Transfection of miRNA Mimic or Inhibitor

Transfection of MicroRNA Mimic or Inhibitor in Regular Cell Line and Primary Cells Derived from Patients

Si Wang¹ , Jing Xu^2,3 , Yuanxu Guo ^2,3 , Yongsong Cai⁴ , Wenhua Zhu^2,3 , Liesu Meng^2,3 , Congshan Jiang ⁵ *, Shemin Lu^2,3*

Author information

1. Clinical Laboratory, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, haanxi, 710061, China.

2. Institute of Molecular and Translational Medicine (IMTM), and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi, 710061, China.

3. Key Laboratory of Environment and Genes Related to Diseases (Xi' an Jiaotong University), Ministry of Education, Xi' an, Shaanxi, 710061, China.

4. Department of Joint Surgery, Xi' an Hong Hui Hospital, Xi' an Jiaotong University Health Science Center, Xi' an, Shaanxi, 710054, China.

5. National Regional Children's Medical Centre (Northwest); Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province; Xi' an Key Laboratory of Children's Health and Diseases, Shaanxi Institute for Pediatric Diseases; Xi' an Children's Hospital, Affiliated Children's Hospital of Xi' an Jiaotong University, Xi'an, Shaanxi, 710003, China

*Corresponding: Congshan Jiang (jiangcongshan@xjtu.edu.cn) and Shemin Lu (lushemin@xjtu.edu.cn)

Background

Transfection is a specialized technique for introducing exogenous genes into cells. With the deepening of research on gene and protein functions, transfection has become a common basic method in laboratory work. Transfection can be roughly divided into three types of pathways: physical mediation, chemical mediation, and biological mediation. Electroporation, microinjection and gene gun are very promising physical methods. While, there are also many chemical methods, such as the classical calcium phosphate coprecipitation method, liposome transfection method; Biological methods include primitive protoplast transfection and various virus mediated transfection techniques [1].

    Lipofection refers to the cationic liposome with a positive charge on the surface, which can interact with the phosphate group of nucleic acid through electrostatic interactions to encapsulate DNA or RNA molecules, forming lipid complexes. It can also be adsorbed by negatively charged cell membranes on the surface, and then enter cells through fusion or endocytosis. Lipofection is suitable for transfecting DNA or RNA into suspension or adherent cultured cells, and is currently one of the most convenient and efficient transfection methods in the laboratory [2].

    MicroRNAs (miRNAs) are a class of non-coding small RNAs composed of 18-25 nucleotides. Mostly, they regulate the expression of various protein coding genes at the post-transcriptional level by inhibiting translation or inducing mRNA degradation, and are closely related to physiological processes such as cell differentiation, proliferation, apoptosis, and hematopoiesis during normal physiological processes. While under pathological conditions, abnormal expression of miRNA participates in the occurrence and development of diseases by regulating multiple genes [3, 4].

    The excessive proliferation and limited apoptosis of rheumatoid arthritis synovial fibroblasts (RASF) are considered as the pathological basis of RA, which can directly promote joint destruction and cartilage damage by enhancing the production of matrix degrading enzymes [5, 6]. Mimic and inhibitor of miRNAs were transfected into the regular SW982 cell line and the primary RASF cells from patients to study the molecular mechanism of these miRNAs during gain and loss of their function.

Abstract

The transfection of miRNA mimics and inhibitors can lead to the gain and loss of intracellular miRNA function, helping us better determine the role of miRNA during gene expression regulation within specific physical condition. Our previous research has confirmed the efficiency and convenience of using liposomes to transfect miRNA mimics or inhibitors [7]. This work takes miR-424 and miR-497 as examples to provide a detailed introduction of transfection process of miRNA mimics and inhibitors in regular SW982 cell line and primary RASF cells from patients by using lipofection, which can also serve as a reference for miRNA transfection in other cell lines.

Keywords: miRNA, mimic and inhibitor, lipofection

Materials and Reagents

1.  Lipofectamine™ 3000 (Invitrogen, Catalog No. L300001)

2.  Mimics and inhibitors (GenePharma company)

3.  0.25% Trypsin with EDTA (Hyclone, Catalog No. SH30042.01B)

4.  Fetal bovine serum (FBS) (Sangon biotechnology, Catalog No. E51002)

5.  DMEM high glucose culture medium (Hyclone, Catalog No. SH30022.01B)

6.  Penicillin-Streptomycin 100x (HyClone, Catalog No. SV30010)

7.  Saline (CR double-crane, Catalog No.H20054037)

8.  PBS (Biosharp, Catalog No.BL601A)

9.  DNase (Sigma-Aldrich, Catalog No. AMPD1)

10. Hyaluronidase (Sigma-Aldrich, Catalog No. 37259-53-3)

11. Type I collagenase (Sigma-Aldrich, Catalog No. SCR103)

12. TRIzol reagent (Invitrogen, Catalog No.15596-026)

13. 100 mm sterile polystyrene culture dish (Nunc, Catalog No. 150340)

14. 6-well sterile polystyrene cell culture plate (Nunc, Catalog No. 150239)

15. 15mL sterile centrifuge tube (KIRGEN, Catalog No. KG2611)

16. 1.5mL centrifuge tube (KIRGEN, Catalog No.KG2211S)

Equipments

1. Cell culture incubator: 37 ℃ and 5% CO₂ (Thermo, THM#3427)

2. Centrifuge (Eppendrof, 5810R)

Procedure

1. Cell preparation:

SW982 cells are regularly cultured in DMEM high glucose medium supplemented with 10% FBS and 0.1% Penicillin-Streptomycin and incubated at 37°C in humid conditions provided with 5 % CO₂.RASF cells were isolated from synovial tissues of the knee joints from four RA patients. The procedures for RASF isolation are as follows.

1) Cut off excess adipose tissue from the RA synovial tissue specimen, rinse the synovial tissue with sterile saline until there are no obvious blood stains, select suitable tissues, place it in a sterile culture dish, and use ophthalmic scissors to cut it as small as possible.

2) Add 5mL of saline, soak the tissues, and then transfer it into a 15mL centrifuge tube, centrifuge it for 5 mins with 500g, discard the supernatant.

3) Add 5mL of 0.25% trypsin-EDTA for resuspension, add type I collagenase (final concentration 1mg/mL), hyaluronidase (final concentration 0.15mg/mL), DNase (final concentration 0.15mg/mL), mix them well, digest at 37 ℃ for 60 to 90 min, and shake

the centrifuge tube occasionally.

4) After tissues digestion, add 5mL of culture medium and resuspended. Standstill until the remaining tissue block in the 15mL centrifuge tube settles at the bottom, the supernatant is transferred to another 15mL centrifuge tube for centrifugation to obtain

cell pellets.

5) Wash the pellets with 5mL of culture medium, resuspend, centrifuge, and discard the supernatant. Add 4mL of culture medium again and repeat the resuspension centrifugation step to collect the cells as many as possible.

6) Count the collected cells with a cell counting chamber. Seed 4 million cells every 25 cm² of the culture bottle. The newly isolated cells are highly diverse. In addition to RASF, there are lymphocytes, osteoclast, macrophages, and macrophage-like synovial cells. Therefore, the abundant cell density at the very beginning is crucial for the growth of synovial cells.

7) Place the culture flask inoculated with cells in a 5% CO₂ cell incubator at 37 ℃, and observe the cell adhesion for any contamination on the next day. On the third day, change the fluid and discard the other types of cells or dead cells which are not

adhered to the bottom of the flask. Passage the cells when the confluence reaches 90%.

2. Cell seeding:

1) Wash thoroughly with PBS buffer 3 times. After discarding all the liquid, add 1mL of trypsin, gently move the culture flask to make it even, and incubate it at 37 ℃ for about 2 minutes. Observing the cell condition and avoid excessive digestion, which

may affect the cell morphology.

2) Add 4mL of complete culture medium to terminate the reaction, gently detach the cells from the bottom with a pipette, transfer the cells to a 15mL centrifuge tube, centrifuge 1000 g for 5 minutes to collect cell pellets, discard the supernatant, and add 1 ml of the culture medium and fully resuspend the cells. According to the required number of cells in the experiment, inoculate SW982 cells and RASF cell in 6-well plate overnight, and make sure they grow to 80% confluence.

3. Preparation of the transfection reagents:

1) For each well in the 6-well plate, add 5µL Lipofectamine™ 3000 reagent and mix well in a 1.5mL centrifuge tube containing 250 µl serum-free DMEM medium and incubate at room temperature for 5 minutes.

2) Add miRNA mimics or inhibitors and mix them in another centrifuge tube containing 250 µl serum-free DMEM medium.

3) Combine the above-mentioned two mixture, gently mix for 15 times and incubate at room temperature for 20 minutes.

4) Prepare the cells when transfection reagents were incubated during this 20 minute. Wash the cells in the 6-well plate with PBS and add 1.5mL of serum-free culture medium. After the incubation of the transfection reagent is completed, add 500μl of the mixture from the previous step to each well, mix thoroughly and incubate in CO₂ incubator. Four-six hours later, change the medium with the complete culture medium.

4. Validation of the transfection efficiency

The validation of transfection efficiency can be achieved by detecting the intracellular miRNA level following the transfection of exogenous miRNA mimic or inhibitors. Collect the cell lysate with TRIzol-reagent, and perform RT-qPCR. In our published results, it is show that miRNA expression can increase up to a thousand times of its previous intracellular level after transfection of the mimics, and expression of some miRNAs can also be significantly reduced after transfection with inhibitors. This indicates the success of the liposome transfection method, which is simple and highly reproducible.

Notes

1. MiRNA mimics and inhibitors were synthesized by the company. Mother solution at 20 µM were prepared before use, and stored at -20 ℃.

2. Mock group with no mimics or inhibitors, but only Lipofectamine 3000, as well as the negative control (NC) group with miRNA should be included. And all the experiments should be carried out in triplicates as a minimum.

3. According to our previous experience, for miRNA transfection, the final concentration of the 10 nM mimic substance is enough for most miRNA experiments, while for the inhibitors, a 100 nM should be OK. The optimal concentration could be obtained through pilot experiments.

Recipes

SW982 cell culture medium: Add 10% fetal bovine serum and 0.1% 100 U/mL penicillin and streptomycin to DMEM high glucose medium, adjust the pH with NaOH or HCl until 7.2-7.4.

Acknowledgments

We are grateful for the National Science Foundation of China (grant No. 81671629, 81701619, 81970029 and 81902249) and Shaanxi Province Natural Science Foundation (Project No. 2021JQ-024 and 2018JM7057).

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