CN101804208A - Application of miR-451 in preparing medicine for treating non-small cell lung cancer - Google Patents

Abstract

The invention belongs to the field of genetic engineering, in particular to the application of miR-451 in preparing a medicine for treating non-small cell lung cancer. Since the recombinant of Pre-miR-451 has influence on propagation, apoptosis, migration and drug resistance of non-small cell lung cancer cells, it is proved that the miR-451 prohibits the propagation, migration and drug resistance of the non-small cell lung cancer cells and promotes the apoptosis of the non-small cell lung cancer cells; and living body level also indicates that the miR-451 has excellent anti-cancer effect.

Description

Application of miR-451 in the preparation of drugs for the treatment of non-small cell lung cancer

technical field

The invention belongs to the field of genetic engineering, and particularly relates to the application of miR-451 in the preparation of drugs for treating non-small cell lung cancer.

Background technique

RNA is one of the most important material bases in organisms, and together with DNA and proteins, it constitutes the framework of life. But for a long time, RNA was thought to be just a "transition" between DNA and protein, from which it obtained its sequence and then converted genetic information into protein. However, a series of studies have shown that these small RNAs actually manipulate many cellular functions, and they can turn off or regulate gene expression by reacting against DNA through the binding of complementary sequences. A recently discovered non-protein-coding RNA family, microRNA (miRNA, microRNA), can regulate gene expression by binding to specific mRNA or regulating the protein translation process of specific mRNA. At present, there are more than one thousand miRNAs identified in the human genome, which may be involved in regulating the expression of 30% of human protein-coding genes.

MicroRNA, referred to as miRNA for short, usually has a length of 19-25 nucleotides, widely exists in various animals, plants and even single-celled eukaryotes, and is a kind of highly conserved small molecule single-stranded RNA in evolution. miRNA is located in the intron region of the gene, and the gene encoding miRNA is transcribed into pri-microRNA (pri-miRNA) in the nucleus. Pri-miRNA is cut into a miRNA precursor (pre-miRNA) with a stem-loop structure of about 70 nucleotides in length under the action of a Drosha RNase. Pre-miRNA is transported from the nucleus to the cytoplasm under the action of the Ran-GTP-dependent nucleoplasm/cytoplasm transport protein Exportin 5. Under the action of Dicer enzyme, pre-miRNA is cut into double-stranded miRNA:miRNA* with a length of 21-25 nucleotides. Then the double-stranded miRNA:miRNA* unwinds, and the mature miRNA enters the cytoplasmic RISC (RNA-induced silencing complex) to exert gene silencing function. Complementary base pairing between miRNA and the 3' untranslated region (3'UTR) of the messenger RNA (mRNA) of a specific downstream gene causes the degradation of the mRNA or inhibits its translation, resulting in the failure of protein expression. We refer to these downstream genes as target genes of miRNAs, which usually have important biological functions in cells. miRNAs perform important functions in cells by regulating the expression of target genes, and are related to embryonic development, growth and development, and cell growth. Proliferation and differentiation are closely related. Scientists have confirmed that the nucleotide sequence of miRNA is highly conserved among different species, the mutation rate is extremely low, and its expression has gene clustering phenomenon, time-space and tissue specificity. miRNA regulates the expression profile of protein genes at the post-transcriptional level to determine the process and diversity of a series of important life activities such as cell differentiation and embryonic development. It has been found that miRNAs are not only important regulators of cell proliferation, differentiation, and apoptosis, but more than 50% of miRNAs are located in tumor-related genomic regions, including LOH regions, chromosomal amplification regions, and fragile sites. It has been identified that the expression profiles of miRNAs in various hematological tumors and solid tumors are changed, which provides a theoretical basis for us to reveal the mechanism of tumorigenesis from the perspective of RNA regulation, and provides new gene targets and molecules for tumor diagnosis and treatment mark.

In the human genome sequence, miR-451 is a MicroRNA located at 17q11.2, rat and zebrafish genomes, located on chromosome 11 and chromosome 5, respectively. The sequence of miR-451 is 5'-aaaccguuaccauuacugaguu-3' (GeneBank: GeneID: 574411). miR-451 is highly conserved in evolution, and there is homology among different species. According to the data released by the World Health Organization (WHO), the morbidity and mortality of lung cancer are on the rise in countries all over the world, especially in industrially developed countries. In developed countries, lung cancer is one of the most common malignant tumors, ranking first among male malignant tumors and ranking second and third among female common malignant tumors. At the end of the 20th century, lung cancer accounted for the first place in the death of malignant tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers and can be divided into squamous cell carcinoma, adenocarcinoma, and large cell lung cancer. Many lung cancer patients have metastasized at the time of diagnosis, and often lose the opportunity for surgery. The chemotherapy regimens for lung cancer are also ineffective, and patients are prone to drug resistance. The five-year survival rate is less than 5%. There is an urgent need to find new and effective treatments approach to lung cancer. With the deepening of genetic engineering research, scientists have shown strong interest in using genetic engineering to develop tumor drugs. The inventors found that miR-451 has a good application prospect in the treatment of non-small cell lung cancer.

Contents of the invention

technical purpose

The purpose of the present invention is to provide the application of miR-451 in the preparation of medicines for treating non-small cell lung cancer.

The object of the present invention is achieved through the following steps:

1. Amplify Pre-miR-451: Pre-miR-451, that is, the gene sequence encoding miR-451 precursor pre-miR-451, its RT and PCR primers are synthesized by Invitrogen, and the primer sequence is shown in the specific examples RT -PCR. RT-PCR amplified Pre-miR-451, and the purified product was shown by agarose gel electrophoresis, and a band with the expected size was amplified at about 72bp.

2. Connection: pcDNA ^TM 6.2-GW/EmGFP-miR vector (purchased from Shanghai Gemma Company, see Figure 1 for the vector, where EmGFP is a green fluorescent protein reporter gene for detecting transfection efficiency), through BamHI and Xho I enzymes After cutting, a linear vector was obtained, and the PCR product was connected to the vector to form a pcDNA ^™ 6.2-GW-Pre-miR-451 recombinant. 10ul ligation system: 2ul PCR product, 1ul linear vector, 1ul 10*T ₄ ligase buffer, 1ulT ₄ DNA ligase, 5ul ddH ₂ O.

3. Conversion:

A. Prepare fresh Escherichia coli competent cells (JM109), and mix the competent cells.

B. Take 20ul of competent cells into a new centrifuge tube, add 1ul of the connection system, and let it stand in an ice bath for 30min.

C. Competent cells were heated at 42°C for 42 seconds, and immediately placed in ice water at 4°C for 2 minutes after taking them out.

D. Add 400ul blank LB culture medium, shake at 37°C, incubate at constant temperature for 30min, resuspend and spread on the agar plate with a sterilized glass rod. After the plate is dry, invert the plate and incubate at 37°C for 12-16h.

4. Amplification: According to the characteristics of blasticidin resistance carried by the plasmid vector, select positive clones and shake in 5ml LB culture medium at a constant temperature of 37°C for 12-16h to amplify pcDNA ^TM 6.2-GW-Pre-miR in large quantities -451 recombinant. For the extraction and purification of recombinants, refer to the instructions of the BIOMIGA Non-endotoxic Plasmid Mini-extraction Kit (PD1214).

5. The vector pcDNA ^TM 6.2-GW-Pre-miR-451 was digested with BamHI and Xho I to obtain two fragments, which were the 72bp Pre-miR-451 fragment and the 5.7kb linear pcDNA ^TM 6.2-GW vector. The results were compared with The expected results were consistent, and the sequencing results of pcDNA ^TM 6.2-GW-Pre-miR-451 were completely consistent with the sequence in GenBank.

6. The correct sequenced recombinants were used to transfect tumor cells using the liposome method, and the transfection steps were referred to the instructions of LipofectamineTM2000. The successfully transfected cells expressed green fluorescent protein, and the transfection efficiency was analyzed by flow cytometry, and the transfection efficiency of different cell lines could reach 60%.

7. After Pre-miR-451 is transfected into tumor cells with the plasmid, the CMV promoter expresses Pre-miR-451 rapidly, efficiently and continuously in tumor cells, and forms pre-miR with a stem-loop structure through the action of Drosha (RNaseIII) -451 (about 72nt), and then through the action of Dicer to form mature miR-451 (22nt), which exerts the function of gene silencing.

1. Basic verification test of miR-451 against non-small cell lung cancer

(1): The expression profile of miR-451 in non-small cell lung cancer tissues and adjacent normal tissues:

1. Sample preparation: 60 cases of surgical resection specimens of non-small cell lung cancer (NSCLC) were collected, all of which belonged to stage IIIb and IV after clinical and pathological analysis. The specimens were provided by the Pathology Department of the Second Affiliated Hospital of Nanjing Medical University.

2. Chip preparation and analysis: According to the requirements of LC Sciences in the United States, lung cancer tissue samples were prepared, and the chip was prepared by LC Sciences, and Sanger miRBase V10.0 version was used to complete the chip analysis.

3. Chip results: The experimental data comes from LC Sciences, and the results are shown in Figure 2 and Table 1.

4. Analysis of results: the signal of miR-451 in normal tissue (Sample A) was 26,894.73, the signal in tumor tissue (Sample B) was 157.30, and the log2 (Sample B/Sample A) was -7.41, indicating that miR-451 was in normal tissue (Sample B) It is highly expressed in tissues and significantly low in tumor tissues, with a difference of 150 times, suggesting that miR-451 may play an important anti-tumor role as a tumor suppressor gene in lung cancer.

Table 1 Significant difference list (significantly differentially expressed transcripts with p value<0.01, wherein hsa is the probe for detecting human specimens)

No. Probe_ID Sample ASignal Sample BSignal log2(Sample B/Sample A) 1 hsa-miR-451 26,894.73 157.30 -7.41 2 hsa-miR-196a 33.02 3,362.17 6.67 3 hsa-miR-335* 81.08 4,720.06 5.90 4 hsa-miR-205 94.21 5,112.51 5.79 5 hsa-miR-335 162.56 8,041.86 5.59 6 hsa-miR-483-5p 49.00 1,331.34 4.70 7 hsa-miR-30a 2,588.28 115.34 -4.54 8 hsa-miR-134 5.37 107.00 4.35 9 hsa-miR-126 27,320.62 1,279.92 -4.35 10 hsa-miR-182 68.22 1,297.40 4.10 11 hsa-miR-21 889.51 12,086.79 3.90 12 hsa-miR-30d 4,000.27 319.67 -3.64 13 hsa-miR-574-5p 48.52 546.44 3.54

(2): RT-PCR analysis of the expression profile of miR-451 in non-small cell lung cancer tissues and adjacent normal tissues

1. Methods: Trizol reagent was used to extract tissue total RNA, and RT-PCR was performed using Ambion's SuperTaq polymerase system and mirVana detection kit, and human U6 was used as an internal reference.

2. RT-PCR results: as shown in Figure 3.

3. Analysis of the results: the expression of miR-451 increased in normal tissues and decreased in tumor tissues, which was consistent with the results of the gene chip.

2. Effect of miR-451 on the proliferation, apoptosis, migration and drug resistance of non-small cell lung cancer cells

(1): Cell proliferation experiment

1. MTT assay for cell proliferation: Four NSCLC cell lines were selected, including lung adenocarcinoma cell lines A549, H3255, lung squamous cell carcinoma cell line H157, lung large cell carcinoma cell line H1299, and four cell lines were transfected with Pre-miR- 451 was the experimental group, and the empty vector sequence (negative control, NC) was set as the control group. These cell lines were seeded on 96-well plates at 2500 cells/well. Three days after transfection, cell proliferation was monitored by MTT staining.

2. Measurement of results: as shown in Figure 4.

3. Analysis of results: MTT assay showed that compared with the control group, the proliferation of A549 cells transfected with Pre-miR-451 was significantly reduced. This phenomenon was also observed in three cell lines H157, H1299, and H3255, suggesting that miR-451 can inhibit the growth of tumor cells.

Experiment 2: Apoptosis experiment

1. Caspase3/7 activity method to measure cell apoptosis: NSCLC cell lines A549, H157, H1299, and H3255 were transfected with Pre-miR-451 and empty vector sequences, and the latter was set as the control group. After 72 hours of transfection, these cell lines were cultured in 0 μM DDP (cisplatin, chemotherapeutic drug) and 2 μM DDP medium, and the caspase3/7 activity in the cells was measured by ApoONE Homogeneous Caspase3/7 Assay.

2. Determination of caspase3/7 activity: as shown in FIG. 5 .

3. Analysis of the results: Compared with the control group, after transfection of Pre-miR-451, the activity of caspase3/7 in the four cell lines increased, suggesting that miR-451 can promote the apoptosis of tumor cells. Although the increase of caspase3/7 activity caused by Pre-miR-451 alone was not as obvious as that of DDP alone, the combined use of DDP and Pre-miR-451 significantly increased caspase3/7 activity, which was higher than that of Pre-miR-451 alone And DDP suggest that miR-451 can increase the sensitivity of tumor cells to chemotherapy drugs and can be used as an intervention target for tumor therapy.

Experiment 3: Cell invasion experiment

1. Transwell migration experiment: A549 cell line was transfected with Pre-miR-451 and empty vector sequence respectively, the latter was set as the control, inoculated into the upper chamber of Transwell, and the invasion of A549 cells was observed at 24h, 48h, and 72h after inoculation. .

2. Cell counting: the number of tumor cells in the lower chamber of the Transwell was determined by direct counting. The experimental group was significantly lower than the control group. The counting results are shown in Figure 6.

3. Analysis of results: Compared with the control group, the invasion ability of A549 cells after transfection with Pre-miR-451 decreased, and miR-451 can inhibit the invasion and metastasis of tumor cells.

Experiment 4: Observing the anti-cancer effect of miR-451 at the in vivo level

(1) Subcutaneous tumor experiment

1. In vivo model: We selected four 5-6-week-old female nude mice, and inoculated A549 cells transfected with Pre-miR-451 and empty vector (NC) into the flanks of the same mouse on both sides. In the subcutaneous tissue, the number of cells inoculated on each side was 1*10 ⁵ or 5*10 ⁴ , and the growth of tumor cells was observed.

2. Evaluation of tumor growth: observe once a day for at least five weeks. The length L and width W of the tumor focus were measured, and the volume of the tumor focus was calculated according to the formula (L*W^2)*0.5 to determine the effect of miR-451 on tumor cell growth at the in vivo level.

3. Measurement results: By the end of the observation, the side of the 4 mice transfected with Pre-miR-451 had no obvious tumor focus formation, and 3 of the 4 mice were observed at 22-27 days. The formation of tumor foci can be felt on the side inoculated with NC, and the volume of these tumor foci was 35-145cm ² at the end of the observation. Tumor volumes are shown in Figure 7.

4. Analysis of results: From the results of in vivo experiments, it can be seen that miR-451 can inhibit the growth of lung cancer cells in mice, which is consistent with our previous results obtained at the cell line level, confirming that miR-451 has a tumor suppressor effect.

(2) Lung carcinoma in situ experiment

1. Materials for lung cancer model establishment: 4 Lox-stop-lox K-Ras G12D (LSL K-Ras G12D) transgenic mice were donated by the Mouse Models of Human Cancer Consortium (MMHCC), HEK293 (American Type Culture Collection, cat#CRL.1573) Adenovirus packaging cells were donated by the Department of Biochemistry and Molecular Biology, Third Military Medical University, and ADCre and AdPre-miR-451 recombinant adenovirus were provided by Microbix. The Ad adenoviral vector carries the β-galactosidase operon to detect the transfection efficiency. Lung adenocarcinoma model was established by using Cre recombinase to induce K-Ras gene expression in transgenic mice and promote lung tumorigenesis. References: (1) The let-7microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle 7:6, 759-764.2008 (2) Establishment of animal models of mouse lung adenocarcinoma. Journal of Shanxi Medical University. 1007-6611 (2009) 05-0408-03.

2. Modeling steps and intervention of miR-451: 3-week-old mice were anesthetized by intraperitoneal injection of pentobarbital sodium 45 mg/kg, and then treated with Ad adenovirus: CaPi (Ad adenovirus: CaPi: titer was 2.5×10 Add 50ul of Ad adenovirus of 'pfu to 69ul of MEM, then add 6ul of 2.5mol/L calcium chloride) co-precipitate instilled into the nasal cavity of the mouse, instill about 125ul in total, and instill in 2 times, Repeat after 5 days, a total of two times. Experimental group: AdCre+AdPre-miR-451, n=2, control group: AdCre+AdNC, n=2.

3. Results measurement: After 7 weeks of intranasal instillation, the mice were sacrificed, and the lung tissues of the mice were weighed, embedded in paraffin, sectioned, and stained with HE. The tumor volume of the experimental group (Cre/Pre-miR-451) was significantly smaller than that of the control group (Cre/NC), and the ratio of lung tumor: whole lung tissue is shown in Figure 8.

4. Analysis of the results: In this experiment, the transgenic mouse LSL K-Ras G12D was used to establish a lung adenocarcinoma model, which once again proved that miR-451 can inhibit the occurrence of lung adenocarcinoma and the growth of tumor cells.

The expression vector of miR-451, these expression vectors include but not limited to plasmids, bacteria, host cells.

A pharmaceutical composition comprising a therapeutically effective amount of the miR-451 or its expression vector and pharmaceutically acceptable auxiliary materials.

The pharmaceutical composition is formulated in any pharmaceutically acceptable dosage form, including but not limited to tablets, pills, capsules, injections, oral liquids or liposomes.

Application of the above miR-451 sequences or their expression vectors in the preparation of tumor targeting drugs.

Statistical analysis among the present invention adopts SPSS 11.0 software (Release 11.0, SPSS Inc.) to carry out data processing, and data represent with mean ± standard deviation. Analysis of variance and t test were carried out, and the results were considered significant when P<0.05.

Beneficial effect

1. At present, the more commonly used methods for preparing miRNAs include preparing miRNAs in vitro and transfecting cells with DNA as a template and transcribing miRNAs in vivo. The present invention adopts the latter method, which is simple and convenient, and has been reported in a large number of documents that miRNAs can be successfully prepared without direct manipulation of RNA, which overcomes the disadvantages of easy degradation and high cost of artificially synthesized miRNA. Moreover, since the bacterial strain containing the expression vector can be stored for a long time and amplified in large quantities, it provides great convenience for future research.

2. Because miR-451 generally exists in the form of precursor (pre-miR-451) in the body, and Pri-miR-451 is cut into a length of about 72 nucleotides under the action of a Drosha RNase, with a stem loop Structure of miRNA precursors (pre-miRNAs). Pre-miR-451 is transported from the nucleus to the cytoplasm under the action of the Ran-GTP-dependent nuclear/cytoplasmic transporter Exportin 5. Under the action of Dicer enzyme, pre-miR-451 is cleaved into a 22-nucleotide double-stranded miRNA: miRNA*. Then the double-stranded miRNA:miRNA* unwinds, and the mature miR-451 enters the cytoplasmic RISC (RNA-induced silencing complex) to exert gene silencing function. Since Pre-miR-451 can be successfully processed into mature miR-451 in vivo, the present invention also uses Pre-miR-451 instead of miR-451. In the experiment, a large amount of Pre-miR-451 was obtained by recombinant transfection of Escherichia coli JM109 cells, which was ready for corresponding pharmacological experiments. At the same time, in order to avoid the impact of the carrier on the experiment, an empty carrier was used as a control test, and the results showed that the empty carrier had no effect on the present invention.

3. Through the effect of miR-451 on the proliferation, apoptosis, migration and drug resistance of non-small cell lung cancer cells, it is said that miR-451 can inhibit the proliferation, migration and drug resistance of non-small cell lung cancer cells, and promote the proliferation of non-small cell lung cancer cells. Apoptosis of lung cancer cells, and in vivo levels also indicated that miR-451 has an anticancer effect.

Description of drawings

Figure 1 is a plasmid vector expressing miR-451.

Figure 2 shows the analysis of miRNA expression profile in lung cancer tissue and adjacent normal tissue by gene chip (Sample A on the left is adjacent normal tissue, the signal value is Cy3; the middle Sample B is lung cancer tissue, the signal value is Cy5, and the right one is Cy5). is the signal ratio Cy3/Cy5 of adjacent cancer/cancer; in the Cy3/Cy5 signal ratio diagram, when the Cy3 signal is higher than the Cy5 signal, the color is displayed in green; when the Cy3 signal is equal to the Cy5 signal, the color is displayed in yellow; when When the Cy5 signal is higher than the Cy3 signal, the color is shown in red).

Figure 3 RT-PCR measurement of miR-451 levels in lung cancer tissue (T) and adjacent normal tissue (N), GAPDH was set as an internal reference.

Figure 4 shows the effects of miR-451 on the proliferation of lung cancer cell lines A549, H1299, H3255, and H157 cells measured by MTT method.

Figure 5 is the detection of apoptosis of lung cancer cell lines A549, H1299, H3255, H157 by miR-451 precursor

Figure 6 shows the effect of miR-451 precursor on the migration of lung cancer cell line A549 cells measured by Transwell method

Figure 7 shows the effect of miR-451 on the proliferation of A549 cells measured by in vivo experiments, the vertical axis is the subcutaneous tumor volume, and the horizontal axis is the mouse code

Figure 8 shows the effect of miR-451 on the growth of lung adenocarcinoma observed on the LSL K-Ras G12D transgenic mouse model, where the vertical axis is the ratio of lung tumor (Tumor): total lung tissue (Total Lung Area); the horizontal axis is the transgenic Mouse codes, #1, #2 are the experimental group (Cre/Pre-miR-451), #3, #4 are the control group (Cre/NC).

Detailed ways

The present invention will be further described below by way of examples, but the present invention is not limited.

General Notes:

The experimental method of not specifying specific conditions in the embodiment is basically all according to " Molecular Cloning Experiment Guideline (3rd Edition) " (Molecular Cloning: A Laboratory Manual, ^3rd ed. Huang Peitang et al.) edited by Sambrook, J et al. , Science Press. 2002.8) or in accordance with the conditions and methods suggested by the material provider, and other techniques not described in detail correspond to standard methods well known to those skilled in the art.

Materials of the present invention: the microorganisms, cell lines, plasmids or other expression vectors and culture medium mentioned in this application all have commercial supply or can be obtained by the public in other ways, they are only for example, not unique to the present invention, can Replace with other suitable tools and biological materials, respectively.

Example 1

1. Amplify Pre-miR-451: Pre-miR-451, its RT and PCR primers were synthesized by Invitrogen Company, and the primer sequences are shown in the following RT-PCR. RT-PCR amplified Pre-miR-451, and the purified product was shown by agarose gel electrophoresis, and a band with the expected size was amplified at about 72bp.

RT-PCR

Primers for miR-451 and U6 (as an internal reference) were designed and synthesized by Invitrogen.

1) Primer sequences and reaction conditions

RT primers for MiR-451:

5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAACTCAG-3'

The RT primer of GAPDH is Oligo(dT) ₁₅

Table 1. PCR primer sequences and reaction conditions

2) Synthesis of cDNA

5X RT Buffer 4μl

dNTP 2 μl

Inhibitor 1 μl

Primer 1μl

RNA 1μl (mass less than 1μg)

RT ace 1 μl

H ₂ O (RNase Free) 10μl

             

3) RT-PCR reaction system (50μl system)

Using cDNA as a template, PCR reaction system:

10×Ex Taq Buffer(Mg2+Free) 5μl

MgCl2(25mM) 5μl

dNTP(2.5mM) 5μl

Upstream primer (20pM/μl) 2μl

Downstream primer (20pM/μl) 2μl

Template 2μl

Ex Taq(5U/μl) 0.5μl

ddH ₂ O 28.5 μl

             

Total volume 50μl

The following reaction conditions were employed:

Pre-denaturation at 94°C for 2 minutes, followed by 35 cycles (18S RNA 25 cycles), the conditions for each cycle are: denaturation at 94°C for 30s, annealing at 54°C for 45s, and extension at 72°C for 2 minutes.

4) Identification of PCR products

50×TAE stock solution preparation: Tris 60.5g, glacial acetic acid 14.3ml, 0.5mol/L EDTA 25ml, add ddH2O to 250ml.

1.0% agarose gel preparation: 1.0g agarose, 100ml 1×TAE solution, heat until the agarose melts, add ethidium bromide to 0.5μg/ml when cooled to about 50°C, and use it after the plate is solidified.

Agarose gel electrophoresis: Inject 1×TAE solution into the electrophoresis tank to be higher than the gel surface, take 10 μl of PCR product, add 1 μl of 10× DNA loading buffer, mix and load the sample, use DL2000 DNA Marker as a reference, 80V electrophoresis, Jetta gel imaging system was used to observe and collect images, and the experiment was repeated three times.

2. Connection: The pcDNA ^TM 6.2-GW/EmGFP-miR vector was purchased from Shanghai Gemma Company, (the vector is shown in Figure 1, where EmGFP is the green fluorescent protein reporter gene to detect the transfection efficiency), digested with BamHI and XhoI Afterwards, a linear vector was obtained, and the PCR product was connected to the vector to form a pcDNA ^™ 6.2-GW-Pre-miR-451 recombinant. 10ul ligation system: 2ul PCR product, 1ul linear vector, 1ul 10*T4ligase buffer, 1ulT4DNA ligase, 5ul ddH ₂ O.

3. Transformation: 1. Prepare fresh Escherichia coli competent (JM109) cells, and mix the competent cells. 2. Take 20ul competent cells in a new centrifuge tube, add 1ul connection system, and let stand in an ice bath for 30min. 3. Heat at 42°C for 42 seconds, take it out and place it in ice water at 4°C for 2 minutes. 4. Add 400ul blank LB culture medium, shake at 37°C, incubate at constant temperature for 30min, resuspend and spread on the agar plate with a sterilized glass rod, after the plate is dry, invert the plate and incubate at 37°C for 12-16h.

4. Amplification: According to the characteristics of blasticidin resistance carried by the plasmid vector, select positive clones and shake in 5ml LB culture medium at a constant temperature of 37°C for 12-16h to amplify pcDNATM6.2-GW-Pre-miR in large quantities -451 recombinant. For the extraction and purification of recombinants, refer to the instructions of the BIOMIGA Non-endotoxic Plasmid Mini-extraction Kit (PD1214).

5. The eukaryotic expression vector pcDNATM6.2-GW-Pre-miR-451 was digested with BamHI and XhoI to obtain two fragments, which were the 72bp Pre-miR-451 fragment and the 5.7kb linear ^pcDNATM 6.2-GW vector , the result was consistent with the expected result, and the sequencing result of pcDNA ^TM 6.2-GW-Pre-miR-451 was completely consistent with the sequence in GenBank.

6. The correct sequenced recombinants were used to transfect tumor cells using the liposome method, and the transfection steps were referred to the instructions of LipofectamineTM2000. The successfully transfected cells expressed green fluorescent protein, and the transfection efficiency was analyzed by flow cytometry, and the transfection efficiency of different cell lines could reach 60%.

Example 2

MTT assay for cell proliferation:

1) Cell inoculation: Digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with RPMIt604 culture medium containing 10% fetal bovine serum, inoculate 100-1000 cells per well in a 96-well culture plate, The volume of each well is 200ul. Divided into four experimental groups: empty vector group, Pre-miR-451 group, DDP group, Pre-miR-451+DDP group.

2) Cultivate cells; move the culture plate into a COz incubator, and culture it for 3-5 days at 37° C., 5% CO 2 : and humidity.

3) Color development: After the 2nd to 5th day of culture, add 20ul of MTT solution (5mg/m1) to each well, continue to incubate at 37°C for 4h, terminate the culture, and carefully aspirate and discard the culture supernatant in the well. Add 150 μl DMSO (dimethyl sulfoxide) and shake for 10 min to fully dissolve the crystals.

4) Colorimetry: The optical density (OD) was measured at a wavelength of 490nm, and the experiment was repeated three times with 3 replicate wells in each group. The control group infected with the empty vector was used as the control, and the survival rate was calculated. Survival rate=OD value of experimental group/OD value of control group×100%.

Four non-small cell lung cancer cell lines (NSCLC lines), including lung adenocarcinoma cell lines A549 and H3255, lung squamous cell carcinoma cell line H157, and lung large cell carcinoma cell line H1299 were selected according to the above method, and the four cell lines were transfected with miR-451. Body sequence (Pre-miR-451), empty carrier sequence (negative control) is set as control group.These cell lines are planted on 96-well plate, 2500 cells/well.After transfection 3 days, MTT staining method monitors cell proliferation .MTT assay showed that compared with the control group, the proliferation of A549 cells transfected with Pre-miR-451 was significantly reduced. This phenomenon was also observed in the three cell lines H157, H1299, and H3255, suggesting that miR-451 can inhibit tumors cell growth.

Example 3

Apoptosis assay (determined by western blot)

1. Extraction of total cell protein

2. SDS-PAGE electrophoresis

1) Clean and install glass plates and ceramic sheets.

2) Prepare perfusion separation gel and let it stand at 37°C for 25 minutes.

3) Prepare perfusion stacking gel and let it stand at room temperature for 20 minutes.

4) Add protein samples and molecular weight markers, and the amount of protein loaded in each period is 40 μg.

5) Constant voltage electrophoresis (80V for stacking gel, 160V for separating gel).

3. Semi-dry transfer film

1) After the electrophoresis is over, wear gloves, cut the gel (cut corner marker) according to the molecular weight range of the target protein, and cut one piece of nitrocellulose membrane (cut corner marker) and two filter papers of the same size.

2) Soak the gel, membrane and filter paper in the transfer buffer for 10 minutes.

3) Lay out the membrane transfer apparatus from bottom to top: filter paper, membrane, gel, filter paper, and remove air bubbles, especially pay attention to the air bubbles between the membrane and the gel. Wipe off the excess liquid, transfer to the membrane with a current of 0.8mA×membrane area (cm2) for 1.5 hours.

4. Western Blotting

1) After the transfer to the membrane, check whether the marker has been transferred to the membrane (or Ponceau stained) to determine the transfer effect. After washing the membrane with TBST, 5% skimmed milk powder (prepared by TBST) was blocked overnight at 4°C.

2) Add an anti-Caspase-3/7 (activated) monoclonal antibody (1:500 and 1:500), shake at room temperature for 2 hours.

3) Rinse 3 times with TBST, 10 min each time.

4) Add secondary antibody (1:200), shake at room temperature for 1 h.

5) Rinse 3 times with TBST, 10 min each time.

5. ECL detection

1) Mix the AB solution, and spread it on the membrane after it stops dripping.

2) After reacting for 5 minutes, press in dark room for 5 seconds to 30 minutes according to the fluorescence intensity.

3) Develop for 15-30 seconds, wash with water, and fix for 1-3 minutes.

4) Image scanning and quantitative analysis.

According to the method above, NSCLC cell lines A549, H157, H1299, and H3255 were transfected with Pre-miR-451 and empty vector sequences respectively, and the latter was set as the control group. After 72 hours of transfection, these cell lines were cultured in 0 μM DDP (cisplatin, chemotherapeutic drug) and 2 μM DDP medium, and the caspase3/7 activity in the cells was measured by ApoONE Homogeneous Caspase3/7 Assay. Caspase3/7 activity assay: as shown in Figure 5. Compared with the control group, after transfection of Pre-miR-451, the activity of caspase3/7 increased in all four cell lines, suggesting that miR-451 can promote the apoptosis of tumor cells. Although the increase of caspase3/7 activity caused by Pre-miR-451 alone is not as obvious as that of DDP alone, the combined use of DDP and Pre-miR-451 can significantly increase the activity of caspase3/7, suggesting that miR-451 can increase tumor cell resistance. The sensitivity of chemotherapy drugs can be used as an intervention target for tumor therapy.

Example 4

Transwell (cell invasion assay)

1. Transwell chamber preparation

① Coating basement membrane: Coat the upper chamber surface of the bottom membrane of the Transwell chamber with 50mg/L Matrigel 1:8 dilution, and air-dry at 4°C.

②Hydration of basement membrane: Aspirate the residual liquid in the culture plate, add 50ul serum-free culture solution containing 10g/LBSA to each well, 37℃, 30min.

2. Preparation of Cell Suspension

① Before preparing the cell suspension, the cells can be starved for 12-24 hours with serum removed to further remove the influence of serum. ②Digest the cells, centrifuge to discard the culture medium after the digestion is terminated, wash with PBS 1-2 times, and resuspend in serum-free medium containing BSA. Adjust the cell density to 1-10×105.

3. Inoculation of cells

① Take 100-200 μl of cell suspension and add it to the Transwell chamber,

② Generally add 500 μl medium containing FBS or chemokines to the lower chamber of the 24-well plate,

③Cultivated cells: Routinely cultured for 12-48 hours (mainly depends on the invasion ability of cancer cells).

4. Results statistics:

"Adherent" cell count

The so-called "adherence" here means that after the cells pass through the membrane, they can attach to the lower chamber side of the membrane without falling into the lower chamber. Cells can be counted under a microscope by staining them

①Use a cotton swab to wipe off Matrigel and the cells in the upper chamber, stained with crystal violet.

② Cell counting: We used a Leica DC 300F upright microscope to observe and take pictures. Turn the Transwell chamber upside down and you can clearly see the cells attached to the upper and lower sides of the bottom membrane of the chamber.

"Non-attached" cell count Due to some cell's own reasons or some membrane relations, sometimes cells cannot attach to the membrane after passing through the membrane, but fall into the lower chamber.

According to the above method, the A549 cell line was transfected with Pre-miR-451 or the empty vector sequence, and the latter was set as the control, and inoculated into the upper chamber of Transwell, and the invasion of A549 cells was observed at 24h, 48h, and 72h after inoculation. Cell counting: The number of tumor cells in the lower chamber of the Transwell was determined by direct counting. The experimental group was significantly lower than the control group. The counting results are shown in Figure 6. Compared with the control group, the invasion ability of A549 cells decreased after transfection with Pre-miR-451, and miR-451 can inhibit the invasion and metastasis of tumor cells.

Example 5

Observing the anticancer effect of miR-451 at the living level

Subcutaneous Tumor Experiment

1. In vivo model: We selected four female nude mice at the age of 5-6 weeks, and inoculated A549 cells transfected with miR-451 and empty vector (NC) into the subcutaneous tissue of the ribs and backs on both sides of the same mouse. 1*10 ⁵ or 5*10 ⁴ cells were inoculated on each side, and the growth of tumor cells was observed.

2. Evaluation of tumor growth: observe once a day for at least five weeks. The length L and width W of the tumor focus were measured, and the volume of the tumor focus was calculated according to the formula (L*W^2)*0.5 to determine the effect of miR-451 on tumor cell growth at the in vivo level.

3. Measurement results: By the end of the observation, the side transfected with miR-451 precursor had no obvious tumor focus formation, while 3 of the 4 mice were inoculated with NC on the 22-27 day of observation The formation of tumor foci can be felt, and at the end of the observation, the volume of these tumor foci is 35-145cm ² in size. Tumor volumes are shown in Figure 7.

4. Analysis of results: From the results of in vivo experiments, it can be seen that miR-451 can inhibit the growth of lung cancer cells in mice, which is consistent with our previous results obtained at the cell line level, confirming that miR-451 has a tumor suppressor effect.

Carcinoma in situ lung experiment

1. Materials for the establishment of lung cancer model: 4 Lox-stop-lox K-Ras G12D (LSL K-Ras G12D) transgenic mice were donated by Mouse Models of Human Cancer Consortium (MMHCC) , HEK293 (American Type Culture Collection, cat#CRL.1573) adenovirus packaging cells were donated by the Department of Biochemistry and Molecular Biology, Third Military Medical University, and ADCre, AdPre-miR-451 recombinant adenovirus were provided by Microbix. The Ad adenoviral vector carries the β-galactosidase operon to detect the transfection efficiency. Lung adenocarcinoma model was established by using Cre recombinase to induce K-Ras gene expression in transgenic mice and promote lung tumorigenesis. Related literature: (1) The let-7microRNA reduces tumor growth in mouse models of lung cancer Cell Cycle 7:6, 759-764.2008 (2) Establishment of mouse lung adenocarcinoma animal model. Journal of Shanxi Medical University. 1007-6611 (2009) 05-0408-03.

2. Modeling steps and intervention of miR-451: 3-week-old mice were anesthetized by intraperitoneal injection of pentobarbital sodium 45 mg/kg, and then treated with Ad adenovirus: CaPi (Ad adenovirus: CaPi: titer was 2.5×10 Add 50ul of Ad adenovirus of 'pfu to 69ul of MEM, then add 6ul of 2.5mol/L calcium chloride) co-precipitate instilled into the nasal cavity of the mouse, instill about 125ul in total, and instill in 2 times, Repeat after 5 days, a total of two times. Experimental group: AdCre+AdPre-miR-451, n=2, control group: AdCre+AdNC, n=2.

3. Results measurement: After 7 weeks of intranasal instillation, the mice were sacrificed, and the lung tissues of the mice were weighed, embedded in paraffin, sectioned, and stained with HE. The tumor volume of the experimental group (Cre/Pre-miR-451) was significantly smaller than that of the control group (Cre/NC), and the ratio of lung tumor: whole lung tissue is shown in Figure 8.

4. Analysis of the results: In this experiment, the transgenic mouse LSL K-Ras G12D was used to establish a lung adenocarcinoma model, which once again proved that miR-451 can inhibit the occurrence of lung adenocarcinoma and the growth of tumor cells.

Claims (1)

1.miR-451 application at preparation treatment non-small cell lung cancer drug.

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