| Back ground: Cancer of cervix is the most common one of malignant tumors in thefemale genital system, it is still the first incidence in the women in some developpingcountries. Some documents reported that patients of cancer of cervix increased from378,000to454,000from1980to2010, the mean annual incremental rate was0.6%. In somedevelopping countries,76%of cervical cancer patients take residence in unde-veloppedareas. For the past few years, along with widespread mass screening for cancer of cervix,the prevalence and mortality for cancer of cervix descend obviously in some countriesand regions, but incidence of cancer of cervix for young women takes on increasingtendency. In Xinjiang, compared with Hazakh, the death rate of the Uygur nationalitywas higher about one time than Hazakh (9.67/100,000). Now, operation andchemoradiation are the leading therapeutic tools for cance of cervix. For cervical cancerpatients in the early stage, operation is more effective therapy, chemoradiation has to beused because the cervical cancer patients in the middle or advanced stage have nooperation opportunity, but the side-effects of chemoradiation often make therapies not tokeep going. Metastasis and infiltration are the important factors of mal-curative effectand unfavourable prognosis for cancer of cervix, so further study on the mechanism ofmetastasis and infiltration of cancer of cervix is of the considerable significance for itsclinical treatment. Researches of many years show that enzymatic active and expressionin tumor cells can change to adapt to their fast proliferation, mitoses, infilitration andmetastasis, for example, synthesis of protein, lipids and nucleic acid enhance. All vitalmovements of cancer cells depend on water microenvironment, which furtherly needsfast water transmembrane transport compared with normal cells, and the volume andmorphology of cancer cells will change when they infiltrate surrounding substrates andblood or lymphatic vessels. A lot of studies from knockout mice implicate AQPs to takepart in many expected physiological functions, including urine concentration andexocrine gland secretion, as well as, including brain swelling, neural signal transduction, skin moisturization, and fat metabolism. The aquaporins (AQPs) are a family of small,integrate glucoproteins, facilitating osmotically driven water transport selectively acrosscell plasma membranes. Up to now, there are13members (AQP0～12). Recently somestudies on AQPs indicated that AQPs took part in cell migration and proliferation andenhanced local invasiveness and metastasis via blood and lymph vessels. In this study,preliminarily screening AQP1, AQP3, and AQP8subtypes expressing in cancer of cervixby using flourescent real-time PCR (RT-PCR), immunofluorescence and immunohisto-chemistry, and then selecting AQP8gene as the aim gene, transfecting aim gene AQP8into SiHa cells by lentivirus vector, probing the AQP8influence on migration, prolifera-tion, substrate adherence and local invasiveness of SiHa cells according to AQP8geneoverexpression and expression silence, respectively. Objective: To screen AQPssubtypes idio-expressing in cancer of cervix, and to investigate AQP subset effects onmigration, proliferation, substrate adherence and local invasiveness of SiHa cells in vivoand in vitro. Methods:1) Comparing the difference of osmotic water permeability in4kinds of cervical carcinoma cell lines SiHa, HeLa, Caski, ME-180by osmotic waterpermeability measurement and selecting cervical carcinoma cell line of low waterpermeability for the experiment, and then examining expression of isoforms ofaquaporins by Real-Time PCR and immunofluorescence, furtherly selecting idio-aquaporins expressing in cervical carcinoma tissues by immunohistochemistry;2)Building AQP8overexpression plasmid pLVX-AQP8-IRES-tdTomato, packaginglentivirus particles by calcium phosphate transfection, then infect SiHa cells, screeningstably expressing AQP8SiHa cell line by RFP (red fluorescence protein) reporter gene;building AQP8gene silent plasmids, pLVX-shRNA2-AQP8-1, pLVX-shRNA2-AQP8-2,pLVX-shRNA2-AQP8-3and pLVX-shRNA2-AQP8-4, transfecting293T cells usinglipofectamine2000, screening highest silent rate plasmid by Western Blot, packaginglentivirus particles by calcium phosphate transfection, then infecting SiHa cells,screening stably expressing AQP8gene silence SiHa cell line by GFP (greenfluorescence protein) reporter gene;3) Probing AQP8gene influence on migration andlocal invasion of SiHa cells by transwell migration and invasion assays, wound healingassay, tumor cell growth, substrate adherence assays and subcutaneous xenografts assay.Results:1) In SiHa, osmotic water permeability differences between100mOsm and300mOsm groups,200mOsm and300mOsm groups were not significant (P＞0.05),osmotic water permeability differences between400mOsm and300mOsm groups,500mOsm and300mOsm groups were significant (P＜0.05); In Caski, osmotic water permeability differences between100mOsm and300mOsm groups,200mOsm and300mOsm,500mOsm and300mOsm groups were significant (P＜0.05), osmotic waterpermeability differences between400mOsm and300mOsm groups were not significant(P＞0.05); In HeLa, osmotic water permeability differences between100mOsm and300mOsm groups,400mOsm and300mOsm groups were significant (P＜0.05), osmoticwater permeability differences between200mOsm and300mOsm,500mOsm and300mOsm groups were not significant (P＞0.05); In ME-180, osmotic water permeabilitydifferences among groups were significant (P＜0.05); AQP0, AQP1, AQP2, AQP3,AQP4, AQP5, AQP8were isoforms of aquaporins expressing in SiHa cells; MVD ofAQP1were43.6±17.8,56.2±11.6,70.8±21.1, respectively, in control group, CIN3and cervical carcinoma group. Difference among three groups was significant (P＜0.05);Positive rates of AQP3were13.33%,26.67%,48.57%, respectively, in control group,CIN3and cervical carcinoma group. Difference between cervical carcinoma and controlgroup was significant (P＜0.05); Positive rates of AQP8were46.67%,86.67%,54.29%,respectively, in the three groups. Difference among three groups was significant (P＜0.05);2) Sucessfully building stable infection SiHa cell lines expressing AQP8gene andsilencing AQP8gene;3) Migration and invasion rate of AQP8-SiHa was obviouslyquicker than control groups (P＜0.05); Wound healing speed of AQP8-SiHa wasobviously quicker than control groups (P＜0.05); The difference of proliferation andsubstrate adherence between AQP8-SiHa and control groups was not significant (P＞0.05); The xenografts with AQP8showed finger-like local invasiveness intosubcutaneous adipose and muscle, but had no effects on tumor volumes (P＞0.05);Migration and invasion rate of AQP8shRNA-SiHa was obviously lower than controlgroups (P＜0.05); Wound healing speed of AQP8shRNA-SiHa was obviously lower thancontrol groups (P＜0.05); The difference of proliferation and substrate adherencebetween AQP8shRNA-SiHa and control groups was not significant (P＞0.05); Thexenografts with AQP8shRNA showed no significance in local invasion and tumorvolumes compared with control group (P＞0.05). Conclusions:1) Osmotic waterpermeability of SiHa cells is the lowest, and subsets of aquaporins expressing in Sihacells may be related with their migration and infiltration; Expression of AQP1, AQP3andAQP8in cervical carcinoma may be related with cervical carcinogenesis anddevelopment;2) Stable infection SiHa cell lines expressing AQP8gene and AQP8shRNA are used to study AQP8gene function in cervical carcinoma, and provide theresearch basis for AQP8gene effects on the migration and invasion of SiHa cells in vivo and in vitro;3) Overexpression of AQP8gene facilitates the migration and localinvasiveness potential of SiHa cells, suppression of AQP8gene expression can inhibit themigration and local invasiveness potential of SiHa cells, AQP8gene may participate inthe invasive process of tumors. |
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