Application of long non-coding RNA Ovol2-AS

Abstract

The invention relates to the fields of physiology and biomedicine, in particular to a long-chain non-coding RNA (lncRNA) for regulating the proliferation and function of pancreatic beta cells and its application. A pregnant mouse model was established, islets of pregnant and non-pregnant mice were isolated to detect the differential expression levels of lncRNA, and a new lncRNA Ovol2‑AS that regulates the proliferation and function of islet β cells was screened and discovered. Overexpression of Ovol2‑AS in the primary disaggregated mouse islet cells and the mouse islet β cell line Min6 can significantly promote the proliferation of β cells and inhibit cell apoptosis. The long-chain non-coding RNA Ovol2‑AS can be used for inhibiting the apoptosis of islet cells and promoting the proliferation of islet cells; it can also be used for preparing or screening drugs for treating diabetes.

Description

Technical field

[0001] The invention relates to the fields of physiology and biomedicine, in particular to long-chain non-coding RNA (lncRNA) for regulating the proliferation and function of pancreatic β cells and its application. Background technique

[0002] Diabetes mellitus is a metabolic disease with multiple etiologies, characterized by chronic hyperglycemia, accompanied by disorders of sugar, fat and protein metabolism caused by defects in insulin secretion and / or action. In the development of diabetes, the number and function of pancreatic β-cells play an important role in its clinical process. In recent years, the incidence of diabetes in the world, especially in China, has increased rapidly. In 2010, my country conducted a long-term follow-up survey of nearly 100,000 people. The results showed that in the samples of adults aged 18 and above in my country, the diagnosis was made according to the latest international clinical diagnostic standards. The estimated prevalence...

Examples

Embodiment 1

[0030] 1. Main experimental materials: primary islet cells of pregnant mice and control group

[0031] Main reagents: HBSS (10×), (1×) (Invitrogen); Collagenase XI (Sigma); Trizol lysate (Invitrogen).

[0032] 2. Experimental method

[0033] (1) Establish a pregnant mouse model

[0034] Purchase SPF-grade C57BL / 6 female mice and male mice, and keep them in SPF-grade animal room: rearing in separate cages (4 mice per cage), and the mice can eat and drink freely. The environmental temperature of the animal room is 21±1 degrees, the humidity is 55±10%, and the circadian rhythm is 12-12 hours. Part of the female mice served as the control group, and the other part of the female mice and the male mice were caged at a ratio of 2:1. The mice at 14.5 days of gestation were taken. The pregnant mice were again confirmed pregnancy through embryo phenotype observation when the islets were isolated. time.

[0035] (2) Isolating islets from the control group and C57BL / 6 female mice at 14.5 days of...

Embodiment 2

[0064] Example 2 Ovol2-AS (AK007144) regulates the proliferation and apoptosis of pancreatic β-cells

[0065] Further in vitro experiments were conducted to study the effect of Ovol2-AS on the proliferation and apoptosis of pancreatic β-cells. Ovol2-AS plasmid and lentivirus-packaged Ovol2-AS were overexpressed in the mouse Min6 cell line and isolated and cultured primary pancreatic islets, and the cell proliferation was detected by CCK-8, Edu, ki67 and other methods, and applied AnnexinV / 7-AAD kit and TUNEL kit were used to detect cell apoptosis. In situ and flow cytometry were used to analyze whether transfection of Ovol2-AS would affect the proliferation and apoptosis of mouse pancreatic β-cells.

[0066] (1) Construction of Ovol2-AS expression vector

[0067] (1) Obtain the full-length fragment of Ovol2-AS According to the full-length fragment sequence of Ovol2-AS on NCBI, design primers and select restriction sites, the 5'end restriction site is HindⅢ, and the 3'end restriction...

Technical field

[0001] The invention relates to the fields of physiology and biomedicine, in particular to long-chain non-coding RNA (lncRNA) for regulating the proliferation and function of pancreatic β cells and its application. Background technique

[0002] Diabetes mellitus is a metabolic disease with multiple etiologies, characterized by chronic hyperglycemia, accompanied by disorders of sugar, fat and protein metabolism caused by defects in insulin secretion and / or action. In the development of diabetes, the number and function of pancreatic β-cells play an important role in its clinical process. In recent years, the incidence of diabetes in the world, especially in China, has increased rapidly. In 2010, my country conducted a long-term follow-up survey of nearly 100,000 people. The results showed that in the samples of adults aged 18 and above in my country, the diagnosis was made according to the latest international clinical diagnostic standards. The estimated prevalence...

Examples

Embodiment 1

[0030] 1. Main experimental materials: primary islet cells of pregnant mice and control group

[0031] Main reagents: HBSS (10×), (1×) (Invitrogen); Collagenase XI (Sigma); Trizol lysate (Invitrogen).

[0032] 2. Experimental method

[0033] (1) Establish a pregnant mouse model

[0034] Purchase SPF-grade C57BL / 6 female mice and male mice, and keep them in SPF-grade animal room: rearing in separate cages (4 mice per cage), and the mice can eat and drink freely. The environmental temperature of the animal room is 21±1 degrees, the humidity is 55±10%, and the circadian rhythm is 12-12 hours. Part of the female mice served as the control group, and the other part of the female mice and the male mice were caged at a ratio of 2:1. The mice at 14.5 days of gestation were taken. The pregnant mice were again confirmed pregnancy through embryo phenotype observation when the islets were isolated. time.

[0035] (2) Isolating islets from the control group and C57BL / 6 female mice at 14.5 days of...

Embodiment 2

[0064] Example 2 Ovol2-AS (AK007144) regulates the proliferation and apoptosis of pancreatic β-cells

[0065] Further in vitro experiments were conducted to study the effect of Ovol2-AS on the proliferation and apoptosis of pancreatic β-cells. Ovol2-AS plasmid and lentivirus-packaged Ovol2-AS were overexpressed in the mouse Min6 cell line and isolated and cultured primary pancreatic islets, and the cell proliferation was detected by CCK-8, Edu, ki67 and other methods, and applied AnnexinV / 7-AAD kit and TUNEL kit were used to detect cell apoptosis. In situ and flow cytometry were used to analyze whether transfection of Ovol2-AS would affect the proliferation and apoptosis of mouse pancreatic β-cells.

[0066] (1) Construction of Ovol2-AS expression vector

[0067] (1) Obtain the full-length fragment of Ovol2-AS According to the full-length fragment sequence of Ovol2-AS on NCBI, design primers and select restriction sites, the 5'end restriction site is HindⅢ, and the 3'end restriction...

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