The Tissue Distribution Of RSK4in Human Normal Tissues And Its Expression And Function In Renal Cell Carcinoma

【Background】Renal cell carcinoma is one of the most common malignant tumors inurinary system and accounts for approximately four percent of epithelial cancersin adult. Clinically, RCC can remain occult for the majority of its course,leading to the fact that about thirty percent patients present with metastasis wheninitially diagnosed. Besides, RCC is resistant to radiation and chemotherapy, andmany patients who undergo curative surgical resection experience recurrenceduring subsequent follow-up, which bring great difficulties to the diagnosis andtreatment of RCC in administration. Therefore, it is highly essential to explorenew specific and effective factors for diagnosis, prognosis prediction as well asthe therapeutic targets of RCC. RSK4is a member of ribosomal s6kinase (RSK) family and consisted of746amino acids, but its functions remain largely unknown. There are only fewstudies about the distribution of RSK4mRNA in human normal tissues;however, the expression of RSK4protein in human normal and tumorous tissueshave not been.reported systematically. In most studies, RSK4is considered as apotential tumor suppressing gene, and it is reported that RSK4expression isreduced in some tumors and RSK4overexpression could be against the invasionand metastasis of the tumor cells. To our knowledge, the expression and functionof RSK4in RCC is rare, and there was only one article reported that RSK4mRNA level of fresh RCC tissues is lower than that in adjacent tissues, of whichthe possible mechanism is that RSK4may modulate senescence induction andcontributing to cell proliferation control. However, so far the studies of RSK4are very limited and its tissue distribution remains unclear. Only few articleshave reported the expression of RSK4mRNA in human tissues, nevertheless,there is no systemic study about the expression of RSK4protein in humannormal tissues and all kinds of tumorous tissues. Therefore, RSK4expression inRCC remains unclear. Whether it acts as a pro-or an anti-tumor gene needs tobe determined, and this is exactly what we try to clarify in our research.【Objectives】1. To study the distribution of RSK4protein in different kinds of humannormal and tumorous tissues, and provid a basis for further functionalresearch;2. To analyze the expression of RSK4protein in RCC and its relationship withthe clinocopathologic features and prognosis of RCC patients;3. To preliminarily explore the expression of RSK4and its molecular mechanisms of function in RCC cell line.【Methods】1. Tissue micoarray was used to detect the expression of RSK4protein inthirty types of human normal tissues and fifty-four types of tumoroustissues by immunohistochemistry;2. The expression of RSK4protein was detected in thirty-one RCC microarraytissues and seventy surgical specimens by immunohistochemistry;3. Chi-square test was used to analyze the relationship of RSK4expressionand the clinicopathologic features of RCC patients;4. Kaplan–Meier method and Cox hazard regression model were employed toanalyze the relationship of RSK4expression and survival in RCC patients;5. The expression of RSK4in RCC cell line RPC and GRC-1were detected byquantitative real-time PCR (qRT-PCR) and western blot;6. Up-and down-regulation of RSK4were studied by lipofectamine-mediatedgene transfection, the results of which were confirmed by qRT-PCR andwestern blot at the mRNA and protein level, respectively;7. The regulation of RSK4level on the cell cycle of RCC cell lines wasstudied by flow cytometry;8. The affect of RSK4level on the ability of invasion and metastasis of RCCcell lines was detected by transwell method.【Results】1. The positive expression of RSK4was found in twelve types of humannormal tissues with different intensity, including pancreatic ductal epithelialcells, salivary epithelial cells, sweat gland epithelial cells and Blymphocytes in germinal centre of tonsil, and the expression of RSK4in renal tubular epithelial cells, myocardial cells and hepatic cells, etc.,wasweak. Among fifty-four type tumors, positive expression of RSK4wasobserved in fourteen types of human tumorous tissues, of which sometumors showed strong positivity of RSK4, including clear cell renal cellcarcinoma, uterus clear cell carcinoma, ovarian serous papillarycystadenocarcinoma and gastric adenocarcinoma, while some tumorsmanifested with weak positivity such as breast cancer and hepatocellularcarcinoma, etc．2. The positive rates of RSK4in RCC microarray tissues and surgicalspecimens were74%（23/31）and55%（39/70）, respectively;3. For thirty-one RCC microarray tissues, RSK4expression was not correlatedwith the age, gender, tumor size, pathological subtypes, lymph nodeinvolvement and distant metastasis（P>0.05）; instead, its expressionsignificantly correlated with the Fuhrman grading （P=0.032）and staging（P=0.029）of tumors, and the RSK4positivity was getting stronger withthe increase of tumor grading and staging;4. For seventy surgical specimens, RSK4expression was not correlated withthe age, gender, tumor size and distant metastasis（P>0.05）; however, therewas a significant positive correlation between the expression of RSK4andtumor Fuhrman grading （P<0.001）, staging （P=0.006）and lymphnodeinvlovment（P=0.012）; in additon, RSK4expression varied in differentpathological subtype of RCC（P=0.038）, and clear cell RCC（77.8%，14/18）showed higher positivity of RSK4than papillary RCC （50%，21/42）andchromophobe RCC（16.7%，1/6）; moreover, in papillary RCC, the RSK4positivity is much higher in type2（71.4%，20/28） than that in type1 （7.1%，1/14）（P<0.001）;5. Uni-and multivariate survival analysis showed the positive expression ofRSK4predicted a poor survival（P=0.001）in all RCC patients andespecially the papillary RCC patients（P=0.032）;6. The results of qRT-PCR and western blot showed that the expression ofRSK4in RCC cell lines was higher than that in normal renal epithelial cellline; furthermore, the RSK4level varied in different RCC cell lines;7. Flow cytometry analysis showed that compared with untreated RPC cells,RSK4-overexpressed RPC cells showed decreased proportion of G0-G1phase cells （71.08±0.51vs.73.46±0.24，P<0.05）but increased proportionof S phase（17.95±0.18vs.15.69±0.36，P<0.05）, however, there was nosignificant difference for the proportion of G2-M phase cells in the twogroup (10.97±0.49vs.10.85±0.39，P>0.05); on the contrary, compared withuntreated GRC-1cells, siRSK4GRC-1cells had significant increase of theG0-G1phase cells （75.39±0.25vs.59.68±0.21，P<0.05）and decrease ofS and G2-M phase cells （19.85±0.87vs.27.92±0.87；4.76±0.14vs.12.40±0.27，P<0.05）.8. Transwell studies found that RSK4-overexpressed RPC cells showedenhanced ability of invasion and migration, which was about two timeshigher than that in untreated RPC cel（l2.2±0.11vs.1，P<0.05）; instead, theability of invasion and migration in siRSK4GRC-1cells was reduced toone-third of the normal GRC-1cells（0.36±0.05vs.1，P<0.05）.【Conclusions】1. For the first time, we systematically reveal the distribution of RSK4proteinin different kinds of human normal and tumorous tissues; 2. The expression of RSK4protein varies in different pathologic subtype ofRCC, indicating that it could be helpful for the differential diagnosis ofRCC subtypes, especially the type1and type2papillary RCC;3. RSK4could be a potential independent prognostic prediction factor forRCC;4. RSK4could promote the cell cycle progression and the ability of invasionand migration in RCC cell lines, indicating that RSK4might be one of thekey molecules in RCC progression and could serve as a new potentialtherapeutic target for RCC patients. Based on the above results, wedemonstrate the fact for the first time that RSK4may act as an oncogene inthe tumorigenesis of RCC.