Silencing of LncRNA PVT1 inhibits the proliferation, migration and fibrosis of high glucose-induced mouse mesangial cells via targeting microRNA-93-5p
Authors: Jianzhou Li
Jian Song
Qing Zhao
Xiaohong Jin
Yanhua Li
Journal: Bioscience reports (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: LncRNA-PVT1; PI3K/Akt/mTOR pathway; diabetic nephropathy; miR-93-5p; mouse mesangial cells.
Affiliations: Department of Endocrinology, Caoxian People's Hospital, East Qinghe Road, South Fumin Avenue, Caoxian Development Zone, Heze City 274400, Shandong Province, China., Department of Medical, First People's Hospital of Jinan City, No. 132, Daminghu Road, Lixia District, Jinan City 250011, Shandong Province, China., Department of Nephrology, Qilu Hospital of Shandong University, No. 107, Wenhua West Road, Jinan City 250012, Shandong Province, China.

Abstract:  Objective: The present study aimed to investigate the regulatory role of long non-coding RNA plasmacytoma variant translocation 1 (PVT1) on high glucose (HG)-induced mouse mesangial cells (MMCs). Methods: PVT1 expression in diabetic nephropathy (DN) mice and HG-induced MMCs was detected by qRT-PCR. EdU and Colony formation, Annexin V-PI staining, Muse cell cycle, Scratch, and Transwell assays were performed to detect the cell proliferation, apoptosis, cell cycle, migration, and invasion, respectively. The contents of fibrosis factors in cell-culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was performed to detect the expression of factors involved in apoptosis, cell cycle, migration and invasion, fibrosis, and PI3K/Akt/mTOR pathway. The targeting relation between miR-93-5p and PVT1 was predicted by StarBase3.0 (an online software for analyzing the targeting relationship) and identified by Dual-luciferase reporter (DLR) assay. Results: PVT1 was overexpressed in DN kidney tissues and HG-induced MMCs. HG-induced MMCs exhibited significantly increased EdU-positive cells, cell colonies, S and G2/M phase cells, migration and invasion ability, and contents of fibrosis factors, as well as significantly decreased apoptosis rate compared with NG-induced MMCs. HG significantly up-regulated Bcl-2, CyclinD1, CDK4, N-cadherin, vimentin, Col. IV, FN, TGF-β1 and PAI-1, and down-regulated Bax, cleaved caspase-3, cleaved PARP, and E-cadherin in MMCs. Silencing of PVT1 eliminated the effects of HG in MMCs and blocked PI3K/Akt/mTOR pathway. MiR-93-5p was a target of PVT1, which eliminated the effects of PVT1 on HG-induced MMCs. Conclusions: PVT1 silencing inhibited the proliferation, migration, invasion and fibrosis, promoted the apoptosis, and blocked PI3K/Akt/mTOR pathway in HG-induced MMCs via up-regulating miR-93-5p. 

Retracted: Yes

PubPeer comments:
 [And here is data for plasmid (including Cas9) integration ] - showing there has been DSBs. [Of course, biorxiv had to reject this ] Submitted to F1000,[ like this work on AAV+Cas9 integration in data from Massachusetts General Hospital] , which biorxiv had rejected. If you use plasmids (ie DNA) you will have transgenic humans. There is no workaround for that. , 
 [And if there is 0 citation of a paper saying 10 21 deaths (CRISPR PD0-1 Tcell) in 18 months (July 2018 - Nov 2019)] and subsequently [dont want to publish results from Dr Wu ] after eventually 20 21 people have died, [And Dr Wu doesnt want to work with CRISPR anymore ] what chance do I stand to publish the dangers of this hype. , 
 [And their data also shows that dCas9 still have low endonuclease activity left.] , 
 Won t the primer binding site at the 3 end of the pegRNA get sequestered by the spacer in the 5 end of pegRNA as they are complementary sequence? Why should we do so much optimisation on the pegRNA s primer binding site and donor template length to get one target working when a simple ODN can give good efficiency for small deletions, inserting and point mutation. Finally the author who made xCas9 (PAMless) agrees that Nickase is better than Cas9. , 
 And should you not retract a paper that says in the title  genome editing without double-strand breaks - when there is clear proof of double-strand breaks (DSB). Or give an explanation of the [plasmid integrations?] . How does that happen without DSB? We worry so much about image manipulations and duplications - why not this? , 
 This is now a [pre-print in OSF] - the sequences with [integrations can be found here] . , 
 NEED TO MENTION THAT THE ENZYME CUTTING SITE FOR pU6-GG-accept is wrong. IT SHOULD BE BsaI. ,