CN111996259B

CN111996259B - Application of micro RNA-3677-3p in medical and diagnostic fields

Info

Publication number: CN111996259B
Application number: CN202010949735.4A
Authority: CN
Inventors: 李延飞; 金月玲; 李觉; 沈君炜
Original assignee: Shanghai University of Medicine and Health Sciences
Current assignee: Semico Hangzhou Biotechnology Co ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2021-08-10
Anticipated expiration: 2040-09-10
Also published as: CN111996259A

Abstract

The invention provides an application of miRNA-3677-3p in preparing a liver cancer clinical detection marker and an inhibitor thereof in preparing a medicament for treating liver cancer.

Description

Application of micro RNA-3677-3p in medical and diagnostic fields

Technical Field

The invention relates to application of micro RNA-3677-3p in the fields of medicine and diagnosis. In particular, the invention relates to application of miR-3677-3p in cancer treatment and as a histopathology and detection marker.

Background

Micro RNA (microRNA, miRNA) is a non-coding regulation RNA family which has phosphate groups at the 5 'end and hydroxyl groups at the 3' end and is twenty-few nucleotides in length, and the micro RNA and the miRNA form a novel gene regulator. In animals, mirnas play a regulatory role by binding to imperfect complementary sites within the 3 '-untranslated region (3' UTR) of their target mrnas.

Tumors are considered to be a complex disease with overexpression of oncogenes and/or loss of expression of oncogenes in almost all processes of development or progression of tumors. Calin et al found that more than 50% of miRNA genes are located in tumor-associated regions or fragile regions, and are related to various human cancers. The miRNA participates in the maintenance of the stable state of a normal cell by regulating the translation or the stability of mRNA, so the abnormal expression of the miRNA can cause the abnormality of a corresponding target gene, but the classification of all the miRNA into two types of miRNA with carcinogenic effect and cancer-inhibiting effect is also impossible, because the cell also has a strictly evolved supervision mechanism to maintain the stability of the normal cell, therefore, the same miRNA can be considered as an oncogene or a cancer-inhibiting gene in different cell types, and generally, only one target gene of any miRNA is contained in a certain cell type, and each target gene can interact with a plurality of miRNAs to form an intricate regulation network, so that the various genes are subjected to complex regulation after transcription and participate in the generation and development of tumors. The intensive research on miRNA is helpful for understanding various physiological and pathological mechanisms of organisms, promotes the research on pathogenesis of human stubborn diseases such as cancer and the like, and finally provides new thought and theoretical basis for diagnosis and treatment of cancer.

The first tumor suppressor miRNA was found in B cells of Chronic Lymphocytic Leukemia (CLL) patients. Approximately 40% of CLL patients have chromosomal 13q14 deletion, while the miR-15a and miR-16-1 genes are located just within the LEU2 intron, resulting in the loss or downregulation of expression of these two genes in approximately 68% of CLL patients. bcl-2 is an anti-apoptosis gene and participates in the generation of various tumors, and Cimmino and the like find that the deletion of miR-15a and miR-16-1 causes the over-expression of bcl-2, so that the damaged cells cannot die, and further the formation of tumors is caused. Research also finds that one C in 7 downstream basic groups of miR-16-1 precursor of 2 CLL patients is mutated into T, so that the expression level of miR-16-1 is reduced, and the effect of the miR-16-1 on cancer suppressor genes is further proved.

Hepatocellular carcinoma (HCC) is a common malignant tumor in our country, and the mortality rate is second. The occurrence of hepatocellular carcinoma is related to the expression and structural abnormality of a plurality of oncogenes, cancer suppressor genes and products thereof, and researches find that miRNA expression profiles of patients with chronic hepatitis B and hepatitis C have no significant difference; miR-182, pre-miR-199b, miR-224 and miR-15b of a patient with chronic hepatitis is up-regulated in expression level compared with a patient with cirrhosis, and miR-28, miR-342, miR-126, miR-199a, miR-145b, miR-143, miR-368 and pre-miR-372 are down-regulated in expression level; miR-18, pre-miR-18 and miR-224 in the liver cancer tissue have higher-level expression than normal tissues, and the expression levels of miR-199a, miR-195, miR-199a, miR-200a and miR-125a are reduced. The discrimination of cancerous from normal tissue with these differentially expressed miRNAs can reach a surprising 97% accuracy. The miRNA is not only found to be abnormally expressed or mutated in various tumors, but also the mutation or ectopic expression of the miRNA plays an important role in the development of the tumors. Defects at any step in the miRNA biosynthesis process may cause down-regulation of its expression, which acts as a cancer suppressor, leading to up-regulation of the expression of downstream target proteins (oncogenes) and ultimately to enhanced cell proliferation and invasiveness, reduced apoptosis and enhanced angiogenesis, promoting tumor formation. Conversely, overexpression of mirna (oncomiR) functioning as an oncogene is also a cause of tumorigenesis, and due to increased expression caused by amplification of the oncomiR gene, persistent promoter activation, and the like, expression of a target gene (oncogene) is decreased, and tumorigenesis is caused. Targeted therapies that induce the loss of function of specific oncomirs in tumor cells to achieve anti-tumor effects are also emerging as an emerging therapeutic strategy. At present, methods for deleting the function of miRNA are mainly divided into two main categories, namely chemically synthesized oligonucleotide analogue inhibitors and vector or virus-based cloning inhibitors. A cloning inhibitor based on a vector or a virus is a method for stably inhibiting miRNA for a long time developed by Phillip Sharp and the like of the science and technology of Mazhou province in 2007. The effective miRNA sponge is an effective miRNA inhibitor, contains a plurality of miRNA binding sites (RBSs), can adsorb corresponding miRNA molecules like a sponge, and can not be combined with target molecules of the miRNA after adsorption, thereby influencing the function exertion of the miRNA. Compared with a chemical synthesis inhibitor, the miRNA sponge can be stably expressed in cells to play a role, and has the advantage of longer action effect. The method is widely applied to the basic research fields of miRNA function loss transgenic animal construction and the like and the research and research of tumor targeted therapy. A plurality of researches respectively reveal that sponge sequences targeting miR-23b, miR-21 and miR-10b all show efficient tumor inhibition after being highly expressed in tumor cells, and the potential of miRNA sponge as an anti-tumor therapy is strongly suggested. It has been shown that miR-3677-3p (named miR-3677) is highly expressed in patients with liver cirrhosis suffering from antral-gastric vasodilation. In addition, miR-3677-3p is reported to be miRNA related to prognosis of liver cancer patients. However, the effect of the micro-RNA in liver cancer is still fragmentary, and the elucidation of the action mechanism of micro-RNA-3677-3 p in the regulation of the formation, the development and the metastasis of hepatocellular carcinoma in the prior art at home and abroad has not been reported yet.

Disclosure of Invention

The invention aims to provide application of miR-3677-3p in treatment and diagnosis of hepatocellular carcinoma, and particularly provides application of miR-3677-3p serving as a hepatocellular carcinoma detection marker and in preparation of a medicine for treating hepatocellular carcinoma.

The invention also aims to provide an in vitro diagnosis product for preparing hepatocellular carcinoma by using miR-3677-3p, and more specifically, the diagnosis product is a kit. In a preferred embodiment, the kit comprises a sponge sequence as set forth in SEQ ID No. 1-2.

The invention also aims to provide a pharmaceutical composition for inhibiting hepatocellular carcinoma, and more specifically, the active ingredient of the drug for inhibiting hepatocellular carcinoma is miR-3677-3p sponge sequence, and the sponge sequence can inhibit the growth of hepatocellular carcinoma cells by targeted inhibition of GSK3 beta. In a preferred embodiment, the sponge sequence is as set forth in SEQ ID No. 1-2.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 11A overexpression of the sponge resulted in a significant down-regulation of miR-3677-3p expression; the MiR-3677-3p sponge obviously inhibits the proliferation of HCC cells; 1C and 1D, miR-3677-3p spongiome EdU level is obviously reduced; 1E and 1F: (ii) colony formation results; the PCNA level of the 1G and 1H miR-3677-3p sponge group is obviously reduced;

FIG. 2 cell cycle analysis and apoptosis analysis;

FIG. 3 luciferase assay;

figure 4 results of in vivo experiments.

Detailed Description

The data analysis of the invention adopts the sps 17.0 software to carry out statistical analysis. Data analysis used t-test and two-way analysis of variance. Error bars were calculated from three independent replicates of experimental data. Data are presented as mean ± SEM. Denotes p <0.05, p <0.01 and p <0.001, respectively.

Experimental materials: two HCC cell lines (Hep3B and Huh7) were purchased from cell sample bank of Shanghai cell biology institute, China academy of sciences. Both cell lines were cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). HCC cells at 37 ℃ with 5% CO₂The humidified incubator of (Thermo Fisher Scientific, Inc.). Animal experimental animals were purchased from SLAC laboratory animals Inc. (Shanghai, China), and the study protocol was approved by the animal care and use committee of the Shanghai health medical college.

Experimental correlation sequence:

wherein, the sequence 1-2 is a miR-3677-3p sponge sequence, and the miR-3677-3p sponge sequence is connected into a pLVX-puro carrier to obtain a carrier containing the miR-3677-3p sponge sequence, and the carrier is provided with a puromycin selective marker. The shRNA sequence is an shRNA sequence taking a human GSK3 beta sequence as a target point.

Example 1 miR-3677-3p as a marker for diagnosing liver cancer and inhibiting effect on liver cancer cells

1. Viral packaging 293T cells were co-transfected with lentiviral plasmids using FuGene HD transfection reagent (Roche Diagnostics). The virus-containing medium was removed 48h after transfection and filtered to remove cell debris. To obtain Hep3B and Huh7 cells with miR-3677-3p sponge sequence over-expression, 2X 10 cells were added 12h before infection⁵Cells were seeded on each well of a 6-well plate. Subsequently, the cell culture medium was changed to 1ml of fresh medium and 1ml of the above medium containing the virus. After 4 days, 1ug/ml puromycin was added to the medium and treated for 1 week. Puromycin resistant cells were then seeded into 6-well plates to form cell lines for further analysis. To generate Hep3B cells using shGSK3 beta,2 x 10 to⁵Cells were seeded into 6-well plates and then infected with virus-containing supernatant (8. mu.g/mL polybrene added). Stably infected cells showed green fluorescence 5 days after infection and were sorted by flow cytometry (BD Biosciences, Franklin Lakes, United States).

2. Cell proliferation assay: cell proliferation was measured by the CCK-8 (Japan Co., Ltd.). Hep3B and Huh7 cells over-expressed by miR-3677-3p sponge sequence at 1X 10³The density of individual cells/well was seeded in 96-well plates. After 12, 36, 60 and 84 hours of incubation, 10. mu.l of CCK-8 reagent was added to each well, followed by incubation at 37 ℃ for 2 hours. Absorbance at 450nm was measured using SpectraMax M5(Thermo Fisher Scientific, Unite States).

3. Clone formation experiments: culture of 1X 10 in 6-well plates per well³HCC cells (Hep3B and Huh7 cells with miR-3677-3p sponge sequence over-expression). Then cultured in a cell culture chamber for 10 days. Then, the cells were fixed with methanol for 20 minutes, finally stained with crystal violet, and photographed with a camera (Nikon, Japan).

4. And (3) immunofluorescence staining: the cell proliferation assay used the EdU-555 proliferation kit (c075l, Beyotime). Hep3B and Huh7 cells over-expressed by miR-3677-3p sponge sequence at 1X 10⁴The density of individual cells/well was cultured in 24-well plates. After 48h, the old medium was replaced with fresh medium containing 10. mu.M EdU. After 2h of cell culture, the cells were treated with 4% paraformaldehyde for 20min, infiltrated with 0.25% TritonX-100 for 5min, and incubated for 3min at room temperature for probe cross-linking. Subsequently, nuclei were counterstained with hoechst33342 for 5min, finally washed with PBS and imaged with a fluorescence microscope (Nikon).

5. Western blot analysis: all cells were washed twice with frozen PBS. Then lysed using a strong RIPA buffer containing protease inhibitors (Thermo, Waltham, MA) for 1h at 4 ℃. The lysate was then centrifuged at 12000rpm for 15 minutes at 4 ℃ and the protein concentration was determined using the BCA protein assay kit (Beyotime). The cell lysates (total protein 20. mu.g) were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane (Merck). Cell membranes were blocked with 3% bovine serum albumin for 2 hours at room temperature, then incubated with different primary antibodies: anti-GSK3 beta (ab65740, Abcam), anti-GSK3 beta (phosphorylated Y216) (ab75745, Abcam), anti-PCNA (ab18197, Abcam) and anti-GAPDH (ab181602, Abcam). Then incubated with the appropriate secondary antibody for 1h at room temperature. Protein bands were visualized using an ECL color kit (Yazyme) with a LAS4000mini imager (GE).

6. Reverse transcription and quantitative PCR reactions: total RNA from Hep3B and Huh7 cells over-expressed by miR-3677-3p sponge sequence was extracted with Trizol reagent (TaKaRa), and 500ng of RNA was reverse transcribed into cDNA using Prime-Script RT kit (TaKaRa) for each sample. The miR-3677-3p and U6 primers were purchased from Ribobio. The obtained cDNA was amplified using Takara-Ex-Taq-PCR kit (Takara). Quantitative PCR was performed using Stratagene Mx3000QPCR system (Stratagene) and analyzed by the Δ CT method. The qPCR primer sequence is shown in SEQ ID No. 9-14.

The experimental results are as follows:

we detect the influence of a miR-3677-3p sponge sequence on the expression of miR-3677-3p in liver cancer cells and the effect of miR-3677-3p on HCC in vitro. miR-3677-3p is highly expressed in HCC, and by over-expressing a sponge sequence of miR-3677-3p in Hep3B and Huh7 cells, the fact that the over-expression of the sponge leads to the significant down-regulation of miR-3677-3p expression can be observed, and the sponge sequence is suggested to be effective (FIG. 1A). The MiR-3677-3p sponge significantly reduced the CCK-8 absorbance levels of Hep3B and Huh7 cells, indicating that the MiR-3677-3p sponge significantly inhibited the proliferation of HCC cells (fig. 1B). Furthermore, in the miR-3677-3p sponge group, EdU levels were also significantly reduced, indicating a significant reduction in the number of cells in the mitotic phase (fig. 1C and 1D). Colony formation results showed that miR-3677-3p sponge caused a significant reduction in clone numbers (fig. 1E and 1F). We examined genes, such as PCNA, that are closely related to cell proliferation. PCNA levels were also significantly reduced in the miR-3677-3p sponge group (FIGS. 1G, 1H). The results show that the miR-3677-3p sponge inhibits the proliferation of liver cancer cells, and the miR-3677-3p can be used as one of clinical detection markers of liver cancer.

Example 2 cell cycle analysis and apoptosis analysis

Cell cycle analysis: cell cycle and apoptosis detection kit (Beyotime) was used to detect cell cycle. Hep3B and Huh7 cells with miR-3677-3p sponge sequence over-expression are inoculated on a 6-well plate respectively. The cells were then washed twice with cold PBS and collected in 1.5ml Eppendorf tubes. Subsequently, the cells were treated with 70% alcohol. Then treated with RNase A and PI and finally analyzed by flow cytometry (BD). Data was collected and analyzed using FlowJo analysis software.

Apoptosis assay: the annexin v-FITC/PI apoptosis kit (Nanjing Kaiyu) is adopted to detect apoptosis. HCC cells from the control group (HCC cells not over-expressed) and the sponge group were seeded in 6-well plates. The HCC cells were then washed with PBS and then collected in a 1.5ml Eppendorf tube. Cells were treated with annexinv-FITC/PI and analyzed by flow cytometry (BD). Finally, data was collected and analyzed using FlowJo analysis software.

The experimental results are as follows: we examined the role of miR-3677-3p in cell cycle and apoptosis. Consistent with previous results, miR-3677-3p inhibition decreased the percentage of cells in the mitotic phase and increased the percentage of cells in the resting phase (fig. 2A and 2B). However, the miR-3677-3p sponge is over-expressed, and has no influence on the apoptosis of HCC cells (FIGS. 2C and 2D). These results indicate that miR-3677-3p does not affect apoptosis of HCC cells.

Example 3 luciferase assay

The GSK3 gene is a cancer suppressor gene closely related to tumor cell proliferation. We predicted by miRDB that miR-3677-3p might directly target GSK3 β (fig. 4A). And (3) verifying whether the miR-3677-3p can directly regulate and control the GSK3 beta by using a luciferase reporter gene experiment. The GSK3 beta 3'UTR and GSK3 beta 3' UTR mutant primers are shown in SEQ ID NO. 5-8. These sequences were ligated into the pMIR-REPORT luciferase reporter vector at the splice points SacI and HindIII. 293T cells grown in 24-well plates were transfected with 50 nmiRNA (negative control, nonsense sequence given by Ribbio), miR-3677-3p mimic (as an overexpression positive control, Ribbio, product number: miR10018101-1-5) and miR-3677-3p inhibitor (positive control, Ribbio, product number: miR20018101-1-5), respectively. Mu.g of pMIR reporter luciferase reporter vector (containing GSK 3. beta.3' UTR) and 0.02. mu.g of blank Renilla luciferase vector (pRL-TK; Promega) were transfected into the corresponding culture wells using Fugene HD. After 24 hours of transfection, cell lysates were assayed for fluorescence and Renilla luciferase activity using the dual luciferase reporter system (Promega).

The experimental results are as follows:

we found that miR-3677-3p regulates GSK3 β by binding to the 3' UTR region (FIGS. 3B and 3C). To further confirm the relationship of miR-3677-3p to GSK3 β cells, we examined the situation when the 3' UTR of GSK3 β was mutated. We found that miR-3677-3p only targets cells that act on the normal UTR of GSK3 β (fig. 3D). Significant increases in mRNA, protein, and phosphorylated protein expression of GSK3 β were detected in miR-3677-3p sponge-treated cells (fig. 3E, 3F, and 3G). These results indicate that miR-3677-3p can inhibit the expression of GSK3 beta cells by targeting the 3' UTR, thereby ultimately affecting the proliferation of liver cancer cell lines.

Example 4 animal experiments

Control group (Hep3B cells not overexpressing sponge sequence) and Hep3B cells (5X 10) of miR-3677-3p sponge group⁶One cell/one) were injected subcutaneously into the right forelimb axilla of Balb/c nude mice, respectively. Tumor volume was observed during tumorigenesis. 28 days after inoculation, mice were sacrificed by intraperitoneal injection of sodium pentobarbital (120mg/kg), and tumors were removed and weighed.

The experimental results are as follows:

in vivo experiments show that the inhibition of miR-3677-3p can obviously inhibit the tumor growth of an HCC cell line, and Hep3B cells of a control group and a miR-3677-3p sponge group are subcutaneously injected into Balb/c nude mice. Mice were sacrificed on day 28 post injection and solid tumors were excised. Tumor volume and weight were measured (fig. 4A). After miR-3677-3p sponge is over-expressed, the tumor weight is obviously reduced (figure 4B). Statistical analysis revealed that tumor volume was significantly reduced after over-expression of miR-3677-3p sponge (FIG. 4C). Then, the mRNA expression level of GSK3 β protein and PCNA were examined, and it was found that the mRNA expression level of GSK3 β protein was significantly up-regulated in the miR-3677-3p sponge group (fig. 4D), the mRNA level of PCNA was significantly down-regulated, and the protein level was also significantly down-regulated (fig. 4D and 4E). These results indicate that inhibition of miR-3677-3p can significantly inhibit the in vivo development of HCC. In order to confirm the regulation effect of miR-3677-3p on GSK3 beta, in the shGSK3 beta experimental group, the expression of GSK3 beta is obviously reduced (FIG. 4F), and the proliferation of HCC cells is promoted (FIG. 4G). These data indicate that miR-3677-3p inhibits HCC progression through targeting effect GSK3 β.

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

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.

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Claims

1. An application of miRNA-3677-3p sponge sequence in preparing GSK3 beta expression promoter is characterized in that the miRNA-3677-3p sponge sequence is shown in SEQ ID NO. 1-2.

2. The application of the miRNA-3677-3p sponge sequence as claimed in claim 1 in preparing a GSK3 beta expression promoter, wherein the GSK3 beta expression promoter can be used for inhibiting the growth of liver cancer cells without mutation in the 3' UTR region of GSK3 beta protein.