The Expression Of Hiwi Gene And Its Effect On The Growth Of Breast Cancer Celts

Breast cancer is a common kinds malignant tumours in women,with an increasing incidence, especially among younger woman. Atpresent, the etiology and pathogenesis of breast cancer are not clear. TheHiwi gene is the human homolog of the piwi family, locating in12q24.33and encodes Hiwi protein of98.5kDa, known to be requiredfor stem cell self-renewal. Recently, accumulating reports have revealedthat abnormal Hiwi expression is associated with poorer prognosis ofhuman malignant tumors, such as human esophageal squamous cellcarcinoma, human adenocarcinoma of the pancreas, human gastriccancer, and colorectal cancer, and the high degree of Hiwi expressionwas significantly correlated with the lower degree of differentiation,deep invasion and metastasis. Therefore, Hiwi gene is closely related tothe tumor occurrence and development.But whether the high expressionof Hiwi in breast cancer tissue, whether Hiwi affects breast canceroccurrence and development, there is no related research has been doneat home and abroad.In present study, we evaluated the expression of Hiwi in humanbreast cancer specimens, and in breast cancer cell lines, and observed theHiwi gene effect on growth of human breast cancer cells byoverexpression and suppression expression of Hiwi gene then elaboratethe relativity between Hiwi gene and breast cancer occurrence anddevelopment. Firstly, Overexpression of Hiwi in breast cancer specimens andbreast cancer cell lines, and is associated with the cancergrade malignancy.47human breast cancer specimens and47peritumor specimens wereresected from47breast cancer patients, breast cancer cell lines,MDA-MB-231, MCF-7and MCF-12A were cultured in vitro。TotalRNA from the intra-tumor, peritumor specimens, or the breast cancercell lines was extracted and detecting mRNA expression of Hiwi gene byquantitative real-time PCR（RT-PCR）； Proteins of breast cancerspecimens and cultured breast cancer cells were collected and analyzedfor Hiwi expression by western Blot. Then the correlation of Hiwiexpression with clinico-pathological variants were analyzied.First aspect, the RT-PCR revealed a significant high Hiwi level inthe intratumor specimens than in the peritumor specimens from47breast cancer patients. That the Hiwi expression was not associated withthe age and menopause. However, there was a significant association ofHiwi overexpression with the tumor size, lymph node metastasis andhistological grade. The Hiwi expression in patients with larger size,axillary lymph node metastasis or higher histological grade wassignificantly higher. Second aspect, we re-evaluated the Hiwi expressionin protein level by western blot amalysis in15intratumor specimens and15peritumor specimens. The protein level of Hiwi in the intratumorspecimens was also significantly higher than in the peritumor specimens.And the significant high Hiwi mRNA level was also found in breastcancer cell lines, MDA-MB-231, MCF-7, but not in MCF-12A, comparedto the primary breast cells there was a significant high level of Hiwiexpression in protein level in both MDA-MB-231and MCF-7cells, but notin MCF-12A cells. Taken together, we confirmed the overexpression of Hiwi in breast cancer specimens and breast cancer cell lines.Secondly, Hiwi over expression promotes the growth of breastcancer cells, MCF-7pcDNA3.1（+） and theHiwi-2A-eGFP-pcDNA3.1（+） vectors were transfected into MCF-7cells respectively by liposome.The positive cell clones were selected inthe presence of G418. Quantitative real-time polymerase chain reactionand Western Blot analysis for Hiwi expression.The growth of breastcancer cell in vitro was assessed by cell count assay at8thday andcolony formation assay at6thday.A significantly higher level of Hiwi mRNA was observed in variouspassages of the MCF-7cells with Hiwi gene transfection compared tothe control cells，and the Hiwi overexpression in protein level wasstabilized in the MCF-7cells with Hiwi gene transfection。The cell count assay was performed for cell counting every otherday for three times post cell inoculation. MCF-7cells with Hiwi genetransfection grew more efficiently than MCF-7control cells, particularlyat4or6day post inoculation, with a significant difference Furthermore,there was more colonys formed by MCF-7with Hiwi gene transfectionthan MCF-7control cells.Thus, we confirmed the promotory role of Hiwi to the growth ofbreast cancer cells by both kinds of assays.Thirdly, RNAi-mediated Hiwi knockdown blocks thepromotion of overexpressed Hiwi to the MCF-7cell growth.To further confirm the promotion by Hiwi to the growth of breastcancer cells, we knockdown the Hiwi with Hiwi specific siRNAs in theMCF-7Hiwi (+) cells, and then to determine the influence of Hiwiknockdown on the breast cancer cell growth. indicated that both siRNAssignificantly blocked the Hiwi expression in both mRNA and protein levels, compared to the siRNA-Con We then evaluated the influence ofHiwi knockdown on the growth of breast cancer cells. By cell countassay and colony formation assay at6thday. The cell count assay wasperformed for cell counting every other day for three times post theinoculation of103/mL MCF-7Hiwi (+) cells.Both siRNAs significantlyinhibited the MCF-7Hiwi (+) cell growth at4or6day post inoculation,compared to the siRNA-Con. The numbers of MCF-7Hiwi (+) formedcolonies in both siRNA-Hiwi1and siRNA-Hiwi2groups weresignificantly less than in the control siRNA group. Thus, we reconfirmedthe promotory role of Hiwi to the growth of breast cancer cells by theloss-of-function strategy.In summary, present study confirmed the overexpression of Hiwi inbreast cancer specimens and breast cancer cell lines, identified thepromotion of Hiwi to the growth of breast cancer cells, using both thegain-of-function and loss-of-function approaches: Overexpressed Hiwipromoted MCF-7cell growth, while Hiwi knockdown by RNAi methodinhibited the promotion by Hiwi. It implied the oncogenic role of Hiwiin breast cancers. Hiwi gene will be new target for breast cancer therapy.