TITLE:
Transcriptome Profiles and Gene Expression of Min6 Cells Are Altered by Pancreatic Stellate Cells
AUTHORS:
Ratnakar Reddy Bynigeri, Sasikala Mitnala, Ravikanth Vishnubhotla, Rupjyoti Talukdar, Surya Satyanarayana Singh, Nageshwar Reddy Duvvuru
KEYWORDS:
β-Cells, Gene Expression, Islets, Pancreatic Stellate Cells, Tran-scriptome
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.9 No.6,
June
28,
2018
ABSTRACT:
Aim: To identify the influence of pancreatic stellate
cell (PSCs) secretions on gene expression profiles of Min6 cells by whole
transcriptome sequencing. Methods: Pancreatic stellate cells (PSCs) were isolated from C57BL6J mice and
propagated in vitro to acquire the activated phenotype. Total RNA was
isolated from monocultured (MC) and PSC cocultured (CC) Min6 cells to prepare cDNA libraries, which
were subjected to whole transcriptome sequencing for identifying differential
expression of β-cell transcription
factors (Pdx-1, Rfx6 and NeuroD1) related
to insulin gene transcription and GSIS related genes such as Glut2, Gck, Abcc8, Kcnj11 and L-type Ca2+ channels (Cacnb2, Cacna1c). qRT-PCR was used to validate the
gene expression. GSIS of Min6 cells was examined by estimating insulin levels
in response to high glucose challenge. Results: Transcriptome analysis of discovery set revealed
that coculture of Min6 cells with PSCs caused increased expression of β-cell specific genes (Ins1, Rfx6 and NeuroD1)
concomitant with decreased expression of Pdx-1, MafA and Nkx2-2. Expression of GSIS associated genes (Glut2, Gck, Abcc8, Kcnj11 and Cacnb2) was
decreased in such conditions. Validation by qRT-PCR in Min6 cells cocultured
with PSCs revealed increased significant expression of Ins1 (2.1 ± 0.22 folds; p ≤ 0.001), Rfx6 (1.68 ± 0.23 folds; p ≤ 0.002) and NeuroD1 (0.96 ± 0.11 folds; p ≤ 0.01), accompanied by downregulation of Cacnb2 (-0.93 ± 0.57 folds; p ≤ 0.05). PSC
secretions did not restore the GSIS from glucose unresponsive higher passage
Min6 cells (MC: 1.33 ± 0.42; CC: 1.55 ± 0.72 pmol/mg protein; p = ns) upon
high glucose stimulation. However, glucose responsive higher passage Min6 cells
cocultured with PSCs presented increased insulin secretion (MC: 7.025 ± 0.64;
CC: 14.84 ± 1.01 pmol/mg protein; p ≤
0.04) concomitant with marginal increase of insulin contents. Conclusion: PSC secretions increase Ins1, Rfx6 and NeuroD1 gene expression, GSIS from glucose
responsive Min6 cells, but do not restore the GSIS from glucose unresponsive
Min6 cells.