CN110578003A - Application of miR-200a in preparation of liver cancer diagnosis and treatment kit and preparation of anti-tumor drugs - Google Patents
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Abstract
The invention belongs to the technical field of biomedicine, and particularly discloses application of miR-200a in preparation of a liver cancer diagnosis and treatment kit and preparation of an anti-tumor drug. The negative growth relation of miR-200a and liver cancer cells is determined, the effect that miR-200a can enhance the effect of doxorubicin on anti-tumor cells is determined, and the method has important clinical significance.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of miR-200a in preparation of a liver cancer diagnosis and treatment kit and preparation of an anti-tumor drug.
Background
Primary hepatocellular carcinoma is a common malignant tumor of a digestive system, the lethality rate of the primary hepatocellular carcinoma is higher than that of the primary hepatocellular carcinoma in the third place of the malignant tumor, and more than 700000 new cases occur every year worldwide. The main causes are hepatitis virus infection, alcoholic cirrhosis and non-alcoholic fatty liver. Hepatitis B-related hepatocellular carcinoma accounts for more than half of the total number of liver cancers worldwide, and more than 90% of primary hepatocellular carcinoma in China is based on liver cirrhosis caused by chronic hepatitis B. Hepatitis B virus replicates in liver cell and the virus DNA segment is integrated into the host cell DNA to activate the corresponding cancer gene of the cell to start the tumor generating process. The main treatment means for primary liver cancer is tumor resection, liver transplantation, radiofrequency ablation and hepatic artery perfusion chemoembolization. However, the prognosis of the dry disease of most patients is poor, the recurrence rate can reach 35-37% within three years, and the total survival rate of five years is still lower than 10% worldwide. The cure rate of early liver cancer can reach 50-74%. How to improve the early diagnosis rate of liver cancer, targeted treatment according to biological characteristics is a key factor for improving survival and cure rate. Therefore, the specific tumor molecular marker is selected for detection and selection of more accurate treatment, and the method has very important clinical significance. At present, a highly specific tool is still lacked for liver cancer diagnosis, the commonly used auxiliary diagnosticians such as alpha-fetoprotein and the like have a certain false positive rate, and the clinical urgent need for the specific diagnostic tool is strong.
Micro ribonucleic acid (microRNA) is a small molecular non-coding RNA with the molecular weight of 20-25 bases, participates in regulation and control of expression after gene transcription, and regulates gene expression after transcription and translation level. By incomplete complementary combination with a target gene mRNA 3' UTR segment through a base complementary pairing principle, the microRNA can inhibit the translation of target mRNA or promote the degradation of the target mRNA, and the expression of the target gene is regulated. Because the tumor-associated protein can stably exist in tissues and circulating blood and can be obtained by a non-invasive examination means, the expression abnormality of the tumor-associated protein is closely related to tumors, and the tumor-associated protein is very suitable to be used as a tumor biomarker for early diagnosis, relapse judgment and molecular pathology typing. At present, 3% of human genes are known to participate in coding microRNA, and more than 1500 microRNAs participate in regulation and control of cell proliferation, apoptosis, growth, differentiation, transfer and the like. With continuous exploration on the functions of the microRNA, the microRNA is proved to be involved in the occurrence, development and immune escape of various tumors. Such as chronic lymphocytic leukemia, multiple myeloma, gastric cancer, breast cancer, esophageal cancer, liver cancer, cervical cancer, etc. The influence mode of microRNA on the pathological process of the tumor is mainly as follows: 1) the function of oncogenes or cancer suppressor genes is exerted by acting on the difference in target genes. 2) The oncogene or the cancer suppressor gene promotes or suppresses the production of tumors by activating or suppressing the production of the corresponding microRNA. 3) The microRNA gene has sequence mutation, which causes abnormal regulation and control of gene expression. 4) The response of the tumor to chemotherapeutic drugs is affected by affecting the expression of the target gene. Reports on the relevance of microRNA and tumors are many, but the candidate genes with high specificity are still few, and the action mechanism of the candidate genes is not completely clarified.
miR-200a is a member (miR-141, miR-200a, miR-200b, miR-200c and miR-429) of miR-200 family, whether miR-200a has clinical application potential in hepatitis B associated liver cancer as a target marker is not clear at present, and the development of the application of miR-200a in the preparation of liver cancer diagnosis and treatment kits has important clinical significance.
Disclosure of Invention
The invention provides application of miR-200a in preparation of a liver cancer diagnosis and treatment kit and preparation of an anti-tumor medicament, and defines a negative growth relation between miR-200a and liver cancer cells and an effect that miR-200a can enhance an anti-tumor cell effect of doxorubicin.
The invention provides application of miR-200a in preparation of a liver cancer diagnosis and treatment kit.
The invention provides application of miR-200a in preparation of a medicine for enhancing doxorubicin anti-tumor cell effect.
Preferably, the tumor cell is a liver cancer cell line Huh7 or a liver cancer cell line HepG 2.
preferably, the concentration of doxorubicin against tumor cells is 2 μ M and the duration of action is 12 h.
Compared with the prior art, the application of the miR-200a in preparing the liver cancer diagnosis and treatment kit and preparing the anti-tumor medicament has the following beneficial effects:
The invention proves the value of miR-200a in liver cancer diagnosis by detecting the expression level of miR-200a in primary liver cancer tissues, and finds that the change of the expression level of miR-200a in liver cancer cells can influence the anti-tumor sensitivity of a chemotherapeutic drug doxorubicin for treating liver cancer. We find that the expression level of miR-200a in cells can be up-regulated to obviously enhance the anti-hepatoma cell proliferation capacity of doxorubicin, and suggest that miR-200a participates in the reaction of hepatoma cells to chemotherapeutic drugs. We find that the regulation and control of the drug resistance of hepatoma cells to doxorubicin can be realized by regulating the expression level of miR-200 a. We also find that the expression level of miR-200a is up-regulated, so that the hepatoma carcinoma cell apoptosis induction effect of doxorubicin can be enhanced, and the miR-200a is prompted to participate in the effect of hepatoma carcinoma cell drug resistance. The research result has important clinical significance for developing the application of miR-200a in preparing liver cancer diagnosis and treatment kits.
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FIG. 1 shows the expression of miR-200a in liver cancer tissues and normal liver tissues, liver cancer cell lines and normal liver cells;
FIG. 1A shows the expression level of miR-200a in primary liver cancer tissues and paracancer normal liver tissues, and FIG. 1B shows the expression level of miR-200a in human liver cells, liver cancer cell strains Huh7 and liver cancer cell strains HepG 2;
FIG. 2 shows that miR-200a constructed in different liver cancer cell strains through a lentiviral vector is used for up-regulating and down-regulating miR-200a expression levels of stably-expressing cell strains;
FIG. 2A is a Huh7 hepatoma cell line, and FIG. 2B is a HepG2 hepatoma cell line;
FIG. 3 is a diagram showing that the CCK8 cell detection kit detects the influence of miR-200a expression on the proliferation activity of liver cancer cells;
FIG. 3A is a Huh7 liver cancer cell, FIG. 3B is a HepG2 liver cancer cell;
FIG. 4 is a plot of the cell proportion of the different cell cycles of Huh7 following modulation of miR-200a expression levels;
FIG. 5 is the effect of miR-200a expression on the apoptosis of hepatoma carcinoma cells Huh7 in combination with the anticancer drug doxorubicin;
FIG. 5A is the change in the viability of hepatoma cells under the combined effect of miR-200a expression and doxorubicin; FIG. 5B is the half-lethal dose of hepatoma cells required by different groups under the combined action of miR-200a expression and doxorubicin;
FIG. 6 is a flow cytometer derived cell cycle map for different groups;
FIG. 6A is a control group, FIG. 6B is a miR-200a upregulated expression group, and FIG. 6C is a miR-200a downregulated expression group;
FIG. 7 is a graph of flow cytometry derived apoptosis for different groups;
FIG. 7A is a control group, FIG. 7B is a miR-200a upregulated expression group, and FIG. 7C is a miR-200a downregulated expression group; FIG. 7D is control + doxorubicin, FIG. 7E is miR-200a upregulated expression + doxorubicin, and FIG. 7F is miR-200a downregulated expression + doxorubicin;
FIG. 8 is the effect of miR-200a in combination with doxorubicin treatment on the growth phase and apoptosis of cells;
FIG. 8A shows the results of the ratio of different cell phases, and FIG. 8B shows the results of apoptosis;
FIG. 9 is a bar graph of the results of tumors in nude mice in vivo tumor formation experiments and the change in body weight of mice;
FIG. 9A is a graph of tumor mass volume measurements in mice treated with different treatment regimens (without doxorubicin) after different times of tumor implantation; FIG. 9B is a graph of tumor mass volume measurements in mice treated with different treatment regimens (doxorubicin addition) after different times of tumor implantation; FIG. 9C is a graph of tumor mass measurements taken from mice treated with different treatments 25 days after tumor implantation; FIG. 9D is a graph of the change in body weight of mice following tumor implantation for different treatment regimens;
FIG. 10 is a graph of the effect of regulating miR-200a expression levels on cell metabolism;
FIG. 10A is the effect of different treatment modalities on the oxygen consumption rate of hepatoma cells, FIG. 10B is the effect of different treatment modalities on the acidification rate of cells, and FIG. 10C is the effect of different treatment modalities on the oxygen consumption of cells.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention provides application of miR-200a in preparation of a liver cancer diagnosis and treatment kit, and the improvement of the expression level of miR-200a can strengthen the anti-tumor effect of chemotherapeutic drug doxorubicin.
in the following experiments, miR-200a gene is inserted into a vector to construct a lentiviral vector capable of expressing miR-200a, the lentiviral vector is transformed into cells, and a miR-200a up-regulated stably-expressing cell strain (pGC-GFP-miR-200a-mimic) and a miR-200a down-regulated stably-expressing cell strain (pGCSIL-GFP-miR-200a-inhibitor for inhibiting expression of miR-200 a) are respectively constructed. The construction methods of the lentiviral vector capable of expressing the miR-200a, the miR-200a up-regulated stable expression cell strain and the miR-200a down-regulated stable expression cell strain refer to' ElevatedCXCL1 primers for viral expressing and is inhibited bymiRNA-200a.Oncotarget.2016Oct 4; 65052 and 65066. "; the lentiviral vector capable of expressing the miR-200a, the miR-200a up-regulation stable expression cell strain and the miR-200a down-regulation stable expression cell strain can also be designed and prepared by Shanghai Jikai gene company.
the "miR-200 a up-regulation expression group" referred to in the following experiment means that the expression concentration level of miR-200a in cells is up-regulated, the "miR-200 a down-regulation expression group" means that the expression concentration level of miR-200a in cells is down-regulated, and the control group means a cell strain transfected with an empty viral vector. Huh7 and HepG2 hepatoma cell lines were performed according to the lentivirus infection instruction manual. And (4) screening a drug-resistant gene stable cell strain by puromycin.
Related research:
Expression of miR-200a in liver cancer tissue and normal liver tissue, liver cancer cell line and normal liver cell
FIG. 1A shows the expression level of miR-200a in primary liver cancer tissue (right side of FIG. 1A) and paracancer normal liver tissue (left side of FIG. 1A), and it can be seen that the expression level of miR-200a in the paracancer normal liver tissue is higher than that in the liver cancer tissue. FIG. 1B shows that miR-200a is expressed in human hepatocytes, hepatoma cell lines Huh7 and HepG2, and it can be seen that the expression level of miR-200a in human normal hepatocytes is higher than that in hepatoma cell lines. The expression level of miR-200a is proved to be related to the growth negativity of the liver cancer cells.
secondly, inducing or inhibiting the influence of miR-200a expression on the growth of liver cancer cells in the liver cancer cells
FIG. 2 shows that miR-200a constructed in different liver cancer cell strains through a lentiviral vector is used for up-regulating and down-regulating miR-200a expression levels of stably-expressing cell strains; FIG. 2A shows the miR-200a expression level of a cell strain regulated and controlled in a Huh7 liver cancer cell strain by a lentiviral vector, and FIG. 2B shows the miR-200a expression level of the cell strain regulated and controlled in a HepG2 liver cancer cell strain by the lentiviral vector, and the expression level is verified by qPCR. The results show that the expression level of the miR-200a up-regulated expression group in the Huh7 liver cancer cell line and the HepG2 liver cancer cell line is higher than that of the control group and the miR-200a down-regulated expression group.
FIG. 3 is a diagram showing that the CCK8 cell detection kit is used for detecting the influence of miR-200a expression on the proliferation activity of liver cancer cells; FIG. 3A shows that a CCK8 cell detection kit detects the influence of miR-200a expression on the proliferation activity of Huh7 liver cancer cells at different action time points, and 3B shows that the CCK8 cell detection kit detects the influence of miR-200a expression on the proliferation activity of HepG2 liver cancer cells at different action time points, and the results show that miR-200a expression is up-regulated to inhibit the proliferation of Huh7 and HepG2 liver cancer cells, and miR-200a expression level is down-regulated to promote the proliferation of Huh7 and HepG2 liver cancer cells.
FIG. 4 is a plot of the cell proportion of the different cell cycles of Huh7 following modulation of miR-200a expression levels; the results show that the cell ratio of the miR-200a expression level up-regulated group in S phase and G2/M is lower than that in the other two groups, and the cell ratio of G1/G0 is higher than that in the other two groups, and FIG. 4 illustrates that the cell growth is influenced after the miR-200a expression level is regulated because cell cycle arrest is caused in Huh7 cells.
Thirdly, with Huh7 liver cancer cells as objects, the influence of miR-200a expression regulation on the effect of anticancer drug doxorubicin on induction of liver cancer cell apoptosis is researched
(1) Cell viability assay: the CCK kit (purchased from Homon molecular technology company, USA) is used for determining the influence of different drugs on the apoptosis of the liver cancer cells, and the operation is completed according to the kit operation instructions. Half the cytostatic concentration (IC50) was determined by adding doxorubicin (Sigma, USA) at different concentrations, 0, 10-4μM、10-3μM、10-2μM、10-1mu.M, 10 mu.M, 100 mu.M and 1000 mu.M, and observing for 24h, and performing absorbance reading detection by a Biotek microplate reader, wherein the absorbance value of OD450 is used for expressing the cell viability.
FIG. 5 is the effect of miR-200a expression on the apoptosis of hepatoma carcinoma cells Huh7 in combination with the anticancer drug doxorubicin; FIG. 5A is the change in the viability of hepatoma cells under the combined effect of miR-200a expression and doxorubicin; FIG. 5B is the half-lethal dose of hepatoma cells required by different groups under the combined action of miR-200a expression and doxorubicin. The result shows that after the expression level of miR-200a is up-regulated, half lethal dose of doxorubicin required by the hepatoma cell Huh7 is reduced; after the expression level of miR-200a is down-regulated, the hepatoma cell Huh7 needs half lethal dose of doxorubicin to be increased.
(2) Cell cycle experiments: cells of the miR-200a up-regulation expression group, the miR-200a down-regulation expression group and the control group are proliferated to about 80%, washed by PBS (phosphate buffer solution) at 4 ℃, fixed by 70% ethanol, and subjected to measurement grouping by using a loss cell analyzer after being resuspended by PI (American Pierce fly, USA) solution.
Apoptosis assay: cells of a control group, a miR-200a up-regulation expression group and a miR-200a down-regulation expression group are washed by PBS (phosphate buffer solution) at 4 ℃, incubated by Annexin V-APC (bioscience, USA), and subjected to up-flow cytometry to detect the apoptosis rate.
In cell cycle experiments and apoptosis experiments, the final concentration of doxorubicin is 2uM, and the action time is 12 h.
FIG. 6 is a flow cytometry-derived cell cycle map of different groups, FIG. 6A is a control group, FIG. 6B is a miR-200a up-regulation expression group, and FIG. 6C is a miR-200a down-regulation expression group. FIG. 7 is a flow cytometry-derived apoptosis graph of different groups, FIG. 7A is a control group, FIG. 7B is a miR-200a up-regulation expression group, and FIG. 7C is a miR-200a down-regulation expression group; FIG. 7D is control + doxorubicin, FIG. 7E is miR-200a upregulated expression + doxorubicin, and FIG. 7F is miR-200a downregulated expression + doxorubicin. FIG. 8 is the effect of miR-200a in combination with doxorubicin treatment on the growth phase and apoptosis of hepatoma cells; FIG. 8A shows the results of the ratio of different cell phases, and FIG. 8B shows the results of apoptosis. The result shows that the up-regulation of miR-200a expression level and the combination of doxorubicin for treating the liver cancer can cause cell S-phase retardation, induce the apoptosis level and enhance the effect of doxorubicin for treating the liver cancer.
Fourthly, verifying the influence of miR-200a expression level on the anti-tumor effect of doxorubicin in a mouse body
in vivo tumor formation experiment in nude mice:
(1) subject: nude mice of 8 weeks old were selected for subcutaneous tumor formation experiments, and animal experiments were approved by the animal ethics center of university of pittsburgh (IAC-regulated UC).
(2) The treatment method comprises the following steps: control group: the right axilla was injected with the control Huh7 cell line.
MiR-200a upregulated expression panel: right axillary injection of miR-200a up-regulated expression of Huh7 cells.
MiR-200a down-regulated expression panel: right axillary injection of miR-200a down-regulated expression of Huh7 cells.
The observation period was 4 weeks, and when grown to 6d, drug intervention was performed on day 7.
Blank group (including control group without doxorubicin addition, MiR-200a up-regulated expression group, MiR-200a down-regulated expression group): the tail vein is injected with normal saline.
Doxorubicin group (including doxorubicin-added control group, MiR-200a up-regulated expression group, MiR-200a down-regulated expression group): 4mg/kg, tail vein injection of doxorubicin drug.
tumor volume and mouse weight were measured every 5 days, tumor volume is length × width/2, after 25 days of drug intervention, and after the last measurement, the mice were sacrificed and specimens were obtained for further processing.
FIG. 9 is a bar graph of tumor results and mouse body weight change in nude mice in vivo tumor formation experiments, wherein FIG. 9A is a graph of tumor volume measurements in mice implanted with different times and different treatment modalities (no doxorubicin added) in the blank group; FIG. 9B is a graph of tumor mass volume measurements in mice with different treatment regimens (doxorubicin addition) for the doxorubicin group after different times of tumor implantation; FIG. 9C is a graph of tumor mass measurements taken from mice treated in different ways after 25 days of tumor implantation; figure 9D is a graph of the change in body weight of mice following tumor implantation for different treatment regimens.
The results in FIG. 9 show that the miR-200a up-regulation group can inhibit tumor growth, and the miR-200a down-regulation group can promote tumor growth. The miR-200a up-regulation group in the liver cancer cells can enhance the effect of doxorubicin on inhibiting the growth of the liver cancer cells, and the miR-200a down-regulation group antagonizes the effect of doxorubicin on inhibiting the growth of tumors.
Fifth, regulating and controlling influence of miR-200a expression level on energy metabolism of Huh7 cells
The experimental method comprises the following steps: the Huh7 cells were manipulated using an XF cell mitochondrial metabolism assay kit (Agilent technologies) according to the manual, and the cell oxygen consumption rate and extracellular acid concentration were measured and recorded using an XF-24 analyzer. The experiment was repeated three times.
FIG. 10 is a graph of the effect of regulating miR-200A expression level on cell metabolism, wherein FIG. 10A is the effect of different treatment modes on the oxygen consumption rate of Huh7 hepatoma cells, FIG. 10B is the effect of different treatment modes on the acidification rate of Huh7 cells, FIG. 10C is the effect of different treatment modes on the oxygen consumption rate of Huh7 cells, and "1" in FIG. 10C indicates a blank group and "2" indicates a combined doxorubicin treatment group.
The results in FIG. 10 show that the up-regulation of miR-200a expression level can inhibit oxidative phosphorylation of hepatoma cells, reduce adenosine triphosphate production and glycolysis process, inhibit basic energy metabolism of cells, and enhance the anti-tumor effect of doxorubicin.
The extracellular experiments and animal experiments prove that the effect of doxorubicin on inducing liver cancer apoptosis can be enhanced by increasing the expression level of miR-200a, and the sensitivity of liver cancer cells to the treatment effect of doxorubicin is weakened by reducing the expression level of miR-200 a. The negative regulation and control effects of the miR-200a on the growth, proliferation and apoptosis of the liver cancer cells are proved by interfering the expression of the miR-200a in the liver cancer cells. Through cell metabolic energy detection, the miR-200a is found to inhibit the basic metabolic process of cells, the action mechanism is consistent with the apoptosis promotion effect of doxorubicin, and the miR-200a is used for enhancing the sensitivity of doxorubicin to liver cancer cell treatment.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (4)
1. an application of miR-200a in preparing a kit for diagnosing and treating liver cancer.
2. an application of miR-200a in preparing a medicament for enhancing the effect of doxorubicin on anti-tumor cells.
3. The use of claim 2, wherein the tumor cell is the hepatoma cell line Huh7 or hepatoma cell line HepG 2.
4. The use according to claim 2, characterized in that the doxorubicin antitumor cells are present at a concentration of 2 μ M and have an action time of 12 h.
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