Downregulated TFAM Enhances The Mycoplasma Infection To Promote The Migration And Invasion Of Hepatocellular Carcinoma Cells

Background:Microbial infection is considered to be a major factor inducing malignant tumors.Infection with certain viruses,bacteria,and parasites is responsible for approximately16.1% of new cancer cases worldwide.For instance,the major risk factors for hepatocellular carcinoma（HCC）are chronic hepatitis B virus（HBV）and hepatitis C virus（HCV）infection,and they are associated with malignant progression.Therefore,the prevention and treatment of infection have a significant impact on the prevention and treatment of malignant tumors.HCC is one of the most common malignant tumors in humans.Although HBV infection plays an important role in HCC,it may not be the only pathogen of HCC.However,the role of other infectious microorganisms in human HCC has not been extensively studied.Mycoplasmas are the smallest and simplest prokaryotes that can survive independently in an inanimate medium and have double-stranded DNA（dsDNA）.Mycoplasmas are widespread in various hosts.They depend on various growth factors produced by the host that cannot be produced by themselves.Mycoplasmas may cause various diseases in animals.In recent years,the occurrence of mycoplasma infection was observed in many tumor types.In addition,mycoplasma infection has recently been proven to affect the host’s metabolic pathways and considerably facilitates the development of gastric cancer.However,the factors mediating mycoplasma infection and the biological effects of mycoplasma infection in HCC remain unclear.Our previous experiment showed that Mycoplasma infection was significantly increased in HCC cells with stable knockdown of mitochondrial transcription factor A（TFAM）when compared to control cells.Based on this,we hypothesis that the decreased expression of TFAM may enhance mycoplasma infection in HCC cells by damaging mitochondrial function,causing the change of a series of downstream related molecules’ expression,which may lead to the malignant progression of hepatocellular carcinoma.Aims:1.To determine whether the decreased expression of TFAM enhances mycoplasma infection in HCC cells.2.To explore the molecular mechanism of the decreased expression of TFAM enhances mycoplasma infection in HCC cells.3.To investigate the effect of mycoplasma infection enhanced by the decreased expression of TFAM on the migration and invasion of HCC cells.Methods:1.Immunofluorescence staining and Western blot were used to detect mycoplasma infection in HCC cell lines using anti-dsDNA antibody and anti-mycoplasma specific membrane protein p37 antibody;the cell membrane fluorescence probe wheat germ agglutinin（WGA）was used to detect the location of dsDNA and p37.2.MycoAlert ^TM PLUS Mycoplasma detection kit was used to detect whether HCC cells were infected with mycoplasma.3.RNA sequencing of TFAM knockdown HCC cells was carried out to screen the transcriptional changes of previously reported molecules that can interact with mycoplasma protein p37 in TFAM knockdown cells.4.Western blot and QPCR were used to detect the changes of protein and mRNA levels of TFAM and key molecules of mycoplasma infection.5.Immunohistochemical technique was used to analyze the correlation between TFAM and mycoplasma specific membrane protein p37 and the expression of related signal pathway molecules regulated by TFAM in 100 HCC patients.6.MitoSOX mitochondrial superoxide fluorescence probe was used to detect the effect of TFAM on the generation of mitochondrial reactive oxygen species（ROS）of HCC cells.7.Scratch experiment and Transwell migration and invasion experiment were used to analyze the influence of TFAM and mycoplasma infection on the migration and invasion of HCC cells.Results:1.Immunofluorescence staining and Western blot detection showed that the decreased expression of TFAM significantly enhanced mycoplasma infection in HCC cells using anti-dsDNA antibody and anti-mycoplasma specific membrane protein p37 antibody;On the contrary,this phenomenon was reversed after restoration of TFAM expression,and overexpression of TFAM significantly attenuated mycoplasma infection in HCC cells.2.The results of Mycoplasma detection showed that the HCC cells were successfully infected with mycoplasma after they were co-culture.3.The RNA sequencing results showed that ANXA2（annexin A2）was the most up-regulated gene in mRNA level of TFAM knockdown HCC cells,among the previously reported molecules that could interact with mycoplasma protein p37.4.Western blot and QPCR results displayed that decreased expression of TFAM promoted the mycoplasma infection of HCC cells by up-regulating the expression of transcription factor Sp1（specificity protein 1）and then up-regulating the expression of ANXA2.5.The immunohistochemical staining results showed that TFAM was negatively correlated with the expression of p37 and ANXA2 in HCC tissues,and mycoplasma infection was positively correlated with poor prognosis of HCC patients.6.MitoSOX staining showed that the production of mitochondrial ROS of HCC cells with TFAM knockdown increased.7.Migration and invasion experiment showed that mycoplasma infection enhanced by down-regulation of TFAM expression promoted the migration and invasion of HCC cells by activating NF-k B signaling.Conclusions:In our current study,we found that the generation of mitochondrial ROS of HCC cells increased after the down-regulating of TFAM expression,and then increased the expression level of ANXA2 by up-regulating the transcription factor Sp1,enhanced the mycoplasma infection in HCC cells and promoted the migration and invasion of HCC cells by activating the NF-k B signal pathway.