The effects of miR-25 and the potential molecular mechanisms on migration and invasion of triple negative breast cancer / 中华检验医学杂志

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer(TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression.

Expression of miR-145 in breast cancer and its role in invasion and migration of breast cancer cells / 中华检验医学杂志

Objective To investigate the expression level of microRNA-145 in breast cancer cell lines andtissues and its impact on breast cancer invasion and metastasis.Methods MiR-145 expression was detected by FQ-PCR in 5 breast cancer cell lines ( HBL-100, MCF-7, MDA-MB-231, MDA-MB-468 and SK-BR-3)and in breast cancer tissue and paraneoplastic tissues (n=39).The miR-145 expression plasmid ( Psif-miR-145 ) and negative control plasmid were transfected into SK-BR-3 using lipofectamine, respectively.The characteristics of invasion and migration of the transfected SK-BR-3 cells were examined by scratch test and transwell assay.The target genes of miR-145 were predicted by bioinformatics and the ANGPT2 gene were verified as miR-145 target by the dual-luciferase reporter assay.The expression levels of ANGPT2 protein was examined by western blot after pSIF-miR-145 transfection by lipofectamine in breast cancer cell line SK-BR-3.Results FQ-PCR result indicated that miR-145 expression level waslower in breast cancer tissue (45.93 ±22.02)than paraneoplastic tissue [ (182.04 ±56.92), U value was 7, P<0.01].MiR-145 expression level was lower in breast cancer cell lines than normal breast cells.miR-145expression in 4 breast cancer cell lines was 0.51 ±0.05, 0.07 ±0.01, 0.36 ±0.04 and 0.04 ±0.01, respectively.Compare with normal breast cell, miR-145 was lower expressed in all 4 breast cancer cell lines (t value separately was 15.93, 308.17, 25.02, 201.30;P<0.05).Lower expression of miR-145 was observed in the highly invasive breast cancer cells (MDA-MB-231, MDA-MB-468 and SK-BR-3), compared with weakly invasive breast cancer cell (MCF-7) (t value separately was14.18, 3.78, 15.20;P<0.05). Wound healing assay shows that overexpression of miR-145 in SK-BR-3 significantly reducedthe motility as compared with control group (P <0.01).The cell invasion assay indicated the numbers of miR-145 overexpressed SK-BR-3 cells, which invased to lower chamber, was 137 ±37, the numbers of invased cells was 617 ±80 when the negative control was applied. Over-expression of miR-145 could repress the expression levelsof ANGPT2 protein;miR-145 could repress the activity of luciferase reporter carrying a 3′-untranslated region of ANGPT2 mutated the predicted binding site, the activity of luciferase was reversed. Conclusions MiR-145 depressed in breast cancer cell lines and breast cancer tissues.MiR-145 maybe plays an important role in breast cancer invasion and migration by directly target ANGPT2.

Expression of miR-21 in breast cancer and its role in invasion and migration of breast cancer cells / 中华检验医学杂志

Objective To investigate the expression of microRNA-21(miR-21)in breast cancer cell lines and serum of patients with breast cancer and the impact on the invasion and migration of breast cancer cells.Methods From Jan 2013 to Feb 2014, miR-21 expression were determined by fluorescent quantity polymerase chain reaction (FQ-PCR) in 4 breast cell lines (HBL-100, MCF-7, MDA-MB-231 and MDA-MB-468) and in serum from breast cancer patients ( n =56 ) , breast benign disease patients ( n =39 ) andhealth controls ( n =45 ) . The characteristics of cell invasion and migration were examined by transwellinvasion and migration assay afterbreast cancer cell line MDA-MB-231 were transfectedwith miR-21 inhibitor or negative control by lipofectamin.The t test was used to analysis the normal distribution data. Results FQ-PCR results showed that the relative expression of miR-21 in the normal breast epithelial cell line HBL-100 was 1.01 ±0.04, in the breast cancer cell line MCF-7, MDA-MB-231 and MDA-MB-468 were 1.99 ±0.11,4.02 ±0.38 and 3.73 ±0.79 respectively.Compared with the normal controls, miR-21 were highly expressed in the three breast cancer cell lines, the difference was statistically significant (t=9.01, 9.20 and 4.55, respectively, P<0.01); and the miR-21 was highly expressed in invasive and metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-468),compared with weakly invasive breast cancer cell line MCF-7, the difference was statistically significant ( t values were 6.14 and 2.91, P<0. 05), suggesting that miR-21 is highly expressed in breast cancer cells, and is closely related to the invasion and metastasis.The relative expression of miR-21 in serum of breast cancer was 2.63 (1.57-4.59), in benign breast disease group was 1.34 (1.01-1.78), in healthy control group was 0.81 (0.52-1.59), the miR-21 expression in the serum of breast cancer patients was significantly higher than in patients with benign lesions and normal control group (U values were 208 and 279, P<0.01), whereas no significant difference in serum in patients with benign lesions and normal control group, the miR-21 expression in the serum of breast cancer patients with lymph node metastasis (U=95 , P=0.19) was 3.55 (2.44-5.26), significantly higher than those without lymph node metastasis [2.11(1.59-3.25), U=216,P=0.021]. The results of invasion and migration assay showed that cells treated with miR-21 inhibitor invasion was:44 ±18, the number of cell migration was:98 ±22, while the negative control treated cells after invasion was:133 ±44, migration cell number:255 ±35;miR-21 inhibitor treatment compared with the negative control, cell invasion and migration was also significantly decreased( t values were 5.46 and 9.08, P<0. 01) .The cell invasion and migration assay indicated the numbers of MDA-MB-231 cells, which invaded or migrated to lower chamber, were 44 ±18 and 98 ±22 respectively after miR-21 inhibitor was applied, The numbers of invaded or migrated cells were 133 ±44 and 255 ±35 when the negative control was applied.The ability of cell invasion and migration was decreased significantly in the inhibitor group compared with the negative group(tvalue separately was 5.46, 9.08, P<0.01).The capacity of breast cancer cell invasion and migrationwas significantly decreased after transfection ofmiR-21 inhibitor.Conclusions MiR-21 is highly expressed in breast cancer cell lines and breast cancer patients′serum.Altered expression of miR-21 maybeplays an important role in breast cancer invasion and migration.MiR-21 may serve as new biomarker to early detectionand prognosis estimation of breast cancer.

The protection of lianhuaqingwen against acute radiation-induced lung injury in rats / 中华放射医学与防护杂志

Objective To investigate the radioprotective function of lianhuaqingwen (LHQW) in rat acute radiation-induced lung injury.Methods Totally 36 female Wistar rats were randomized into 3 groups as administered group (treated by LHQW plus radiation),radiation group irradiated with a single of 20 Gy in 6 MV X-ray by Elekta Synergy VMAT,and blank control group without radiation.Performance status (PS) was estimated during 31 d of LHQW instragastric administration.After rats being sacrificed at 1,14,28 d of LHQW adminstration,the pathomorphological changes were observed in trauma lung tissue,the cell number in BALF (Bronchoalveolar lavage fluid) was counted,the levels of TNF-α and IL-6 in serum were measured by ELISA,and TNF-α and IL-6 mRNA expressions in lung tissue were assayed by RT-PCR.Results After LHQW treatment,the PS of rat was significantly elevated with less inflammation in morphous,and the cell number in BALF was markedly decreased in compare with radiation alone group.Furthermore,the serum levels of TNF-α and IL-6 were obviously reduced (tTNF-α =7.372,2.891,tIL-6 =6.335,3.257,P ＜ 0.05) and the TNF-α and IL-6 mRNA levels in lung tissue were also decreased (tTNF-αmRNA =3.714,2.144,tIL-6mRNA =3.589,2.883,P＜0.05).Conclusions LHQW plays a protective role against acute radiation-induced lung injury in rats and the down-expressions of TNF-α and IL-6 may be involved.

Lianhuaqingwen Capsules Inhibited the Expression and Effect of MCP-1 in Rats with Radiation-induced Acute Lung Injury / 医药导报

Objective To explore the inhibitory effect and possible mechanisms of lianhuaqingwen capsules on radiation-induced acute lung injury in rats. Methods Rats were randomly divided into control group, radiation group and radiation plus lianhuaqingwen group, the control group and the radiation group rats were given 0. 9% sodium chloride solution, the radiation plus lianhuaqingwen group rats were given lianhuaqingwen 0. 9% chlorine sodium solution. HE staining was applied to test the lung tissue inflammation; quantitative RT-PCR and ELISA were used to measure the content of IL-6, TNF-α and MCP-1 in rats;immunohistochemical assay was taken to detect the infiltration of macrophage in lung tissues. Results The relative mRNA expression of IL-6, TNF-α and MCP-1 in the control, radiation model control and radiation plus Lianhuaqingwen groups were (0. 002 1±0. 000 20),(0. 006 6±0. 000 32),(0. 003 9±0. 000 22); (0. 003 7±0. 000 16),(0. 007 4±0. 000 33),(0. 005 5± 0.000 24);(0.001 4±0.000 15),(0.005 4±0.000 72),(0.003 2±0.000 17),respectively; the concentration (pg·mL-1) of IL-6,TNF-αand MCP-1 in the serum were (35. 2±10. 9),(111. 8±26. 1),(68. 2±15. 2); (229. 3±28. 5),(837. 5±57. 6), (566. 9±39. 8);(96. 85±8. 20),(314. 53±12. 76),(191. 32±10. 97),respectively; and the macrophages at high magnification field in each group were (59. 5±4. 3),(503. 9±25. 8)and (106. 2±12. 6), respectively. Lianhuaqingwen capsules significantly alleviated the lung inflammation in rats with radiation-induced acute lung injury,inhibited the accumulation of macrophage in lung tissue,reduced the expression of IL-6 and TNF-α,and decreased the content of MCP-1 in lung tissues and sera(P<0. 05). Conclusion Lianhuaqingwen capsules attenuated the lung inflammation developed in rats with radiation-induced acute lung injury through inhibiting the expression of MCP-1 and reducing the accumulation of macrophage in lung tissues.

Clinical significance and expression of microRNA-21 in diffuse large B-cell lymphoma cell lines and serum of patients / 中华检验医学杂志

ObjectiveTo study the expression of microRNA-21 ( miR-21 )in serum of patient with diffuse large B cell lymphoma (DLBCL) and DLBCL cell lines and validate the significance of miR-21 in early diagnosis,genotyping and prognosis estimates of DLBCL.MethodsmiR-21 expression were detected by fluorescent quantity polymerase chain reaction (FQ-PCR)in 9 lymphoma cell lines (OCI-Ly1,OCI-Ly3,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly10,OCI-Ly18,OCI-Ly19 and HBL),the serum from DLBCL patients (n =62) and health controls (n =50 ).Kaplan-Meier survival analysis was carried out during the relapsefree survival period of DLBCL patients to explore the relationship between the prognosis and microRNA expression level.ResultsReal time FQ-PCR result indicated that miR-21 expression was higher in DLBCL cell lines than that in normal B cells (BC).miR-21 expression in normal B cell and 9 DLBCL cell lines separately were 1.04 ± 0.02,2.30 ± 0.35,237.97 ± 56.19,5.27 ± 0.83,3.40 ± 0.30,11.22 ± 2.70,133.55 ± 16.78,6.63 ±0.24,4.91 ±0.37 and 81.59 ±6.64.Compared with BC,the expression of miR-21 were higher in all 9 DLBCL cell lines ( t =7.3,13.7,21.0,6.2,8.8,13.6,6.5,39.5,18.1 ;P ＜ 0.01 ).miR-21 expression segregates with specific molecular subgroups of DLBCL The expression was higher in the ABC type cell lines (OCI-Ly3,OCI-Ly10,HBL) than GCB type cell lines (OCI-Ly1,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly18,OCI-Ly19;t =11.18,P ＜ 0.01 ).Consistent with the cell line models,miR-21 expression levels were higher in serum from DLBCL patients [21.38 (10.26-45.21 )] than from controls [1.87 ( 1.05-3.97 ),U =168,P =0.000],and the levels were higher in DLBCL cases with an ABC-type [28.68 ( 14.92-98.44 )] than those in GCB-type [18.30 ( 7.32-33.46 ),U =336,P =0.043].MiR-21 expression levels were different in sera from different clinical stage DLBCL patients.The miR-21 level in serum of patients with subgroup ABC and subgroup GCB in stage Ⅰ and Ⅱ were 47.49( 25.65-295.41 ) and 24.74( 16.08-50.38) respectively and in stage Ⅲ and Ⅳ were 16.66 ( 5.35-44.30 ) and 11.96 ( 4.10-21.05) respectively.The levels were higher in DLBCL cases withⅠ -Ⅱ stage than those with Ⅲ-Ⅳ stage (U =62,P =0.013 in GCB type; U =53,P =0.014 in ABC type).Moreover,compare with relapse-free survival in DLBCL patients,high miR-21 expression was associated with well prognosis ( U =259,P =0.035).ConclusionsMiR-21 is high expression in DLBCL cell lines and DLBCL patients serum.miR-21 level in sera from DLBCL patients is associated with clinical stage,molecular subgroup and prognosis estimates.MiR-21 may serve as a new biomarker to early detection,genotyping and prognosis estimates of DLBCL.

The expression and its clinical significance of microRNA-301 in pancreatic carcinoma / 中华检验医学杂志

Objective To study the expression of microRNA-301 in pancreatic carcinoma andvalidate the significance of miR-301 in invasion and metastasis of pancreatic carcinoma.Methods miR-301 expression were detected by FQ-PCR in 5 pancreatic cancer eell lines(PANC-1,PaCa-2,AsPC-1,Hs766T.BxPC-3).Further immunohistochemistry in pancreatic cancer tissue microarrays was detected miR-301 expression,which contained 60 pancreatic cancer specimens along with 10 normal adjacent tissues and 10 normal pancreas tissues.After high expression of miR-301 in pancreatic carcinoma being confirmed.the clinical significance of high expression of miR-301 in invasion and metastasis of pancreatic carcinoma were studed.Pancreatic cancer cell lines(PANC-1.PaCa-2)were transfected by 100 nmoml/L miR-301 inhibitor(anti-miR-301)or negative eontrol(Anti-miR~(TM) Negative Control#1).COX-2 and MMP-2 protein expression in pancreatic cancer cell lines were detected by WB.and cell migration assays were performed using transwell technology.Results FQ-PCR resuhs indicated that miR-301 expression was higher in pancreatic cancer cell lines than normal pancreatic cells.The relative level of miR-301 in 5 pancreatic cancer cell lines(PANC-1,PaCa-2,AsPC-1,Hs-766T,BxPC-3)and normal pancreatic cell were 33.09± 4.21,30.76±3.18,47.57±3.56,20.20 ±1.21,76.75±13.51 and 1.00±0.08 respectively.The miR-301 level in all 5 pancreatic cancer cells were significantly higher than those of normal pancreatic cell(t=8.86,9.53,6.39,6.77,11.18,P<0.01).Immunohistochemistry results also showed miR-301 expression was higher in pancreatic carcinoma tissues than those in the cancer adjacent tissues and normal pancreatic tissues.The relative levels of miR-301 in pancreatic carcinoma tissues.normal adjacent tissues and normal pancreas tissues were 0.88±0.09,0.22±0.04 and 0.14±0.05 respectively.The miR-301 levels in pancreatic carcinoma tissues were significantly higher than those of normal adjacent tissues and normal pancreatic tissues(t=15.1,10.6,P<0.01).There was no significant difference between normal adjacent tissues and normal pancreas tissues(t=1.32,P=0.22).After miR-301 inhibitor was introduced into pancreatic cancer cells PANC-1 and PaCa-2.miR-301 levels were reduced while the protein levels of COX-2 and MMP-2.which were invasion and metastasis related factors,were down-regulated.The cell migration assay indicated the numbers of PANC-1 and PaCa-2 cells,which migrated to lower chamber.were 587±27 and 363±13 respectively after miR-301 inhibitor was applied.The numbers of migrated cells were 1091 4-15.737±44 when the netative control was applied.The cell invasion ability was decreased significantly in the inhibitor group compared with the negative group(t=7.89,7.56,P<0.01).Conclusions miR-301 is highly expressed in pancreatic cancer cell lines and pancreatic cancer tissues.Inhibition of miR-301 expression can effectively supress the invasion of pancreatic cancer cells.miR-301 may serve as a new biomarker for early detection of pancreatic cancer and molecular target for early treatment of pancreatic cancer.

Clinical analysis of 32 cases of cerebellar hemorrhage in the elderly / 中华老年医学杂志

Objective To study clinical characteristics and CT features of cerebellar hemorrhage in the elderly patients in order to make early diagnosis and treatment. Methods The clinical characteristics, CT scan, sites and causes of hemorrhage, as well as the treatment and the prognosis of 32 elderly patients with cerebellar hemorrhage were analysed. Results The main clinical symptoms were vomiting, vertigo and headache and were observed in 32(100.0%), 26(81.2%) and 18(56.2%) cases, respectively. High motality rate was observed in hemorrhage volume≥25 ml, patients aged over 70 years and accomanied with cerebral hernia and multi-organ failure. Conclusions The clinical manifestations of cerebellar hemorrhage were not typical in the elderly. Cerebral hernia and multiple organ failure were the main causes of death. Hypertension and artherosclerosis were the main etiologies of cerebellar hemorrhage. The early diagnosis is necessary for the treatment of cerebellar hemorrhage. The prognosis was related to the quantity and site of hemorrhage.