4-6 h.

3) Removing the transfection solution, adding serum-containing DMEM medium, and continuing to culture for 72 h. Sucking cell supernatant into a centrifuge tube, centrifuging at low speed, filtering at 0.45 μm, centrifuging at 4 deg.C and 20000r/min at ultra speed for 2 hr, and collecting concentrated solution.

4) Construction of overexpression Stable Strain

The virus solution was added to infect the cells and polybrene was added to a final concentration of 4. mu.g/ml. After 24h the virus-containing medium was aspirated, complete medium was added, and after 48h incubation in an incubator, the stably transduced cell lines were selected by changing the medium to Puromycin at a final concentration of 1. mu.g/ml (two to three days with one change).

(3) Target miRNA mimics transfection hepatoma carcinoma cell

1) Cells were transfected when seeded to 70-90% confluence.

2) Dilution with Opti-MEM Medium

3000 reagent (2 tubes), mix well.

3) MiRNA mics was diluted using Opti-MEM medium and diluted per tube

Diluted miRNA mix (1:1 ratio) was added to the 3000 reagent.

4) Incubate at room temperature for 5 minutes and add dropwise to the cells.

5) Cells were incubated at 37 ℃ for 2-4 days and then transfected cells were analyzed.

(4) Determination of cell growth curves

1) Different kinds of hepatocarcinoma cells are grown according to growth characteristics of 3-5 × 10³The total amount of cells was calculated per 100. mu.l/well, and after digesting the cells sufficiently, the cells were diluted to the desired concentration and seeded in a 96-well plate. Three wells per group per day, cells were inoculated on a 5-7 day basis.

2) And adding sorafenib to observe the state and the number of the cells after the cells are basically attached to the wall. Color reaction was performed with CCK-8 developer (CellCountkit-8, DOJINDO, Japan) by adding 10. mu.l of CCK-8 to 100. mu.l of the culture medium at 37 ℃ with 5% CO₂Placing and incubating in incubator for 1h 15min, and labeling with enzymeThe absorbance at 450 nm was measured and the actual starting density of the cells was recorded.

3) The cell morphology was observed under a microscope, measured at regular time intervals, and the cell growth was recorded.

4) Typically 5 to 7 days. And after the measurement is finished, collecting data, processing and drawing a chart.

(5) Cell clone formation assay

1) The miR-NC, miR-15a and miR-20b overexpression liver cancer stable strains MHCC-LM3 are inoculated on a 6-well plate according to a certain number, after the cells are cultured for 24 hours and adhered to the wall, sorafenib with proper concentration is added to detect the influence of the sorafenib on the long-term treatment of the stable cell strains.

2) Culturing for 2-3 weeks, changing fresh culture solution every 3-5 days and adding sorafenib treatment until macroscopic cell clone is formed.

3) The medium in the dish was aspirated, the 6-well plate was placed on ice and rinsed 2 times with pre-cooled PBS (4 ℃).

Fixation was performed with pre-cooled 100% methanol (4 ℃) for 10 min.

4) The cells were removed from the ice, equilibrated at room temperature, and stained by overlaying the cells with crystal violet staining solution. Incubate at room temperature for 10min, discard crystal violet stain, wash cells with distilled water several times until the stain no longer flows out.

5) Colony formation staining results were photographed and cell clones on each dish were counted according to the same criteria (cell clone size).

(6) Nude mouse tumorigenesis experiment

1) The mice used were male BLAB/c nude mice 5-6 weeks in size, purchased from Shanghai Spiker laboratory animals, Inc.

2) Taking cells of miR-NC, miR-15a and miR-20b overexpression liver cancer stable strain MHCC-LM3, and carrying out treatment by 1 × 10⁶The same number was injected subcutaneously into the abdomen of nude mice.

3) Mice were randomly divided into two groups: mice were subjected to continuous sorafenib gavage in the experimental group and continuous DMSO gavage in the control group.

4) The growth of the tumor was observed, the mice were sacrificed about 4-8 weeks until the maximum tumor diameter reached 15mm, and the tumor was photographed and weighed.

(7) Flow Annexin V-FITC/PI double dyeing method

1) The Binding Buffer (10X) was diluted to 1X Binding Buffer working solution for use (1ml Binding Buffer (10X) was supplemented with 9ml sterile deionized water).

2) For adherent cells, the cells are digested by trypsin without EDTA, the digestion time of the trypsin is not suitable to be too long or too short, and preferably, when the adherent cells can be blown down by gentle blowing, cell culture solution is added, the cells are blown down by gentle blowing and transferred into a centrifugal tube, and the cells are collected by centrifugation at 500-.

3) After the cells are collected, a precooled PBS solution is added to the cells to be gently shaken or gently blown by a liquid transfer device to be washed, and the cells are collected by centrifugation and washed twice.

4) Adding 1 × Binding buffer working solution into the cell sediment, and resuspending the cells to make the cell concentration reach 1 × 10⁶cell/ml。

5) Aspirate 100. mu.l of cell suspension (total number of cells 1X 10)⁵cell) to a new tube, add 5. mu.l annexin V-FITC and 5. mu.l PI, mix gently, incubate for 15min at room temperature in the dark.

6) Flow cytometry detection:

after the staining incubation, 400. mu.l of 1 XBinding Buffer working solution was added to each tube, mixed well and detected by flow cytometry (detection within 1 hour).

Setting 3 control groups of normal cells induced by apoptosis, PI single staining and Annexin V-FITC single staining, wherein the normal cell group can be used as a fluorescence compensation adjustment to remove spectrum overlapping and set the position of a cross gate. The results were analyzed by software such as FlowJo, and a scattergram was drawn with FITC as the abscissa and PI as the ordinate.

(8) Trypan blue staining method

1) Preparing 4% trypan blue mother liquor: weighing 4g trypan blue, adding a small amount of distilled water, grinding, adding double distilled water to 100ml, filtering with filter paper, and storing at 4 ℃. When used, the solution is diluted to 0.4% by PBS.

2) The adherent cells were digested with trypsin to prepare a single cell suspension and diluted appropriately.

3) Dyeing: the cell suspension was mixed well with 0.4% trypan blue solution at a ratio of 9: 1. (final concentration 0.04%)

4) Counting: within 3 minutes, live and dead cells were counted, respectively.

5) Under the observation of the lens, the dead cells were stained in a distinct blue color, while the viable cells were resistant to staining in a colorless transparent state.

6) Counting cell death rate: mortality (%). percent (total dead cells/(total live cells + total dead cells) × 100%.

(9) Cell RNA extraction

The RNA was extracted using TRIzol Reagent (Invitrogen) by the following procedure:

1) extracting cell line RNA, collecting cells in logarithmic growth phase, sucking culture solution, adding TRIzol reagent (1ml TRIzol/10 cm) in corresponding amount according to the area of culture dish²) The cells were lysed, blown several times, and the lysed cells were collected into RNase-free EP tubes and allowed to stand at room temperature for 5 minutes.

2) Chloroform (200. mu.l/1 ml TRIzol) was added to the centrifuge tube in proportion, rapidly and vigorously shaken for 15 seconds, allowed to stand at room temperature for 2 to 3 minutes, and centrifuged at 12000 Xg for 15 minutes at 4 ℃.

3) The upper aqueous phase was transferred to a new RNase-free EP tube as much as possible, isopropanol of equal volume was added, mixed by inversion 5 times, left to stand at room temperature for 10 minutes, and centrifuged at 4 ℃ at 12000 Xg for 10 minutes, whereupon RNA precipitation was observed.

4) The supernatant was decanted, 75% ethanol (1ml/1ml TRIzol) was added, mixed well, RNA washed, centrifuged at 4 ℃ for 5 minutes at 7500 Xg.

5) Discarding the supernatant, removing residual ethanol as much as possible, and naturally drying the precipitate for 5-10min (taking care not to completely dry); adding 30-50 μ l DEPC H₂O, blowing and sucking for several times, and dissolving the RNA precipitate.

6) Measuring the concentration and purity OD 260/280(1.8-2.0) of RNA by an enzyme-labeling instrument; gel electrophoresis was performed to observe whether degradation occurred or not, and the samples were stored at-80 ℃.

(10) Reverse transcription of RNA

Reverse transcription was performed using M-MLV Reverse Transcriptase (Promega).

The primer information used was as follows:

(SEQ ID No.1)R:gtgcagggtccgaggt；

(SEQ ID No.2)miR-15a-F:gagtagcagcacataatgg；

(SEQ ID No.3)miR-15a-RT:

gtcgtatccagtgcagggtccgaggtattcgcactggatacgacacaaac；

(SEQ ID No.4)miR-20b-F:gagcaaagtgctcatagtg；

(SEQ ID No.5)miR-20b-RT；

gtcgtatccagtgcagggtccgaggtattcgcactggatacgacctacct；

(SEQ ID No.6)U6-F：ctcgcttcggcagcaca；

(SEQ ID No.7)U6-RT：aacgcttcacgaatttgcgt。

the specific operation is as follows:

1) the following components were added to the nuclease-free EP tube:

the mixture was placed in a PCR apparatus at 70 ℃ for 5 minutes and then immediately cooled on ice for 5 min.

2) The following components are added into the system:

after mixing gently, the mixture was placed in a PCR instrument at 37 ℃ for 60 min.

The cDNA obtained by the reversion was stored at 4 ℃.

(11) Real-time quantitative PCR

Real-time quantitative PCR reaction use

Premix Ex Taq^TM(Perfect Real Time) kit (TaKaRa Biotechnology Co., Ltd. Dalian, China) using Thermal cycleDicce^TMRealThe Time System (TP800 real-Time fluorescent quantitative PCR instrument, TaKaRa) was used for the operation. The length of the amplification product of the quantitative PCR is preferably 80bp to 150bp (can be extended to 300 bp).

The reaction system is as follows:

reaction conditions are as follows:

dissolution curve analysis step:

95℃ 15sec

60℃ 30sec

95℃ 15sec

the dissociation time was 4 sec.

The fluorescence background signal and the threshold value are default values set by an instrument and are automatically generated after each PCR reaction is finished, the Ct value represents the cycle number of the fluorescence signal in each reaction tube when the fluorescence signal reaches the set threshold value (10 times of the baseline fluorescence intensity), each template is subjected to 3 multitubes, the obtained Ct value is averaged, the average value is subtracted from the Ct average value of an internal reference gene (U6) of the corresponding template, and the expression quantity of miR-15a-5p and miR-20b-5p is calculated by a 2- △△ Ct method through the obtained delta Ct..

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Screening of gene participating in liver cancer sorafenib drug resistance by CRISPR/Cas9 knockout library

Infecting MHCC-LM3 liver cancer cells by using a CRISPR/Cas9 omic lentivirus library, carrying out Puromycin screening, carrying out cell culture for 7-10 generations, adding sorafenib-treated cells after the cytogenetic background is stable, carrying out two rounds of treatment, extracting genomic DNA (the number of the front and rear samples is 3) of the liver cancer cells before and after sorafenib is added, detecting the enrichment condition of sgRNA by PCR/RNA-seq, and carrying out a screening process schematic diagram as shown in figure 1-1.

Finally, through deep sequencing analysis, two candidate target microRNAs with large sgRNA content change difference, namely miR-15a and miR-20b (figure 1-2), are found. Compared with the method before dosing, the sgRNA content of miR-15a in the drug-resistant cell is increased by 38 times, the sgRNA content of miR-20b is increased by 59 times, and because the sgRNA and the target gene thereof are in a negative regulation relationship, the expression of miR-15a and miR-20b is reduced when the sgRNA content is increased. The screening library result shows that the deletion of miR-15a and 20b promotes the resistance of the hepatoma carcinoma cells to sorafenib. On the contrary, the expression of miR-15a and 20b enhances the sensitivity of the hepatoma carcinoma cells to sorafenib.

Example 2

Exogenous expression of miR-15a and miR-20b for promoting liver cancer cell apoptosis induced by sorafenib

And (3) carrying out chemical synthesis on the mimics of miR-15a and miR-20b, and transfecting liver cancer cells Huh7, Focus and MHCC-LM 3. (1) RNA of MHCC-LM3 cells transfected with miR-15a and miR-20b mimics is extracted respectively, and is reversely converted into cDNA, expression conditions of miR-15a and miR-20b transcription levels are detected by utilizing a qPCR technology, and results show that the RNA and the RNA are remarkably improved (figure 2-2). (2) After liver cancer cells MHCC-LM3 and Huh7 transfected by mimics are cultured for 48 hours, the liver cancer cells MHCC-LM3 and Huh7 are divided into two groups, namely: DMSO control group and sorafenib treatment group, 48 hours after drug treatment, cells were trypsinized, trypan blue was formulated to a final concentration of 0.04% for dead cell staining, and cell mortality was counted: mortality (%) (% dead cell count/(total live cell count + total dead cell count) × 100%, cell mortality was found to be significantly increased by miR-15a and miR-20b overexpression in the sorafenib-treated group (fig. 2-2). (3) In addition, the transfected hepatoma carcinoma cells are inoculated in a six-well plate at a density of 70% and divided into two groups (a DMSO control group and a sorafenib treatment group), and the apoptosis condition induced by sorafenib is detected by using a flow cytometer by using an annexinV/PI double staining method after 48 hours, so that the apoptosis of miR-15a and miR-20b overexpressed cells is remarkably increased (figures 2-3 to 2-6).

Example 3

Exogenous expression of miR-15a and miR-20b for inhibiting liver cancer cell proliferation induced by sorafenib

In order to further explore the functions of miR-15a and miR-20b in liver cancer cells induced by sorafenib, growth curves and clonogenic experiments are used for detecting the functions of miR-15a and miR-20b under the condition that the liver cancer cells are treated by short-term and long-term sorafenib.

Taking the liver cancer cells MHCC-LM3 and Focus of mimics transfected by miR-15a and miR-20b respectively, and taking the cells in the logarithmic growth phase at 5 x 10 per hole³The individual cells are inoculated in a 96-well plate, after the cells are completely attached to the wall, sorafenib is added to treat the cells to form an experimental group, and DMSO is treated to form a control group, the CCK8 kit is used for detecting the cell growth condition of 5 days, and the experimental results show that the overexpression of miR-15a and miR-20b inhibits the proliferation capacity of the liver cancer cells induced by sorafenib (figure 3-1 and figure 3-2), which shows that two mimics of microRNA and sorafenib are respectively added to act on the liver cancer cells together, so that the curative effect of sorafenib can be improved.

In addition, overexpression lentiviruses expressing pri-miR-15a and pri-miR-20b are constructed, liver cancer cells are infected, and puromycin is added for screening to obtain stable cell strains; divided into two groups, sorafenib-treated and DMSO-control groups, at 2 x 10³Cells in log phase growth of one/well were seeded in six-well plates and plate colony formation experiments were performed to examine the effect of sorafenib on long-term treatment of these stable cell lines (fig. 3-3 and 3-4).

The expression of miR-15a and miR-20b is detected and found to enhance the sensitivity of the liver cancer cells to sorafenib by using the method for detecting apoptosis and survival of the cells.

Example 4

miR-15a and miR-20b enhance sensitivity of hepatoma carcinoma cells to sorafenib in vivo

In vitro experiments clearly show that miR-15a and miR-20b enhance the sensitivity of liver cancer cells to sorafenib, and then a tumor-bearing nude mouse model is used for researching the influence of the expression of miR-15a and miR-20b on the subcutaneous tumorigenicity capability of liver cancer cells nude mice treated by sorafenib. Will be 1 × 10⁶MHCC-LM3 cells (lentivirus infected to form a stable cell line) and control cells of miR-15a or miR-20b respectively over-expressed were injected subcutaneously into the abdomen of nude mice, and the mice were randomly divided into two groups: the experimental group makes the mice to be continuously gavaged by sorafenib, and the control groupContinuous DMSO gavage. The growth of the tumor was observed, the mice were sacrificed about 4-8 weeks until the maximum tumor diameter reached 15mm, and the tumor was photographed and weighed. Results show that the overexpression of miR-15a and miR-20B effectively inhibits the tumorigenicity ability of liver cancer cells treated by sorafenib (A and B in figure 4) under the skin of a nude mouse, namely miR-15a and miR-20B enhance the sensitivity of the liver cancer cells to sorafenib.

The drug resistance problem of sorafenib is an important factor for restricting prognosis and benefiting of a liver cancer patient. According to the invention, through CRISPR/Cas9 omics screening, miR-15a and miR-20b are identified as candidate targets for enhancing sorafenib sensitivity, and a series of cell and animal experiments are designed to illustrate the role of miR-15a and miR-20b in participating in sorafenib resistance of hepatoma cells, so that a new potential drug target is provided for treating hepatoma and reversing sorafenib resistance. Therefore, the research has stronger theoretical and practical significance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> east Hospital of Shanghai City

Application of <120> MicroRNAs in preparation of reinforcing agent for treating liver cancer by sorafenib

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gtgcagggtc cgaggt 16

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gagtagcagc acataatgg 19

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<213> Artificial Sequence (Artificial Sequence)

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gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacacaaac 50

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<213> Artificial Sequence (Artificial Sequence)

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gagcaaagtg ctcatagtg 19

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gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctacct 50

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aacgcttcac gaatttgcgt 20

Claims (4)

1. Application of MicroRNAs in preparation of a reinforcing agent for treating liver cancer by sorafenib;

   the MicroRNAs comprise miR-15a and/or miR-20 b.
2. Application of MicroRNAs in preparation of drugs for promoting sorafenib to induce liver cancer cell apoptosis;

   the MicroRNAs comprise miR-15a and/or miR-20 b.
3. Application of MicroRNAs in preparation of a medicament for inhibiting sorafenib-induced liver cancer cell proliferation;

   the MicroRNAs comprise miR-15a and/or miR-20 b.
4. 4. The use of claim 2 or 3, wherein the medicament is in a dosage form comprising one or more of tablets, granules, capsules and emulsions.

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