The Effect Of Escherichia Coli On The Expression Of MUC16 In Lung Epithelial Cells And Its Regulatory Mechanism

Objective:The research team discovered the existence of microorganisms in lung cancer tissues in previous studies,and explored the influence of E.coli on the expression of MUC16 in lung epithelial cells and its regulatory mechanism.Methods:1.Establish a cell-strain co-culture system:the lung adenocarcinoma cell line NCI-H1734,the lung squamous cell carcinoma cell line SK-MES-1 and the normal lung bronchial epithelial cell BEAS-2B were amplified and passaged with the standard strain of Escherichia coli ATCC25922 and knocked out.The ATCC25922 strain with dgcQ gene(ATCC25922 Δ dgcQ)and the ATCC25922 strain with dgcE gene knockout(ATCC25922 Δ dgcE)were co-cultured.At 4h,8h and 15h of co-cultivation,the changes of cell morphology and population density under co-cultivation of cell strains were observed.2.When cells and strains are co-cultured for 4h,8h and 15h,take each group of cell strains co-culture system,fix with neutral formaldehyde,and then stain with eosin and Hoechst 33258 staining solution and take fluorescence photos to observe cell cytoplasmic protein and The state of DNA in the nucleus.3.When the cells were co-cultured with the strain for 4h,8h and 15h,qRT-PCR was used to detect cell MUC16 genes,housekeeping genes(GAPD,ACTB,UBC,TBP,SDHA,HPRT1,YWHAZ)and related genes(LINC00842,MNT,MAX,MAZ,DACH1,TERT)expression situation.4.Cluster analysis of regulatory genes or related genes that are similar to the MUC16 gene change rule.Results:1.The status of each strain is different when cultured separatelyThe growth of different strains is different:ATCC25922 grows fastest,8h and 15h strains have the highest density;ATCC25922Δ dgcQ grows the slowest,8h,15h strains have the lowest density;among the three,the growth rate of ATCC25922 ΔdgcE is in the middle,and the strains at 8h and 15h have the middle density.2.Changes in cell morphology and population density under the condition of co-culture of cell strains1)Co-culture of lung bronchial epithelial cells BEAS-2B and E.coliThe overall mortality(10%-20%)of the BEAS-2B cells added to the strains is not high,and the surviving cells shrink and shrink and have mutual adhesion.The overall performance is better than that of lung cancer cells(NCI-H1734,SK-MES-1).)Tolerance.Comparison of the lethality of strains:ATCC25922 has the strongest lethality to BEAS-2B cells,ATCC25922ΔdgcQ has the lowest lethality to cells,and ATCC25922ΔdgcE has the middle lethality to cells.2)Co-culture of lung adenocarcinoma cell NCI-H1734 and E.coliThe overall tolerance of NCI-H1734 cells added to the strain is in the middle compared to the other two cells:lung adenocarcinoma cell NCI-H1734 has a mortality rate(50%-85%)higher than that of normal lung bronchial epithelial cells BEAS-2B(10%-20%).%),but lower than SK-MES-1(70%～90%)of lung squamous cell carcinoma cells.Comparison of the lethality of strains:The lethality of strain ATCC25922 against NCI-H1734 cells was moderate,the lethality of strain ATCC25922 Δ dgcQ against NCI-H1734 cells was the highest,and the lethality of strain ATCC25922Δagainst NCI-H1734 cells was the lowest.3)Co-culture of lung squamous cell carcinoma SK-MES-1 and Escherichia coliThe SK-MES-1 cell death rate(70%～90%)of the added strains was the highest among the 3 cell lines,and the bacterial proliferation was the most obvious at 15h,and the overall resistance was compared with the other two cells(BEAS-2B,NCI-H1734)The force is the lowest.Comparison of the lethality of strains:ATCC25922 has the strongest lethality on SK-MES-1 cells,ATCC25922 Δ dgcQ has the middle lethality on cells,and ATCC25922ΔdgcE has the lowest lethality on cells.3.Changes in the expression of key cell genes under the condition of co-cultivation of cell strains1)MUC16 gene changes in lung epithelial cells and E.coli co-culturedAfier adding the strain,the MUC16 gene in BEAS-2B cells was significantly increased at all time points,which was 3 to 5 times higher than when cultured alone;after the addition of the strain,the MUC16 gene in NCI-H1734 cells was significantly increased at each time point,which was higher than that in NCI-H1734 cells.It was 4 to 7 times higher when cultured;the MUC16 gene in SK-MES-1 cells was slightly lower than when cultured alone at 4h after adding the strain;both increased significantly at 8h and 15h,which was 4 to 5 times higher than when cultured alone,2)Expression changes of cell MUC16 regulatory genes and related genes under co-culture conditionsThe trend of MUC16 in the co-cultivation system is similar to that of TERT.In the co-culture system of the BEAS-2B group of lung epithelial cells:The common upstream transcription factors of MUC16 and TERT were mainly MAZ and MAX at 4h,MAX,MNT,DACH1 at 8h,and MNT at 15h.Under different grouping conditions,the change law of LINC00842 and DACH1,MNT,MAZ is closeIn the co-culture system of lung adenocarcinoma cell NCI-H1734 group:the common upstream transcription factors of MUC16 and TERT are mainly MAZ and MAX at 4h,MAZ and DACH1 at 8h,and MAZ at 15h.Under different grouping conditions,the change law of LINC00842 is close to that of MAZ and DACH1.Conclusion:1.Escherichia coli dgcQ and dgcE genes can affect the growth rate and exercise capacity of the strain.2.During the co-culture of Escherichia coli(standard strains ATCC25922 ATCC25922 Δ dgcQ,ATCC25922 Δ dgcE)and lung epithelial cells(BEAS-2B,NCI-H1734,SK-MES-1),normal lung bronchial epithelial cells BEAS-2B affect the large intestine Bacillus has the strongest tolerance,followed by lung adenocarcinoma cells NCI-H1734,and lung squamous cell carcinoma cells SK-MES-1 the worst.3.During the co-culture of Escherichia coli and lung epithelial cells(BEAS-2B,NCI-H1734,SK-MES-1),the expression of MUC16 was significantly up-regulated,with lung adenocarcinoma cells NCI-H1734 being the most up-regulated.4.In the co-culture system of lung adenocarcinoma cell NCI-H1734 group,MUC16 and TERT have similar changes.It is possible to regulate MAZ and DACH1 to regulate MUC16.