Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent
A technology of 1. mir-185 and regulator, applied in the field of biomedicine, can solve the problems of cell growth and senescence regulation without miRNAs, so as to promote the telomere disorder and senescence of tumor cells, inhibit the growth of tumor cells, and delay senescence. Effect
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Embodiment 1
[0047] Example 1 Construction of miR-185 overexpression cells and rescue (rescue) experiments
[0048] 1. Experimental method
[0049] Synthesize oligo primers containing the positive and negative sequences of shPOT1, anneal the two primers by gradient cooling, and ligate the annealed double-stranded RNA containing the positive and negative sequences of shPOT1 into the pLKO vector. HEK293T cells were transfected with plasmids expressing miRNA or shPOT1 to prepare viruses. Human fibrosarcoma cells HTC75 were infected with virus to construct a stable transfected cell line. Rescue experiment: The full-length GFP or POT1 was cloned into pBabe-CMV-DEST-SFB, transfected into HEK293T cells, and the virus was prepared. The miR-185 overexpression cell line was infected with virus to construct a rescue (rescue) cell line. Cell lines were tested for protein expression by immunoblotting.
[0050] 2. Experimental results
[0051] attached Figure 5 In B, the expression of POT1 in eac...
Embodiment 2
[0052] Example 2 Western blot experiment
[0053] 1. Experimental materials
[0054] Reagents: POT1 endogenous antibody used in this experiment (purchased from Novus Biologicals, product number NB500-176), diluted 1:1000 with 3% bovine serum albumin (BSA) before use; GAPDH antibody (purchased from Abmart company, product number 3B3), diluted with 3% BSA at 1:5000; secondary antibody (goat anti-mouse, purchased from LI-COR Company, product number 926-68050, goat anti-rabbit, purchased from LI-COR Company, product number 926- 32211), diluted 1:10000 with 3% BSA when used.
[0055] Human fibrosarcoma cell HTC75 was preserved by the Songyangzhou Research Group of the School of Life Sciences, Sun Yat-sen University.
[0056] 2. Experimental method
[0057] S1. Preparation of cell lysate: culture according to conventional human tumor cell culture methods, collect cells, wash once with phosphate buffered saline (PBS); suspend in PBS, add 5X SDS loading buffer to lyse and boil.
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Embodiment 3
[0068] Example 3 Real-time fluorescent quantitative PCR experiment
[0069] 1. Experimental method
[0070] Total cellular RNA was extracted using TRIzol (Invitrogen, Cat. No. 15596-026), treated with DNase I (Invitrogen, Cat. No. 18068015) to remove residual DNA, and then reverse-transcribed into cDNA using TAKARA PrimeScript™ II1st Strand cDNA Synthesis Kit. Using SYBR Green (ABI, Cat. No. 4309155) as the dye, real-time fluorescent quantitative PCR experiments were performed. Set the PCR program: first step, 95°C for 10 minutes; second step: 95°C for 15 seconds, 60°C for 1 minute, 40 cycles. Melting curve program: Step 1, 95°C for 10 minutes; Step 2: 95°C for 10 seconds, 55°C for 10 seconds, each cycle increases 55°C by 0.5°C, and ends after rising to 95°C.
[0071] 2. Experimental results
[0072] The experimental results are attached image 3 A and Figure 5 As shown in A. attached image 3 A shows that in miR-185 overexpression cells, the mRNA level of POT1 is lowe...
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