Abstract:

Objective To explore the effects and possible mechanisms of methyltransferase-like protein 3 （METTL3） on the proliferation and migration of liver cancer cells by regulating the stability of RAB5C. Methods The expression of RAB5C in liver cancer tissues and its relationship with clinicopathological characteristics were were analyzed using the UALCAN database； The liver cancer cells of the logarithmic growth phase were divided into the following groups： si-NC group （transfected with a blank fragment）， si-RAB5C-1 group （RAB5C silenced）， si-RAB5C-2 group （RAB5C silenced）， si-NC+over-NC group （transfected with a blank fragment+a blank vector）， si-METTL3+over-NC group （METTL3 silenced+a blank vector）， and si-METTL3+over-RAB5C group （METTL3 silenced+RAB5C overexpression vector）； RAB5C-WT+si-NC group （RAB5C wild-type vector+control vector） and RAB5C-MUT+si-METTL3 group （RAB5C mutant vector+METTL3 silenced）； EdU experiment was conducted to detect cell proliferation ability； Transwell experiment was conducted to detect cell migration ability； Flow cytometry was used to detect the level of cell apoptosis； GEPIA database was used to analyze the correlation between METTL3 and RAB5C expression； SARMP database was used to predict the m⁶A modification site in RAB5C； The targeted regulation relationship between METTL3 and RAB5C was identified by using dual luciferase reporter gene assay and Me-RIP assay. Results Analysis of the UALCAN database showed that the expression levels of RAB5C mRNA in normal tissues （n=50） and liver cancer tissues （n=371） were 54.1 （44.9， 63.1） and 93.5 （67.1， 120.0）， respectively， with a statistically significant difference （χ²=0.01， P<0.001）. The expression levels of RAB5C mRNA in patients with non-metastatic lymph nodes （n=252） and metastatic lymph nodes （n=4） were 93.9 （65.1， 120.4） and 117.5 （100.3， 142.9）， respectively； the expression levels in patients with TP53 mutation （n=105） and non-mutation （n=105） were 100.2 （80.5， 133.1） and 84.7 （62.3， 115.9）， respectively， with statistically significant differences （χ²=0.72， P=0.020； χ²=0.74， P=0.010）. The EdU assay results indicated that the cell proliferation rates of the HepG2 cells in the si-NC group， si-RAB5C-1 group， and si-RAB5C-2 group were （39.3±2.93）%， （19.98±1.77）%， and （19.98±2.46）%， respectively， with a statistically significant difference （F=63.01， P<0.001）， and the cell proliferation rates of the si-RAB5C-1 group and si-RAB5C-2 group were both lower than that of the si-NC group （both P<0.05）. The results of the Transwell assay showed that the migration numbers of HepG2 cells in the si-NC group， si-RAB5C-1 group， and si-RAB5C-2 group were 94.2±1.2， 26.1±0.5， and 25.1±0.6， respectively， with a statistically significant difference （F=87.26， P<0.001）， and the number of cell migration in the si-NC group was higher than that in the si-RAB5C-1 group and the si-RAB5C-2 group （both P<0.05）. The results of flow cytometry showed that the apoptosis rates of HepG2 cells in the si-NC group， si-RAB5C-1 group and si-RAB5C-2 group were （7.18±1.04）%， （12.56±1.50）%， and （11.68±1.54）%， respectively， with a statistically significant difference （F=13.09， P=0.007）， and the apoptosis rates of the si-RAB5C-1 group and the si-RAB5C-2 group were both higher than that of the si-NC group （both P<0.05）. GEPIA and SARMP database analysis showed that the expression of METTL3 was positively correlated with that of RAB5C （r=0.13， P=0.002）， and there were multiple m⁶A modification sites and corresponding m⁶A binding site motifs in RAB5C. The results of the MeRIP-qPCR experiment showed that the relative expression level of RAB5C in the HepG2 cell of si-NC group was 1.00±0.11， which was higher than that in the si-METTL3 group （0.28±0.18）， with a statistically significant difference （t=6.89， P=0.002）. The results of the dual-luciferase reporter gene assay showed that compared with the RAB5C-WT+si-NC group （1.00±0.16）， silencing the expression of METTL3 in HepG2 liver cancer cells could significantly inhibit the luciferase activity of the wild-type RAB5C promoter （0.50±0.12）， with a statistically significant difference （t=4.26， P=0.010）； while compared with the RAB5C-WT+si-NC group （1.00±0.03）， si-METTL3 treatment had no significant effect on the luciferase activity of the mutant RAB5C promoter （0.97±0.01） （t=1.53， P=0.200）. The results of EdU assay， Transwell experiment， and flow cytometry showed that there were statistically significant differences in cell proliferation， migration， and apoptosis among the si-NC+over-NC group， si-METTL3+over-NC group， and si-METTL3+over-RAB5C group （all P<0.05）. Compared with the si-METTL3+over-NC group， there were statistically significant differences in cell proliferation， migration， and apoptosis in the si-METTL3+over-RAB5C group （all P<0.05）. Conclusions METTL3 may regulate the expression of RAB5C through mediated m⁶A modification， thus inhibiting the occurrence and development of liver cancer.

Key words: Liver neoplasms, Methyltransferase-like protein 3, N⁶-methyladenosine, RAB5C