【原】shRNA稳转细胞株传代后原本敲低的蛋白水平恢复，为什么？怎么办？

昵称71917755 2024-10-21 发布于云南

展开全文

常记溪亭日暮，沉醉不知归路。

公众号：华佗的小斧头

Nature杂志：2023年，超过1万篇SCI高级论文撤搞。

网址：https://pubmed.ncbi.nlm./38087103/

中国作者撤稿占到所有SCI论文撤稿的49.86%！

中国撤稿率23.5%。

前言

无言！

很多人在做RNA干扰的时候，都会遇到一个奇葩的问题：通过慢病毒转染shRNA，基因敲低了，然而传代几次之后，或者把之前冻存的稳转细胞株复苏后，再次检测，发现原本敲低的蛋白质水平，又回到从前，为什么？

终于查到一篇这方面的正式的文献（并非来自论坛留言区了）。

Li W, Shen M, Jiang YZ, et al. Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2. Genes Dev. 2020;34(19-20):1310-1315. doi:10.1101/gad.339804.120

全文：

https://genesdev./content/34/19-20/1310.full.pdf+html

USP20 knockdown inhibits lung colonization by breast cancer cells

USP20敲低抑制乳腺癌细胞肺转移

Given the important role of SNAI2 in metastasis, we next investigated the effect of USP20 on lung metastasis of breast cancer cells.

鉴于SNAI2基因在转移中的重要作用，我们接下来研究了USP20基因对乳腺癌细胞肺转移的影响。

First, we generated cell lines with stable USP20 knockdown using lentiviruses containing USP20-targeting shRNAs.The protein level of SNAI2 was decreased when the cell lines were first generated.

首先，我们使用含有靶向USP20 shRNAs的慢病毒构建了具有稳定USP20敲低的细胞系。当细胞系首次构建时，SNAI2的蛋白质水平降低。

However, the SNAI2 protein level in USP20 knockdown cells recovered after several passages, possibly because SNAI2 is essential for LM2 cells and the cells developed compensating mechanisms to regain SNAI2 expression over time.

然而，USP20敲除细胞株中的SNAI2蛋白水平在几次传代后恢复，这可能是因为SNAI2对LM2细胞（人高转移性乳腺癌细胞系）至关重要，并且随着时间的推移，细胞出现了补偿机制来恢复SNAI2的表达。

Thus, we took an alternative approach of knocking down USP20 using two different siRNAs, and the siRNA knockdown effect was confirmed to last for at least 10 d, which is sufficient for in vivo experimental lung colonization studies (Supplemental Fig. S3A,B).

因此，我们采用了另一种方法，即使用两种不同的siRNA敲除USP20，并证实siRNA敲除效果持续至少10天，这足以进行体内实验性肺转移研究（补充图S3A、B）。

Firefly luciferase-labeled siRNA transfected SUM159-M1a and LM2 cells were injected intravenously into NSG mice, and bioluminescent imaging (BLI) was used to monitor their metastatic seeding and growth in the lung.

将萤火虫荧光素酶标记siRNA转染的SUM159-M1a和LM2细胞通过静脉注射到NSG小鼠体内，并使用生物发光成像（BLI）监测其在肺部的转移接种和生长。

Five days after injection, there was already a significantly lower number of cancer cells seeded in the lung for USP20 or SNAI2 knockdown cells compared with the control cells (Fig. 4A; Supplemental Fig. S4A).

注射后五天，与对照细胞相比，在肺中接种USP20或SNAI2敲低细胞的癌症细胞数量已经显著减少（图4A；补充图S4A）。

This effect was maintained for at least 3 wk for both siRNAs of USP20 after injection of LM2 cells, and then the two siRNAs of USP20 diverged at week 4, possibly due to different knockdown efficiencies in vivo (Fig. 4B).

注射LM2细胞后，USP20的两种siRNA的这种效果至少持续了3周，然后USP20的这两种siRNA在第4周出现分化，这可能是由于体内敲除效率不同造成的（图4B）。

Mice were sacrificed 4 wk later and metastasis nodules on the lungs were counted. Significantly less lung metastasis nodules were observed for USP20 and SNAI2 knockdown groups (Fig. 4C,D), which could result from both reduced lung seeding as well as a modest decrease in proliferation (Supplemental Fig. S4B).

4周后处死小鼠，计数肺部转移结节。USP20和SNAI2敲除组的肺转移结节明显减少（图4C，D），这可能是由于肺接种减少和增殖适度减少造成的（补充图S4B）。