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

Inactive Publication Date: 2016-12-14
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no studies and reports on miRNAs in the regulatio

Method used

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  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent
  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent
  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent

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Experimental program
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Example Embodiment

[0047] Example 1 Construction of miR-185 overexpressing cells and rescue experiment

[0048] 1. Experimental method

[0049] Synthesize oligo primers containing the positive and negative sequences of shPOT1, anneal the two primers by gradient cooling, and connect the annealed double-stranded RNA containing the positive and negative sequences of shPOT1 into the pLKO vector. Transfect HEK293T cells with plasmids expressing miRNA or shPOT1 to prepare viruses. The human fibrosarcoma cell HTC75 was infected with the virus to construct a stable transfected cell line. Rescue experiment: clone the full-length GFP or POT1 into pBabe-CMV-DEST-SFB, transfect HEK293T cells to prepare virus. The miR-185 overexpression cell line was infected with the virus to construct a rescue cell line. Western blotting was performed on the cell line to detect protein expression.

[0050] 2. Experimental results

[0051] Attached Figure 5 In B, the expression of POT1 in each group indicates that the cell lin...

Example Embodiment

[0052] Example 2 Western blotting experiment

[0053] 1. Experimental materials

[0054] Reagents: The POT1 endogenous antibody used in this experiment (purchased from Novus Biologicals, product number NB500-176), diluted 1:1000 with 3% bovine serum albumin (BSA); GAPDH antibody (purchased from Abmart, product number 3B3), use 3% BSA diluted 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] The human fibrosarcoma cell HTC75 was preserved by Songyangzhou Research Group, 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.

[0058] The composition of the medi...

Example Embodiment

[0068] Example 3 Real-time fluorescent quantitative PCR experiment

[0069] 1. Experimental method

[0070] Use TRIzol (Invitrogen, article number 15596-026) to extract total cellular RNA, treat the sample with DNase I (Invitrogen, article number 18068015) to remove residual DNA, and then use TAKARA PrimeScript™ II1st Strand cDNA Synthesis Kit to reverse transcribed into cDNA. Use SYBR Green (ABI, article number 4309155) as the dye to perform real-time fluorescent quantitative PCR experiments. Set up the PCR program: the first step, 95°C for 10 minutes; the second step: 95°C for 15 seconds, 60°C for 1 minute, 40 cycles. Dissolution curve program: the first step, 95°C for 10 minutes; the second step: 95°C for 10 seconds, 55°C for 10 seconds, 55°C is increased by 0.5°C per cycle, and it ends when it reaches 95°C.

[0071] 2. Experimental results

[0072] The experimental results are attached image 3 A and Figure 5 Shown in A. Attached image 3 A shows that in miR-185 overexpressing...

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Abstract

The invention provides application of human microRNAs miR-185 to the preparation of a cell growth and/or senescence regulation agent. The miR-185 can directly act in a 3'-untranslated region (3'-UTR) of a messenger RNA (mRNA) of human telomere POT1, then mRNA and protein expression of POT1 can be inhibited, telomere damage and telomere length disorder can be caused, cell aging can be accelerated, and cell growth velocity can be inhibited. Therefore, miR-185 has significant regulation functions in the cell growth and aging process, effective cell growth and aging regulation agents can be developed for miR-185, for example, an anti-aging medicine can be developed and used in the cell aging delaying process, a tumor growth inhibitor can be developed for inhibiting growth of tumor cells, and the medicines can be used in anticancer treatment and all have great significance.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of human microRNAs (microRNAs) miR-185 in the preparation of cell growth and aging regulators. Background technique [0002] Telomere is a highly ordered special structure composed of double-stranded TTAGGG repeat sequence and single-stranded 3'overhang composed of DNA sequence and its interacting proteins at the end of eukaryotic chromosome. It can protect chromosome ends, maintain genome stability and participate in biological processes such as cell aging regulation. Every time a normal human cell divides, the length of the telomere will gradually shorten due to the problem of terminal replication. When the telomere shortens to a certain limit, the cell will not be able to continue to divide and enter the process of aging and death. Telomere-binding protein is a special protein complex that specifically binds to telomeres. It has the functions ...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/7088A61P35/00A61P39/00
CPCA61K45/00A61K31/7088
Inventor 松阳洲黄燕黄军就李婷婷
Owner SUN YAT SEN UNIV
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