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Bomber - Bob Flash Game
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Bomber - Bob was added on:
Aug 24, 2008
Bomber - Bob has been played 3,279 times
Bomber - Bob Description
Flight a bomber against enemy aircraft and sunk ships.
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Find here the Air Canada delayed flights which have a right to compensation
When your Air Canada flight was delayed or cancelled, you could be entitled to compensation. In our 'problem list' below you find the flights of which passengers are entitled to this. We review over 13 million flight, weather and sociopolitical data every day. This data is one of the reasons we have a 97% success rate in court.
With Air Canada to North America
Canada's biggest Airline operates flights throughout the world. Connecting North America to the rest of the globe. Air Canada connects the United Kingdom to Canada on a Daily bases from a range of different airports. Sometimes this results in flight delays and/or cancellations.
Recently delayed flight Air Canada
AC 5216Wed 11 December 2019
Heathrow Airport (LHR)
1800 hourNewark Liberty Intl. Airport (EWR)
2125 hour
AC 9944Wed 11 December 2019
Oslo Airport (OSL)
1500 hourHeathrow Airport (LHR)
1625 hour
AC 6363Wed 11 December 2019
Toulouse Blagnac Airport (TLS)
0700 hourBrussels Airport (BRU)
0845 hour
AC 9901Wed 11 December 2019
Düsseldorf Airport (DUS)
0605 hourCopenhagen Airport (CPH)
0725 hour
AC 6388Tue 10 December 2019
Heathrow Airport (LHR)
1300 hourAhmedabad (AMD)
0240 hour
AC 5659Tue 10 December 2019
Munich Airport (MUC)
1155 hourSan Francisco Intl. Airport (SFO)
1500 hour
AC 5685Tue 10 December 2019
Frankfurt Intl. Airport (FRA)
1125 hourNewark Liberty Intl. Airport (EWR)
1415 hour
AC 5983Tue 10 December 2019
Frankfurt Intl. Airport (FRA)
0835 hourO'Hare Intl. Airport (ORD)
1100 hour
AC 6377Tue 10 December 2019
Oslo Airport (OSL)
0640 hourBrussels Airport (BRU)
0850 hour
AC 6970Mon 9 December 2019
Heathrow Airport (LHR)
1445 hourHamburg Airport (HAM)
1735 hour
Can't find your flight ?
Does your delayed flight not appear on the list below? Check your flight and within a few minutes you will know if you are entitled to a compensation.

subset: commoncrawl


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Search homes for sale in Palm Desert, La Quinta, & Indio, CA. Search all Real Estate in the Coachella Valley here.
Ronald Christopher & Associates is a boutique Real Estate & Mortgage office that was established in 2013, Veteran owned & operated by Ron & Tina Saporito. Meeting within the business many years ago, Ron & Tina discovered they both had the same drive and passion for the real estate & mortgage industry but wanted to offer their clients a more enhanced, personalized experience. Together, the two of them put their heart and soul into their careers and offer their clients a more personalized, professional, and less stressful transaction.
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How to Buy a House - Basic First Timer Tips
I would definitely recommend Tina as a realtor! This is my first family home purchase, she answered all my questions promptly and when I felt nervous she put me at ease by responding everything I wanted to know. Very very happy and pleased with our first home Tina and her husband made sure we found a home that fulfilled our needs and expectations.
-Esperanza Vega, Client
We are so fortunate to have found Ron and Tina Saporito when we were looking at properties in Palm Desert. Not only did they help us find our dream home, they helped sell our previous home as well. They are knowledgeable, hardworking, and happy to assist in any way possible. They made our buying and selling process amazing and I would highly recommend them to anyone with …
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"Tina was awesome. Absolutely listened to what we wanted in a new home. She was involved with every detail through out the escrow process as well. Emails and telephone messages were answered promptly. Would recommend Tina and will use her in the future as well."
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Ron is a true professional. He knows the market, how to advertise and follows through on his promises. He sold two of my property investments and when another opportunity to sell arises, he is my man. Ron goes above and beyond what is expected.
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Tina was great to work with start to finish, providing steady calm guidance throughout our purchase. She was quick to respond whenever we contacted her and worked hard to keep escrow on track. She is a realtor who will help and not pressure. We're glad we found her!
-Peter Chadwick, Client


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EU ITN granted to MANIC
06 December 2019
Materials NL Challenges grant awarded to Professor Loi
05 December 2019
Materials NL Challenges grant for Dr. Maresca
05 December 2019
Professor Katja Loos wins Research Award of the Alexander von Humboldt Foundation
29 November 2019
Red deer hardly react to wolf urine in the Netherlands and Poland
28 November 2019
Master's student Remi Brandt wins KNVI Thesis Prize for Computing Science and Engineering
26 November 2019
NWO awards € 4 million to DIGITAL TWIN programme
07 November 2019
Astronomers map new emission line to trace most common molecule in the Universe
07 November 2019
NWO ENW-KLEIN grant of EUR 304,000 for Prof. Fraternali
05 November 2019
Prof. Faraji elected to Q-Chem board of directors
05 November 2019
Ramping up to divide: An unstable protein is master switch for cell division
04 November 2019
Migratory birds are worse off in West Africa
29 October 2019
Evolution is resetting the annual clock in migratory birds
24 October 2019
Zoology 2019: International meeting on Zoology and Behavioral Biology in Groningen
23 October 2019
Demonstrating that it can be done
14 October 2019
Down Under with Top Dutch (update)
11 October 2019
Kick-off Young Science and Engineering Network (YSEN)
11 October 2019
Lecture by Nobel Laureate Christopher Pissarides
03 October 2019
Amina Helmi presents plans for Spinoza prize
02 October 2019
Prof. Daniël Boer elected Fellow of American Physical Society
26 September 2019
UMCG and UG aim to functionally accommodate particle accelerator at UMCG
26 September 2019
Start of MOSAiC – the Greatest Arctic Research Expedition of All Time
20 September 2019
Imagining Science
20 September 2019
New microscope shows the atomic structure of materials in stunning detail
18 September 2019
When the wind blows from the wrong direction
17 September 2019
Getting to the roots of Parkinson's disease
17 September 2019
1.5 million euros for imaging functionality of nanomaterials
04 September 2019
1.5 million euros for computational modeling from super-fast MRI
03 September 2019
Gravitation Grant for designing Hybrid Intelligence
30 August 2019
University of Groningen in two of the six Gravitation grants for groundbreaking research
30 August 2019
Data Science: a breath of fresh air for physics, genetics and history
19 August 2019
Cold winters not caused by Arctic climate change
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Amina Helmi appointed as Deputy Scientific Director NOVA
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NWO START-UP Grant Marcos Guimarães
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Closing the door: breaking new ground related to a potential anticancer drug target
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The University of Noorderzon
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Wageningen and Groningen scientists research nature-inclusive agriculture in Northern Netherlands
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Thirteen Veni grants for young Groningen researchers
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11 July 2019
Spinoza Prize 2019 for astronomer Amina Helmi
21 June 2019
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61 million euros for social and scientific breakthroughs
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12 June 2019
Pastoor Schmeitsprijs voor de Sterrenkunde voor dr. Selma de Mink
27 May 2019
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27 May 2019
Three UG researchers win Vidi grants
24 May 2019
The North of the Netherlands goes down under during Solar Challenge
22 May 2019
"Worms man" Dr. Jeroen Onrust wins Science Prize Campus Fryslân 2019
17 May 2019
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From paperclip to patent
23 April 2019
Why lightning often strikes twice
17 April 2019
Still going strong after four decades
16 April 2019
EU awards COFUND grant for 18 Post-docs working on the Origin and Evolution of Life
03 April 2019
Staatssecretaris Keijzer bezoekt RUG-robots op Hannover Messe
02 April 2019
Royal Decoration for Prof. Jeff De Hosson
29 March 2019
Introducing..... Sahar El Aidy
25 March 2019
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25 March 2019
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Molecular motors run in unison in a metal-organic framework
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Amina Helmi to receive Suffrage Science award
08 March 2019
First Waddenplastic.nl research outcomes
08 March 2019
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08 March 2019
Amina Helmi to receive Suffrage Science award
08 March 2019
Wadden Mosaic project starts underwater sampling
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The unusual heredity of telomere length
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Maarten Loonen's climate change grief
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Faculty of Science and Engineering wins NNV Diversity Award 2018
22 January 2019
Two young researchers head to top institutes with Rubicon grant
16 January 2019
Smoking gun
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Yeast makes ethanol to prevent metabolic overload
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Toekenning ECHO subsidie aan Wouter Roos
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18 December 2018
Pollution poses threat to Arctic goose
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NTU Singapore and UG sign Double Doctorate agreement
30 November 2018
Suspicious genes, fast legs
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Anouk Goossens receives Shell Award for researching building blocks of the electronic brain
26 November 2018
Large Wadden Fund grant for researching underwater nature in Wadden Sea
23 November 2018
Support from the Government for the development of sustainable, nature-inclusive agriculture in the Northern Netherlands
16 November 2018
The lift between life and death
14 November 2018
Honorary professor Van Roon awarded Senior Jan Glerum Education Prize
09 November 2018
Celebrating Excellence in Research: 100 Women of Chemistry
08 November 2018
Grants awarded call Data Science 2018
06 November 2018
TOP NWO grant for Prof. Slotboom
05 November 2018
Astronomers discover the giant that shaped the early days of our Milky Way
31 October 2018
Eric de Vries wins NWO Physics Valorisation Chapter Prize
30 October 2018
New generation of smart computers inspired by our brains
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Photo report: the Ocean Grazer
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Temperate Wadden Sea helps bar-tailed godwit adapt to climate change in the Arctic
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Opening Symposium of Bernoulli Institute for Mathematics, Computer Science and Artificial Intelligence on 1 Nov
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4 Million Euro for EU-funded MARIE CURIE INNOVATIVE TRAINING NETWORK
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Gosens wins the Prix Galien Research Award 2018
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Young Academy Groningen welcomes eight new members
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Zpannend Zernike: Backstage in the wondrous world of science and technology
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Heike Kamerlingh Onnes chair and Rudi Drent chair for Groningen professors
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Small galaxy caused 'snail shell' structure in our part of the Milky Way
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UG Students help young students with logistics
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Thousands of deaths prevented by vaccinations
21 August 2018
University of Groningen and MEYER WERFT sign collaboration agreement
25 July 2018
Nature has the answer
24 July 2018
Three young researchers to top institutes abroad on Rubicon grants
23 July 2018
Super-telescope bring astronomers closer to understanding dark matter
18 July 2018
Dr Marthe Walvoort wins Gratama Prize
06 July 2018
Feringa honoured with bronze bust and book
08 June 2018
Nine UG researchers win Vidi grants
01 June 2018
New all round research institute for mathematics, computer science and artificial intelligence: Bernoulli Institute
01 June 2018
SRON in finals with successor to Herschel satellite
30 May 2018
Why we need algae
16 May 2018
Nanoscience student Eleveld wins audience award national Famelab
14 May 2018
Petra Rudolf next EPS President-elect
09 May 2018
Sijbren Otto wins prestigious Royal Society of Chemistry Award
08 May 2018
Photo report: in Oscar Kuipers' lab
03 May 2018
Professor Maria Antonietta Loi receives Physics Prize 2018
13 April 2018
UG researchers roll up their sleeves on NICO Expedition to the Atlantic Ocean
04 April 2018
Spotlight on engineering in Groningen
21 March 2018
Drier soil leads to more extreme heat waves
19 March 2018
'ZAP biobased research will be changing society'
13 March 2018
Practical spin wave transistor one step closer
02 March 2018
Princess Beatrix and Princess Mabel to attend 4th Prince Friso Engineering Award ceremony in Groningen
19 February 2018
Artificial intelligence selects best candidate
30 January 2018
ERC grants help three UG scientists head to the top
28 November 2017
UG and Northern Online Entrepreneurs launch Groningen Digital Business Centre
25 November 2017
Birds without own brood help other birds with parenting, but not selflessly
23 October 2017
Daydreams in a computer model
02 October 2017
Five Veni grants of 250,000 euros
28 July 2017
Van Leersum grant for international comparative study of medicine use in children with ADHD
24 July 2017
Lung Foundation supports research on lung tissue repair
20 July 2017
Mudflats are the solar panels of the Wadden Sea
05 July 2017
Countering the effects of sleep deprivation by resetting the molecular clock?
05 July 2017
Astronomers see mysterious nitrogen area in a butterfly-shaped star formation disk
15 June 2017
Two Vidi grants awarded worth €800,000
30 May 2017
Award for outstanding PhD abroad granted to He
18 May 2017
Ben Feringa delivers Nobel lecture in De Oosterpoort
04 April 2017
Hightech onderneming DEMCON opent vestiging op Zernike Campus
14 March 2017
Researchers create a high-performance, low-energy artificial synapse for neural network computing
24 February 2017
Marie Sklodowska Curie COFUND grant for DSSC!
13 February 2017
Prize for Fraaije's video on enzyme research
03 February 2017
Efficient demand and supply matching by incentivizing end-users in buildings (MatchIT)
01 February 2017
Two young UG science talents will go abroad with NWO Rubicon grants
24 January 2017
How carbohydrates make infant formula more like human breast milk
18 January 2017
Joint knowledge helps to control conflicts in the Wadden Sea region
11 January 2017
DTEC grant awarded for Visual Storytelling of Big Imaging Data
21 December 2016
University of Groningen strengthens technological profile with new centre
21 October 2016
Veni grants awarded
15 July 2016
European Control Award for Ming Cao
30 June 2016
PhD Rozema Dutch candidate for ECCOMAS award
14 April 2016
PhD student creates recyclable rubber (video)
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New technique reveals causes of ageing in yeast
01 December 2015
Better steel thanks to nanotechnology
17 November 2015
Saving energy and money with smart systems
05 October 2015
Follow the expedition!
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Darwin's finches have reached their limits on the Galápagos
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Sustainable Buildings spin-off wins Pioneers prize
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Unifocus video: Debunking the myths of left and right-handedness
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Astronomers use MUSE to see into the deep Universe like never before
26 February 2015
Virtual spring keeps robots in formation
16 February 2015
University of Groningen Nerdalize start-up receives Ministerial funding
14 January 2015
Three young researchers to top institutes abroad on Rubicon grants
13 January 2015
Mow even later to save the godwit
06 January 2015
Ocean Grazer – Catching waves
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ERC Starting Grant for top researchers Thorben Cordes, Lude Franke and Mladen Popović
17 December 2014
Has car manufacturer taken the corner too fast with the boxfish design?
17 December 2014
Prof. Bart van Wees elected APS Fellow
11 December 2014
Prestigious ERC Starting Grant for Mladen Popović
11 December 2014
Gronings succes van E.coli met koperbindend 21e aminozuur
05 November 2014
1,5 million Euro for effect exercising on Alzheimer
03 November 2014
Unilever Research Prize for Top Master student Machteld Kamminga
30 October 2014
Colloquium Mathematics, Professor Rafael Ortega
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'FOM Projectruimte' for Prof. Bart van Wees
27 June 2014
Robotic fish, cruise control and Johann Bernoulli
24 June 2014
ZonMw Parel voor toxicoloog Geny Groothuis
23 June 2014
Spectacular inauguration of the Infoversum
19 June 2014
Funding for 'Playing with Urban Complexity'
11 June 2014
University of Groningen offers resit training on 14 and 15 June
11 June 2014
Theunis Piersma awarded Spinoza Prize
06 June 2014
Research on therapy for loved ones of missing persons
05 June 2014
National Student Survey results: University of Groningen student satisfaction grows
04 June 2014
Order of the Star of Italy for Prof. Morganti
03 June 2014
Production of radioactive isotopes for medical application
02 June 2014
Culture and Academia reign supreme this weekend in Groningen
21 May 2014
Colloquium Computer Science, Professor Roberto Battiti, University of Trento
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Nieuwe prijs voor jong talent in de sterrenkunde
15 May 2014
The ultimate microscope: a computer
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Canceled!!!Colloquium Computer Science, Professor Jan Sijbers (University Antwerpen)
13 May 2014
Sweet surprise for scientists
06 May 2014
Ambassador of Mongolia to deliver keynote speech at CEASG Launch Conference
01 May 2014
Large family shortens lives of jackdaw parents
14 April 2014
Inschrijving examentraining VWO en HAVO loopt storm
13 March 2014
Professors and porters go 'on the road' together to visit schools
13 March 2014
More micro-grids expertise for Groningen
07 March 2014
Hedy d'Ancona prepares the way for international Aletta Jacobs prize giving ceremony
27 February 2014
Groningen Centre of Energy Law awarded two NWO projects for research on Smart Grids
12 February 2014
Study shows independent association between diabetes and depression and impulse control disorders including binge-eating and bulimia
31 January 2014
Groningen economists predict medal table for Sochi: 6 gold medals for the Netherlands
24 January 2014
Professor Renata Kallosh
01 July 2013
ERC Advanced Grant for astronomer Rafaella Morganti
17 October 2012
Innovative inhaler: the Novolizer
18 April 2010
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2017 Jeep Compass Latitude
26,961 miles / $14,982
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This is a complete list of all the current tutorials on ScreenCastsOnline
Date: 4th October 2019
Duration: 27:05
Show ref: SCOM0879
There are so many new features in iOS and iPadOS 13 that we needed a second show to dive deeper and show you more of the essentials.
In this episode, Rosemary shows you some of the new features in core applications, including operating system changes, to allow us to use our devices more effectively and efficiently with all that iOS 13 and iPadOS 13 has to offer.
Date: 1st October 2019
Duration: 06:52
Show ref: SCOM0878
In this tip video, Lee shows off Picsew, a fantastic image editor for iOS that focuses on stitching together multiple images, either from the camera, or using screenshots.
Using Picsew, you can also crop images, add annotations and watermarks, capture images from a web browser, and much more.
Date: 27th September 2019
Duration: 35:33
Show ref: SCOM0877
In this tutorial, Lee walks you through a visually stunning calendar application on iOS called Timepage, by Moleskine. Whilst focusing on looking amazing, it doesn't miss out on functionality and allows you to work with contacts, maps and even the weather, all from a single interface.
In this episode, you learn about navigating the interface, using the HeatMap view, and adding and editing events. Lee dives a bit deeper to reveal Timepage's Smart Alerts, how to configure notifications, as well as its Siri Shortcuts integration.
Date: 24th September 2019
Duration: 05:33
Show ref: SCOM0876
A very neat menu bar app that gives you direct access to a host of utilities and system functions.
In this tip video, Don explores some of the options and demonstrates just how useful One Switch can be.
Date: 19th September 2019
Duration: 38:45
Show ref: SCOM0875
For the first time, the iPhone and the iPad each have their own version of iOS: iOS 13 and the brand new iPadOS.
In this episode, Don takes a first look at these operating systems and highlights some of the new and changed features across both devices.
In the upcoming months, we will take a look at many more new features for both iOS and iPadOS
Date: 17th September 2019
Duration: 12:37
Show ref: SCOM0874
In this video, Lee walks you through Mosaic, a fully customisable window management utility for macOS. This useful app allows you to keep your desktop tidy and efficient, while keeping everything you need to get your work done easily accessible.
Date: 13th September 2019
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BioMed Research International
Volume 2019, Article ID 9287804, 13 pages
The Effect of MicroRNA-331-3p on Preadipocytes Proliferation and Differentiation and Fatty Acid Accumulation in Laiwu Pigs
1College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271018, Shandong, China
2Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai'an 271018, Shandong, China
3Weifang University of Science and Technology, Weifang 261000, Shandong, China
Correspondence should be addressed to Yongqing Zeng; nc.ude.uads@gnezqy and Wei Chen; nc.ude.uads@nehcw
Received 12 June 2019; Revised 3 October 2019; Accepted 14 November 2019; Published 4 December 2019
Academic Editor: Rosario Caltabiano
Copyright © 2019 Tao Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective. The proliferation and differentiation of preadipocytes are regulated by microRNAs (miRNAs), hormones, and other factors. This study aimed to investigate the effects of miR-331-3p on the proliferation and differentiation of preadipocytes in addition to fatty acid metabolism. Methods. Preadipocytes were transfected with miR-331-3p mimics, miR-NC, or miR-331-3p inhibitor to explore its effect on cell proliferation and fatty acid accumulation. Furthermore, preadipocytes were transfected with pre-miR-331-3p, pcDNA3.1(+), or miR-331-3p inhibitor to explore its effect on differentiation. Results. It was observed that miR-331-3p could inhibit preadipocytes proliferation. Furthermore, miR-331-3p was highly expressed during cellular differentiation and appeared to promote the process. In addition, dual fluorescein analysis showed that dihydrolipoamide S-succinyltransferase (DLST) is a target gene of miR-331-3p, and overexpression of miR-331-3p could regulate the metabolism of fatty acids in the citrate pyruvate cycle by targeting DLST expression. Conclusion. In summary, these findings indicated that miR-331-3p exerts contrasting effects on the processes of fat deposition.
1. Introduction
The Laiwu pig is a Chinese breed with many characteristics that make it favorable for the commercial market. The meat has a high level of intramuscular fat, averaging approximately 10.32%. This is significantly higher than Yorkshire pigs (2% intramuscular fat) as well as many others Chinese breeds that have around 5% intramuscular fat on average. Although some studies have been carried out on the fat deposition of Laiwu pigs [1–3], the actual mechanism remains elusive.
The amount of fat deposited in an animal depends on body's balance of synthesis and catabolism rates. Recent studies have shown that animal fat deposition is the result of not only an increase in the number of fat cells but also an increase in the volume and accumulation of lipid droplets. Factors affecting the differentiation and growth of fat cells may also affect the formation of fat. Preadipocytes, which have the ability to proliferate and differentiate into adipocytes in vivo, have become an important model helping to complete the current understanding of adipose tissue formation and proliferation. It has been reported that the proliferation, differentiation, and fat deposition of preadipocytes are regulated by various factors [4].
In recent years, studies have shown that miRNAs are involved in the regulation of many biological processes, such as cell proliferation and differentiation, biological metabolism, and adipogenesis [5–7]. MicroRNAs mainly interact with the 3′-UTR of the target gene, which often leads to degradation of the target gene mRNA or inhibition of translation. In other words, they exert posttranscriptional regulation of target genes. Many miRNAs play an important role in the regulation of fat formation [8, 9]. For example, miR-143 promotes adipogenesis by acting on the target gene ERK5 [10, 11], miR-21 promotes adipogenesis by acting on the target genes TGFBR2 and STAT3 [12, 13], and miR-519d promotes fat deposition by acting on the target gene PPARα [14]. It has also been demonstrated that both miR-27 and miR-130 inhibit adipogenesis by acting on the target gene PPARγ [15–17]. Another miRNA that inhibits adipogenesis is miR-224, which exerts the effect by acting on the EGR2 gene [18]. However, there are still many mechanisms of miRNA action, and the role of miRNAs in adipocyte proliferation, differentiation, and lipid metabolism requires further research. With the development of high-throughput sequencing technology, an increasing number of miRNAs have been discovered to be involved in the fat metabolism pathway, thus promoting the study of miRNAs and their target genes involved in adipose tissue function. Xie et al. used the Illumina sequencing technology to analyze differential expression of miRNAs in the livers of Tongcheng and Yorkshire pigs [19]. They were able to identify 58 differentially expressed miRNAs. Furthermore, high-throughput sequencing was employed to analyze subcutaneous fat of 7- and 240-day-old Rongchang pigs. A total of 93 upregulated and 33 downregulated miRNAs were discovered at 240 days of age [20]. Similarly, Chen et al. also identified 9 differentially expressed miRNAs in Meishan pig back fat, indicating that miRNAs may regulate fat deposition in pigs [21].
Currently, numerous studies have reported that miR-331-3p plays an important role in the proliferation and differentiation of cancer cells, as well as the occurrence and development of cancer [22–24]. Furthermore, miR-331-3p is able to directly target the cell cycle-associated gene, E2F1. Upregulation of miR-331-3p has the ability to inhibit the growth and clonal formation of gastric cancer cells [25]. The expression of miR-331-3p in prostatic tissue is low. Transfection of miR-331-3p can inhibit the expression of ERBB-2 gene and inhibit the downstream PI3/Akt sex hormone receptor signaling pathway. It can inhibit the expression of prostate-specific antigen (PSA) by reducing the activity of androgen to stimulate the promoter of PSA. It is also found that the expression of miR-331-3p may be specific in the nervous system [26–28]. In addition, miR-331-3p has also been used as a marker for the detection of liver cancer in serum [29]. Sequencing of subcutaneous adipose tissue (by RNA-seq) of Laiwu and Yorkshire pigs showed that 17 upregulated and 22 downregulated miRNAs were identified in Laiwu pigs [30]. Chen et al. use both Laiwu and Yorkshire pigs as research subjects, and high-throughput sequencing analysis of the longissimus dorsi muscle of the 2 pig breeds was performed using the RNA-seq technology [31]. Consequently, 19 significantly differentially expressed miRNAs were detected and selected for further screening. Among them, relative to Yorkshire pigs, 7 differentially expressed miRNAs, including miR-331-3p, were upregulated, whereas 12 were downregulated in Laiwu pigs. In conclusion, miR-331-3p may influence the formation of adipose tissue as well as intramuscular fat deposition in pigs.
Through bioinformatics prediction, we found that DLST and solute carrier family 25 member 1 (SLC25A1) are the target genes of miR-331-3p, and these two genes are related to fat deposition. DLST encodes dihydrolipoyl succinyltransferase, which is a core component of KGDHC, which is the rate-limiting enzyme for the second oxidative decarboxylation of the Krebs cycle [32]. The SLC25A1 gene encodes a mitochondrial citrate transporter (CTP) that regulates citric acid transport bidirectionally between the mitochondria and cytoplasm [33]. Both DLST and SLC25A1 are present in the citrate pyruvate cycle pathway, which transports the acetyl-COA necessary for the synthesis of fatty acids from the mitochondria into the cytoplasm. In the present study, the interaction of DLST and SLC25A1 with miR-331-3p and their role in fat deposition were researched.
2. Materials and Methods
2.1. Experimental Animals and Sample Collection
Two breeds of pigs, Laiwu (weight = 100 kg, n = 6) and Yorkshire (weight = 100 kg, n = 6), served as the research subjects in this study. The pigs were raised in different groups at the Laiwu and Yorkshire farm (Laiwu city, Shandong, China) and were raised in the same environment. All groups were also given identical diets, which met their nutrient requirements as previously described (NRC, 1998). Subsequently, all pigs were ethically sacrificed and processed for market. Tissue samples including heart, liver, kidney, spleen, lung, muscle (longissimus dorsi), and fat (back fat) were collected and stored at −80°C. In addition, ear tissue samples from the Laiwu pigs were collected and stored at −20°C. All animal experiments were performed in accordance with the Institutional Animal Care as well as the National (GB 13078–2001 and GB/T 17237–1998) and the Agricultural Standards (NY 5148-2002-NY 5151-2002) of the People's Republic of China.
2.2. Cell Culture
PK15 cells (a porcine kidney epithelial cell line) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified 5% CO2 atmosphere. Cells used in experiments were in the logarithmic phase of growth and cell viability before treatment was >95%.
2.3. Isolation, Culture, and Differentiation of Laiwu Porcine Primary Preadipocytes
Laiwu pigs (<7 days of age) were obtained from the Laiwu and Yorkshire farm. Adipose tissue was isolated from porcine back and neck under sterile conditions. Adipose tissue was digested with 1 mg/mL collagenase 1 (Solarbio, Beijing, China) at 37°C for 60 to 90 min. Then, FBS-supplemented medium was added to terminate the digestion, and all the liquids were sifted through a 100 μm cell strainer. The culture medium was removed by centrifugation (100 ×, 10 min). Subsequently, Red Blood Cell Lysis Buffer (Solarbio, Beijing, China) was added and incubated at room temperature for 10 min and was centrifuged at 200 × for 10 min. Finally, the primary preadipocytes were resuspended in Dulbecco's modified Eagle's medium DMEM/F12 (Hyclone, Shanghai, China) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Pen-strep, Solarbio, Beijing, China).
Porcine preadipocytes were cultured in growth medium for 3 days to reach approximately 80% confluence (day 0). Then, the growth-arrested cells were cultured in differentiation medium (DMEM/F12, 10% FBS, 1% Pen-strep, 5 ug/mL insulin (Solarbio, Beijing, China), 1um/mL dexamethasone (DEX, Solarbio, Beijing, China), and 0.5 mM/mL 3-isobutyl-1-methylxanthine (IBMX, Solarbio, Beijing, China)) for 2 days (Day 2). The cells were then treated with maintenance medium (DMED/F12, 10% FBS, 1% Pen-strep, and 5 ug/ml insulin) for an additional 6 days. During this period, the maintenance medium was replaced every 2 days.
2.4. Bioinformatics Analysis
Target sequences for miR-331-3p were predicted using TargetScan (http://www.TargetScan.org), MiRBase (http://www.mirbase.org), and miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/).
2.5. Construction of 3′-UTR Luciferase Reporter Plasmids
Wild-type DLST and SLC25A1 3′-UTR fragments containing the binding site of miR-331-3p were amplified by PCR from total RNA extracted from the back fat of Laiwu pigs. Products of the PCR reactions were directionally subcloned into the pGL3 vector downstream of the firefly luciferase open-reading frame (Promega, USA). Mutation of the seed sequence (the miR-331-3p target site) was accomplished by overlap extension PCR using the general primer and mutagenic primer, which are listed in Table 1. First, the wild-type DLST and SLC25A1 3′-UTR fragments were used as templates, including miR-331-3p target site. The mutant DLST 3′-UTR fragment's upstream and downstream regions were amplified with primers F1, R3 and F3, R1 (the same as above), respectively. The mutant SLC25A1 3′-UTR fragments were amplified using primers F2, R4 and F4, R2. Next, the wild-type and mutant DLST and SLC25A1 3′-UTR fragments were both inserted into the pGL3 vector. Positive clones were identified through bacterial-liquid PCR and were confirmed by sequencing (Sangon Biotech, Shanghai, China). The wild-type and mutant DLST 3′-UTR vectors were named as pGL3-DLST-wt and pGL3-DLST-mut, respectively. The wild-type and mutant SLC25A1 3′-UTR vectors were named as pGL3-SLC25A1-wt and pGL3-SLC25A1-mut, respectively.
Preadipocytes were fixed for 1 h in 10% formalin, washed with PBS, and stained for 30 min by complete immersion in a working solution of Oil Red O. The cells were then washed twice in water, and data were collected by imaging the cells. In order to quantify fatty acids, excess water was evaporated by placing the stained cultures at a temperature of about 32°C for 15 min. Next, 1 ml of isopropyl alcohol was added to the stained culture dish, and the extracted dye was immediately removed. The absorbance of the resulting solution was monitored spectrophotometrically at 510 nm.
2.6. Dual-Luciferase Reporter Assay
PK-15 cells (Procell, Wuhan, China) cultured in 24-well plates were transfected using Lipofectamine™ 3000 Transfection Reagent (Thermo, USA) according to the manufacturer's instructions. The transfection mixture for each well contained 250 ng 3′-UTR luciferase reporter vector, 12.5 ng of pGL4.74 (Promega, USA) for normalization, and either 15 pmol miR-331-3p mimic expression plasmids, miR-331-3p mimic, or negative control mimic (GenePharmn, Shanghai, China). Each group of experiments was repeated at least 3 times. After 48 h of transfection, the luciferase activity was measured using the Dual-luciferase Reporter Gene Assay System Kit (Promega).
2.7. RNA Extraction and Gene Expression Assay Using Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted from the cell and tissue samples using the MicroElute Total RNA Kit (Omega Bio-Tek, USA) according to the manufacturer's instructions. The quantity of the isolated RNA was determined by measuring the UV260/280 absorbance ratio using a biophotometer (Eppendorf, Germany). For miRNA reverse transcription, Mir-XTM miRNA First-Strand Synthesis Kit (Takara Biotechnology, Dalian, China) was used. Quantitative Real-Time PCR (qRT-PCR) was performed using the SYBR Premix Ex Taq Kit (Takara Biotechnology, Dalian, China) on a LightCycler® 96 (Roche). The miRNA-specific primers for the qRT-PCR included the forward primer GGTATGGGCCTATCCTAGAA and universal primer used for the reverse primer. The housekeeping gene U6 was used as an internal control.
For mRNA reverse transcription, PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Biotechnology, Dalian, China) was used according to the manufacturer's instructions. qRT-PCR was performed using the SYBR Premix Ex Taq Kit (Takara Biotechnology, Dalian, China) on a LightCycler® 96 (Roche). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control for the other genes. Primers used to amplify the mRNAs are listed in Table 2. The quantitative fluorescence results were calculated by 2−ΔΔCt method.
2.8. Transfection of Porcine Preadipocytes
Preadipocytes were seeded in 96-, 24- or 6-well plates. Once cell densities reached 80% confluence, they were transfected with either the mimic, inhibitor, or negative control for miR-331-3p (Sangon, Shanghai, China), respectively. The miR-331-3p mimic and inhibitor were transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at concentrations of 3 pmol, 15 pmol, and 75 pmol/well for 4 h. After that, the growth medium was replaced.
2.9. Detection of Cell Proliferation Activity by Cell Counting Kit 8 (CCK-8 Assay)
Preadipocytes were inoculated into 96-well plates at a density of 2 × 103. Five replicates were used for each group of cells, and 5 blank controls were used to remove background signals. After culture for 24, 48, 72, and 96 h, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance of each well was determined at 450 nm using a microplate reader (Labsystem). The cell proliferation curve was plotted, and the experiment was repeated in triplicate.
2.10. Flow Cytometry
Preadipocytes were seeded in 96-well plates and incubated for 24 h. By this time, the cells reached confluence and were transfected with either miR-331-3p mimic, miR-NC, or miR-331-3p inhibitor (GenePharma, Shanghai, China). Preadipocytes were harvested by digestion with 0.25% trypsin and washed 3 times with PBS to remove cell debris. The cells were then fixed in cold 70% ethanol overnight and treated with 1 mg/mL RNaseA and propidium iodide (PI) for 30 min at 37°C. The cells were then filtered through a 0.75 μm membrane prior to analysis on a FACScan argon laser cytometer (BD, USA).
2.11. Western Blotting
Preadipocytes were washed with PBS, lysed using RIPA buffer (Beyotime, Shanghai, China) on ice, and centrifuged at 10,000 × at 4°C. The whole protein concentration for each sample was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China). All protein samples and SDS-PAGE Sample Loading Buffer (Beyotime, Shanghai, China) were mixed at a ratio of 4 to 1 and boiled for 5 min. Next, all protein samples were diluted to the same concentration. Protein bands were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% Blocking Buffer (Beyotime, Shanghai, China) for 1 h. Next, primary antibodies directed against SLC25A1, DLST, or actin (Cell Signaling Technology, CST, USA) were incubated on the membranes at 4°C overnight. The membranes were then treated with HRP-conjugated secondary antibodies (Cell Signaling Technology, CST, USA) at room temperature for 2 h. Signals were detected using the enhanced chemiluminescence system reagents (Amersham Pharmacia Biotech, USA). All experiments were repeated in triplicate.
2.12. Statistical Analysis
All data are presented as means ± standard deviation (SD). Statistical significance was assessed by Student's t-test. Differences were considered statistically significant at .
3. Results
3.1. Expression Analysis of miR-331-3p in Different Tissues of Laiwu and Yorkshire Pigs
To investigate the differences in miR-331-3p expression between Laiwu and Yorkshire pigs, RNA was extracted from various tissues, and the expression levels were tested by qRT-PCR (Figures 1(a) and 1(b)). Significant differences were observed between the 2 breeds. In the liver, muscle, and back fat of Laiwu pigs, miR-331-3p expression was more than that of Yorkshire pigs (). However, the opposite was observed in the spleen, lung, and kidney ().
Through a biological website analysis, 2 candidate target genes of miR-331-3p were predicted (DLST and SLC25A1). Expression levels of the 2 genes were assessed in various tissues between the 2 breeds of pigs by qRT-PCR. The expression trend of DLST gene is opposite to the expression trend of miR-331-3P, whether in Laiwu pigs' or in Yorkshire pigs' liver, kidney, muscle, and back fat (Figure 1(c)) (). However, except in liver tissue, the expression of SLC25A1 was significantly elevated in the muscle and back fat (Figure 1(d)) ().
3.2. miR-331-3p Suppressed Proliferation of Porcine Preadipocytes
Preadipocyte proliferation, differentiation, and fatty acid accumulation form the basis of adipogenesis. To elucidate whether miR-331-3p regulates adipogenesis, the effects of miR-331-3p on preadipocyte proliferation were first explored. Synthetic miR-331-3p mimics, miR-331-3p inhibitors, and the negative control (miR-NC) are shown in Figure 2(a), which were transfected into preadipocytes, respectively. Additionally, miR-331-3p mimics significantly increased the expression levels of miR-331-3p in preadipocytes while endogenic expression of miR-331-3p in preadipocytes was remarkably inhibited by transfecting with miR-331-3p inhibitors. Subsequently, cellular proliferation was detected by the addition of the dye, CCK-8. As presented in Figure 2(b), miR-331-3p expression significantly suppressed cell proliferation at 24 h, 48 h, and 72 h (). In contrast, the miR-331-3p inhibitor significantly promoted cell proliferation at 48 h and 72 h. To verify the accuracy of this result, a series of marker genes' expression were quantified (Figure 2(c)), including CDK2, CDK3, CDK4, and Cyclin B. These genes were significantly downregulated in the group receiving the miR-331-3p mimic. The mRNA expression of CDKN1A was significantly upregulated after transfection of miR-331-3p mimics (Figure 2(d)).
Moreover, the cell cycle distribution in miR-331-3p transfected preadipocytes was investigated. In comparison with miR-NC group, the miR-331-3p mimic treated group prominently increased the number of cells in G0/G1 phase; both of S phase and G2/M phase decreased significantly (, Figure 2(e)). Taken together, these data indicate that transfection with miR-331-3p could effectively increase the proliferation of adipocytes.
3.3. miR-331-3p Promoted Differentiation of Preadipocytes
In our previous research, we successfully constructed the eukaryotic expression vector of miR-331-3p (pre-miR-331-3p) [34]. To investigate the relationship between the expression of miR-331-3p and preadipocyte differentiation, preadipocytes induced differentiation. This day is defined as the 0th day. Expression levels of miR-331-3p were measured by qRT-PCR on days 0, 2, 4, 6, and 8. The level of miR-331-3p was investigated during porcine preadipocyte differentiation. miRNA expression profiling revealed that the level of miR-331-3p was upregulated firstly and then downregulated during adipogenesis of porcine preadipocyte cells (0 d–8 d) (Figure 3(a)). Even on day 8 after cell differentiation, the expression of miR-331-3p was still higher than that on day 0. In addition, preadipocytes were transiently transfected with pre-miR-331-3p, pcDNA 3.1(+), or miR-331-3p inhibitor prior to differentiation. Two days later, complete medium was replaced with differentiation induction medium. Expression of a marker gene for differentiation, PPARγ, was detected (Figure 3(b)). The expression level of PPARγ was upregulated in the pre-miR-331-3p group relative to the pcDNA 3.1(+) and miR-331-3p inhibitor treated groups. Therefore, it could be reasonably hypothesized that miR-331-3p may promote preadipocyte differentiation.
3.4. The Determination of Target Gene of miR-331-3p
To confirm target gene for miR-331-3p, 2 different bioinformatics software programs were used to predict the target genes. Interestingly, the 2 programs (Targetscan, miRWalk) produced different results. The genes were analyzed using gene ontology and pathway analysis to confirm the likely targets of miR-331-3p. It was determined that DLST and SLC25A1 were both associated with fatty acid metabolism and are components of the citric acid pyruvate cycle. To verify direct interactions between miR-331-3p and the 3′-UTR of DLST and SLC25A1, the 3′-UTR regions were cloned into a luciferase reporter vector (Figure 3(c)) and were used to transiently transfect PK-15 cells. Luciferase activity was observed after 2 days (Figure 3(d)). miR-331-3p mimics were cotransfected with wild-type DLST 3′-UTR vector, and luciferase activity was decreased relative to the NC or mut-type DLST 3′-UTR vector groups. However, the groups were cotransfected with miR-331-3p mimics and wild-type SLC25A1 3′-UTR vectors, and luciferase activity was increased relative to the NC or mut-type SLC25A1 3′-UTR vector groups. It can be concluded that DLST was the direct target gene of miR-331-3p, which may regulate DLST. Meanwhile, SLC25A1 is more likely to be an indirect target of miR-331-3p.
3.5. miR-331-3p Regulates the Gene Expressions of DLST and SLC25A1 in Porcine Preadipocytes
To further study the mechanism by which miR-331-3p regulates DLST and SLC25A1 gene expressions, the miR-331-3p and RNA expression level of DLST and SLC25A1 were measured 2 days after transfection with miR-331-3p mimics, miR-NC, and miR-331-3p inhibitor. The gene expression of miR-331-3p was significantly upregulated () in the experimental group and the contrary result was observed in the inhibitor group. Meanwhile, the expression of DLST was significantly downregulated () in the experimental group and the contrary result was observed in the inhibitor group, but the expression of SLC25A1 had opposite tendency with the expression of DLST, which means that the gene expression of SLC25A1 was significantly upregulated in the mimics treatment group () (Figure 4(a)).
To further clarify whether the miR-331-3p regulates the protein expression of DLST and SLC25A1, total protein was extracted for Western blot analysis. The level of DLST protein was downregulated in the miR-331-3p mimic group compared with the negative control and inhibitor groups () (Figures 4(b) and 4(c)). In contrast, the protein expression of SLC25A1 in the mimics group was significantly upregulated compared with the inhibitor group () and was also upregulated compared with the negative control ().
3.6. The Role of miR-331-3p in Adipocyte Fatty Acid Metabolism
Both DLST and SLC25A1 are important components of citrate pyruvate cycle pathway (Figure 4(d)). The DLST gene encodes the dihydrolipoamide succinyl transferase, a core enzyme of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC). The KGDHC catalyzes α-ketoglutarate to form succinyl coenzyme A. The citrate pyruvate cycle not only results in the synthesis acetyl-CoA but also functions to transport it from the mitochondrial to the cell matrix. This is the only source of acetyl-CoA, which is indispensable for all fatty acid synthesis. To further study the mechanism of action, mRNA expressions of some known genes involved in the citrate pyruvate cycle were analyzed, the results of which are presented in Figure 4(e). The expressions of citrate lyase (CS), SLC25A1, ATP citrate lyase (ACLY), and fatty acid synthase (FASN) were upregulated in the miR-331-3p mimic treated group relative to the NC and inhibitor treated groups (). However, the expressions of DLST, dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase complex flavoprotein subunit A (SDHA), fumarate hydratase (FH), malic enzyme 1 (ME1), pyruvate carboxylase (PC), and malate dehydrogenase 1 (MDH1) were all downregulated in the miR-331-3p mimic treated group ().
Preadipocytes were transfected with either miR-331-3p mimic, miR-331-3p inhibitor, or miR-331-3p NC. The preadipocytes were then stained with Oil Red O (Figure 4(f)), and fatty acid contents of the cells were assessed (Figure 4(g)). It can be clearly seen that the fatty acid content of the miR-331-3p mimic treated group is substantially higher than that in the other treatment groups ().
4. Discussion
In the present study, miR-331-3p was identified as a novel gene that regulates proliferation, differentiation, and fatty acid accumulation of porcine preadipocyte. The organization of miR-331-3p in Laiwu and Yorkshire pigs was analyzed. The expression of this miR-331-3p was observed to be consistent with the previously published sequencing results [30, 31], which is suggestive of the important role it plays in the regulation of fat formation. The expression of miR-331-3p in different tissues was also analyzed. Compared with Yorkshire pigs, the expression of miR-331- 3p was significantly elevated in the liver, the muscle, and the back fat of Laiwu pigs. The liver is an important organ for lipid metabolism and plays an important role in the digestion, absorption, synthesis, decomposition, and transport of lipids [35, 36]. Fat is the main tissue of lipid storage. The free fatty acids produced by the hydrolysis of triglycerides in fat can be transported to the tissues through oxidative decomposition and energy supply after being combined with plasma albumin [37]. The muscle itself contains adipose tissue, which is referred to as intramuscular fat. The intramuscular fat mainly refers to the fat deposited among the fibers in the muscle connective tissue and the muscle bundle. Intramuscular fat has certain specificities in lipid metabolism and cell differentiation, which is related to the special location of IMF [38]. It has been well established that Yorkshire pigs are the leanest breeds, and its liver, back fat, and muscle are important sites for the proliferation and differentiation of preadipocytes and significant on fatty acid accumulation. Therefore, the high expression of miR-331-3p observed in Laiwu pigs may be due to the fact that miR-331-3p has the ability to orchestrate these processes.
Two candidate target genes of miR-331-3p were predicted through a biological website analysis, and its expression levels were assessed in various tissues between the 2 breeds of pigs by qRT-PCR. Compared with Yorkshire pigs, the expression of DLST was significantly reduced in the liver, the muscle, and the back fat of Laiwu pigs. However, except in liver tissue, the expression of SLC25A1 was significantly elevated in muscle and back fat. This may indicate that there is a negative regulatory targeting relationship between miR-331-3p and DLST, but a positive regulatory targeting relationship between miR-331-3p and SLC25A1 was uncertain.
The formation of adipose tissue is a process in which preadipocytes grow, proliferate, undergo terminal differentiation to form fat cells, and grow by hypertrophy. In the present study, preadipocytes were isolated from Laiwu pigs. Above all, CCK8 assay and flow cytometry analysis were used to detect cell growth when preadipocytes were transfected with miR-331-3p mimics, miR-331-3p inhibitors, or miR-NC. Results of CCK8 assay showed that cell proliferation rate of cells when transfected with miR-331-3p mimics was significantly lower than that in cells transfected with miR-331-3p inhibitors, or miR-NC, and flow cytometry analysis showed that after transfection with miR-331-3p mimics, the proportion of cells in G0/G1 phase increased significantly, while the proportion of cells in S phase and G2/M phase decreased significantly compared with cells transfected with miR-331-3p inhibitor and miR-NC. These results were consistent with the results of CCK8 analysis, which indicated that miR-331-3p could inhibit cell proliferation. In order to further elucidate the mechanism of action, qRT-PCR was used to detect several proliferation-related genes, including CDK2, CDK3, CDK4, Cyclin B, and CDKNIA. Cyclin-dependent kinases (CDKs), such as CDK2, CDK3, and CDK4, have been recognized as key regulators of cell growth and proliferation in eukaryotic cells and are required for G1-S phase transitions in mammalian cells [39, 40]. Cyclin B is required for cells to enter and exit the M phase of the cell cycle [41]. Conversely, overexpression of cellular CDKN1A may be an inhibitor of cell proliferation [42]. Levels of CDK2, CDK3, CDK4, and Cyclin B in preadipocytes transfected with miR-331-3p were significantly lower than those in cells transfected with miR-NC; however, the difference of CDKN1A expression is contrary to that of other genes. Therefore, miR-331-3p may inhibit the cells by affecting the expression of cell cycle-related genes. Taken together, these data indicate that miR-331-3p is likely an inhibitory regulator of the proliferation of porcine adipocyte progenitor cells.
Subsequently, overexpression of miR-331-3p was able to induce differentiation of preadipocytes. Furthermore, miR-331-3p expression was detected at days 0, 2, 4, 6, and 8 of cell differentiation. Trends in expression were increasing initially and then gradually decreasing. The expression of the PPARγ gene also showed the same trend. The PPAR family of transcription factors is important regulators of the differentiation of precursor adipocytes into mature adipocytes. The PPARs family contains 3 subtypes, alpha, beta, and gamma. Among them, PPARγ is the most important marker gene in the process of precursor adipocyte differentiation [43]. The expression of the PPARγ gene was increased significantly on days 2 and 4 after the overexpression of miR-331-3p. The expression trend of binding to miR-331-3p is suggestive of a role in the promotion of the differentiation process in preadipocytes.
Genes related to fatty acid metabolism (DLST and SLC25A1) were predicted bioinformatically. Next, these predicted target genes were verified experimentally by a Dual-Luciferase Reporter Assay. Luciferase activity of wt-DLST-3′-UTR was significantly suppressed by ectopic expression of miR-331-3p. However, luciferase activity of wt-SLC25A1-3′-UTR was significantly upregulated. This may be due to the fact that the SLC25A1 gene is not a direct target of miR-331-3p; rather it may regulate the expression indirectly by other means. The results of qRT-PCR and Western blot of DLST and SL25A1 genes also proved this conclusion. After transfection of miR-331-3p mimics, miR-NC, and miR-331-3p inhibitor, induced mature adipocytes were stained by Oil Red O and its lipid droplet quantified. Those results showed that overexpression of miR-331-3p indeed increased fatty acid synthesis, which in turn corroborates the hypothesis that miR-331-3p can promote fatty acid synthesis through the modulation of the citrate pyruvate cycle.
5. Conclusions
In summary, miR-331-3p functions as a regulator of the proliferation and differentiation of adipocytes and fatty acid metabolism. Overexpression of miR-331-3p can inhibit cellular proliferation and was observed to promote preadipocytes differentiation. Furthermore, miR-331-3p can also promote the synthesis of fatty acids by regulating the target gene DLST to promote acetyl-CoA translocation.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors certify that there are no conflicts of interest with any financial organization regarding the material discussed in the manuscript.
This study was supported by the Shandong Provincial Natural Science Foundation (ZR2018BC046), Funds of Shandong "Double Tops" Program (SYL2017YSTD12), and Shandong Provincial Modern Pig Technology and Industry System Project (SDAIT-08-02).
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