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Article

Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1α axis

  • Authors:
    • Pampana Sandhya Venkata Lakshmi
    • Jagrati Parashar
    • Biswajit Biswas
    • Ramesh Ummanni
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    Affiliations: Department of Applied Biology, Indian Institute of Chemical Technology, Hyderabad 500007, India
  • Article Number: 74
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    Published online on: February 16, 2026
       https://doi.org/10.3892/or.2026.9079
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Abstract

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PCa), associated with poor prognosis and resistance to androgen receptor (AR)‑targeted therapies. Hypoxia is a well‑established driver of lineage plasticity and has been implicated in promoting NE differentiation (NED) of tumors. However, the underlying molecular mechanisms linking hypoxia to NED remain unclear. In the present study, miR‑135b‑5p was identified as a critical regulator of hypoxia‑induced NED through modulation of the hypoxia‑inducible factor alpha‑1 subunit alpha inhibitor (HIF1AN)‑HIF1α axis. Exposure of androgen‑dependent PCa cell lines (LNCaP and VCaP) to hypoxia induced neurite outgrowth and increased expression of NE markers, concurrent with upregulation of miR‑135b‑5p. Target prediction followed by experimental validation in luciferase reporter assays confirmed that HIF1AN is a direct target of miR‑135b‑5p. Suppression of HIF1AN results in the stabilization of HIF1α, which in turn activates the AKT/mTOR signaling pathway, facilitating NE trans differentiation. Functional studies demonstrated that overexpression of miR‑135b‑5p by mimics promotes NED in LNCaP cells, while inhibition of miR‑135b‑5p reverses the NE features in NE‑LNCaP and NCI‑H660, NE cells. Furthermore, pharmacological inhibition of HIF1α using PX‑478 abrogated hypoxia‑induced NED and attenuated activation of AKT/mTOR signaling, further underscoring the significance of the miR‑135b‑5p‑HIF1AN‑HIF1α axis in NED of PCa cells. Collectively, the findings of the present study reveal a novel miR‑135b‑5p‑HIF1AN‑HIF1α signaling axis that is involved in hypoxia‑induced NED via AKT/mTOR activation and identify miR‑135b‑5p and HIF1α as potential therapeutic targets for NEPC.
View Figures

Figure 1

Hypoxia-induced NE differentiation in
prostate cancer cells. (A) LNCaP cells were exposed to a hypoxic
environment for different durations. (B) Neurite outgrowth was
assessed using bright-field microscopy and quantified with ImageJ
(1.50i; National Institutes of Health). Bar graphs represent the
mean ± SD from three independent experiments (C) and (E) Expression
of NE markers was evaluated in hypoxia-induced LNCaP and VCaP
cells. (D) Expression of NE markers in LNCaP, in vitro
derived NE-LNCaP cells, and hypoxia-treated LNCaP cells was
determined by western blotting. The data represent the mean ± SD
for three independent experiments, with *P<0.05, **P<0.01 and
***P<0.001. NE, neuroendocrine.

Figure 2

miR-135b-5p is overexpressed in
hypoxia-induced NE prostate cancer. (A) Expression levels of
miR-135b-5p in various cancers were analyzed using the
CancerMIRNome database which derives its expression data from The
Cancer Genome Atlas database, an open access resource. (B)
miR-135b-5p expression was assessed by RT-qPCR in WPMY-1, LNCaP,
NE-LNCaP, and also hypoxia-treated LNCaP and VCaP cells. (C) Venn
diagram showing the predicted common target genes of miR-135b-5p.
(D) Relative expression of HIF1α was measured in LNCaP and NE-LNCaP
cells using RT-qPCR. (E) NE-LNCaP cells were transfected with a
miR-135b-5p mimic or inhibitor, and HIF1AN expression was analyzed
by RT-qPCR. (F) and (G) Luciferase reporter assays were performed
to confirm direct binding of miR-135b-5p to the 3′UTR region of
HIF1AN. Data are presented as the mean ± SD from three independent
experiments. The data represent mean ± SD for three independent
experiments, with *P<0.05, **P<0.01 and ***P<0.001. miR,
microRNA; NE, neuroendocrine; RT-qPCR, reverse
transcription-quantitative polymerase chain reaction; ns, not
significant.

Figure 3

Inhibition of miR-135b-5p attenuates
the progression of NE prostate cancer. (A) LNCaP and NE-LNCaP cells
were transfected with a miR-135b-5p mimic or inhibitor, and neurite
outgrowth was determined by ImageJ. (B and C) Expression of NE and
a hypoxia-associated marker was evaluated by western blotting. (D)
LNCaP and VCaP cells were transfected with the miR-135b-5p
inhibitor under hypoxic conditions and neurite length was
determined by ImageJ. (E) Expression of NE and hypoxia-associated
markers was assessed by western blotting. (F and G) VCaP cells
under hypoxia and NCI-H660 cells were transfected with the
miR-135b-5p inhibitor, and the expression of NE and
hypoxia-associated markers was determined by western blotting. Data
are presented as the mean ± SD from three independent experiments.
***P<0.001. NE, neuroendocrine; miR, microRNA; HIF1α, hypoxia
inducible factor alpha-1; HIF1AN, hypoxia inducible factor alpha-1
subunit alpha inhibitor; CH-A chromogranin A.

Figure 4

HIF1α downregulation attenuates NE
differentiation of prostate cancer cells under hypoxia. (A and B)
HIF1α was downregulated using siRNA in NE-LNCaP cells, and
phenotypic changes were assessed by measuring neurite length and
expression of NE markers was evaluated by western blotting. (C and
D) HIF1α was silenced in LNCaP cells under hypoxia, and
morphological changes were assessed by quantifying neurite
outgrowth using bright-field microscopy and ImageJ, and expression
levels of NE markers and HIF1α were determined by western blotting.
(E and F) VCaP cells under hypoxia and NCI-H660 cells were
transfected with HIF1α siRNA. Expression of NE markers was analyzed
by western blotting. The data are presented as the mean ± SD from
three independent experiments. **P<0.01 and ***P<0.001. si-,
small interfering; NE, neuroendocrine; HIF1α, hypoxia inducible
factor alpha-1; CH-A chromogranin A.

Figure 5

Inhibition of miR-135b-5p attenuates
NE prostate cancer progression via the AKT/mTOR signaling pathway.
(A) Expression and activation of AKT/mTOR pathway components and
hypoxia-associated markers were evaluated in LNCaP, NE-LNCaP and
hypoxia-treated LNCaP cells by western blotting. (B-E) NE-LNCaP,
LNCaP, VCaP under hypoxia, and NCI-H660 cells were transfected with
a miR-135b-5p inhibitor, and the expression of AKT/mTOR pathway and
hypoxia-associated markers was analyzed by western blotting. miR,
microRNA; NE, neuroendocrine; HIF1α, hypoxia inducible factor
alpha-1; HIF1AN, hypoxia inducible factor alpha-1 subunit alpha
inhibitor; p-, phosphorylated.

Figure 6

Downregulation of HIF1α hampers
AKT/mTOR-mediated induction of NE differentiation. (A) NE-LNCaP
cells were transfected with HIF1α siRNA, and expression of HIF1α
and AKT/mTOR pathway markers was analyzed by western blotting.
(B-D) Hypoxia-treated LNCaP and VCaP, and also NCI-H660 cells, were
transfected with HIF1α siRNA, and the expression of HIF1α and
AKT/mTOR pathway components was assessed by western blotting. NE,
neuroendocrine; siRNA, small interfering RNA; HIF1α, hypoxia
inducible factor alpha-1; p-, phosphorylated.

Figure 7

PX-478, a HIF1α inhibitor, suppresses
NE differentiation of PCa cells. (A and B) NE-LNCaP cells were
treated with PX-478, and the expression of NE markers, AKT/mTOR
pathway components, and HIF1α was assessed by western blotting.
(C-F) LNCaP and VCaP cells were treated with PX-478 under hypoxic
conditions, and changes in NE markers, AKT/mTOR pathway proteins,
and HIF1α were analyzed by western blotting. (G and H) NCI-H660
cells were treated with PX-478, and the expression of NE markers,
AKT/mTOR pathway components, and HIF1α was determined by western
blotting. PX-478, S-2-amino-3-[4′-N, N,-bis (chloroethyl) amino]
phenyl propionic acid N-oxide dihydrochloride; NE, neuroendocrine;
HIF1α, hypoxia inducible factor alpha-1; CH-A chromogranin A.

Figure 8

Schematic representation of
hypoxia-driven NED via miR-135b-5p in PCa. Under hypoxic
conditions, miR-135b-5p promotes NED in PCa cells by directly
targeting and downregulating HIF1AN, an inhibitor of HIF1α. This
leads to stabilization of HIF1α and subsequent activation of the
AKT/mTOR signaling pathway, driving the neuroendocrine phenotype.
NED, neuroendocrine differentiation; miR or miRNA, microRNA; PCa,
prostate cancer; HIF1α, hypoxia inducible factor alpha-1; HIF1AN,
hypoxia inducible factor alpha-1 subunit alpha inhibitor; p-,
phosphorylated.
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Lakshmi PS, Parashar J, Biswas B and Ummanni R: Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis. Oncol Rep 55: 74, 2026.
APA
Lakshmi, P.S., Parashar, J., Biswas, B., & Ummanni, R. (2026). Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis. Oncology Reports, 55, 74. https://doi.org/10.3892/or.2026.9079
MLA
Lakshmi, P. S., Parashar, J., Biswas, B., Ummanni, R."Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis". Oncology Reports 55.4 (2026): 74.
Chicago
Lakshmi, P. S., Parashar, J., Biswas, B., Ummanni, R."Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis". Oncology Reports 55, no. 4 (2026): 74. https://doi.org/10.3892/or.2026.9079
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Spandidos Publications style
Lakshmi PS, Parashar J, Biswas B and Ummanni R: Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis. Oncol Rep 55: 74, 2026.
APA
Lakshmi, P.S., Parashar, J., Biswas, B., & Ummanni, R. (2026). Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis. Oncology Reports, 55, 74. https://doi.org/10.3892/or.2026.9079
MLA
Lakshmi, P. S., Parashar, J., Biswas, B., Ummanni, R."Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis". Oncology Reports 55.4 (2026): 74.
Chicago
Lakshmi, P. S., Parashar, J., Biswas, B., Ummanni, R."Hypoxia‑induced miR‑135b‑5p promotes neuroendocrine differentiation of prostate cancer cells through HIF1AN‑HIF1&alpha; axis". Oncology Reports 55, no. 4 (2026): 74. https://doi.org/10.3892/or.2026.9079
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