Regulation Mechanism Of Oncogenic Microorganisms HPV And H.pylori In Mediating Malignant Transformation By Activating MTOR Signaling Pathway

BackgroundContinuous chronic infection of oncogenic microorganisms(including viruses or bacteria)is one of the main causes of uncontrolled cell proliferation and subsequent malignant transformation.A World Health Organization/International Center for Cancer Research(WHO/IARC)report in 2012 pointed out that about 17%of cancers in the world are directly related to certain tumor-associated virus or bacterial infections.Studies have confirmed that high-risk human papillomavirus(High-risk Human papillomavirus,HPV)and Helicobacter pylori(H.pylori)are Class A carcinogenic pathogens that cause human cancer(HPV and H.p can cause humans The proportion of pathogens of cancer is 31.5%and 22.0%respectively).The proliferation disorder and malignant transformation of cervical squamous epithelium and gastric mucosal epithelial cells are the result of the interaction between environmental factors(external factors)and host regulatory responses(intrinsic factors).Therefore,it is an important scientific issue to analyze the mechanism of inflammatory cancer transformation related to HPV and H.pylori infection,and it is of great significance to effectively reduce the occurrence of malignant transformation related to the cervix and stomach.Persistent infection of high-risk HPV is the main initiating factor of cervical cancer.HPV transforming genes E6 and E7 are continuously expressed in tissues,and mediate the degradation of host tumor suppressor protein p53 and degenerative retinoblastoma protein pRb.In addition,HPV infection can participate in the process of cervical cell malignant transformation by leading to increased host gene instability,host gene methylation patterns,and non-coding RNA disorders.H.pylori colonization of gastric mucosal epithelial cells can promote malignant transformation and increase the risk of gastric cancer by regulating tumor-related molecules such as RUNX3,P53,E-cadherin and β-catenin.In addition,CagA,the virulence factor of H.pylori infection,injects peptidoglycan into gastric mucosal epithelial cells through type Ⅳ secretion system,which triggers the inflammation microenvironment of the stomach area and stimulates the mutation of host genetic material,which is the early motility mediating malignant transformation.Exploring the pathogenesis of HPV and H.pylori is the basic research work for early identification and intervention of tumors,HPV and H.pylori infection can induce Malignant transformation through interaction with different pattern recognition receptors and signaling pathways in host cells.In addition to inhibiting the expression of p53 and pRb genes,HPV also interacts with the main upstream pathways:growth factor receptors,Notch receptors,Ras and PI3KCA,etc.to activate and change the mTOR signaling pathway,stimulate the survival and proliferation of host cells,and induce infection Malignant transformation of cells.In addition to stimulating gastric mucosal epithelium to release cytokines,H.pylori infection can also activate NF-κB,Wnt,JNK and mTOR signaling pathways by recognizing multiple pattern recognition receptors such as NOD-like receptors,Notch receptors and Toll-like receptors.It forms a positive feedback loop for H.pylori to promote the expression of inflammatory factors,participates in regulating the host immune response and inflammatory response of pathogenic microorganism infection,and mediates uncontrollable inflammation to increase the risk of inflammatory cancer transformation.Research evidence shows that HPVand H.pylori infection can activate the mTOR pathway by affecting gene mutations in the mTOR signaling pathway and the activation of upstream signaling molecules,and the activation of this pathway can further lead to genetic instability,uncontrolled proliferation,apoptosis resistance,and changes in metabolic characteristics,Which eventually leads to the malignant transformation of infected cells.The mTOR signaling pathway may play an important role in the malignant transformation induced by HPV and H.pylori infection.Therefore,further exploration of the molecular mechanism of HPV and H.pylori infection activating the mTOR signaling pathway and the biological role of this pathway in HPV and H.pylori-induced malignant transformation will help improve disease monitoring and prevention and provide new ideas for targeted therapy.Part Ⅰ:HPV-16 E7 up-regulates miR-106a targeting LKB1 and activates mTOR signaling pathway to regulate proliferation and autophagy of cervical squamous cell carcinoma cellsHigh-risk HPV-16 infection is most closely related to cervical squamous cell carcinoma,and the continuous expression of its transforming genes E6 and E7 is the main cause of cervical malignant transformation.E6 and E7 degrade the tumor suppressor p53 and pRb,respectively,leading to cell proliferation disorder and inducing cell transformation.E6 and E7 can also regulate host microRNAs(miRNAs)by up-regulating transcription factors to affect host gene expression,thereby destroying the stability of cell growth and survival mechanisms,leading to cervical malignant transformation and even cervical cancer.miR-106a is a member of the miR-17 family,and its role in the.occurrence and development of cancer seems to be tissue or cell specific.At present,it is unclear whether miR-106a can regulate the expression of host miR-106a in HPV-related cervical cancer,especially whether HPV and its transforming genes can regulate the expression of host miR-106a.Therefore,studying the role and mechanism of miR-106a in HPV-positive cervical cancer is of great value in revealing the molecular regulation mechanism of HPV carcinogenesis and selecting effective therapeutic targets.It has been reported in the literature that the activation of mTOR(Mammalian target of rapamycin)may be related to HPV virus infection.Studies have shown that HPV-16 E7 in cervical cancer can reduce the expression of the tumor suppressor gene LKB1,and LKB1 is an important key node upstream of mTOR.A number of studies,including previous studies of our research group,have reported that the LKB1/AMPK/mTOR signaling pathway plays an important role in cervical cancer.The tumor suppressor LKB1 activates its downstream AMPK,negatively regulates mTOR,and inhibits cell proliferation and transformation.And migration.LKB1 can also affect the PI3K/Akt/mTOR signaling pathway by regulating PTEN and inhibit tumor progression.However,what is the role of miR-106a in HPV-positive cervical cancer?Is its molecular mechanism related to the LKB 1/AMPK/mTOR pathway?It is not clear that if high-risk HPV-16 infection regulate the expression of miR-106a in host cells.Objective1.To explore the expression and clinical significance of miR-106a in HPV-16 positive cervical squamous cell carcinoma tissues and cell lines.2.To investigate the effects of miR-106a on the proliferation and autophagy of cervical cancer cells and its molecular regulation mechanism.3.To explore the regulatory effect and mechanism of HPV-16 transformed gene E7 on miR-106a.Results1.Elevated expression of miR-106a associated with malignant clinicopathologic features in HPV-16-induced patients with cervical squamous cell carcinoma.In the 91 cases of CSCC,72 samples tested positive for HPV-16(79.1%).RT-qPCR showed that miR-106a expression was significantly upregulated in HPV-16-positive cervical squamous cell carcinoma tissue samples.The ROC curves for miR-106a reflected strong separation between CSCC tissues and nontumorous tissues.the elevated miR-106a was highly correlated with large tumor size and poor differentiation,high-grade FIGO staging,and lymph node metastasis of cervical squamous cell carcinoma,but there was no obvious correlation with the patient’s age,vascular invasion,vaginal wall stump invasion and other parameters;multivariate analysis showed that the high expression of miR-106a is related to tumor size,lymph node metastasis,lymphatic invasion and other factors.It suggests that that miR-106a may be involved in cervical carcinogenesis and tumor progression.2.LKB1 is a.new target of miR-106a in HPV-16 positive cervical cancer.The miRNA prediction algorithm Targetscan,micro-RNA.org,and miRDB interactions and the luciferase reporter gene assay showed that LKB1 was a direct target of miR-106a.RT-qPCR results showed that miR-106a expression was increased in cervical cancer cell lines(Caski Siha and HeLa)compared with control cells HaCaT,especially in HPV-16 positive Caski cells,the increase was most significant.RT-qPCR and Western blot results showed that miR-106a could negatively regulate the expression of LKB 1.Spearman correlation analysis showed that the expression of LKB1 was significantly decreased in HPV-16-positive cervical squamous cell carcinoma tissue samples,and the correlation analysis of clinicopathological parameters showed that the low expression of LKB 1 was correlated with the malignant pathological characteristics of cervical cancer patients.3.miR-106a promotes proliferation of cervical cancer cells by targeting LKB1.The results of CCK-8,clone formation and EDU experiments showed that miR-106a significantly promoted the proliferation of cervical cancer Caski cells,but the proliferation of cervical cancer cells was promoted/inhibited respectively after the knockdown/overexpression of LKB 1 in Siha and HeLa cells(both of which lacked LKB1 expression),indicating that LKB1 inhibited the proliferation of cervical cancer cells.The LKB1 rescure experiment in CaSki cells was further verified,both plate cloning and EDU results showed that overexpression of LKB 1 could partially reverse the proliferation promotion effect of miR-106a-mimics on cervical cancer cells.However,knockdown of LKB 1 reversed the reduction of cell proliferation induced by miR-106a-inhibitor,indicating that LKB1 could reverse the proliferation promotion effect of miR-106a on cervical cancer Caski cells.The above experiments confirmed that miR-106a promoted the proliferation of cervical cancer Caski cells by targeting LKB1.4.miR-106a negatively regulates autophagy in HPV-16 positive cervical cancer cells by targeting LKB1.Western blot results showed that miR-106a could significantly reduce autophagy in CaSki cells;in SiHa and HeLa cells without LKB1,miR-106a has no obvious effect on autophagy-related proteins.Further testing results showed that miR-106a in CaSki cells also inhibited rapamycin and starvation-induced autophagy.Western blot results showed that LKB1 promoted the level of autophagy.LKB1 rescue experiments o proved that the overexpression of LKB1 can reverse the miR-106a on autophagy.The above results indicate that miR-106a inhibits autophagy of HPV-16-related cervical cancer by targeting LKB1.Autophagy flow test results showed that miR-106a could inhibit the number of autophagosomesn(yellow spots)and autolysosomes(red spots)in CaSki cells,and miR-106a significantly inhibits rapamycin and starvation autophagy flow,while LKB1 promotes the autophagic flux of cervical cancer cells.The above results indicate that miR-106a inhibits autophagy in CaSki cells by targeting LKB1.5.miR-106a affects AMPK-mTOR signaling pathway by targeting LKB1.Western blot results showed that miR-106a could inhibit the expression of p-AMPK and promote the expression of p-mTOR.The rescue experiment of LKB1 showed that overexpression of LKB1 weakened the effect of miR-106a on the inhibition of p-AMPK expression and the promotion of p-mTOR expression;in addition,knocking down LKB1 could partially reverse the effect of miR-106a-inhibitor on p-AMPK The effect of p-mTOR levels.It shows that miR-106a activates AMPK-mTOR signaling pathway by targeting LKB1 in HPV-16 positive CaSki cells.6.HPV-16 E7 activates the expression of miR-106a and regulates its function.RT-qPCR results showed that the expression of miR-106a was significantly increased in the HPV-16 E7 stable expression cell lines RPE1-E7 and HaCaT-E7,and As compared with CaSki-siE6 cells,the expression of miR-106a was significantly decreased in CaSki-siE6E7 cells.In addition,in HPV-16 E7-positive cervical squamous cell carcinoma tissues,the expression of miR-106a was positively correlated with the expression of E7.RT-qPCR and Western blot results showed that compared with the blank vector(Vector),the expression of LKB1 in the above two E7-expressing cells was reduced;however,when E7 cells were transfected with miR-106a inhibitor,LKB1 expression could be partially restored.It shows that E7 could inhibit the expression of LKB1 by activating miR-106a.Among E7 expressing cells,the proliferation activity of CCK-8 cells was significantly higher than that of blank vector cells.After transfection with miR-106a inhibitor,the cell proliferation rate of E7 cells decreased.It shows that HPV,-16 E7 can activate the expression of yimiR-106a and regulate its proliferation function.7.HPV-16 E7 up-regulates miR-106a expression through activation of transcription factor c-Jun.Predict the transcription factor c-Jun could mediate miR-106a,the CHIP assay showed that miR-106a is a direct target of c-Jun.RT-qPCR and Western blot results show that E7could mediate the expression of c-Jun.The rescue of c-Jun with reduced E7 expression shows that c-Jun can partially reverse the reduction of miR-106a after E7 knockdown.The above experimental results prove that HPV-16 E7 promotes miR-106a in cervical cancer by enhancing the expression of the transcription factor c-Jun expression.ConclusionsIn this study,we systematically discussed the role of miR-106a in HPV-16 positive cervical cancer and its molecular mechanism.miR-106a is highly expressed in HPV-16-positive cervical squamous cell carcinoma tissues and cells,and is related to the malignant pathological characteristics of patients.Studies have found that the tumor suppressor gene LKB1 is a new target of miR-106a,and miR-106a promotes the proliferation of cervical cancer cells and inhibits autophagy by targeting LKB1 to activate the AMPK-mTOR signaling pathway,thereby exerting carcinogenic effects.In addition,HPV-16 E7 could up-regulate the expression of miR-106a and promote cell proliferation through the transcription factor c-Jun.This study provides a new experimental basis for further elucidating the molecular mechanism of HPV-16 carcinogenesis and finding new therapeutic targets for HPV-related cervical cancer.Part Ⅱ:H.pylori mediates gastric malignant transformation through NF-κB up-regulation of MY ADM and activation of mTOR signaling pathwayInflammation caused by Helicobacter pylori(H.pylori)infection is an important basis for mediating malignant transformation.H.pylori colonizes gastric mucosal epithelial cells by up-regulating transcription factors such as NF-κB,AP-1 and IRFs,and further induces IL-1β,IL-8 and TNF-α and other inflammatory cancer cross-cytokine production,and activates downstream NF-κB,Wnt/β-Catenin,EGFR,FAK,and mTOR signaling pathways form a positive feedback loop that promotes the expression of inflammatory factors by H.pylori,increasing the risk of inflammatory cancer transformation.MYADM(Myeloid-associated differentiation marker,myeloid-associated differentiation marker gene)is a hematopoietic-related marker gene that is expressed in pluripotent progenitor cells and is up-regulated during myeloid differentiation.Related studies have shown that MYADM can encode the FAK subfamily of protein tyrosine kinases,which are abnormally expressed in a variety of inflammations and tumors.For example,in the evolution of non-alcoholic fatty liver disease from simple fatty liver to steatosis,inflammation and necrosis to cirrhosis,the expression of MYADM gradually increases.MYADM is highly expressed in tumors such as melanoma,hormone-resistant prostate cancer and colorectal cancer and is related to tumor metastasis and poor prognosis.However,the expression change and biological function of MYADM in malignant transformation of stomach,and whether it is related to H.pylori infection has not been reported yet.Studies have shown that H.pylori infection can activate the PI3K/Akt/mTOR signaling pathway and promote the proliferation of gastric epithelial cells.However,what is the expression and function of MYADM in H.pylori-infected gastric mucosal epithelial tissues and cells?Could H.pylori infection regulate the expression of MYADM in host cells and what is the regulation mechanism?It is not clear that the downstream molecular mechanism of MYADM related to the PI3K/Akt/mTOR pathway.Objective1.To explore the expression and clinical significance of MYADM in the malignant transformation related to H.pylori infection.2.Explore whether H.pylori could regulate the expression of MYADM and its molecular mechanism.3.Reveal the role and molecular mechanism of MYADM in gastric malignant transformation.Methods and results.1.MYADM is upregulated in GC tissues and cells.Oncomine,TCGA and GEO.analyses showed that MYADM expression was significantly increased in tumor samples.RT-qPCR showed that,the expression of MYADM was also significantly increased in human GC samples.Moreover,The MYADM mRNA level was also significantly increased in human GC samples when compared with those in gastritis samples.Consistently,compared with GSE-1,the expression of MYADM was significantly increased in GC cell lines.The protein expression of MYADM in human GC biopsies was visibly increased.2.MYADM expression is correlated with gastric Pathological process and its high expression predicts poor GC prognosis.IHC staining suggested that MYADM expression was strikingly increased in GC tissues in the progress of gastric malignant transformation.Furthermore,compared with the normal corresponding adjacent tissues,MYADM were also significantly increased in human GC samples.Kaplan-Meier analyses showed that high expression of MYADM significantly correlated with the poor overall survival(OS),disease-free survival(DFS)and recurring progression survival(RPS)of GC patients.These results strongly indicated that MYADM had an oncogenic role in GC.3.MYADM promotes GC cells proliferation in vitro and tumor formation in vivo.GC cells were transfected with MYADM small interfering(siRNA)or Overexpression plasmid.Colony formation,CCK-8 and EdU assays suggest that MYADM contributes to GC cells proliferation in vitro.The MYADM stably knocked down in the LV-shMYADM-transfected MKN-45 cells were then subcutaneously injected into the nude mice.The growth of the xenograft tumors in the LV-shMYADM group was significantly slower and with lighter tumor weight.RT-qPCR showed that,MYADM mRNA levels were also significantly decreased.Moreover,the subcutaneous xenograft tumors in the LV-NC group showed more easily local tumor invasion,with cancer cells invading into the surrounding muscle tissue.In addition,IHC staining showed that MYADM and Ki67 expression was significantly reduced in LV-shMYADM group.Altogether,we confirmed that MYADM could promote GC cells proliferation in vitro and tumorigenicity in vivo.4.MYADM facilitates the invasion and metastasis of GC cells in vitro and in vivo.Transwell and wound healing experiments showed that MYADM the invasion,migration and EMTof GC cells.The hematogenous metastasis of the nude mice with tail vein,in vivo imaging and hematoxylin and eosin(HE)staining showed that less and smaller volumes of metastatic loci occur in the lungs of the mice in the LV-shMYADM group than those in the LV-NC group.Moreover,some of the metastatic loci invaded the lung capsule in the LV-NC group,whereas none was observed in the LV-shMYADM group.5.MYADM activates mTOR signaling pathway in GC cells.Western blot results showed that Knock down of MYADM reduced the expression of p-PI3K,p-Akt and p-mTOR in GC cells,but overexpression of MYADM increased the expression of p-PI3K,p-Akt and p-mTOR in GC cells while the change of the total proteins were not obvious.The results were further confirmed in LV-shMYADM and LV-shNC transfected MKN-45 cells.The above results suggest that MYADM regulates the PI3K/Akt/mTOR pathway.6.H.pylori upregulates MYADM expression.We tested whether MYADM expression could be regulated by H.pylori infection in GC cells.Both MYADM mRNA and protein levels were significantly increased by H.pylori infection,in a dose and time-dependent manner.Indeed,the MYADM mRNA expression was higher in H.pylori-positive human AG samples.Consistent with these results,MYADM protein expression was also increased in H.pylori-positive human AG samples.IHC staining in the gastritis mouse model infected with H.pylori indicated that,MYADM levels were higher in H.pylori infection group when compared with the non-infected control group.IHC staining of the MNU(N-methyl-N-nitrosourea)-treated gastritis mouse model with H.pylori infection showed that,compared with the control group,MYADM expression was slightly increased in the non-infected MNU group,but dramatically increased after H.pylori infection.These results confirmed the important role of MYADM in H.pylori-related malignant transformation.7.MYADM is transcriptionally activated by c-Jun.As predicted by PROMO3.0.2,MYADM promoter contains nine c-Jun binding site.Luciferase reporter assays and ChIP-PCR assays showed that c-Jun could directly bind to the MYADM promoter region.Moreover,RT-qPCR and western blot results showed c-Jun could mediate the expression of myadm.our data strongly indicated that MYADM was a target gene of c-Jun.We examined whether H.pylori could upregulate MYADM expression via c-Jun.RT-qPCR and western blot results that H.pylori-mediated upregulation of MYADM was relieved by c-Jun knockdown both in mRNA and protein levels,suggesting that c-Jun played an important role in the overexpression of MYADM triggered by H.pylori.8.H.pylori upregulates MYADM expression by the NF-κB pathway.In order to determine how H.pylori infection upregulated MYADM expression,five key signaling pathways inhibitors were added before infection with H.pylori in GES-1,AGS and MKN-45 cells.Western blot showed that the upregulation of MYADM protein expression induced by H.pylori infection was blocked by NF-κB inhibitor strongly.In addition,RT-qPCR showed that induction of MYADM mRNA expression by H.pylori was also blocked by Bay 11-7082 treatment.western blot also showed that,c-Jun and MYADM expression by H,pylori was also blocked by Bay 11-7082 treatment.Our results indicated that infection of H.pylori increased MYADM expression through activation of the NF-κB pathway.ConclusionsIn this study,we systematically studied the role and molecular mechanism of MYADM in the malignant transformation associated with H.pylori infection.MYADM is related to the evolution of gastric malignant transformation associated with H.pylori infection,and MYADM is highly expressed in H.pylori-positive gastritis and gastric cancer tissues and cells,which is related to the poor prognosis of gastric cancer patients.Studies have found that H.p activates the NF-κB signaling pathway to up-regulate the expression of the transcription factor c-Jun to promote MYADM transcription,which in turn activates the PI3K/Akt/mTOR signaling pathway to promote the ability of gastric cancer cells to proliferate,invade and metastasize in vivo and in vitro.It shows that MYADM may be a potential target of Helicobacter pylori-related gastric malignant transformation.The above studies help to clarify the new mechanism of H.pylori-induced gastritis and cancer transformation,and provide experimental evidence for the development of effective early diagnosis,treatment and discovery of new targets for H.pylori-related gastric diseases.