The Role Of Rab31 In The Invasion And Metastasis Of Cervical Cancer

BackgroundCervical cancer(CC)is one of the most common gynecological malignant tumor,75%?85%are squamous cell carcinoma,followed by adenocarcinoma.The main risk factor for this cancer is the high-risk human papillomavirus.HPV is a DNA virus that infects human epidermis and mucous membrane squamous epithelium.High-risk HPV encodes three early proteins,of which E5 has a weak transformation effect.E6,E7 are viral oncogenes,which are involved in regulating the expression and replication of viral genes in host cells,resulting in the inactivation of tumor suppressor genes P53 and Rb,and cause cells are infinite proliferation.HPV is mainly transmitted sexually.The patient's symptoms are not obvious in the early stage and vaginal bleeding occurs in the later stage.In recent years,cervical cancer vaccines have been marketed.Vaccination has greatly reduced the incidence of cervical cancer.The popularity of regular cervical cancer screening also detects precancerous lesions early and treats them in a timely manner.However,there are still difficulties in the treatment of patients with cervical cancer,especially advanced cervical cancer invades and oppresses adjacent tissues and organs,and transfers to other parts through the lymph and blood,causing a poor prognosis.Cancer invasion and metastasis is an important cause of patient death.Cervical cancer metastasis is mainly spread directly to adjacent tissues and lymphatic metastasis.Cervical cancer invade the myometrium,paravaginal tissues and tissues around the pelvis,advanced cancer cells drain to all levels of lymph nodes with lymph fluid.Rab protein is a number of small GTP binding proteins.It is mainly localized in the trans-Golgi network and endosomes,which responsible for regulating the material transport,vesicles and material transport.Many studies have shown that Rab31 plays a role in the development of cancer and is related to the prognosis of various cancers.Rab31 is highly expressed in breast cancer tissues and promotes cancer cell proliferation,which is an independent prognostic factor for breast cancer;Rab31 expression is upregulated in glioblastoma multiforme and affects its development;Rab31 is highly expressed in pancreatic cancer and is a prognostic marker of low survival rate;Rab31 is highly expressed in osteosarcoma tissues and promotes the proliferation,invasion and metastasis of cancer cells;Rab31 is highly expressed in gastric cancer tissues and promotes the proliferate and inhibits the apoptosis.However,the role and mechanism of Rab31 in cervical cancer invasion and metastasis have not been specifically studied.Our previous mass spectrometry results show that HPV E6 and E7 oncogenes up-regulate the expression of Rab31.Therefore,this experiment investigated the effect and mechanism of Rab31 on cancer invasion and metastasis in cervical cancerObjectiveExplore the role and mechanism of Rab31 in cervical cancer invasion and metastasis.Methods and results1.Rab31 is highly expressed in cervical cancer tissues and cell linesUsing qRT-PCR and Western Blot assay,the mRNA and protein expression levels of Rab31 were detected in HaCaT cells and cervical cancer cell lines HeLa,SiHa,CaSki,and C33a.The results showed that Rab31 was highly expressed in cervical cancer cells and higher in HPV-positive cells.The expression of Rab31 in non-cancerous and cervical cancer tissues was detected by qRT-PCR,and the result showed that the expression of Rab31 in cervical cancer was higher than that of non-cancer tissues.It can be seen that Rab31 showed high expression level in cervical cancer.2.Decreasing the expression of Rab31 can inhibit the invasion and metastasis of cervical cancer cells.Overexpression of Rab31 can promote the invasion and metastasis of cervical cancer cells.HPV18-type HeLa cells and HPV 16-type SiHa cells with high expression of Rab31 were used as research objects.The expression of Rab31 was knocked down in two types of cells using lentiviral infection technology.Wound-healing assay and transwell test were used to investigate the effect of low expression of Rab31 on the invasion and metastasis of cervical cancer cells.The results showed that knocking down the expression of Rab31 reduced the number of invasion and metastasis of the two cells and inhibited the invasion and metastasis capacity.Rab31 was overexpressed with the plasmid in relatively low-expressing C33a cells,Overexpression of Rab31 promoted the invasion and metastasis ability of cervical cancer cells.To the contrary,Rab31 overexpression promoted the ability of cervical cancer cells to invade and migrate.3.Reducing the expression of Rab31 inhibites cervical cancer cells invasion and metastasis,which through inhibits the cancer cells EMT pathway and reduces the aggregation of F-actin.In HeLa and SiHa cells,the expression of EMT pathway-related biomarkers was detected by WB.After knocking down the Rab31,the expression of epithelial marker E-cadherin was increased,and the mesenchymal markers N-cadherin,Vimentin,snail and slug expression were reduced.The matrix metalloproteinases MMP2 and MMP9 that degrade the extracellular matrix are reduced,and the ability of cells to invade and migrate is reduced.Cellular immunofluorescence assay also showed that after reducing Rab31 expression,E-cadherin expression increased and Vimentin expression decreased.In addition,F-actin staining of cytoskeleton with ghost pen cyclic peptide showed that after down-regulating the expression of Rab31,F-actin expression was reduced and pseudo-foots are reduced,the ability of cells to invade and migrate is decreased.After Rab31 was overexpressed,the expression of mesenchymal markers Vimentin,Snail,and Slug increased,and the cells transformed to mesenchymal type,which promoted the invasion and metastasis of cancer cells.4.Rab31 interacts with MAPK6 to promote the invasion and metastasis of cervical cancerThe results of CO-IP experiments showed that there was an interaction between Rab31 and MAPK6,and WB results showed that after interfering with Rab31 expression,the protein expression of MAPK6 were decreased.HeLa and SiHa cells were transfected with small interference to interfere with the expression of MAPK6.CCK8 and Transwell tests showed that the proliferation rate of cancer cells slowed down and inhibited cancer cell invasion and metastasis.In two types of cells,we interfered with Rab31 and overexpressed MAPK6 at the same time.We found that overexpression of MAPK6 partially reversed the inhibition of Rab31 low expression on cell invasion and metastasis,indicating that the effect of Rab31 expression on the cancer cell invasion and metastasis depends on the MAPK6 molecule partly.ConclusionsRab31 molecule is highly expressed in cervical cancer tissues and cells.Rab31 increases the expression of EMT-related interstitial marker proteins,reduces the expression of epithelial marker proteins,promotes the degradation of extracellular matrix,and causes EMT in cells.And by reducing the aggregation of F-actin,it reduces the formation of pseudopods and the migration of cervical cancer cells.Finally,we demonstrate that Rab31 interacts with MAPK6 to jointly promote the invasion and metastasis of cervical cancer.