Effectand Mechanism Of Silencing HAS2by RNA Interference On Biologic Activity Of Breast Cancer Cell

PART IEXPRESSION AND CLINICAL SIGNIFICANCE OF HAS2IN BREAST CANCERObjective: To investigate whether the HAS2proteins expresseddifferentially in breast cancer tissues and normal breast tissues. This studyfocused on relationship between the abnormal expression of HAS2andclinical pathological features of breast cancer. The abnormal expression ofHAS2in breast cancer was important theoretical basis for early diagnosisand prognosis of breast cancer patients.Methods: In this section we detected the expression level of HAS2protein using immunohistochemistry in55invasive ductal breast carcinomaspecimens and10normal breast tissue samples for preliminary screeningand testing the abnormal expression of HAS2protein in breast cancer. Thedifferent expression level of HAS2protein was observed in breast cancerand normal breast tissues. The relationship between abnormal expression of HAS2and clinical pathological features of breast cancer patients wasexplored. Detecting the expression of HAS2was significant for earlydiagnosis and prognostic of breast cancer patients.Results: The expression level of HAS2protein was high in breastcancer tissues, the positive rate was56.36%, while the rate was10%innormal breast tissue. The expression level of HAS2protein wassignificantly higher in breast cancer than normal breast tissue (P <0.05),and the difference was statistically significant. The expression levels ofHAS2protein was correlated with tumor size, TNM stage and lymph nodemetastasis, but there was no significant correlation with age, ER, PR,CerB-2, P53in breast cancer patients.Conclusion: The expression level of HAS2protein was significantlyhigher in breast cancer than normal breast tissue. The expression levels ofHAS2protein was correlated with tumor size, TNM stage and lymph nodemetastasis.HAS2may be involved in invasion and metastasis of breastcancer, and HAS2detection can be used as a diagnostic marker of breastcancer. PART IITHE SPECIFIC HAS2-SHRNA INHIBITED THE CELLPROLIFERATION AND INVASION OF BREASTCANCER AND THE MECHANISM ABOUT ITObjective: To study the effect of knockdown of expression of HAS2on cell proliferation, invasion and other malignant behavior in breast cancerand to explore the mechanisms about it. To clear whether HAS2wasinvolved in the invasion and metastasis of breast cancerMethods: In vitro experiment, we detected the expression levels ofHAS2mRNA and protein in breast cancer cell lines (BT549, Hs578T,MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3and T47D) by usingRT-PCR, Real-time PCR and Western blot method. RNA interferencetechnology was used to detect the function of HAS2in breast cancer. Thetargeting HAS2-shRNA was transfected into Hs578T human breast cancercells using liposome Lipofectamine2000transfection reagent. Real-timePCR were used to test the silencing efficiency about HAS2gene. Next, wescreened the stable lines to obtain the Hs578T cell lines stably transfectedwith HAS2-shRNA. In the end, western blot was used to verify the stabilityof strains. In this part, we used the clonogenic assay and CCK-8experiment to test the impact of knockdown of HAS2on cell proliferationcapability of breast cancer; used the scratch assay to test the impact of knockdown of HAS2on cell migration capability of breast cancer; usedtranswell chamber for model to test the impact of knockdown of HAS2oncell invasion ability of breast cancer; used HUVEC cells angiogenesisassay to test the impact of knockdown of HAS2on angiogenesis of breastcancer; used flow cytometry to detect changes in the breast cancer cellcycle and apoptosis after transfected the HAS2-shRNA; Finally, we usedthe ELISA-like assay to detect expression level of hyaluronic acid (HA)after silencing HAS2gene.Results: The HAS2mRNA and protein levels were significantlyup-regulated in breast cancer cells compared with the non-malignant breastepithelial cells, and the difference was statistically significant (P <0.05),and further studies have shown that HAS2mRNA, protein level in thebreast cancer cells Hs578T were higher than other breast cancer cell lines.To target different regions of HAS2mRNA sequences, we constructed fiveshRNA (HAS2-shRNA1, HAS2-shRNA2, HAS2-shRNA3, HAS2-shRNA4and negative-shRNA). After transfected with five groups of shRNA,Quantitative real-time PCR test results show that expression level of HAS2mRNA in four experimental groups were inhibited relative to the controland negative-shRNA transfected group. The HAS2mRNA and proteinlevels were significantly up-regulated in breast cancer cells compared withthe non-malignant breast epithelial cells, and the difference was statisticallysignificant (P <0.05), and further studies have shown that HAS2mRNA, protein level in the breast cancer cells Hs578T were higher than otherbreast cancer cell lines. To target different regions of HAS2mRNAsequences, we constructed five shRNA (HAS2-shRNA1, HAS2-shRNA2,HAS2-shRNA3, HAS2-shRNA4and negative-shRNA). After transfectedwith five groups of shRNA, Quantitative real-time PCR test results showthat expression level of HAS2mRNA in four experimental groups wereinhibited relative to the control and negative-shRNA transfected group. Aseries of functional experiments associated with malignant behavior ofbreast cancer were conducted after HAS2-shRNA3was stably transfectedinto Hs578T cell lines. The results of clonogenic assay and CCK-8showedthat knockdown of HAS2could inhibit the proliferation of breast cancercell; the results of scratch experimental showed that the migration abilitywas inhibited breast cancer cell after HAS2was reduced; the results oftranswell chamber model showed that the invasion ability of breast cancercell was also inhibited after knockdown of HAS2; the results ofangiogenesis of HUVEC cells experiment indicated that knockdown ofHAS2could inhibit the formation of blood vessels; the results of flowcytometry indicated that after transfected HAS2-shRNA3, breast cancercells were stayed in G0/G1phase, the number of cells in S phase werereduce and the number of apoptotic cells were increased. Finally, the resultsof ELISA-like analysis showed that after the HAS2gene was silenced, theexpression level of hyaluronic acid (HA) was also decreased. Conclusion: The HAS2gene was effectively silenced byHAS2-shRNA, and breast cancer cell lines Hs578T was stably transfectedwith HAS2-shRNA was obtained, which laid the foundation for thefunction research of HAS2. Knockdown of HAS2expression, themalignant behavior of breast cancer cells were inhibited, and themechanism may be downregulation of HAS2will cause the reduction ofHA synthesis. In other words, HAS2-HA dynamic system may participatein the malignant behavior of breast cancer cells. PART ⅢTHE SPECIFIC HAS2-SHRNA INHIBITED THEGROWTH AND DEVELOPMENT OF XENOGRAFTFROM BREAST CANCER CELL IN NUDE MICEObjective: To investigate the impact of downregulation of theexpression of HAS2on the tumor size, weight, and presence of distantmetastasis of breast cancer cells in nude mice and on the nutritional statusof nude mice. To clarify the specific HAS2-shRNA inhibited thetumorigenesis of breast cancer cells in nude mice.Methods: Hs578T cells stayed in logarithmic growth phase, Hs578Tcells stably transfected with negative-shRNA and Hs578T cells stablytransfected with HAS2-shRNA3were collected and injected in nude micesubcutaneously, get to the nude mice xenograft model. We observed thetumors growth conditions dynamically in nude mice. Tumorigenesis time,the changes in tumor volume and weight were regularly recorded after theappearance of visible tumor.28days after inoculation, mice were killed.We cut the skin, stripped the whole tumor, weighed the tumor, tookpictures of nude mices sacrificed. Tumor tissues were fixed, embedded,sliced, and the pathology of tumor tissue is detected byimmunohistochemistry.Results: The forth day, visible tumor nodules of various sizes were observed at the injection site in nude mice of three groups, and theobservation was finished after28days. Experimental results showed thatthe tumor volume and weight of blank control group, negative-shRNAtransfected group and were significantly bigger than the HAS2-shRNA3transfected group, the nutritional status of the nude mice in the controlgroup and negative-shRNA transfected group were significantly worse thanHAS2-shRNA3transfected group. Anatomy of nude mice, the transfer wasnot found in the lungs, abdominal cavity or other parts of body.Histopathological results confirmed that the HAS2expression level oftumor tissue in HAS2-shRNA3group was significantly lower thancontrols.Conclusion: HAS2gene was still silenced by HAS2-shRNA in vivoexperiments. After downregulation of HAS2expression levels inhibited thegrowth in nude mice. The volume were reduced,. These results wereconsistent with those in vitro.