Knockdown of lncRNA ACTA2-AS1 reverses cisplatin resistance of ovarian cancer cells via inhibition of miR-378a-3p-regulated Wnt5a

Figures & data

Table 1. Association between ACTA2-AS1 expression level and clinicopathological features of patients with OC

Clinicopathological features	ACTA2-AS1		P value
	Low (n = 51)	High (n = 51)
Age, y			0.642
≤50	26	27
>50	25	24
Tumor size, cm			0.028
≤8	39	15
>8	12	36
FIGO stage			0.032
I to II	30	11
III to IV	21	40
Histological subtype			0.691
Serous	18	24
Others	33	27
Histological grade			0.754
G1 to G2	19	25
G3	32	26
Lymph node metastasis			0.016
Absent	35	10
Present	16	41

Table 2. The primer sequences included in this study

Name	primer sequences (5’-3’)
ACTA2-AS1
forward	5′-GTGGTTCTGGTTTGCCTGAT-3′
reverse	5′-CTGGCCCTGTAACACCAGAT-3′
miR-382-5p	5′-CTGGACTTGGAGTCAGAAGGAA-3′
Wnt5a
forward	5′- GTTTCGGCTACAGACCCAGA-3′
reverse	5′- CCCCAGTTCATTCACACCACA-3′
GAPDH
forward	5′- GCACCACCAACTGCTTAGCA-3′
reverse	5′- GTCTTCTGGGTGGCAGTGATG-3′
U6
forward	5′-CTCGCTTCGGCAGCACA-3′
reverse	5′-AACGCTTCACGAATTTGCGT-3′

Figure 1. ACTA2-AS1 is up-regulated in DDP‑resistant OC cell lines and tissues (a) ACTA2-AS1 expression levels in tissues from OC patients (DDP-sensitive and DDP-resistant). (b) Kaplan-Meier survival curves for patients with DDP-sensitive and DDP-resistant. (c) Expression of ACTA2-AS1 in DDP-resistant cell lines. (d) CCK-8 assay of the viability and IC50 value of OC/DDP cells. (e) ACTA2-AS1 from the nuclear and cytoplasmic fractions of OC/DDP cells. (f) ROC curves of ACTA2-AS1. *p < 0.05.

Figure 2. ACTA2-AS1 knockdown inhibits DDP resistance of OC/DDP cells in vitro. (a) RT-qPCR analysis of ACTA2-AS1 expression in OC/DDP cells. (b) CCK-8 assay of the viability and determination of IC₅₀ value of OC/DDP cells following exposure to DDP as a series dose. *p < 0.05.

Figure 3. Silencing ACTA2-AS1 sensitizes OC cells to DDP by targeting miR-378a-3p. (a) For miR-378a-3p, ACTA2-AS1 is thought to act as a molecular sponge. (b) Luciferase assays in HEK293T cells co-transfected with mutant or wild-type ACTA2-AS1 and miR-378a-3p. (c) In HEK293T cells, anti-AGO2 RIP was conducted, followed by RT-qPCR for the detection of the expression of ACTA2-AS1 or miR-378a-3p linked to AGO2. (d) Expression of miR-378a-3p in DDP-resistant cell lines. (e) The effect of ACTA2-AS1 knockdown on the expression of miR-378a-3p in OC/DDP cells was investigated using RT-qPCR. (f) MiR-378a-3p expression levels in tissues from individuals suffering from OC (DDP-sensitive and DDP-resistant). (g) A negative correlation between ACTA2-AS1 expression and miR-378a-3p expression in DDP-resistant OC tissues. (h) RT-qPCR analysis of miR-378a-3p in OC/DDP cells transfected with NC inhibitor or miR-378a-3p inhibitor. (i) CCK-8 assay of the viability and determination of IC₅₀ value of OC/DDP cells upon exposure to DDP as a series dose. *p < 0.05, ^nsp > 0.05.

Figure 4. By targeting Wnt5a, MiR-378a-3p reduces DDP resistance in GC cells. (a) Wnt5a was predicted to function as a molecular sponge for miR-378a-3p. (b) Luciferase assays in HEK293T cells co-transfected with mutant or wild-type Wnt5a and miR-378a-3p. Expression of Wnt5a in DDP-resistant cell by RT-qPCR (c) and Elisa assay (d). Detection of expression of Wnt5a in OC/DDP cells that have been transfected with NC inhibitor, miR-378a-3p mimics, miR-378a-3p inhibitor, or NC mimics using RT-qPCR (e) and Elisa assay (f). (g) Wnt5a expression levels in tissues from OC patients (DDP-resistant and DDP-sensitive). (h) In DDP-resistant OC tissues, there is a negative connection between Wnt5a expression and miR-378a-3p expression. Expression detection of Wnt5a in OC/DDP cells transfected with si-NC or si-Wnt5a using RT-qPCR (i) and Elisa assay (j). (k) CCK-8 assay of the viability and determination of IC₅₀ value of OC/DDP cells after exposure to DDP.as a series dose *p < 0.05, ^nsp > 0.05.

Figure 5. ACTA2-AS1 positively regulates Wnt5a through sponging miR-378a-3p. (a) Correlation analysis between ACTA2-AS1 and Wnt5a expression in DDP-resistant OC tissues. Expression detection of Wnt5a in OC/DDP cells using RT-qPCR (b) and Elisa assay (c). *p < 0.05.

Figure 6. Summary of ACTA2-AS1/miR-378a-3p/Wnt5a axis.