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. 2021 Dec;12(1):3101-3112.
doi: 10.1080/21655979.2021.1944019.

LncRNA MYLK-AS1 acts as an oncogene by epigenetically silencing large tumor suppressor 2 (LATS2) in gastric cancer

Juan Luo  1 , Huifei Xiang  2
Affiliations

LncRNA MYLK-AS1 acts as an oncogene by epigenetically silencing large tumor suppressor 2 (LATS2) in gastric cancer

Juan Luo et al. Bioengineered. 2021 Dec.

Abstract

Extensive studies showed the vital function of long noncoding RNAs (lncRNAs) in the pathological and physiological progression of tumors. Previous evidence has indicated that lncRNA MYLK Antisense RNA 1 (MYLK-AS1) acts as an oncogene to facilitate the progression of several tumors. Nevertheless, little is known about its biological role in gastric cancer (GC). Our report intended to probe the underlying mechanism and function of MYLK-AS1 in GC. Results revealed that MYLK-AS1 showed an upregulated level in GC. It was worth mentioning that upregulated MYLK-AS1 caused the unfavorable clinical outcome in GC patients. Functional assays indicated that MYLK-AS1 silencing retarded the proliferation, cell cycle, migration, and invasion in GC. Besides, in vivo assay validated that MYLK-AS1 deficiency also restrained tumor growth. Through in-depth mechanism exploration, MYLK-AS1 was uncovered to bind with wnhancer of zeste homolog 2 (EZH2), an epigenetic inhibitor, to inhibit the level of Large Tumor Suppressor 2 (LATS2), thereby exerting carcinogenicity. Conclusively, our research highlighted the importance of MYLK-AS1 in GC, indicating that MYLK-AS1 might be an effective biomarker for GC.[Figure: see text].

Keywords: EZH2; LATS2; MYLK-AS1; gastric cancer.

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Figures

Figure 1.
Figure 1.
Relative MYLK-AS1 expression in GC tissues and cells, and its clinical significance. (A) RT-qPCR analysis for MYLK-AS1 expression in GC and adjacent normal tissues. (B) Relative MYLK-AS1 expression in GC cell lines and human normal gastric mucosal cell line by RT-qPCR analysis. (C-E) The expression of MYLK-AS1 was analyzed in GEO datasets (GSE13911, GSE54129 and GSE79973). (F) Overall survival of GC patients with high or low MYLK-AS1 expression by Kaplan-Meier method. **p< 0.01
Figure 2.
Figure 2.
Function of MYLK-AS1 on GC cell growth and invasion in vitro. (A) Relative MYLK-AS1 expression in AGS and MKN45 cells transfected with sh-MYLK-AS1 and sh-NC. (B) The viability of AGS and MKN45 cells transfected with sh-MYLK-AS1 and sh-NC was determined by CCK-8 assay. (C) The cell cycle of transfected AGS and MKN45 cells was assessed by flow cytometry analysis. (D and E) Transwell assay was carried out to evaluate the migratory and invasive abilities after AGS and MKN45 cells were transfected with sh-MYLK-AS1 and sh-NC. (F and G) The protein levels of E-cadherin and N-cadherin were measured by western blot. *p < 0.05, **p< 0.01
Figure 3.
Figure 3.
Effect of MYLK-AS1 on GC tumor growth in vivo. (A) The tumors after being removed from the mice. (B) Tumor volume was calculated. (C) Tumor weight was determined after tumors were harvested. (D) IHC staining was conducted to determine Ki67 expression of tumors from sh-MYLK-AS1 and sh-NC groups. **p< 0.01
Figure 4.
Figure 4.
MYLK-AS1 inhibited LATS2 transcription in GC cells. (A-B) The LATS2 mRNA and protein levels were determined by RT-qPCR and western blot in transfected AGS and MKN45 cells. (C) LATS2 transcriptional activities of transfected AGS and MKN45 cells. **p< 0.01
Figure 5.
Figure 5.
MYLK-AS1 decreased LATS2 transcriptional expression through recruiting EZH2. (A) MYLK-AS1 distribution in AGS and MKN45 cells was confirmed by subcellular fractionation assay. (B) RIP assay was performed in AGS and MKN45 cells, and the relative enrichment of MYLK-AS1 was detected by RT-qPCR. (C) The sh-MYLK-AS1 plasmid was transfected into AGS and MKN45 cells, and ChIP assay were conducted by the use of specific anti-EZH2 antibodies. (D) EZH2 mRNA and protein levels were tested by RT-qPCR and western blot in AGS and MKN45 cells transfected with sh-EZH2 or sh-NC. (E) RT-qPCR and western blot analyses for LATS2 mRNA and protein levels in transfected AGS and MKN45 cells. **p < 0.01, ***p< 0.001
Figure 6.
Figure 6.
The effect of MYLK-AS1 knockdown on GC cell growth and invasion was rescued by silencing LATS2. (A) The transfection efficiency of sh-LATS2 in AGS and MKN45 cells. (B) Cell viability was evaluated by CCK-8 assay in AGS and MKN45 cells transfected with sh-NC, sh-MYLK-AS1 or sh-MYLK-AS1+sh-LATS2. (C) The cell cycle of transfected AGS and MKN45 cells. (D and E) Transwell assay was performed to assess the migration and invasion of transfected AGS and MKN45 cells. (F and G) The protein levels of E-cadherin and N-cadherin were measured by western blot. *p < 0.05, **p< 0.01
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