Malignant Transformation Of Het-1A Cells Induced By Combined HPV And MNNG

Esophageal cancer is an environment-related tumor with multiple factors and stages of development.Tumor cells have strong ability of proliferation and anti-apoptosis.The epithelial-mesenchymal transformation is usually observed in tumorigenesis progression.In the process of understanding the complex etiology of esophageal cancer,environmental risk factors have attracted much attention.Among them,exposure to nitrosamines is one of the early identified risk factors.More and more evidence shows that HPV may play an important role in the occurrence and development of esophageal cancer.Previous research showed that there was a certain correlation between esophageal cancer and HPV type 18 infection in Huai'an area with esophageal cancer high incidence,and there was also a certain risk of exposure to nitrosamines in this area.Based on the previous research results,we established a model of malignant transformation of Het-1A cells by transfection of HPV18 E6 and E7 genes and exposure to MNNG.The malignant cell model was validated by soft agar cloning and tumorigenesis in nude mice.At the same time,we observed the changes of cell morphology,proliferation,apoptosis,invasion and migration under different exposure conditions.The possible molecular mechanism of HPV combined with MNNG inducing malignant transformation of Het-1A cells was preliminarily explored.?.Establishment of a model of malignant transformation of Het-1A induced by combined action of HPV18 and MNNG and expression of related functional proteins1.Establishment of a model of malignant transformation of Het-1A induced by combined action of HPV18 and MNNG?1?Het-1A cells were divided into four groups according to different factors:HPV18combined with MNNG?HPV+MNNG?,HPV18 alone?HPV?,MNNG alone?MNNG?and empty carrier control?control?.The concentration of MNNG was determined to be 2micromol/L.Morphological observation showed that with the increase of MNNG exposure time,the cell morphology of HPV+MNNG group and MNNG group changed obviously,the cell volume became larger,loose,slender,with pseudopodia,which was significantly different from the control group.?2?CCK8 assay showed that the cell proliferation of?HPV+MNNG?and MNNG groups changed in stages.With the prolongation of exposure time,the cell growth was stimulated first,then inhibited,and finally the proliferation was out of control.The sensitivity of cells to toxicants under combined action was stronger than that of MNNG alone,and the change of proliferation rate was more obvious than that of MNNG alone.?3?Flow cytometry showed that the cell cycle distribution was consistent with the proliferation results,showing a phased change.Cell cycle arrest occurred in both groups during exposure.After malignant transformation,the proliferation index PI=?S+G2/M?/?G0/G1+S+G2/M?of?HPV+MNNG?group was higher than that of MNNG group?P<0.05?.The results showed that HPV could affect cell cycle,promote DNA synthesis and mitosis,and synergistically promote cell proliferation.?4?The results of apoptotic double staining showed that the apoptotic rate of MNNG-treated cells increased first and then decreased during malignant transformation.The change of apoptotic rate in?HPV+MNNG?group was consistent with that of MNNG alone,but the change was more obvious.The anti-apoptotic ability of?HPV+MNNG?-treated cells after 35generations was significantly enhanced compared with MNNG-treated cells?P<0.05?.?5?The Transwell experiment showed that the invasion and migration ability of?HPV+MNNG?group and MNNG group was significantly higher than that of control group?P<0.05?,and the invasion and migration ability of HPV+MNNG group was stronger than that of MNNG group?P<0.05?..?6?The results of soft agar colony formation test and nude mice tumorigenesis test showed that HPV-MNNG combined group and MNNG alone group could form obvious positive clones?the number of cells in each colony was more than 50?.This indicated that HPV-MNNG combined group and MNNG alone induced cells to have the ability to grow independently without anchorage.Nude mice tumorigenesis experiment showed that the tumorigenesis rate of the above two groups of cells inoculated into nude mice was 100%,and there was no significant difference in the size of the tumors between the two groups?P>0.05?.HE staining of the tumors showed similar characteristics.The results showed that HPV combined with MNNG group and MNNG alone group had malignant transformation in 35^th generation,and had the ability of tumorigenicity in nude mice.2.Expression of function-related proteins in the progression of malignant transformationIn the progression of malignant transformation,cycle,apoptosis,interstitial transformation and autophagy play an important role,affecting the occurrence and development of tumors,which is also the main focus of cancer mechanism research.On the basis of observing the changes of cell function,we detected the tumor-related proteins during malignant transformation,which laid a foundation for the study of the mechanism of malignant transformation of Het-1A cells induced by HPV combined with MNNG.?1?In the progression of malignant transformation,cyclin D1 expression in?HPV+MNNG?group was first inhibited and then increased,and the expression of cyclin D1 was higher than that in MNNG group after the 35^th generation.The expression of c-Myc protein in?HPV+MNNG?group and MNNG group increased with the infection generation,and the expression of c-Myc protein in HPV transfected cells increased significantly.?2?The expression of anti-apoptotic protein Bcl-2 in malignant transformation Het-1A cells induced by HPV and MNNG was significantly higher than that in control group,and the anti-apoptotic ability of HPV alone group was also enhanced.The apoptotic protein Bax of each group increased with the generation,which may be caused by aging.The anti-apoptotic ability of cells under the combined action of HPV and MNNG was stronger than that of MNNG group.?3?The expression level of E-cadherin in?HPV+MNNG?group and MNNG group decreased with the increase of exposure algebra,and the expression of N-cadherin protein increased significantly compared with the initial exposure,but there was no significant difference between the two groups.The level of interstitial transformation in the four groups changed during the culture process.Combining with the results of cell invasion and migration test,the level of interstitial transformation has an important influence on the ability of cell invasion and migration?4?P62 was overexpressed in the cells of the combined group,and autophagic flow was inhibited.The expression of mTOR protein decreased first and then increased,but it was still lower than that of the control group,while the expression of LC3II protein increased compared with that of the control group,indicating that the combined effect of HPV and MNNG at the initial stage stimulated the occurrence of autophagy.However,with the accumulation of P62expression,autophagy flow was blocked and the autophagy function was impaired.? Mechanisms of malignant transformation of Het-1A cells induced by combined action of HPV18 and MNNG1.Effect of P62 gene on malignant transformation of Het-1A cells induced by HPV18 combined with MNNGWestern blot was used to detect the abnormal expression of autophagy-related protein P62in Het-1A cells during malignant transformation.The biological function of Het-1A cells and the expression of related functional protein were detected by knocking out P62 gene.Transfection of siRNA P62 reduced the proliferation of malignant transformation?HPV+MNNG?cells,markedly blocked cell cycle,decreased cyclin D1 protein expression and increased c-Myc protein expression.Apoptosis-related protein Bcl-2 was down-regulated and Bax was up-regulated,autophagy-related protein LC3II was up-regulated,Beclin1 protein was up-regulated,P62 was down-regulated,apoptosis rate and autophagy level were up-regulated,and cell anti-tumor ability was weakened.The PI3K/AKT signaling pathway was blocked with the inhibition of P62 gene expression.P62 gene intervention provides a direction for cancer treatment and prognosis.2.Effect of P62 on mTOR in HPV18-MNNG co-acting cellsHPV can upregulate the expression of mTOR and P62 proteins in Het-1a cells.P62 gene is closely related to mTOR expression.Knockout of P62 gene in Het-1a cells transfected with HPV leads to decreased mTOR expression and enhanced autophagy function.Knockout of the overexpressed P62 gene in Het-1A cells that were upended under the combined effect of HPV and MNNG reduced the expression of mTOR protein,increased the expression of Beclin1 and LC3II protein,and increased the level of autophagy.3.The mechanism of P62-Keap1-NRF2 signaling pathway in malignant transformation induced by combined action of HPV and MNNGOxidative stress is an important link in the progression of tumorigenesis and development.Abnormal expression of P62 is of great significance to the change of oxidative stress ability of cells.Overexpression of P62 may enhance the antioxidant capacity of tumor cells and promote the development of tumors by activating the antioxidant NRF2 system that scavenges ROS.It is showed that the expression of NRF2 protein in nucleoproteins of malignant transformed cells treated with HPV and MNNG was higher than that of MNNG alone,and the expression of NRF2 gene and its downstream antioxidant genes NQO1 and HO-1 in combined group was higher than that in MNNG alone group?P<0.05?.The antioxidant capacity of combined group was stronger than that in MNNG alone group?P<0.05?.By inhibiting the expression of P62 gene in malignant transformed cells,the expression of NRF2 gene and its downstream antioxidant genes NQO1 and HO-1 in the nucleus of malignant transformed cells decreased significantly?P<0.05?.In situ synthesis of nanoclusters was used to detect the degree of oxidative damage.It was found that inhibition of P62 gene expression resulted in increased fluorescence intensity of nanoclusters accumulated in cells,indicating that inhibition of P62gene could weaken the antioxidant capacity of cells.The interaction between P62 and Keap1protein was confirmed by immunoprecipitation.The malignant transformed Het-1A cells under the combined action of HPV and MNNG exerted antioxidant effect through P62-Keap1-NRF2signaling pathway.Highly expressed P62 protein could promote the continuous activation of NRF2 through competitive binding with Keap1,thus enabling malignant Het-1A cells to acquire stronger antioxidant capacity and promote the occurrence and development of tumors.4.The mechanism of PI3K/AKT signaling pathway in the malignant transformation of combined action of HPV and MNNGPI3K/AKT,a classical pathway,plays a role in many tumors and is involved in the regulation of cell proliferation,cell cycle,apoptosis,interstitial transformation and other progressions.In this study,the above biological functions of Het-1A cells were significantly changed under the combined action of HPV and MNNG,and the PI3K/AKT pathway-related proteins changed significantly during malignant transformation.The PI3K/AKT pathway may play a role in promoting malignant transformation of Het-1A cells under the combined action of HPV and MNNG.PI3K inhibitor LY294002 acted on malignant transformed cells at concentrations of 0?mol/L,10?mol/L,20?mol/L,50?mol/L,respectively.The rate of growth inhibition increased with the increase of concentration?P<0.05?,but the growth inhibition rate decreased with the increase of LY40029 concentration?P<0.05?.When 20?M LY294002 acted on malignant transformed cells,the cell cycle was obviously blocked,the G1 phase increased?P<0.05?,the process of cell cycle was slowed down,the apoptotic rate was increased?P<0.05?,and the anti-apoptotic ability was weakened.Under the intervention of LY294002,the expression of cyclinD1 and P21 was down-regulated,cell cycle was blocked by flow cytometry.LY294002 hindered the progress of cell cycle and inhibited cell proliferation.Bax protein expression was up-regulated,Bcl-2 protein expression was down-regulated,Annexin-V APC-PI double staining was used to detect the increase of cell apoptosis rate.Compared with the control group,the expression of N-cadherin protein was significantly decreased and the expression of snail protein was slightly increased,suggesting that the inhibition of PI3K pathway led to the decrease of the degree of interstitial transformation,the increase of LC3II protein expression,the decrease of P62 protein and the increase of autophagy.Under the action of LY294002,the expression of PI3K,p-AKT and p-mTOR protein was down-regulated by Western blot,and the expression of PTEN protein was up-regulated after the inhibition of PI3K/AKT pathway.These results suggest that this signaling pathway may play an important role in regulating malignant transformation of Het-1A cells under the combined action of HPV and MNNG,and affect the progression of malignant transformation of Het-1A cells.?.Main conclusions as follow:1 The combination of HPV and MNNG can promote malignant transformation of Het-1A cells.HPV plays a synergistic role in promoting cancer and accelerates the progression of malignant transformation of Het-1A cells.2 The expression of P62 gene can affect the malignant transformation progression of Het-1A cells induced by HPV and MNNG.Inhibition of expression of P62 gene can cause cell proliferation to decrease,cell cycle arrest,cell apoptosis rate to increase,and the degree of oxidative damage of cells to increase,antioxidant capacity to weaken.3 HPV transfection up-regulates the expression of P62 and mTOR in Het-1A cells.The expression of mTOR is proportional to the expression of P62.Intervention of P62 gene expression can regulate the mTOR-related signaling pathway.4.During the combined action of HPV and MNNG,the malignant transformed Het-1A cells overexpressed P62 protein,which increased the transcription of antioxidant genes HO-1 and NQO-1 by activating P62-Keap1-NRF2 signaling pathway,enhanced the antioxidant capacity of cancer cells,and further promoted the occurrence and development of cancer..5.Under the action of PI3K inhibitor LY294002,cell proliferation was inhibited,cell cycle was blocked,apoptotic level was increased,and related functional proteins also changed significantly.The blockade of PI3K/AKT signaling pathway can inhibit the development of cancer cells and weaken the anti-apoptotic ability of cells.