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. 2022 Sep 30:2022:5932512.
doi: 10.1155/2022/5932512. eCollection 2022.

The Drosha-Independent MicroRNA6778-5p/GSK3 β Axis Mediates the Proliferation of Gastric Cancer Cells

Mingjun Ren  1   2 , Li Xing  3 , Wanping Wang  3 , Wanying Bi  3 , Wanjun Wu  4 , Gui Jiang  4 , Weiji Wang  5 , Xingdong Liang  1   2 , Manran Liu  6 , Shifu Tang  1   2
Affiliations

The Drosha-Independent MicroRNA6778-5p/GSK3 β Axis Mediates the Proliferation of Gastric Cancer Cells

Mingjun Ren et al. Comput Intell Neurosci. .

Abstract

Background: Gastric cancer (GC) is a primary cause of cancer death around the world. Previous studies have found that Drosha plays a significant role in the development of tumor cells. Soon after, we unexpectedly found that the expression of microRNA6778-5p (miR6778-5p) is unconventionally high in the gastric cancer cells low-expressing Drosha. So, we designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. We aimed to explore the effect of microRNA6778-5p on the proliferation of gastric cancer cells with Drosha knockdown and its intrinsic mechanism.

Methods: We designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. After transfecting miR6778-5p mimics and inhibitor into gastric cancer cell lines with Drosha knockdown, the expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. The Cell Counting Kit-8 (CCK-8) experiment was used to detect the proliferation ability of gastric cancer cells after overexpression or knockdown of miR6778-5p and bioinformatics predicted the relationship between miR6778-5p and glycogen synthase kinase-3β (GSK3β).

Results: After infection with the Drosha knockdown lentivirus, Drosha's mRNA and protein levels were significantly downregulated in gastric cancer cells. The expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. Overexpression of miR6778-5p significantly enhanced the proliferation ability of Drosha low-expression gastric cancer cells; on the contrary, knocking down miR6778-5p weakened the proliferation ability of Drosha low-expression gastric cancer cells. Bioinformatics predicted that miR6778-5p targeted glycogen synthase kinase-3β (GSK3β) and the mRNA and protein levels of GSK3β decreased significantly after overexpression of miR6778-5p.

Conclusion: miR6778-5p promotes the proliferation of Drosha low-expressing gastric cancer cells by targeting GSK3β.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Drosha-independent miR6778-5p expression increases in gastric cancer cells. (a) The mRNA expression levels of Drosha were determined by qRT-PCR in gastric cell lines with Drosha knockdown (∗∗P < 0.05). (b) The protein expression levels of Drosha were verified by WB in gastric cell lines with Drosha knockdown (∗∗P < 0.05). (c) The expression levels of miR6778-5p were detected by qRT-PCR in gastric cancer cells low-expressing Drosha (∗∗P < 0.05).
Figure 2
Figure 2
Overexpression of miR6778-5p promotes the proliferation of gastric cancer cells with Drosha knockdown. (a) The expression levels of miR6778-5p were evaluated by qRT-PCR (∗∗P<0.05). (b) Growth curves were observed using the CCK8 assay (∗∗P < 0.05).
Figure 3
Figure 3
miR6778-5p silence inhibits the proliferation of gastric cancer cells with Drosha knockdown. (a) The expression levels of miR6778-5p were evaluated by qRT-PCR (∗∗P<0.05). (b) Growth curves were observed using the CCK8 assay (∗∗P<0.05).
Figure 4
Figure 4
The miR6778-5p/GSK3β axis mediates the proliferation of Drosha low-expressing gastric cancer cells. (a) miR6778-5p acts on the 275–281 sites in the UTR of GSK3β mRNA through its seed sequence. (b) The mRNA and protein expression levels of GSK3β were detected by qRT-PCR or WB in MGC-803/Drosha KD or SGC-7901/Drosha KD cell lines after being transfected with miR6778-5p mimics (∗∗P < 0.05). Immunoblotting analyses were performed with the indicated antibodies. (c) The mRNA and protein expression levels of GSK3β were determined by qRT-PCR or WB in MGC-803/Drosha KD or SGC-7901/Drosha KD cell lines after being transfected with miR6778-5p inhibitor (∗∗P < 0.05). Immunoblotting analyses were performed with the indicated antibodies.
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