| Colorectal cancer(CRC)is one of the most common gastrointestinal tumors.In terms of tumor treatment,not only are treatment methods such as radiation and chemotherapy utilized,but oncolytic virus therapy is also employed as a relatively safe treatment method,which can selectively destroy tumor cells.Newcastle disease virus(NDV)is a type of oncolytic virus,and the anti-tumor effect of the HBUN/LSRC/F3 strain of NDV(NDV F3)has been preliminarily confirmed in the laboratory.Mammalian STE20-like kinase 1(MST1)encodes a serine/threonine kinase that is widely expressed in human cells.MST1 plays an important role in regulating embryonic development,cell differentiation,maintaining immune system stability,promoting apoptosis,and inhibiting tumor cell growth.Micro RNAs(mi RNAs)are a class of conserved non-coding small RNAs that can regulate gene expression after transcription.This study used bioinformatics to select mi R-708-5p,which targets MST1,and demonstrated its role in regulating MST1 expression by binding to the non-coding region at the 3’ end of MST1 m RNA.Previous studies have shown that mi R-708-5p is involved in tumor cell proliferation,invasion,and apoptosis.However,the relationship between mi R-708-5p and MST1,and whether NDV induces apoptosis of colorectal cancer cells through the mi R-708-5p/MST1 axis,has not been reported and requires further investigation.First,HCT116 cells were divided into control and experimental groups.The experimental group was infected with the NDV F3 strain and subjected to subsequent experiments.Results of the CCK-8 assay showed that NDV F3 significantly inhibited the proliferation of HCT116 cells(P<0.05)after treatment.Results of the Hoechst 33258 staining experiment indicated that NDV F3 induced apoptosis in HCT116 cells.The extent of DNA damage was assessed using single-cell gel electrophoresis,which revealed that as the infection time of NDV on HCT116 cells increased,the length of the cell tail became longer(P<0.01)and the proportion of tail DNA content increased(P<0.01).Transwell assay showed that NDV F3 significantly inhibited the invasion of HCT116 cells(P<0.01).q RT-PCR results demonstrated that NDV F3 decreased the expression of mi R-708-5p gene while increasing the expression of MST1 gene(P<0.05).Furthermore,Western blot results showed an increase in the expression of Caspase3,cleaved-Caspase3,MST1 and their activated forms compared to the control group(P<0.01).Next,a search was conducted through bioinformatics to predict mi R-708-5p as a micro RNA that targets and regulates MST1.Relevant databases were used to determine the expression level of mi R-708-5p in colorectal cancer.The regulation relationship between the two was validated through a dualluciferase reporter gene experiment,which showed that mi R-708-5p directly targeted the 3’-UTR site of MST1,regulating the expression of MST1 m RNA.Mi R-708-5p mimic and mi R-708-5p inhibitor were transfected into HCT116 cells,and the transfection efficiency of mi R-708-5p was detected through q RT-PCR.Then,the transfected cells were infected with NDV F3.The effect of NDV on the proliferation and invasion of HCT116 cells was determined using the CCK-8 and Transwell assays.The results showed that inhibiting the expression of mi R-708-5p increased the pro-apoptotic effect of NDV on HCT116 cells,while overexpression of mi R-708-5p reversed the proapoptotic effect of NDV on HCT116 cells(P<0.05).The Transwell assay revealed that the number of invasive cells in the mi R-708-5p inhibitor+NDV group was significantly reduced compared to the NDV group(P<0.01).q RTPCR results showed an increase in MST1 gene expression in the mi R-708-5p inhibitor+NDV group compared to the NDV group,while the mi R-708-5p mimic+NDV group showed a decrease in MST1 gene expression(P<0.01).Western blot analysis showed that the expression levels of MST1 and Caspase3 proteins were significantly reduced in the mi R-708-5p mimic+NDV group compared to the NDV group,while the expression of Caspase3 and MST1 proteins was significantly increased in the mi R-708-5p inhibitor+NDV group(P<0.01).In summary,NDV F3 reduces the proliferation and invasion of HCT116 cells,induces apoptosis,and further verifies that NDV F3 may inhibit the proliferation and invasion of HCT116 cells and induce cell apoptosis by targeting MST1 through mi R-708-5p.This could help achieve the goal of inhibiting colorectal cancer. |
